2018 CETA/ACTE & AETA JOINT CONVENTION PROCEEDINGS

Transcription

1 Canadian Embryo Transfer Association American Embryo Transfer Association 2018 CETA/ACTE & AETA JOINT CONVENTION PROCEEDINGS September 27-29, 2018 Montréal, Québec, Canada

2 A Special Thank You to our 2018 Joint Convention Sponsors and Exhibitors SPONSORS DOUBLE PLATINUM SPONSOR Vetoquinol PLATINUM SPONSOR Reproduction Resources GOLD SPONSOR RQR - Réseau Québécois en reproduction SILVER SPONSORS Boviteq MOFA Global Partnar Animal Health Professional Embryo Transfer Supply, Inc. (PETS, Inc.) Vytelle LLC BRONZE SPONSORS ABT 360, LLC Minitube USA, Inc. STgenetics & STgenetics Canada Trans Ova Genetics FRIEND SPONSORS Merck Animal Health Zoetis EXHIBITORS ABT 360, LLC Agtech, Inc. ART Lab Solutions Biogenics, Inc. ChemoMetec A/S Ciaq EastGen E.I. Medical Imaging IMV imaging IMV Technologies USA IVF Bioscience IVFtech Minitube USA, Inc. MOFA Global Oosafe, Inc. Partnar Animal Health Professional Embryo Transfer Supply, Inc. (PETS, Inc.) Reproduction Resources SP Scientific Universal Imaging Veterinary Concepts Vetoquinol Vytelle LLC WTA Technologies, LLC (As of September 5, 2018)

3 2018 Joint Convention Proceedings Pre-Conference Seminar I: Embryo Transfer 101 A 2018 Up-date... R. J. Mapletoft and J. F. Hasler Oviductal and Early Uterine Effects on Pregnancy Success Mario Binelli, Angela Maria Gonella-Diaza, Thiago Martins, Mariana Sponchiado Breed Associated Embryo Freezing Capacity Claude Robert, PhD Student/Technician Session: Damage Prevention at Storage and Transportation of Frozen Bovine Embryos Dr. Angelika E. Stock Delivering IVF Calves: Challenges and Opportunities. Gilles Fecteau, DMV, DACVIM What is Genomics and Epigenomics?. Claude Robert, PhD Sexed Semen Utilization in IVF and ET.. Daniela C. Pereira, Alfredo Castro, Eduardo Benedetti Student/Technician Session: Ultrasonography and Endocrine Parameters in Recipients with Successful Embryo Transfer. Dr. Angelika E. Stock Student/Technician Session: Sanitary Considerations at Collection, Search, Freezing and Transfer of Bovine Embryos Dr. Angelika E. Stock Starting an Oocyte Collection Lab and Incorporating IVF into Regular ET Practice.. Dr. Rob Stables Conception of a Bovine Ovum Pick Up Mobile Unit Lab.. Dr. Jonathan Lehouiller IVP Embryo Evaluation for the Receiving Practitioner.. Patti Anderson and Dr. Jeff Anderson Impact of Early Life Nutrition on the Sexual Development and Fertility of Cattle Dr. David A. Kenny The Au Courant Developments in Sex Sorted Semen and Application in Livestock Improvement Programs.. R (Vish) Vishwanath and J F Moreno Re-evaluating Ureaplasma diversum and its Potential Role in Bovine Embryo Transfer. M Bronwyn Crane and Anna Potter Early Pregnancy Losses in Dairy Cattle: Importance, Predisposing Factors, and Implications for Management. Dr. Eduardo de Souza Ribeiro Speaker Biographies Sponsors & Exhibitors... Inside front cover 1

4 CETA/ACTE Office Canadian Embryo Transfer Association P.O. Box 39 Kemptville, Ontario, Canada K0G 1J0 Tel: Fax: Web site: AETA Office American Embryo Transfer Association 1800 South Oak St., Suite 100 Champaign, Illinois, USA Tel: Fax: Web site: DISCLAIMER: The views and opinions expressed in these proceedings are those of the speakers or authors and do not necessarily reflect or represent the views of CETA/ACTE or AETA. 2

5 Embryo Transfer A 2018 Up-date R.J. Mapletoft and J.F. Hasler* WCVM, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5B4, and *Vetoquinol USA, Fort Worth, Texas Introduction The commercial bovine embryo transfer industry arose in the early 1970's in North America (Betteridge, 2003; Hasler, 2014; Seidel, 1981). European breeds of cattle that had been imported into Canada were valuable and scarce, and embryo transfer offered a means by which their numbers could be multiplied rapidly. Several universities, and a few veterinary practitioners and small embryo transfer companies adopted a research technology for commercial use. A small group of veterinarians and academics founded the International Embryo Technology Society (IETS) in 1974 to facilitate sharing of ideas and technical information which was considered necessary for progress to be made (Carmichael, 1980; Schultz, 1980). The IETS has grown to over 1,000 members and has become the main forum for scientific and regulatory exchange and discussion of embryo transfer and associated technologies. In particular, the Import/Export Committee of the IETS (now referred to as the Health and Safety Advisory Committee; HASAC) was instrumental in gathering and disseminating scientific information on the potential for disease control with bovine embryo transfer. The Manual of the International Embryo Technology Society A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures has become the reference source for sanitary procedures used in embryo export protocols (IETS Manual 4 th Edition 2010). In 2016, cooperating practitioners from around the world reported collecting 632,638 in-vivo-derived (IVD) embryos from 93,815 donors; 195,563 were transferred into recipients immediately after collection (fresh), while the remainder were cryopreserved for potential transfer at a later date (Perry, 2017). North America accounted for 52.5% of IVD bovine embryos, while Europe accounted for 20.4% and South America 7.5%. In addition, 666,215 in vitro-produced (IVP) bovine embryos were produced in 2016, 56.8% of which were in South America, 39.1% in North America and 2.8% in Europe. In the last two decades, IVP has increased markedly in both South and North America. Very briefly, bovine embryo transfer involves the selection, management and treatment of donors and recipients, and the collection and transfer of embryos within a narrow window of time 6 to 8 days after estrus. This technology has been incorporated into dairy and beef cattle operations, and often involves the participation of herd veterinarians. The following draws heavily on material contained in prior reviews (Mapletoft, 1985; Mapletoft and Hasler, 2005) and extensive literature of primary research on the topic. More detailed reviews of bovine embryo transfer are available on-line (Mapletoft and Bó, 2016), in very comprehensive papers by Keith Betteridge (1981, 2003) and in a book chapter (Hasler, 2007). Embryo transfer procedures are thoroughly covered in a book available from Hoard s Dairyman (Seidel, Elsden and Hasler, (2003). General Procedural Steps Donors are typically subjected to a superovulation protocol. Collection of single embryos from donors following natural or induced estrus and mating or AI is also possible but is not frequently utilized. The donor may be inseminated naturally or artificially and embryos are normally collected non-surgically 6 to 8 days after breeding. Following collection, embryos must be identified, evaluated and maintained in a suitable medium prior to transfer. At this point, they may be subjected to manipulations, such as splitting and sexing/genetic analysis, and they also may be cooled or frozen for longer periods of storage. Successful embryo transfer also requires transfer to recipient(s) with ovulations that are synchronous to that of the donor, either during natural cycles or following a cycle-synchronization protocol. 3

6 Superovulation The objective of superstimulation treatments is to obtain the maximum number of transferable embryos with a high probability of producing pregnancies. Wide ranges in superovulatory response and embryo yield have been detailed in several reviews of commercial embryo transfer records (Hasler et al., 1983; Looney, 1986). These reports demonstrate a high degree of unpredictability that affects the efficiency and profitability of bovine embryo transfer. There are several reviews of superovulation in the cow (Armstrong, 1993; Bo et al., 2002; 2008; Bo and Mapletoft, 2014; Foote, 1986; Lerner et al., 1986; Mapletoft et al., 2002; Seidel, 1981) Gonadotrophins and Superovulation Two different types of gonadotrophins have been used to induce superovulation in cattle: equine chorionic gonadotrophin (ecg) and pituitary extracts containing follicle stimulating hormone (FSH; Kelly et al., 1997; Murphy et al., 1984). Equine chorionic gonadotrophin is a complex glycoprotein with both FSH and luteinizing hormone (LH) activity and has been shown to have a half-life of approximately 40 hours in the cow (Schams et al., 1977); thus, ecg is normally administered by a single injection to induce superovulation (Murphy and Martinuk, 1991). Recommended doses range from 1500 to 3000 IU/animal with 2500 IU by intramuscular injection commonly used. The long half-life of ecg also causes protracted ovarian stimulation, abnormal endocrine profiles, large follicles and reduced embryo quality (Mikel-Jenson et al., 1982; Saumande et al., 1978). These problems have been overcome by the intravenous administration of antibodies to ecg at the time of the first insemination (Dieleman et al., 1993; Gonzalez et al., 1994). However, antibodies to ecg are not available commercially, and so ecg is seldom used to superstimulate cattle. Also, ecg is not registered for use in cattle in the USA. Pituitary extracts are most commonly used to superstimulate cattle. As the biological half-life of pituitary FSH in the cow has been estimated to be 5 hours or less (Laster, 1972), it must be injected twice daily to induce superovulation (Monniaux et al., 1983; Walsh et al., 1993). The usual regimen is 4 or 5 days of twice daily intramuscular treatments with FSH. Forty-eight to 72 hours after initiation of treatment, prostaglandin F2α (PGF) is administered to induce luteolysis. Estrus (and preovulatory LH release) occurs in 36 to 48 hours, with ovulation 24 to 36 hours later (Reviewed in Bó and Mapletoft, 2014). Purified pituitary extracts (with LH removed) are now available; Folltropin-V (Vetoquinol) is a porcine pituitary extract with approximately 84% of the LH removed (Gonzalez-Reyna et al., 1990). It has been used successfully in constant or decreasing dose schedules with PGF given either 48 or 72 hours after initiating treatment. Recombinant bovine FSH (rbfsh) has also been used to induce superovulation in cattle. Wilson et al., (1993) reported high superovulatory responses following twice daily administration of rbfsh, and more recently, Carvalho et al., (2014) reported the successful superstimulation of Holstein heifers with a single administration of a longacting rbfsh. Although there are no commercial products available for cattle currently, rfsh is used in human medicine suggesting that recombinant FSH is likely to be used in cattle, provided it gains registration and is cost effective. Follicular Wave Dynamics and Superovulation A greater superovulatory response occurred when gonadotrophin treatments are initiated on Day 9 of the estrous cycle (Day 8 post-ovulation) as compared to Days 3, 6 or 12 (Lindsell et al., 1986). Ultrasonography has now shown that the second follicle wave emerges 8.5 to 10.5 days after ovulation (Adams, 1998; Ginther et al., 1989; Pierson and Ginther, 1987). It has also been shown that superovulatory response is greater when FSH treatments are initiated at the time of follicle wave emergence (Nasser et al., 1993). While initiation of FSH treatments in the presence of a dominant follicle resulted in a 40 to 50% decrease in superovulatory response (Bungartz and Niemann, 1994; Guilbault et al., 1991; Kim et al., 2001; Shaw and Good, 2000), the presence of a large number of follicles 3 to 6 mm in diameter 8 to 10 days after ovulation, in the presence of a large follicle, provides evidence for dominant follicle regression and emergence of a new follicle wave (Singh et al., 2004). Manipulation of the Follicular Wave for Superstimulation The conventional protocol of initiating ovarian superstimulation during mid-cycle (8 to 12 days after estrus) has now been supported by ultrasonographic evidence indicating that mid-cycle is the approximate time of emergence of the second follicular wave (Adams et al., 2008). However, the day of emergence of the second follicular wave differs among individuals within wave type, and is 1 or 2 days later in two- than three-wave cycles (Adams, 1994). 4

7 In addition, the necessity of waiting until mid-cycle to initiate superstimulatory treatments implies monitoring estrus and an obligatory delay, making it difficult to superstimulate large numbers of donors at the same time. An alternative approach is to initiate superstimulation treatments subsequent to the synchronization of follicular wave emergence. There are three methods of synchronizing follicle wave emergence for superstimulation. Follicle Ablation The most efficacious approach to the synchronization of follicle wave emergence involves transvaginal ultrasoundguided ablation of all follicles 5 mm, regardless of stage of the estrous cycle (Bergfelt et al., 1994; Garcia and Salaheddine, 1998). This removes the suppressive effects of follicular products (estradiol and inhibin) on FSH release, resulting in an FSH surge and emergence of a new follicular wave 1 day later (Adams et al., 1992a). Superstimulatory treatments are then administered, beginning 1 or 2 days after ablation (Bergfelt et al., 1997). The timing of estrus was more synchronous when a progestin device was inserted for the period of superstimulation and two injections of PGF were administered on the day of device removal., Transvaginal ultrasound-guided ablation of only the dominant follicle (Bungartz and Niemann, 1994; Shaw and Good, 2000) during mid-diestrus, followed in 2 days by superstimulation, also resulted in a higher superovulatory response than when the dominant follicle was not ablated. We have also shown that ablation of the two largest follicles at random stages of the estrous cycle was efficacious in synchronizing follicular wave emergence for superstimulation (Baracaldo et al., 2000). Unfortunately, follicle ablation is difficult to utilize under field conditions. Estradiol and Progesterone Treatment of progestin-treated cattle with estradiol results in synchronous emergence of a new follicle wave (Bó et al., 1995; 1996). The mechanism apparently involves suppression of FSH, and possibly LH, which results in regression of gonadotropin-dependent follicles. Once follicle regression begins and the estradiol is metabolized, FSH surges and a new follicle wave emerges, 1 day later (Adams et al., 1992a). The use of estradiol-17β in progestin-treated cattle was followed by the emergence of a new wave in 3 to 5 days, regardless of the stage of follicular growth at the time of treatment. Estradiol-17β is normally injected with 50 to 100 mg of progesterone at placement of a progestin device to prevent estrogen-induced LH release in animals without a functional corpus luteum (CL). Data from experimental (Bó et al., 1996) and commercial (Bó et al., 2002) embryo transfer records show that the superovulatory response of donors given estradiol-17β and progesterone at unknown stages of the estrous cycle was comparable to those superstimulated 8 to 12 days after estrus. A protocol for synchronization of follicle wave emergence with estradiol-17 β for superstimulation is shown in Table 1. Unfortunately, estradiol-17β is not available commercially in many countries (Lane et al.,, 2008), and so the use of estrogen esters has been investigated. Treatment with 2.5 mg estradiol benzoate plus 50 mg progesterone at the time of progestin device insertion resulted in emergence of a new follicular wave 3 to 4 days later. Superstimulatory treatments initiated 4 days after treatment resulted in responses comparable to the use of estradiol-17β plus progesterone or superstimulation initiated 8 to 12 days after estrus (Bó et al., 2002). It should be noted that since 2006 estradiol cannot be used in cattle for reproductive protocols because of FDA regulations in the USA. Table 1. Protocol for follicle synchronization with estradiol-17 β and progesterone that can be used to superstimulate cows every 30 days, without the need for estrus detection and without compromising results. Day 0 - Insert CIDR and inject 5 mg estradiol-17β plus 100 mg progesterone Day 4 - FSH b.i.d. by deep intramuscular injection Day 5 - FSH b.i.d. by deep intramuscular injection Day 6 - AM - FSH by deep intramuscular injection; PGF - PM FSH by deep intramuscular injection; PGF and remove CIDR Day 7 - FSH b.i.d. by deep intramuscular injection Day 8 - PM - AI Day 9 - AM - AI Day 15 - Embryo collection, freezing and/or transfer; PGF Day 30 Insert CIDR and inject 5 mg estradiol-17β mg progesterone 5

8 Gonadotrophin Releasing Hormone (GnRH) The administration of GnRH or porcine LH (plh) has been shown to induce ovulation of a dominant follicle present at the time of treatment followed by emergence of a new follicle wave in 2 days (Martinez et al., 1999; Pursley et al., 1995; Thatcher et al., 1993). However, neither GnRH nor plh always induces ovulation, and if ovulation does not occur, follicle wave emergence will not be synchronized (Martinez et al., 1999). The reported asynchrony of follicular wave emergence (range, 3 days before treatment to 5 days after treatment) suggested that GnRH-based approaches may not be feasible for superstimulation. However, three clinical reports revealed no differences in the numbers of transferable embryos when donors were superstimulated 2 to 3 days after treatment with GnRH as compared to treatment with estradiol (Hinshaw, 1999; Steel and Hasler, 2009; Wock et al., 2008). It is noteworthy that in these studies, GnRH was administered 2 to 3 days after insertion of a progestin device which may have resulted in an unovulated dominant follicle which would be more responsive to GnRH treatment. Bó et al., (2008) reported on another protocol for superstimulation following the administration of GnRH. It was based on a study in which a persistent follicle was induced by the administration of PGF at the time of insertion of a progestin device 7 to 10 days before GnRH (Small et al., 2009); ovulation and follicle wave emergence occurred 1 to 2 days after the administration of GnRH in >90% of cows, indicating that this approach could be used in groups of randomly cycling donors. As superovulatory responses following administration of GnRH 2 vs 7 days after insertion of a progestin device were not significantly different (Hinshaw et al., 2015), either approach would appear to be efficacious Superstimulation of donors with abnormal ovarian function Cows with abnormal ovarian function are difficult to superstimulate because they usually do not have a functional CL, show estrus at unpredictable times, and stage of follicle development is difficult to predict. It was the need to superstimulate cows with abnormal ovarian function that led to the use of estradiol prior to the administration of FSH. In a retrospective study, embryo production did not differ between 190 cows with abnormal ovarian function which were superstimulated 7 days after receiving a norgestomet implant and an injection of norgestomet and estradiol valerate and 260 Control cows superstimulated between Days 8 and 12 of the estrous cycle. Subsequently, it was shown that estradiol valerate treatment resulted in emergence of a new follicle wave (reviewed in Mapletoft and Bó, 2004). Follicle Numbers and Superovulation The numbers of antral follicles in the ovary as determined by ultrasonography has been shown to vary, and superstimulatory response has been shown to be correlated with the numbers of small antral follicles at the time of initiating FSH treatments (Romero et al., 1991; Ireland et al., 2007; Singh et al., 2004). In humans, circulating antimullerian hormone (AMH) concentrations have been found to be an informative serum marker for ovarian follicle reserve (Toner and Seifer 2013), and information is accumulating that circulating AMH concentrations may be a reliable marker for predicting antral follicle numbers in cattle (Batista et al., 2014; Ireland et al., 2011; Monniaux et al., 2013). There was high repeatability across different phases of the estrous cycle, days in milk, levels of milk production, and parities making AMH determinations particularly useful to select potential donors or to predict superovulatory response in selected donors (Souza et al., 2014). Also, ultrasonic follicle counts by an experienced operator are highly correlated with AMH levels. Reducing the Need for Multiple Treatments with FSH Because the half-life of pituitary FSH is short in the cow (Laster, 1972), traditional superstimulatory treatment protocols have consisted of twice daily intramuscular injections over 4 or 5 days. This requires constant attention and increases the possibility of failures due to non-compliance. Twice daily treatments may also cause stress in donors with a subsequent decreased superovulatory response and/or altered preovulatory LH surge (Edwards et al., 1987; Stoebel and Moberg, 1982). Therefore, simplified protocols may be expected to reduce donor handling and improve response. A single subcutaneous administration of FSH has been shown to induce a superovulatory response equivalent to the traditional twice daily treatment protocol in beef cows in high body condition i.e., body condition score of >3 out of 5 (Bó et al., 1994; Hiraizumi et al., 2015), but results were not repeatable in Holsteins which presumably had less adipose tissue. However, superovulatory responses were improved in Holsteins when the FSH dose was split 6

9 into two; 75% administered subcutaneously on the first day of treatment and the remaining 25% administered 48 hours later when PGF is normally administered (Lovie et al., 1994). An alternative in inducing superovulation with a single administration of FSH is to utilize agents that cause FSH to be released over several days. These are commonly referred to as polymers which are biodegradable and nonreactive in tissues facilitating use in animals (Sutherland 1991). In a series of experiments, FSH was diluted in a 2% hyaluronan solution and administered as a single intramuscular injection (to avoid the effects of body condition); a similar number of ova/embryos was produced as with the twice-daily intramuscular FSH protocol (Tríbulo et al., 2011). However, 2% hyaluronan was viscous and difficult to mix with FSH. More dilute preparations (1% or 0.5% hyaluronan) were easier to mix with FSH, but were less efficacious in a single administration protocol. Their use was improved by splitting the total dose of FSH into two injections administered 48 hours apart (Tríbulo et al., 2012). When compared to the twice daily treatments, the numbers of transferable embryos with the two-injection protocol did not differ. A report derived from commercial embryo transfer data confirmed these results in beef cattle in North America (Hasler and Hockley, 2012). However, the single or two-injection protocol of FSH in hyaluronan is not recommended for lactating dairy cattle where results have been inconsistent, and generally unsatisfactory. Fixed-time AI of Superstimulated Donors Bó et al. (2006) developed a protocol for fixed-time AI in beef donors, without the need for estrus detection, by monitoring the timings of ovulations ultrasonically. Basically, the time of progestin device removal was delayed to prevent early ovulations and allow late developing follicles to catch-up and ovulation was induced with GnRH or plh. In this protocol, follicular wave emergence was synchronized with estradiol and a progestin device on random days of the estrous cycle (Day 0) and FSH treatments were initiated on Day 4. On Day 6, PGF was administered in the AM and PM and the progestin device was removed on Day 7 AM (24 hours after the first PGF). On Day 8 AM (24 hours after the removal of the progestin device), GnRH or plh was administered and fixed-time AI were done 12 and 24 hours later. Delaying the removal of the progestin device from Day 6 PM to Day 7 AM resulted in a higher number of ova/embryos and fertilized ova. From a practical perspective, fixed-time AI of donors has been shown to be useful in eliminating estrus detection for busy embryo transfer practitioners with no adverse effect on embryo production (Larkin et al., 2006). Studies in high-producing Holstein cows in Brazil have indicated that it is preferable to allow an additional 12 hours before removing the progestin device (i.e., Day 7 PM) followed by GnRH or plh 24 hours later i.e., Day 8 PM (Martins et al., 2012). Baruselli et al. (2006) also reported that it is preferable to remove the progestin device on Day 7 PM in Bos indicus beef breeds, followed by GnRH 12 hours later (i.e., Day 8 AM). Baruselli et al. (2006) also reported that it is possible to use a single insemination with high quality semen 16 hours after plh. This protocol has also been used successfully with sex-selected semen, except that inseminations were delayed by an additional 6 hours i.e., 18 and 30 hours after GnRH (Soares et al., 2011). Semen and Semen Quality Superstimulated donors are normally inseminated 12 and 24 hours after onset of estrus (around 60 and 72 hours after injection of PGF; Bó et al., 2006). However, superstimulation places extraordinary pressure on the capacity of frozen/thawed semen to fertilize multiple oocytes. As ovulation rate increases, the number of accessory sperm decreases, and unfertilized oocytes from superovulated cattle seldom have sperm attached to the zona pellucida (DeJarnette et al., 1992). However, viability may also be compromised; Saacke et al. (1988) showed that the number of viable sperm in the lower isthmus of the oviduct is less for a shorter period of time in super-stimulated than in single ovulating cattle. Thus, two inseminations would appear to be warranted. In 1988, Hawk et al. (1988) reported that insemination of superstimulated cattle with 4.4 billion fresh sperm resulted in a greater number of fertilized ova and higher fertilization rates than 70 million frozen-thawed sperm. To investigate this observation, we selected three bulls with normal spermiograms and cryopreserved their semen in insemination dosages of 20, 50 or 100 million sperm in 0.25 ml or 0.5 ml straws. When used as a single insemination at 12 and 24 hours after onset of estrus in superstimulated heifers, there were no differences in fertilization rates (Garcia et al., 1994). We concluded that the key was normal spermiogram when dosages of at least 10 million motile sperm (20 million pre-freeze) were used. 7

10 Semen used in superstimulated cattle should exceed the minimum standards established by the American Society for Theriogenology for frozen/thawed semen (Barth, 1993). Briefly, these are a minimum of 70% morphologically normal sperm, and immediately after thawing, 25% directional motility with a rate of 3/5 and a minimum of 60% intact acrosomes. After a 2 hour stress test, directional motility must exceed 15% (rate 2) and percentage of intact acrosomes must exceed 40%. It is worth noting that the choice of sires is usually made by the cattle owner; although the practitioner can give advice, it may not be followed. Also, the source and storage conditions of frozen semen are highly variable factors that can adversely affect the success of fertilization in superovulated cattle. This is true regardless of the fertility of the sire or the AI organization that froze the semen. Stroud and Hasler (2006) reported that a higher percentage of semen was of good quality when shipped directly from a bull stud to an in-house embryo transfer facility compared to semen that had been stored and shipped from a cattle owner. Superstimulation for Ovum Pick-up (OPU) Although Bos indicus cattle have high antral follicle counts and are not normally superstimulated prior to OPU, most Bos taurus breeds are treated with a half dose of FSH prior to oocyte aspiration. The common approach is to synchronize follicle wave emergence and administer four or six intramuscular injections of FSH over 2 or 3 days. Following a coasting period (with no FSH treatments) of approximately 40 hours, oocytes are recovered from antral follicles by ultrasound-guided oocyte aspiration (Blondin et al., 2002). Superstimulation prior to OPU has resulted in a significant increase in blastocyst production in Holstein donors (Viera et al., 2014), and dilution of FSH in 0.5% hyaluronan prior to a single intramuscular administration has been shown to be equally efficacious (Viera et al., 2015). Lengthened Superstimulation Protocols Based on the notion that exogenous gonadotropins can overcome the wave pattern and result in subordinate follicle break-through, attempts have been made to increase the superovulatory response by adding ecg treatment prior to initiating FSH treatments. Pre-treatment with ecg 2 days before the conventional FSH treatment protocol resulted in a numerically greater number of ovulations and transferable embryos in an unselected group of donors and a significantly greater number of transferable embryos in donors that were defined as poor responders (Bó et al., 2008). More recently, we evaluated the superovulatory response and embryo recovery in donors treated with either a 4- day or a 7-day FSH superstimulatory treatment protocol (García Guerra et al., 2012). Twenty-four beef cows were blocked by number of follicles 5 mm at the time of wave emergence and placed into either a 4-day or 7-day FSH protocol utilizing the same total dose of 400 mg FSH (Folltropin-V; Vetoquinol). The mean number of ovulations detected by ultrasonography was greater in the 7-day treatment group, consistent with a numerically greater number of follicles 10 mm just prior to ovulation. Moreover, ovulations occurred more synchronously in the 7- day group (93% of ovulations occurred 12 to 36 hours post-lh as compared to 66% in the 4-day group) suggesting that the superstimulated follicles were more mature and capable of responding to an LH stimulus. Although the total number of ova/embryos, fertilized ova and transferable embryos did not differ statistically, all end-points favoured the 7-day group. In another study (Dias et al., 2013), a 7-day superstimulation protocol was used to investigate the effects of the length of the follicle growth phase under the influence of progesterone on follicle growth, ovulation and oocyte competence. Beef cows were superstimulated with Folltropin-V (25 mg twice-daily) for 4 or 7 days. Again, the superstimultory response (number of large follicles just prior to insemination) was greater in the 7-day group, and the numbers of ovulations and embryos were numerically higher in the 7-day group. The duration of treatment appears to be responsible for the increase in the superstimulatory response rather than the FSH dose. In the two studies referred to above, the number of ovulatory-sized follicles just prior to ovulation was greater following 7 days of superstimulation than 4 days, whether the total dose of FSH was greater (Dias et al., 2013) or the same (García Guerra et al., 2012). However, further study is needed to determine the optimal dose of FSH when an extended superstimulatory treatment is used. It would appear that lower dosages over 7 days may result in insufficient FSH to maintain follicle growth toward the end of treatment. 8

11 Embryo Recovery Non-surgical embryo recovery involves the passage of a cuffed catheter through the cervix and into the uterus on Days 6 to 8 after estrus (Drost et al., 1976; Elsden et al., 1976; Rowe et al., 1976). The catheter may be placed either into a uterine horn or just inside the cervix. Once the catheter is in place, the cuff is inflated with saline or flushing medium. When inserted into a uterine horn, care must be taken not to over-distend the cuff as the endometrium may split causing loss of collection medium and embryos into the broad ligament. Original reports of non-surgical ova/embryo collection involved the use of two and three-way Foley catheters. Although Foley catheters are inexpensive and readily available, the distance from the cuff to the catheter tip is short, sometimes interfering with drainage of the collection medium. In addition, their rubber or latex composition prevents them from being conveniently sterilized. Autoclavable silicone catheters, specifically designed for embryo recovery, are now widely available in sizes from ranging from 12 to 20-gauge and with 5 or 30 ml cuffs. Some practitioners prefer using the two-way Rusch catheter, which has Luer-Lok fittings (Schneider and Hahn, 1979). Rusch catheters are 67 cm in length and are available in 14- or 18-gauge. The tip in front of the cuff measures 5.5 cm and has four holes. All recovery catheters are stiffened for passage through the cervix by a stainless steel stilette. There are basically two methods of embryo collection (Mapletoft, 1985; 1986): 1) the continuous or interrupted flow, closed-circuit system, and 2) the interrupted-syringe technique. However, any combination of these techniques is possible. It must be recognized that each system has advantages and disadvantages relative to the other. For closed-circuit collection, a bag or flask of collection medium is suspended above the donor and gravity provides the pressure for the inflow of medium. A total of 1 to 2 liters of medium is often used with this technique and the catheter may be positioned either in the uterine body or into each uterine horn separately. The tubing carrying the outflow of collection medium is usually connected directly to an embryo filter with pores that are approximately 50 to 70 µm in diameter (Mapletoft, 1986). For the interrupted-syringe technique, a 35 to 60 ml syringe provides inflow pressure. The syringe also provides negative pressure for recovering the medium. However, excessive aspiration pressure may cause the endometrium to block holes in the catheter tip. The recovered medium is then either filtered or placed directly into a large Petri dish so that embryos can be located. Disposable equipment for all methods of embryo collection is available commercially. Collection media containing no animal products and not requiring refrigeration are available commercially in 1 and 2 liter disposable IV bags. Some practitioners prefer to prepare Dulbecco's phosphate-buffered saline (PBS). This can be prepared and stored ready for use in 500 to 1000 ml containers. If syringes are used in the flushing procedure, it is recommended that those with rubber plungers be washed and heat sterilized before use. Holding media prepared in the lab are normally passed through a disposable 0.22 µm Millipore filter prior to use; the first few ml of medium through a disposable Millipore filter should be discarded as it may be harmful to embryos. Although embryo collection and holding media are now available commercially, they must be kept refrigerated if they contain animal products e.g., serum or BSA. Within a reasonable range, temperature is not critical to bovine embryo survival. Holding embryos during the period from collection to transfer in temperatures ranging from cool (10ºC) to room temperature has proven to be very satisfactory. However, holding embryos at extremely high temperatures (above 38ºC) should be avoided. Embryo collection and transfer cannot be conducted under strictly sterile conditions, but every attempt should be made to be as clean as possible. Sterilization with chemicals may be as likely to kill embryos as bacterial contaminants. Thorough washing of embryos with sterile medium has been shown to remove most infectious agents (Singh, 1985). As a routine, embryos should be passed through 10 washes of fresh medium prior to transfer or freezing (IETS Manual, 2010). Embryo Handling Following collection and filtration of the collection medium, embryos are located under 6-10X magnification with a stereomicroscope. Although embryos are usually transferred to recipients as soon as possible after collection, it is possible to maintain embryos in holding medium for 12 to 18 hours at room temperature (Haslet et al., 1987). It is also possible to cool bovine embryos in holding medium and to maintain them in the refrigerator for 2 to 3 days (Palasz and Mapletoft, 1996). For long term storage, embryos must be cryopreserved. 9

12 Embryos are normally held in a medium similar to that in which they were collected. Media must be buffered to maintain a ph of 7.2 to 7.6 and have an osmolarity around 275 mos. Dulbecco's PBS or commercially-prepared media with Hepes buffer, BSA and antibiotics are normally used in the field. More complex media with a bicarbonate buffer generally yield superior results for long term culture of bovine embryos, but they must be maintained in an incubator with an elevated CO 2 atmosphere. As indicated earlier, embryo collection, holding and freezing media which are free of animal products have become available, avoiding the need for refrigerated storage and increasing biosecurity. Biosecurity To date, none of the infectious diseases studied in the bovine species have been transmitted by embryos when embryo handling procedures were conducted correctly. Several large studies have now shown that zona intact, washed bovine embryos do not transmit infectious diseases (Stringfellow et al., 2004; Wrathall et al., 2004). Embryos must be examined at a magnification of at least 50X to ensure that the zona pellucida is intact and free of adherent material, and embryos must be washed (and trypsin-treated if required) according to the recommendations of the IETS Manual (2010). In addition, every effort is made to ensure that collection and holding media are not contaminated by the environment. Recommendations to ensure that embryos are not contaminated by the environment follow (Reviewed in Steel, 2007). Laboratory In embryo transfer practices with a laboratory, it should be separated from animal holding areas and embryo collection and transfer areas. Only personnel involved in embryo production/manipulation procedures should have access. Floors and sinks should be decontaminated routinely with disinfectant which has been shown to be nontoxic to embryos (e.g. Savlon) and immediate working areas, such as the bench-top and the laminar flow hood should be cleaned with 70% alcohol. Under field/farm conditions, efforts should be made to use clean (preferable sterile) paper sheet covers on the embryo handling surfaces. Eating, drinking or smoking should be prohibited in all embryo handling areas, and personal hygiene (use clean lab coats, shoes and clean hands, disinfected before embryo handling with alcohol) must be strictly in place. Equipment Only necessary equipment, microscopes, warming stage, incubator, refrigerator, biological freezer, scale, ph meter and osmometer should be in the laboratory. Special care should be taken to keep the exterior surfaces of all equipment clean; dust collectors e.g., cooling units and vents in refrigerator which should be cleaned weekly. Covers should protect other equipment such as microscopes and micromanipulator and the stages of microscopes should be cleaned with 70% alcohol with adequate time before use allowed for the alcohol to evaporate. Special attention should be given to the incubator. The elevated temperature and high humidity provides ideal conditions for bacteria to grow. Routine daily quality control and aseptic working habits will prevent contamination of the incubator. It has been known for some time that some types of equipment may be toxic to various cell types, especially after certain methods of sterilization. Therefore, it has become widely accepted practice to test for toxicity prior to using materials that come in contact with gametes or embryos. The rubber plunger of certain types of plastic syringes has been found to be problematic for both semen and embryos and therefore, plastic on plastic syringes are commonly used to hold the handling medium for embryos. Washing and sterilization of equipment Most equipment is now disposable and can be purchased, pre-sterilized from a number of suppliers. All glassware and other reusable equipment used for embryo production/handling should be rinsed once in the distilled water and then soaked for 24 hours in a 1% solution of non-toxic detergent (e.g., Alconox; Alconox Inc. 9E 40th St, New York, N.Y 10016), rinsed 5 times in distilled water, dried and wrapped for sterilization. Sterilization is the process of destroying microorganisms (bacteria, fungi, and viruses) by heat or chemical reaction. There are many methods of sterilization such as gas, radiation, and chemicals, but saturated steam is the most effective and complete method. All non-disposable items should be sterilized with one of the following alternatives: 10

13 Dry heat: items should be held at C for 2 hours. Steam sterilization: items must be held at C and 104 kilopascals pressure for 30 min. Ethylene oxide vapor (gas sterilization): items should be wrapped in material that is readily permeable to ethylene oxide and then exposed to a minimum of 500 mg of ethylene oxide (EO) per 1000 cm 3 for 30 min. Sterilized equipment should be disassembled in order to allow better gas penetration. Adequate time must be allowed for aeration of sterilized equipment (up to 30 or 40 days) or residual gas will kill embryos. Various studies indicate that EO sterilization has the potential of killing embryos. Under these experimental conditions, a period of 144 hours aeration was insufficient to render straws non-toxic to mouse embryos (Hagele et al., 1987). EO is used far less today in the US ET industry than in the past. This is due to increased concerns by OSHA regarding carcinogenic and other health issues and, as a consequence, the necessary safeguards, including an approved EO ventilation system, make it impractical for handling and using EO in most veterinary clinics. Membrane filtration: culture/holding media should normally be filtered through a membrane with pores of 0.22 mm (Millipore filter) prior to use using positive pressure. Serum substitutes and surface tension The surface tension of culture media has been shown to be an important factor in the in vitro culture of mammalian embryos (Palasz et al., 2000). This has been met traditionally by the addition of serum or BSA. However, biological products are potentially infectious and they are variable in constituents and function. Although synthetic polymers can replace surface-active properties successfully, they will not replace the embryotrophic properties of serum or BSA in culture media. These must be substituted for by growth factors in chemically defined media. The effectiveness of serum or BSA in embryo collection, short- or long-term culture and freeze/thaw media (very different biological and physicochemical processes) suggests that both possess common properties, other than nutritional components. At the same time, polyvinylpyrrolidone (PVP) (Leibo and Oda, 1993; Saeki et al., 1991), polyvinyl alcohol (PVA) (Kestintepe et al., 1995), sodium hyaluronate (SH) (Joly et al., 1992; Palasz et al., 1993) and ET surfactant (pluronic) (Palasz et al., 1995) have been used successfully as a replacement for serum/bsa in collection, culture and freezing media for mammalian germ cells. Although some of these defined macromolecules may resemble albumin, they do not provide for the potential reactions that can occur in serum; they are physiologically inert (The Merck Index, 1989). Embryo Evaluation Bovine embryos should be evaluated in small Petri dishes at 50 to 100 X magnification. It is important to be able to recognize the various stages of development and to compare this with the developmental stage that the embryo should be, based on the days from estrus (Table 2). Often a decision as to whether an embryo is worthy of transfer will depend on the availability of a recipient. Fair quality embryos should be transferred fresh, if recipients are available. The IETS considers the export of poor and fair quality embryos to be improper (IETS Manual, 2010). Table 2. Stages of Embryo Development in Cattle Morula: A mass of at least 16 cells. Individual blastomeres are difficult to discern from one another. The cellular mass of the embryo occupies most of the perivitelline space. Day 6. Compact Morula: Individual blastomeres have coalesced, forming a compact mass. The embryo mass occupies 60 to 70% of the perivitelline space. Day 7. Early Blastocyst: An embryo that has formed a fluid-filled cavity or blastocoel and gives a general appearance of a signet ring. The embryo occupies 70 to 80% of the perivitelline space. Early in this stage of development, the embryo may be difficult. Day 7 to 7.5. Blastocyst: Pronounced differentiation of the outer trophoblast layer and of the darker, more compact inner cell mass is evident. The blastocoel is highly prominent, with the embryo occupying most of the perivitelline space. Visual differentiation between the trophoblast and the inner cell mass is possible at this stage of development. Day 7.5 to 8. 11

14 Expanded Blastocyst: The overall diameter of the embryo dramatically increases, with a concurrent thinning of the zona pellucida to approximately one-third of its original thickness. Day 8 to 8.5. Hatched Blastocyst: Embryos recovered at this developmental stage can be undergoing the process of hatching or may have completely shed the zona pellucida. Hatched blastocysts may be spherical with a well defined blastocoel or may be collapsed. Identification of embryos at this stage can be difficult unless it re-expands. Day 9. Classification Embryos are evaluated and classified (i.e., as degenerate, poor, fair or good and excellent) by morphological examination at 50 to 100 X magnification according to the IETS Manual (2010). The overall diameter of the bovine embryo is 150 to 190 um, including a zona pellucida thickness of 12 to 15 μm. The overall diameter of the embryo remains virtually unchanged from the one-cell stage until blastocyst stage. The best predictor of an embryo's viability is its stage of development relative to what it should be on a given day after ovulation. An ideal embryo is compact and spherical. The blastomeres should be of similar size with even density and texture. The cytoplasm should not be granular or vesiculated. The perivitelline space should be clear and contain no cellular debris. The zona pellucida should be uniform, neither cracked nor collapsed and should not contain debris on its surface. Embryos of good and excellent quality and at the developmental stages of late morula to blastocyst yield the highest pregnancy rates. It is advisable to match the stage of embryo development to the day of the cycle of the recipient. Quality Evaluation Excellent: An ideal embryo, spherical, symmetrical and with cells of uniform size, color and texture. Good: Small imperfections such as a few extruded blastomeres, irregular shape and a few vesicles. Fair: Problems that are more definite are seen, including presence of extruded blastomeres, vesiculation, and a few degenerated cells. Poor: Severe problems, numerous extruded blastomeres, degenerated cells, cells of varying sizes, large and numerous vesicles but an apparently viable embryo mass. These are generally not of transferable quality. IETS Recommended Quality Codes The codes for embryo quality range from "1" to "4" as follows: Code 1: Excellent or good. Symmetrical and spherical embryo mass with individual blastomeres (cells) that are uniform in size, color and density. This embryo is consistent with its expected stage of development. Irregularities should be relatively minor and at least 85% of the cellular material should be an intact, viable embryo mass. This judgment should be based on the percentage of embryo cells represented by the extruded material in the perivitelline space. The zona pellucida should be smooth and have no concave or flat surfaces that might cause the embryo to adhere to a Petri dish or a straw. Code 2: Fair. Moderate irregularities in overall shape of the embryo mass or size, color and density of individual cells. At least 50% of the cellular material should be an intact, viable embryo mass. Code 3: Poor. Major irregularities in shape of the embryo mass or size, color and density of individual cells. At least 25% of the cellular material should be an intact, viable embryo mass. Code 4: Dead or degenerating. Degenerated embryos, oocytes or 1-cell embryos; non-viable. The Manual of the International Embryo Transfer Society also states It should be recognized that visual evaluation of embryos is a subjective evaluation of a biological system and is not an exact science. Furthermore, there are other factors such as environmental conditions, recipient quality and technician capability that play important roles in obtaining pregnancies from transferred embryos. It is also recognized that many different systems are used for "grading" embryos and that some are more sophisticated than are others. The criteria for assigning a "quality code" in the standardized forms were simplified to be "user friendly". Generally, unless otherwise specified, only Code 1 embryos should be utilized in international commerce. 12

15 In the superovulated cow, there is likely to be a considerable range of embryo stages on any given day during development. On Day 7 after estrus, there may be morulae and blastocysts within the same flush. At the same time, there may be embryos of excellent quality and unfertilized and degenerate embryos. Generally, wide variations in embryo quality and stages of development are signals that normal-appearing embryos may be stressed or compromised and that pregnancy rates may be disappointing. Embryos of excellent and good quality, at the developmental stages of compact morula to blastocyst yield the highest pregnancy rates, even after freezing. Fair and poor quality embryos yield poor pregnancy rates after freezing and should be transferred fresh. It is advisable to select the stage of the embryo for the synchrony of the recipient. It would also seem that fair and poor quality embryos are most likely to survive transfer if they are placed in the most synchronous recipients. Estrus Synchronization in Recipients High pregnancy rates are partially dependent upon the onset of estrus in recipients being within 24 hours of synchrony with that of the embryo donor (Hasler et al., 1987; Hasler, 2001). Recipients may be selected for embryo transfer by estrus detection of untreated animals or after drug-induced estrus synchronization. Regardless of the method used, timing and critical attention to estrus detection are important. Recipients synchronized with PGF must be treated 12 to 24 hours before donors because PGF-induced estrus occurs in 60 to 72 hours in single ovulating cattle (Kastelic et al., 1990) and in 36 to 48 hours in superstimulated donors (Bó et al., 2002; 2006). The success of estrus synchronization programs is dependent on an understanding of estrous cycle physiology, pharmacological agents and their effects on the estrous cycle, and herd management factors that reduce anestrus and increase conception rates. Treatment alternatives are discussed below. Prostaglandin (PGF) Prostaglandin has become the most common treatment for estrus synchronization in cattle (Folman et al., 1990; Larson and Ball 1992; Odde 1990), but PGF is not effective in inducing luteolysis in the first 5 days of the cycle and when luteolysis is effectively induced, the ensuing estrus is distributed over a 6-day period (Kastelic et al., 1990). This is due to the status of the dominant follicle at the time of treatment. In a two-dose PGF protocol, an interval of 10 or 11 days between treatments has been used because all animals should have a responsive CL at the time of the second PGF. However, a 14-day interval is usually preferred for AI in lactating dairy cattle (Folman et al., 1990). Progestins Various progestins (progesterone and progesterone-like compounds) have been utilized for estrus synchronization (Mapletoft et al., 2003). Progesterone prevents ovulation in cattle, and suppresses LH pulse frequency, which causes suppression of the growth of LH-dependent follicles (i.e., dominant follicle), but it does not suppress FSH secretion (Adams et al., 1992b). Thus, follicle waves continue to emerge in the presence of a functional CL. Progestins given for longer than the CL life-span (i.e., for 14 days or more) result in synchronous estrus upon withdrawal, but fertility is low (Revah and Butler 1996). Progestins used to control the estrous cycle in cattle have relatively less suppressive effects on LH secretion than the CL and are associated with the development of "persistent" follicles, which contain aged oocytes with low fertility. Although an early study indicated no effect of a CL resulting from a persistent follicle on pregnancy rates in recipients (Wehrman et al., 1997), Mantovani et al. (2005) reported reduced pregnancy rates. To synchronize estrus, progestin devices are normally placed in the vagina for 7 days; PGF is given 24 hours before device removal and estrus detection begins 48 hours later. Because of the short period of progestin treatment (7 days), the incidence of persistent follicles is reduced. Progesterone releasing vaginal devices are also well suited to protocols used to synchronize follicular development and ovulation (Mapletoft et al., 2003). Estrus Detection The estrous cycle in cattle averages 21 days, with 84% lasting from 18 to 24 days. Behavioral estrus lasts approximately 12 to 16 hours; ovulation normally occurs 24 to 36 hours after the onset of estrus (Kastelic, 2001). Estrous behavior waxes and wanes, but nearly all cattle will be detected in estrus if observation is continuous. Therefore, the incidence of true silent estrus is negligible. Causes of anestrus (lack of observed estrus) include pregnancy, cystic ovaries, ovarian atrophy, pyometra, embryonic death, free-martinism and white-heifer disease. Most anestrous dairy cows that are non-pregnant are cycling and have a normal genital tract. Dairy heifers and 13

16 postpartum suckled beef cattle often have a prolonged interval of anestrus due to ovarian inactivity. Increasing energy intake and/or 7 to 10 day of progestins will hasten resumption of ovarian activity. The primary sign of estrus is a cow standing firmly when mounted. Secondary signs of estrus include mounting other cows, mucus discharge, swollen vulva, hyperactivity, and bellowing. It is recommended that >80% of inseminations be based on standing estrus behavior. The two principal causes of estrus-detection problems are missed estrus and estrus detection errors. Indicators of missed estrus include prolonged intervals from calving to breeding, prolonged intervals between breedings, higher than usual non-pregnant at pregnancy examination, and <50% of potential estrous periods detected. Several factors can contribute to missed estrus. Often the observer does not spend adequate time observing the cattle for estrus, does not observe frequently enough during the day, or tries to combine estrus detection with other activities (e.g., feeding). If many cattle are in estrus at the same time, they will congregate and form a 'sexually active group, which facilitates estrus detection. However, if only a single animal is in estrus, mounting activity will be much less frequent. Slippery or hard surfaces will also reduce mounting activity. Indicators of estrous detection errors include high concentrations of progesterone in milk or blood at breeding and interbreeding intervals <17 d or >25 d. In several studies, up to 20% of cattle had high progesterone concentrations at the time of insemination, and therefore were not in estrus. Factors contributing to estrus detection errors include misinterpretation of signs of estrus, misinterpretation or misuse of estrus detection aids, and standing estrus in pregnant cows. Means by which estrus detection can be improved include inducing estrus at a predetermined time, allocating adequate time for observation, using estrus detection aids, and predicting the next estrus. Estrus detection aids include mounting detectors such as tail-head devices, chalk or paint, chin-ball markers on teaser bulls or androgen-treated marker animals, pedometers, and electronic estrus detection systems (Kastelic, 2001). These methods should be utilized in addition to, and not as a substitute for, visual observation of estrous behavior. Marker animals may be given several treatments with testosterone to initiate mounting activity, followed by periodic treatments to maintain activity. It has been reported that freemartin heifers implanted with Synovex-H (four implants in each ear) were effective marker animals. The duration of effectiveness of the implants was approximately 3 months. Fixed-time Embryo Transfer (FTET) In recipients, the need for estrus detection can be eliminated by utilizing protocols that have been developed for fixed-time AI in cattle (Mapletoft et al., 2003). Basically, two approaches have been used: the so-called Ovsynch or Cosynch protocols utilizing GnRH (Pursley et al., 1995; Wiltbank 1997) or plh (Martinez et al., 1999), with or without a progestin device (Lamb et al., 2001; Martinez et al., 2002), or estradiol and progesterone to synchronize follicle wave emergence and ovulation in progestin-treated animals (Bó et al., 2012; Mapletoft et al., 2003). GnRH If treatment of cattle with GnRH induces ovulation of a growing dominant follicle (Macmillan and Thatcher, 1991; Thatcher et al., 1993), emergence of a new follicular wave occurs approximately 2 days later (Martinez et al., 1999). Treatment with PGF 7 days after GnRH results in luteal regression and ovulation of the new dominant follicle, especially when a second GnRH injection is given 36 to 56 hours later (referred to as the Ovsynch protocol; Pursley et al., 1995). However, the Ovsynch protocol has been more efficacious in lactating dairy cows than in heifers. The cause for this variability is not known, but ovulation to the first GnRH occurred in a higher percentage of cows than heifers (Martinez et al., 1999; Pursley et al., 1995), and Wiltbank (1997) reported that 19% of heifers showed estrus before the injection of PGF making fixed-time AI difficult. However, the addition of a CIDR to a 7- day GnRH-based protocol improved pregnancy rates after fixed-time AI in heifers and improved pregnancy rates in non-cycling, lactating beef cows (Lamb et al., 2001). GnRH-based protocols have been used to synchronize ovulation in recipients that received IVD (Baruselli et al., 2000; 2010) or IVP (Ambrose et al., 1999) embryos. In these studies, more recipients received embryos than when estrus detection was used because GnRH-based protocols do not depend on expression of estrus; thus, pregnancy rates are higher than in controls. Prevention of early ovulations by addition of a progestin-releasing device to a 7- day GnRH-based protocol is usually used for FTET; Hinshaw (1999) treated 1637 recipients with GnRH plus a progestin-releasing device and transferred IVD embryos, without estrus detection, with an overall pregnancy rate of 59.9%. 14

17 Recent studies have shown that reducing the period of follicle dominance (by removing the progestin device 5 days after insertion) and increasing the time from progestin device removal to GnRH improves pregnancy per AI in GnRH-based protocols (Bridges et al., 2008; Lima et al., 2011; Santos et al., 2010). However, due to a shorter interval between the first GnRH and induction of luteolysis in the 5-day protocol, two injections of PGF are necessary to induce complete regression of the GnRH-induced CL. However, Colazo and Ambrose (2011) showed that when the first GnRH in the 5-day Cosynch protocol was not given in heifers and a single PGF was administered, pregnancy rate to FTAI was not affected. We have preliminary evidence indicating that this modification of the 5-day GnRH-based protocol results in a comparable proportion of recipients receiving an embryo and becoming pregnant per embryo transfer as with other FTET protocols (Bó et al., 2012) or estrus detection (Sala et al., 2016). Estradiol and Progesterone As indicated earlier, treatment with estradiol and progestins has been used to synchronize estrus in cattle, but Bó et al. (1995) demonstrated that estradiol treatment also synchronizes follicle development. In fixed-time AI protocols, a second, lower dose of estradiol is usually given 24 hours after progestin device removal to induce LH release, which occurs approximately 16 to 18 hours later, with ovulation in approximately 24 hours (Mapletoft et al., 2003). Estradiol treatment protocols are the most commonly used treatment to synchronize follicle wave emergence and ovulation in beef and dairy recipients in South America (Baruselli et al., 2010; 2011). The progestin device is usually removed on Day 8 and ovulation is induced by the administration of 0.5 or 1 mg of estradiol cypionate at that time, or 1 mg of estradiol benzoate 24 hours after progestin removal, or administration of GnRH or plh 48 to 54 hours after progestin removal (reviewed in Bó et al., 2002; 2012; Baruselli et al., 2010; 2011). As estrus detection is usually not preformed, Day 9 is considered to be the day of estrus. When estrus detection is done, all the recipients not in estrus by 48 hours after progestin device removal receive GnRH. All recipients with a functional CL on Day 17 receive an embryo; conception rates were comparable to embryo transfer 7 days after observed estrus. Use of ecg to Improve Pregnancy Rates in Recipients A common strategy to increase pregnancy rates in pasture-managed beef recipients in South America is the addition of 400 IU of ecg on either Day 5 or Day 8 of the estradiol/progestin treatment protocol. Overall, 75 to 85% of the recipients treated with ecg receive an embryo (compared to 50% or less with simple PGF synchronization), progesterone concentrations at the time of embryo transfer are high, and conception rates following transfer exceed 50% (reviewed in Baruselli et al., 2010; 2011; Bó et al., 2012). The efficacy of the estradiol benzoate, progestin and ecg treatment protocol for FTET has been confirmed in several different parts of the world in more than 15,000 recipients (Argentina - Bó et al., 2005; Brazil - Nasser et al., 2011; China - Remillard et al., 2006; Mexico - Looney et al., 2010). In each of these studies, treatment with ecg increased the number of recipients receiving an embryo resulting in higher pregnancy rates. The use of ecg in GnRH-based protocols has also been evaluated. In a Canadian study designed to evaluate the potential use of ecg in beef cattle recipients synchronized with GnRH/progestin for FTET (Small et al., 2007), recipient selection rates did not differ, but in a Colombian study (Mayor et al., 2008), ecg significantly increased pregnancy rates following FTET in recipients treated with the GnRH/progestin protocol. In summary, the addition of ecg to estradiol- or GnRH-based protocols which included the use of progestin devices resulted in increased pregnancy rates depending on the type, body condition and cyclicity of the recipients. However, treatment with ecg may not improve pregnancy rates in Bos taurus recipients managed under more optimal conditions. Other Treatments to Increase Pregnancy Rates in Recipients Several studies have investigated the relationship between circulating progesterone concentrations and pregnancy rates in recipients (reviewed in Baruselli et al., 2010; Carter et al., 2008). However, the use of supplementary progesterone has resulted in inconsistent effects on pregnancy rates. Generally, the beneficial effects of increasing circulating concentrations of progesterone seem to be more evident when pregnancy rates in untreated recipients were lower than expected. An alternative strategy is to create an accessory CL by induction of ovulation of the first wave dominant follicle, around the time of embryo transfer (reviewed in Thatcher et al., 2001). Again, results have 15

18 been inconsistent; in Bos indicus recipients, treatment with human chorionic gonadotropin (hcg) on Day 7 increased progesterone concentrations (Marques et al., 2002) and treatment with GnRH (Rodrigues et al., 2003) or GnRH, hcg, plh or a progestin device (Marques et al., 2003) at the time of embryo transfer resulted in increased pregnancy rates. However, in another study involving Bos indicus-cross recipients synchronized with the progestin/estradiol plus ecg protocol, pregnancy rates were not affected by treatment with hcg or GnRH at the time of FTET (Tríbulo et al., 2005). Small et al. [2004] were also unable to improve pregnancy rates in Bos taurus recipients treated with GnRH or plh on Days 5 or 7 after estrus. In a more recent study, administration of 1,000 IU hcg at the time of embryo transfer, resulted in higher serum progesterone concentrations in recipients with lower body condition scores but not with higher body condition scores (Wallace et al., 2011). The authors concluded that giving hcg at the time of embryo transfer increased the incidence of accessory CL and higher serum progesterone concentrations which resulted in higher pregnancy rates in recipients with lower body condition scores. Lower embryonic losses were also observed in recipients that received GnRH 2 days prior to receiving IVP embryos (Garcia Guerra et al., 2016). Management Factors The two management factors that determine the success or failure of an estrus synchronization program are nutrition (body condition score) and post-partum interval. If cows lose weight during pregnancy, the onset of estrous cycles after calving will be delayed, while cows that are fed adequately during pregnancy but fail to gain weight between calving and breeding will cycle but have reduced conception rates (Carvalho et al., 2014a) and may also have reduced pregnancy rates after receiving a viable embryo by embryo transfer (Bó et al., 2005). In a field study, pregnancy rates were significantly higher in beef recipients scoring 3 and 4 than in those scoring 1, 2 (thin) or 5 (obese; reviewed in Mapletoft, 1986). Therefore, the nutritional status of recipients must be evaluated before use in embryo transfer. Some specific donor management problems affecting pregnancy rates were described by Stroud and Hasler (2006). Embryo Transfer Non-surgical embryo transfer techniques utilized today involve the use of specialized embryo transfer pipettes (Mapletoft, 1985; Rowe et al., 1980; Wright, 1981). After confirming synchrony of estrus, the recipient is restrained and the rectum is evacuated of feces. At the same time, the presence and side of a functional CL is confirmed by rectal palpation or ultrasonography. Care is taken to prevent ballooning of the rectum with air. An epidural anesthetic is administered and the vulva is washed with water (no soap) and dried with a paper towel. The embryo is loaded in 0.25 ml straw between at least two air bubbles and the straw is loaded in the embryo transfer pipette. Care must be taken to insure that the straw engages the sheath tightly so as to avoid leakage. The sheath is coated with sterile, non-toxic obstetrical lubricant and the sheathed pipette is passed through the vulvar labia while avoiding contamination. The embryo is placed in the uterine horn adjacent to the ovary bearing the CL by passing the pipette through the cervix, very similar to AI. However, an attempt is usually made to pass the transfer pipette at least half-way down the uterine horn. The uterine lumen should be lined-up prior to passage so as to prevent trauma to the endometrium. The embryo is deposit slowly and firmly while slightly withdrawing the tip of the transfer pipette. Practice and dexterity seem to improve one's ability to achieve high pregnancy rates suggesting that trauma to the endometrium may be a limiting factor with nonsurgical embryo transfer. Stimulation of the cervix and inadvertent introduction of bacterial contaminants do not seem to be major determinants of pregnancy rates under normal circumstances. With practice and attention to detail, pregnancy rates with nonsurgical transfers can equal those of surgical transfers. In summary, with existing technology, an average of 9 to 12 ova/embryos will be collected from each superstimulated donor cow and 5 to 7 embryos will be transferred in recipients, resulting in 3 to 4 pregnancies. It must be emphasized that most donors do not produce this average number of embryos. The variance in embryo production by cattle is enormous, with more than half of donors usually producing fewer embryos than the average, while ~ 20% produce 15 or more and a few donors may produce as many as 60 or more embryos (Seidel, et al., 2019). Pregnancy rates are generally around 60% with fresh embryos and range from 50% to 60% with frozen/thawed embryos. One can anticipate a fetal loss rate of 10% from the time of pregnancy diagnosis until the calf is 6 months old. It is worthy of note that this is not different from that of the normal cattle population and that embryo transfer procedures have been shown to result in no increase in calf mortality or abnormalities. 16

19 Embryo Freezing Basic Principles The freezing of a living cell constitutes a complex physiochemical process of heat and water transport between the cell and its surrounding medium. There exists an optimum cooling-rate for each type of cell. It is dependent on the size of the cell, its surface to volume ratio, its permeability to water, and the temperature coefficient of that permeability (Leibo and Mazur, 1978; Palasz and Mapletoft, 1996). Normally, the medium that contains the embryos cools below its freezing point without ice crystal formation, a phenomenon referred to as super-cooling. Then, at some lower temperature ice nucleation occurs, followed by a rapid rise in temperature due to the release of latent heat of fusion. To avoid extensive super-cooling, ice crystallization is induced in the extracellular medium some 2 C below its freezing point (4 to 7 C) by seeding the medium with an ice crystal (Palasz and Mapletoft, 1996). Water in the cells of the embryo and between the ice crystals outside the embryo does not freeze at this temperature because of solute concentrations lowering its freezing point. During further cooling and enlargement of ice crystals, the solute concentration rises and the embryo cells respond osmotically by losing water into the extracellular unfrozen medium. Cells are injured during freezing and thawing primarily by solution effects and intracellular ice formation (Leibo and Mazur, 1978; Palasz and Mapletoft, 1996). The latter is especially detrimental when relatively large amounts of large ice crystals form. To avoid intracellular freezing, embryos must be cooled at less than 1 C/min. However, very slow cooling rates can also damage cells by what has been referred to as the solution effect. This is especially harmful if cells are not allowed to rehydrate during very rapid thawing. The required thawing rate depends on the freezing regimen used. When embryos are cooled slowly to temperatures between -27 and -40 C and then rapidly to -196 C (liquid nitrogen), thawing must be rapid, e.g., about 200 C/min. Cells treated in this way may contain some intracellular ice, and thawing has to be rapid to prevent injury from the recrystallization of that ice. On the other hand, if embryos are cooled slowly for a longer time and to a lower temperature (below -60 C) before transfer to liquid nitrogen, then thawing is normally done slowly at about 20 C/min (Leibo and Mazur, 1978). Although both systems result in similar rates of embryo survival, the techniques of faster freezing and rapid thawing are preferred in the field. Embryos are normally stored in liquid nitrogen at -196 C. The only reactions that occur at -196 C are direct ionizations from background radiation. Consequently, storage times of more than 200 years are unlikely to produce any detectable reduction in survival or cause genetic change of frozen embryos. Cryoprotectants such as glycerol and ethylene glycol in concentrations ranging from 1.0 to 2.0 M in the freezing medium are required to ensure embryo survival during and after freezing. It is thought that cryoprotectants act by reducing the amount of ice present at any temperature during freezing, thereby moderating the changes in solute concentration. Recommended criteria for a cryoprotectant include high solubility, low toxicity at high concentrations, and a low molecular weight both for easier permeation and to exert a maximum colligative effect (Palasz and Mapletoft, 1996). In recent years, glycerol has largely been replaced by ethylene glycol, which has gained preference because it can be used for "Direct Transfer" i.e., transfer into a recipient without prior removal of the cryoprotectant from the embryo (Leibo and Mapletoft, 1998; Voelkel and Hu, 1992). During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes resulting in swelling or contraction (Palasz and Mapletoft, 1996). Consequently, if the initial addition, or dilution, in particular, following thawing, is carried out inappropriately, the viability of cells can be affected. Glycerol can be added to embryos in a single step but there is clear evidence that the rate of glycerol removal post-thaw is more critical., The standard empirical method was to dilute glycerol out by the step-wise addition of PBS or to move the embryos through decreasing concentrations of glycerol, e.g., 0.25 M steps (Palasz and Mapletoft, 1996). However, Leibo and Mazur (1978) suggested a modification in the procedure of cryoprotectant removal by including nonpermeable solutes like sucrose into the dilution medium. The sucrose acts as an osmotic counterforce to restrict water movement across the membranes. The embryo will shrink in response to the extracellular hypertonic dilution medium and the cryoprotectant will leave the embryo by moving down a concentration gradient. It regains its normal volume when at the end of the process the embryo is placed in normal isotonic culture medium. Using this information, practical methods of quickly removing glycerol from thawed embryos have been devised. As a result, a "one-step straw" was developed so that embryos could be thawed, solutions mixed within the straw 17

20 and transfer to the recipient done non-surgically, all in the field (Leibo, 1984). More recently, this method has given way to "Direct Transfer" utilizing highly permeating cryoprotectants, such as ethylene glycol, which do not harm the embryo osmotically if not removed prior to transfer. Pregnancy results for "Direct Transfer" in Canada, with more than 19,000 embryos, were not different from those achieved with glycerol removal by dilution (Leibo and Mapletoft, 1998). Freeze - Thaw Procedures Utilizing Glycerol The following protocol has been proven successful for the cryopreservation of Day 7 bovine embryos in PBS supplemented with 0.4% BSA and 1.5 M glycerol (Palasz and Mapletoft, 1996). Embryos are pipetted into the freezing medium at room temperature (20 C) and left for 8 to 10 minutes to permit the glycerol to equilibrate within the embryo cells. During this equilibration period the embryo(s) are transferred in freezing medium into French straws that are then securely sealed. The samples can be immediately transferred into the freezing chamber at -6 or -7 C and held for 5 min. Ice crystallization (seeding) of the extracellular medium is initiated by touching the outside wall of the straw with a forceps or Q-tip pre-cooled in liquid nitrogen (do not touch the column of medium that contains the embryo(s). However, the ice crystal in synthetic media (not containing serum or BSA) often does not reliably move across air bubbles and so seeding has to be done in the column containing the embryo; special care must be taken (Hasler, 2010). The samples are held at the seeding temperature for an additional 10 min to allow the ice crystal in the medium to progress to equilibrium. Next, embryos are cooled at 0.3 to 0.8 C/min to a temperature between -30 and -40 C, at which time they are immersed into liquid nitrogen at -196 C) and stored. Thawing is carried out by placing the straw into a water-bath at a temperature between 20 and 35 C. It has been reported that the incidence of cracked zona pellucida was reduced in an air-thaw or when straws were thawed in air for 10 to 15 seconds prior to being submerged into a 35 C water bath; the thaw rate should be around 200 o C/minute. Glycerol, which is a slowly permeating cryoprotectant, must be removed without causing osmotic damage. The method of choice is the use of sucrose solution between 1.0M and 0.5M in a single step for 10 min or 0.3M in a 3- step dilution of 5 min each (0.75M glycerol and 0.3M sucrose; 0.375M glycerol and 0.3M sucrose; 0.3M sucrose; Mapletoft, 1985). The embryos are then transferred into holding medium, washed and evaluated prior to transfer. Direct Transfer of Frozen-Thawed Embryos In 1992, Voekel and Hu (1992) first reported the use of the highly permeating cryoprotectant, ethylene glycol, for the Direct Transfer of frozen/thawed bovine embryos without the necessity of microscope examination and cryoprotectant removal prior to transfer. With this approach, the embryo straw is thawed in a water-bath, much like semen, and the contents of the straw are deposited into the uterus of the recipient, much like artificial insemination. There is no need of a microscope or complicated dilution procedures. The cryoprotectant leaves the embryo in the uterus. The commercial embryo transfer industry rapidly adopted the use of ethylene glycol/direct Transfer. In a 1998 census of Canadian embryo transfer practitioners, Leibo and Mapletoft reported that the Direct Transfer of 19,000 bovine embryos in Canada resulted in an overall pregnancy rate of 58% which was not different to that achieved with glycerol and dilution prior to transfer. In the US, more than 95% of bovine embryos are frozen in ethylene glycol compared to glycerol. The Direct Transfer of these frozen/thawed bovine embryos is very similar to the use of frozen/thawed semen in AI, with no need for a microscope or experience embryologist and is often conducted by technicians. The time interval between collection and cryopreservation is also important to take into consideration, with an elapsed time up to 180 minutes of storage at room temperature posing no problem (Hasler, 2001). However, a storage time of 6 hours or more, at either room temperature or 5 C resulted in a decrease in post-thaw survival in another study (Jousan et al., 2004). Vitrification Freezing Procedures The freezing of bovine embryos is now commonplace and pregnancy rates are only slightly less than that achieved with fresh embryos. However, freezing and thawing procedures are time consuming and require the use of biological freezers and a microscope. A more rapid method of freezing, known as vitrification (Rall and Fahy, 1985), does not require the use of a freezer. High concentrations of cryoprotectants and a very small volume are 18

21 used so that the rate of cooling is very fast. The embryo in its cryoprotectant solution is placed directly into liquid nitrogen. Because of the high concentration of cryoprotectants and the very rapid cooling, ice crystals do not form; the frozen solution forms a glass. As ice crystal formation is one of the most damaging processes in freezing, vitrification has much to offer in the cryopreservation of had to freeze oocytes and embryos. However, its greatest advantage is its simplicity in application. Vitrification procedures are now widely used experimentally and with IVP embryos. A procedure for the Direct Transfer of vitrified bovine embryos with pregnancy rates that did not differ from that of traditional techniques was reported (van Wagtendonk et al., 1996) but pregnancy rates were rather low. Vitrification is appealing because it is fast and simple, but only one embryo can be cryopreserved at a time. Thus, the cryopreservation of a large number of embryos may very well take longer than traditional slow, controlled procedures. In addition, the precise timing of addition of the cryoprotectant is very critical, as is the removal of the cryoprotectant. Consequently, vitrification procedures are not yet being widely utilized in the field. Identification, Certification and Registration of Offspring Records for the accurate identification of parentage and of embryo transfer offspring is of vital importance for both domestic and international application of embryo transfer technology. The IETS has developed three forms for certification of embryo recovery, embryo freezing, and embryo transfer. In addition, a fourth form (certificate D) is recommended for use in embryo exports. The IETS also allocates embryo freezing codes that must appear on all embryo containers and all documentation so that the organization freezing embryos can be identified. Finally, standard procedures for labeling embryo freezing containers are also recommended e.g., embryos frozen for Direct Transfer are to be frozen in yellow straws and placed in yellow goblets. Examples of the above forms and specific instructions on their use, the labeling of embryo freezing containers and the identification of embryo developmental stages and quality grades are available in the Manual of the IETS (2010). References Adams GP, Matteri RL, Kastelic JP et al (1992a) Association between surges of follicle stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fert 94: Adams GP, Matteri RL, Ginther OJ (1992b) The effect of progesterone on growth of ovarian follicles, emergence of follicular waves and circulating FSH in heifers. J Reprod Fert 95: Adams GP. (1994) Control of ovarian follicular wave dynamics in cattle; Implications for synchronization and superstimulation. Theriogenology, 4: Adams GP. (1998) Control of ovarian follicular wave dynamics in mature and prepubertal cattle for synchronization and superstimulation. Proceeding of the XX Congress of the World Association of Buiatrics; Sydney, Australia; Pp Adams GP, Jaiswal R, Singh J et al (2008) Progress in understanding ovarian follicular dynamics in cattle. Theriogenology 69:72-80 Ambrose JD, Drost RL, Monson RL et al (1999) Efficacy of timed embryo transfer with fresh and frozen in vitro-produced embryos to increase pregnancy rates in heat-stressed dairy cattle. J Dairy Sci 82: Armstrong D (1993) Recent advances in superovulation of cattle. Theriogenology 39:7-24. Baracaldo MI, Martinez M, Adams GP, Mapletoft RJ. (2000) Superovulatory response following transvaginal follicle ablation in cattle. Theriogenology 53: Barth AD (1993) Evaluation of frozen bovine semen by the veterinary practitioner. Reviewers: WG Parker, EG Robertson, RG Saacke, WH Cardwell, JR Mitchell, GW McKay. In: Society for Theriogenology Handbook B-9. Baruselli PS, Marques MO, Carvalho NAT et al (2000) Ovsynch protocol with fixed-time embryo transfer increasing pregnancy rates in bovine recipients. In: Arq Fac Vet UFRGS, Porto Alegre, Brazil 28:205 (Abstract) Baruselli PS, Sá Fhilo M, Martins CM et al (2006) Superovulation and embryo transfer in Bos Indicus cattle. Theriogenology 65:77-88 Baruselli PS, Ferreira RM, Sá Filho MF et al (2010) Bovine embryo transfer recipient synchronization and management in tropical environments. Reprod Fertil Dev 22:67 74 Baruselli PS, Ferreira RM, Sales JNS et al (2011) Timed embryo transfer programs for the management of donor and recipient cows. Theriogenology 76:

22 Batista EOS, Macedo GG, Sala RV et al (2014) Plasma anti-mullerian hormone as a predictor of ovarian antral follicular population in Bos indicus (Nelore) and Bos taurus (Holstein) heifers. Reprod Domest Anim 49: Bergfelt DR, Lightfoot KC, Adams GP. (1994) Ovarian dynamics following ultrasound-guided transvaginal follicle ablation in heifers. Theriogenology 42: Bergfelt DR, Bo GA, Mapletoft RJ, Adams GP. (1997) Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle. Anim Reprod Sci 49:1-12. Betteridge KJ (1981) An historical look at embryo transfer. J Reprod Fert 62:1-13. Betteridge KJ (2003) A history of farm animal embryo transfer and some associated techniques. Anim Reprod Sci 79: Blondin P, Bousquet D, Twagiramungu H et al (2002) Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol Reprod 66:38-43 Bó G A, Hockley DK, Nasser LF et al (1994) Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle. Theriogenology 42: Bo GA, Adams GP, Pierson RA, et al. (1995) Exogenous control of follicular wave emergence in cattle. Theriogenology, 43: Bo GA, Adams GP, Pierson RA, Mapletoft RJ. (1996) Effect of progestogen plus E-17β treatment on superovulatory response in beef cattle. Theriogenology 45: Bo G.A., Baruselli P.S., Moreno D., Cutaia L., Caccia M., Tribulo R., Tribulo H. & Mapletoft R.J. (2002) The control of follicular wave development for self-appointed embryo transfer programs in cattle. Theriogenology 57, Bó GA, Cutaia L, Chesta P, Balla E, Picinato D, Peres L, Maraña D, Moreno D, Veneranda G, Baruselli PS. (2005) Application of fixed-time artificial insemination and embryo transfer programs in beef cattle operations. In: Proc Joint Mtg AETA & CETA, Minneapolis, MN pp Bó GA, Baruselli PS, Chesta P et al (2006) The timing of ovulation and insemination schedules in superstimulated cattle. Theriogenology 65: Bó GA, Guerrero DC, Adams GP (2008) Alternative approaches to setting up donor cows for superstimulation. Theriogenology 69:81-87 Bó GA, Coelho Peres L, Cutaia LE et al (2012) Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates. Reprod Fert Dev 24: Bó GA, Mapletoft RJ (2014) Historical perspectives and recent research on superovulation in cattle. Theriogenology 81:38-48 Bridges GA, Helser LA, Grum DE et al (2008) Decreasing the interval between GnRH and PGF2α from 7 to 5 days and lengthening proestrus increases timed-ai pregnancy rates in beef cows. Theriogenology 69: Bungartz L, Niemann H. (1994) Assessment of the presence of a dominant follicle and selection of dairy cows suitable for superovulation by a single ultrasound examination. J Reprod Fert 101: Carmichael RA (1980) History of the International Embryo Transfer Society Part I. Theriogenology 13:3-6 Carter F, Forde N, Duffy P et al (2008) Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers. Reprod Fert Dev 20: Carvalho PD, Souza AH, Amundson MC et al (2014a) Relationships between fertility and postpartum changes in body condition and body weight in lactating dairy cows. J Dairy Sci 97:1-18 Carvalho PD, Hackbart KS, Bender RW et al (2014b) Use of a single injection of long-acting recombinant bovine FSH to superovulate Holstein heifers: a preliminary study. Theriogenology 82: Colazo MG, Ambrose DJ (2011) Neither duration of progesterone insert nor initial GnRH treatment affected pregnancy per timed-insemination in dairy heifers subjected to a Co-synch protocol. Theriogenology 76: DeJarnette JM, Saacke RG, Bame J et al (1992) Accessory sperm: Their importance to fertility and embryo quality, and attempts to alter their numbers in artificially inseminated cattle. J Anim Sci 70: Dias FCF, Costa E, Adams GP, Mapletoft RJ, Kastelic J, Dochi O, Singh J. (2013a) Effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle. Reprod Fertil Dev, 25:

23 Dieleman S, Bevers M, Vos P et al. (1993) PMSG/anti-PMSG in cattle: A simple and efficient superovulatory treatment. Theriogenology 39:25-42 Drost M, Brand A, Aaarts MH. (1976) A device for nonsurgical recovery of bovine embryos. Theriogenology 6: Edwards L, Rahe C, Griffin J et al (1987) Effect of transportation stress on ovarian function in superovulated Hereford heifers. Theriogenology 28: Elsden RP, Hasler JF, Seidel GE Jr. (1976) Non-surgical recovery of bovine eggs. Theriogenology 6: Folman Y, Kaim M, Herz Z, Rosenberg M. (1990) Comparison of methods for the synchronization of estrous cycles in dairy cows. 2. Effects of progesterone and parity on conception. J Dairy Sci 73:2817. Foote RH. (1986) Superovulation practices and related current research. In: Proc Ann Am Embryo Trans Assoc. Fort Worth, Texaspp2-15. Garcia A, Mapletoft RJ, Kennedy R (1994) Effect of semen dose on fertilization and embryo quality in superovulated cows. Theriogenology 41:202 (Abstract) Garcia A, Salaheddine M. (1998) Effects of repeated ultrasound-guided transvaginal follicular aspiration on bovine oocyte recovery and subsequent follicular development. Theriogenology 50: García Guerra, A., Tribulo, A., Yapura, J., Singh, J., Mapletoft, R.J. (2012) Lengthening the superstimulatory treatment protocol increases ovarian response and number of transferable embryos in beef cows. Theriogenology, 78, García Guerra A, Sala RV, Baez GM et al (2016) Treatment with GnRH on Day 5 reduces pregnancy loss in heifers receiving in vitro-produced expanded blastocysts. Reprod Fert Dev 28:185 (Abstract) Ginther OJ, Knopf L, Kastelic JP. (1989) Temporal associations among ovarian events in cattle during oestrous cycles with two or three follicular waves. J Reprod Fert 87: Gonzalez-Reyna A, Lussier JG, Carruthers TD et al (1990) Superovulation of beef heifers with Folltropin: A new FSH preparation containing reduced LH activity. Theriogenology 33: Gonzalez A, Wang H, Carruthers TD et al (1994) Increased ovulation rates in PMSG - stimulated beef heifers treated with a monoclonal PMSG antibody. Theriogenology 41: Guilbault LA, Grasso F, Lussier JG et al (1991) Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fert 91:81-89 Hagele WC, Moker JS, Mapletoft RJ. (1987) The effect of ethylene oxide gas sterilization of semen straws on mouse embryo survival., Theriogenology 27:236. Hasler JF, McCauley AD, Schermerhorn EC, et al. (1983) Superovulatory responses of Holstein cows. Theriogenology 19: Hasler JF, McCauley AD, Lathrop WF, et al. (1987) Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program. Theriogenology 27: Hasler JF. (2001) Factors affecting frozen and fresh frozen embryo transfer pregnancy rates in cattle. Theriogenology 56: Hasler JF (2007) Embryo Transfer and In Vitro Fertilization. In: Comparative Reproductive Biology, Heide Schatten and Gheorghe M. Constantinescu, eds. Blackwell Publishing, Ames, Iowa. pp Hasler JF (2010) Synthetic media for culture, freezing and vitrification of bovine embryos. Reprod Fert Dev 22: Hasler J, Hockley D (2012) Efficacy of hyaluronan as a diluent for a two injection FSH superovulation protocol in Bos taurus beef cows. Reprod Dom Anim 47:459 (Abstract) Hasler JF. (2014). Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America, and personal reminisces. Theriogenology 81: Hawk HW, Conley HH, Wall RJ, et al. (1988) Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm. Theriogenology 29: Hinshaw RH (1999) Formulating ET contracts. In: Proc Soc Therio, Nashville, TN, pp Hinshaw RH, Switzer ML, Mapletoft RJ et al (2015) A comparison of two approaches for the use of GnRH to synchronize follicle wave emergence for superovulation. Reprod Fert Dev 27:263 (Abstract) 21

24 Hiraizumi S, Nishinomiya H, Oikawa T et al (2015) Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle stimulating hormone treatment or six intramuscular treatments over three days. Theriogenology 83: Ireland JJ, Ward F, Jimenez-Krassel F et al (2007) Follicle numbers are highly repeatable within individual animals but are inversely correlated with FSH concentrations and the proportion of good-quality embryos after ovarian stimulation in cattle. Human Reprod 22: Ireland JJ, Smith GW, Scheetz D et al (2011) Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of anti-mullerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle. Reprod Fertil Dev 23:1-14 Joly T, Nibart M, Thibier M. (1992) Hyaluronic acid as a substitute for proteins in the deep-freezing of embryos from mice and sheep: an in vitro investigation Theriogenology 37: Jousan FD, Utt MD, Whitman SS, Hinshaw RH, Beal WE. (2004) Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro. Theriogenology 61: Kastelic JP, Knopf L, Ginther OJ. (1990) Effect of day of prostaglandin F treatment on selection and development of the ovulatory follicle in heifers. Anim Reprod Sci 23: Kastelic JP. (2001) Computerized heat detection. Advances in Dairy Technology 13: Kelly P, Duffy P, Roche JF et al. (1997) Superovulation in cattle: Effect of FSH type and method of administration on follicular growth, ovulatory response and endocrine patterns. Anim Reprod Sci 46:1-14 Keskintepe L, Clay A, Burnley and Brackett BG. (1995) Production of viable bovine blastocysts in defined in vitro conditions Biol Reprod 52: Kim HI, Son DS, Yeon H, Choi SH, Park SB, Ryu IS, Suh GH, Lee DW, Lee CS, Lee HJ, Yoon JT. (2001) Effect of dominant follicle removal before superstimulation on follicular growth, ovulation and embryo production in Holstein cows. Theriogenology 55: Lamb GC, Stevenson JS, Kesler DJ et al (2001) Inclusion of an intravaginal progesterone insert plus GnRH and prostaglandin F2α for ovulation control in postpartum suckled beef cows. J Anim Sci 79: Lane EA, Austin EJ, Crowe MA (2008) Estrus synchronisation in cattle-current options following the EU regulations restricting use of estrogenic compounds in food-producing animals: A review. Anim Reprod Sci 109:1-16 Larkin S, Chesta P, Looney C et al (2006) Distribution of ovulation and subsequent embryo production using Lutropin and estradiol-17β for timed AI of superstimulated beef females. Reprod Fertil Dev 18:289 (Abstract) Larson LL, Ball PJH. (1992) Regulation of estrous cycles in dairy cattle: a review. Theriogenology, 38: Laster DB (1972) Disappearance of and uptake of 125 I FSH in the rat, rabbit, ewe and cow. J Reprod Fert 30: Leibo SP. (1984) A one-step method for direct non-surgical transfer of frozen-thawed bovine embryos. Theriogenology21: Leibo SP, Mazur P. (1978) Methods for the preservation of mammalian embryos by freezing. In: Daniel JC Jr, ed. Methods in Mammalian Reproduction. NY: Academic Press, Leibo SP, Oda K. (1993) High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryo-Letters 14: Leibo SP, Mapletoft RJ. (1998) Direct transfer of cryopreserved cattle embryos in North America. Proc. Ann Mtg. AETA, San Antonio, TX, pp Lerner SP, Thayne WV, Baker RD, et al. (1986) Age, dose of FSH and other factors affecting superovulation in Holstein cows. J Anim Sci 63: Lima FS, Ayres H, Favoreto MG et al (2011) Effects of gonadotropin releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers. J Dairy Sci 94: Lindsell CE, Murphy BD, Mapletoft RJ. (1986) Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle. Theriogenology 26: Looney CR. (1986) Superovulation in beef females. In: Proc 5th Ann Conv AETA Fort Worth, Texas; Looney CR, Nelson JS, Schneider HJ et al (2006) Improving fertility in beef cow recipients Theriogenology 65:

25 Looney CR, Stutts KJ, Novicke AK et al (2010) Advancements in estrus synchronization of Brahman-influenced embryo transfer recipient females. In: Proceedings of the Joint Meeting of the American Embryo Transfer Association & Canadian Embryo Transfer Association,, Charlotte, NC, pp Lovie M, Garcia A, Hackett A ET AL (1994) The effect of dose schedule and route of administration on superovulatory response to Folltropin in Holstein cows. Theriogenology 41:241 (Abstract) Macmillan KL, Thatcher WW. (1991) Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol Reprod 45: Manual of the International Embryo Transfer Society. (2010) Fourth Edition. DA Stringfellow & MD Givens (eds). Savoy, IL: IETS, - Available from amazon.com. Mantovani AP, Reis EL, Gacek F et al (2005) Prolonged use of a progesterone-releasing intravaginal device (CIDR ) for induction of persistent follicles in bovine embryo recipients. Anim Reprod 2: Mapletoft RJ. (1985) Embryo transfer in the cow: General procedures. Rev sci tech Off int Epiz 4: Mapletoft RJ. (1986) Bovine Embryo Transfer. In: DA Morrow, ed. Current Therapy in Theriogenology II. Philadelphia: WB Saunders Co. pp Mapletoft Reuben J, Bennett Steward Kristina, Adams Gregg P. (2002) Recent advances in the superovulation of cattle. Reprod Nutr Dev 42:1-11. Mapletoft RJ, Martinez MF, Colazo MG, Kastelic JP. (2003) The Use of Controlled Internal Drug Release Devices for the Regulation of Bovine Reproduction. J Anim Sci 81(E. Suppl. 2):E28 E36. Mapletoft RJ, Bó GA. (2004) The control of ovarian function for embryo transfer: Superstimulation of cows with normal or abnormal ovarian function. Proceedings of 23 World Buiatrics Congress, Quebec City, QC 34(1&2):67-68 Mapletoft RJ, Hasler JF (2005) Assisted reproductive technologies in cattle: A review. Rev sci tech off int epiz 24: Mapletoft RJ, Bó GA (2016) Bovine Embryo Transfer. In: I.V.I.S. (Ed.), IVIS Reviews in Veterinary Medicine. Ithaca: International Veterinary Information Service ( ; Document No. R S Marques MO, Madureira EH, Bó GA et al (2002) Ovarian ultrasonography and plasma progesterone concentration Bos taurus x Bos indicus heifers administered different treatments on Day 7 of the estrous cycle. Theriogenology 57:548 (Abstract). Marques MO, Nasser LF, Silva RCP et al (2003) Increased pregnancy rates in Bos taurus x Bos indicus embryo recipients with treatments that increase plasma progesterone concentrations. Theriogenology 59:369 (Abstract). Martinez MF, Adams GP, Bergfelt D, Kastelic JP, Mapletoft RJ. (1999) Effect of LH or GnRH on the dominant follicle of the first follicular wave in heifers. Anim Reprod Sci 57: Martinez MF, Kastelic JP, Adams GP et al (2002) The use of a progesterone-releasing device (CIDR) or melengestrol acetate with GnRH, LH or estradiol benzoate for fixed-time AI in beef heifers. J Anim Sci 80: Martins CM, Rodrigues CA, Vieira LM et al (2012) The effect of timing of the induction of ovulation on embryo production in superstimulated lactating Holstein cows undergoing fixed-time artificial insemination. Theriogenology 78: Mayor JC, Tribulo HE, Bó GA (2008) Pregnancy rates following fixed-time embryo transfer in Bos indicus recipients synchronized with progestin devices and estradiol or GnRH and treated with ecg. Reprod Domest Anim 43(suppl 3):180 (abstract). Mikel-Jenson A, Greve T, Madej A et al (1982) Endocrine profiles and embryo quality in the PMSG-PGF 2α -treated cow. Theriogenology 18:33-34 Monniaux D, Chupin D, Saumande J (1983) Superovulatory responses of cattle. Theriogenology 19:55-82 Monniaux D, Drouilhet L, Rico C et al (2013) Regulation of anti-mullerian hormone production in domestic animals. Reprod Fertil Dev 25:1-16 Murphy BD, Mapletoft RJ, Manns J et al (1984) Variability in gonadotrophin preparations as a factor in the superovulatory response. Theriogenology 21: Murphy B, Martinuk S (1991) Equine chorionic gonadotropin. Endocrine Rev 12:27-44 Nasser LF, Adams GP, Bo GA, et al. (1993) Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 40:

26 Nasser LFT, Penteado L, Rezende CR et al (2011) Fixed time artificial insemination and embryo transfer programs in Brazil. Acta Sci Vet 39(Suppl 1):s15-s22 Odde KG. (1990) A review of synchronization of estrus in postpartum cattle. J Anim Sci 68: Palasz AT, Alkemade S, Mapletoft RJ. (1993) The use of sodium hyaluronate in freezing media for bovine and murine embryos Cryobiology 30: Palasz AT, Tornesi MB, Archer J and Mapletoft RJ. (1995) Media alternatives for the collection, culture and freezing of mouse and cattle embryos. Theriogenology 44: Palasz AT, Mapletoft RJ. (1996) Cryopreservation of mammalian embryos and oocytes: Recent advances. Biotech Advan 14: Palasz AT, Thundathil, J, Verall RE, Mapletoft RJ. (2000) The effect of macromolecule supplementation on the surface tension of bovine oocyte maturation and embryo culture media and on oocyte/embryo development. Anim Repro Sci 58: Perry George (2017) 2016 statistics of embryo collection and transfer in domestic farm animals. Embryo Technology Newsletter, 35(4):8-23. Pierson RA, Ginther OJ. (1987) Follicular populations during the estrous cycle in heifers I. Influence of day. Anim Repro Sci 14: Pursley JR, Mee MO, Wiltbank MC. (1995) Synchronization of ovulation in dairy cows using PGF2α and GnRH. Theriogenology 44: Rall WF, Fahy GM. (1985) Ice-free cryopreservation of mouse embryos at -196 o C by vitrification. Nature 313, Remillard R, Martínez MF, Bó GA et al (2006) The use of fixed-time techniques and ecg to synchronize recipients for frozen-thawed bovine IVF embryos. Reprod Fertil Dev 18:204 (Abstract) Revah I, Butler WR (1996) Prolonged dominance of follicles and reduced viability of bovine oocytes. J Reprod Fert 106: Rodrigues CA, Mancilha RF, Dalalio M et al (2003) Increase of conception rates in IVF embryo recipients treated with GnRH at embryo transfer moment. Acta Sci Vet 33: (Abstract). Romero A, Albert J, Brink Z, Seidel GE, Jr. (1991) Numbers of small follicles in ovaries affect superovulation response in cattle. Theriogenology 35:265. Rowe RF, Del Campo MR, Eilts CL, French LR, Winch RP, Ginther OJ. (1976) A single cannula technique for nonsurgical collection of ova from cattle. Theriogenology 6: Rowe RF, Del Campo MR, Critser JK, Ginther OJ. (1980) Embryo transfer in cattle: Nonsurgical transfer. Am J Vet Res 41: Saacke RG, Nebel RL, Karabius DS et al (1988) Sperm transport and accessory sperm evaluation. Proceedings of 12th NAAB Technical Conference on Artificial Insemination pp.7-14 Saeki K, Hoshi M, Leibfried-Rutledge ML, First NL. (1991) In vitro fertilization and development of bovine oocytes matured in serum-free medium. Biol Reprod 44: Sala LC, Sala RV, Fosado M et al (2016) Factors that influence fertility in an IVF embryo transfer program in dairy heifers. Reprod Fert Dev 28:183 (Abstract) Santos JEP, Narciso CD, Rivera F et al (2010) Effect of reducing the period of follicle dominance in a timed AI protocol on reproduction of dairy cows. J Dairy Sci 93: Saumande J, Chupin D, Mariana J et al (1978) Factors affecting the variability of ovulation rates after PMSG stimulation. In: Sreenan JM (ed) Control of Reproduction in the Cow. Martinus Nijhoff, The Hague pp Schams D, Menzer D, Schalenberger E et al (1978) Some studies of the pregnant mare serum gonadotrophin (PMSG) and on endocrine responses after application for superovulation in cattle. In: Sreenan JM (ed) Control of Reproduction in the Cow. Martinus Nijhoff, The Hague pp Schiewe MC, Looney CR, Johnson CA, Hill KG, Godke RA. (1987). Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle. Theriogenology 28: Schneider U, Hahn J (1979) Embryo transfer in Germany. Theriogenology 11:

27 Schultz RH (1980) History of the International Embryo Transfer Society - Part II. Theriogenology 13:7-12 Seidel GE Jr. (1981) Superovulation and embryo transfer in cattle. Science 211: Seidel GE Jr., Elsden RP, Hasler JF (2003) Embryo Transfer in Dairy Cattle. W.D. Hoard & Sons Co., Ft. Atkinson, WI, 96 pages. Shaw DW, Good TE. (2000) Recovery rates and embryo quality following dominant follicle ablation in superovulated cattle. Theriogenology 53: Singh EL. (1985) Disease control: procedures for handling embryos. Rev sci Tech Off Int epiz 4: Singh Jaswant, Dominguez Manuel, Jaiswal Rajesh, Adams Gregg P. (2004) A simple ultrasound test to predict superstimulatory response in cattle. Theriogenology 62: Small J, Colazo M, Ambrose D et al (2004) Pregnancy rate following transfer of in vitro- and in vivo-produced bovine embryos to LH-treated recipients. Reprod Fertil Dev 16:213 (Abstract) Small JA, Colazo MG, Kastelic JP et al (2007) The effects of CIDR and ecg treatment in a GnRH-based protocol for timed- AI or embryo transfer on pregnancy rates in lactating beef cows. Reprod Fertil Dev 19: (Abstract) Small JA, Colazo MG, Kastelic JP et al (2009) Effects of progesterone pre-synchronization and ecg on pregnancy rates to GnRH-based, timed-ai in beef cattle. Theriogenology 71: Soares JG, Martins CM, Carvalho NAT et al (2011) Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors. Anim Reprod Sci 127: Souza AH, Rozner A, Carvalho PD et al (2014) Relationship between circulating AMH (anti-mullerian hormone) and embryo production in superovulated high producing donor cows. In: Proceedings of the Joint Meeting of the American and Canadian Embryo Transfer Associations, Madison, WI pp Steel Trevor. (2007). Cleaning and sterilization of equipment in ET and IVF. Techniques, times and recommendations. Proc. Ann Mtg CETA and CLGA. Pp Steel R, Hasler J (2009) Comparison of three different protocols for superstimulation of dairy cattle. Reprod Fertil Dev 21:246 (Abstract) Stoebel D, Moberg G (1982) Repeated acute stress during the follicular phase and luteinizing hormone surge of dairy heifers. J Dairy Sci 65:92 96 Stringfellow DA, Givens MD, Waldrop JG. (2004) Biosecurity issues associated with current and emerging embryo technologies. Reprod Fert Dev 16: Sutherland W (1991) Biomaterials - Novel material from biological sources. In: Byrom D (ed) Stockton Press, New York, NY pp Stroud B, Hasler JF. (2006) Dissecting why superovulation and embryo transfer usually work on some farms but not on others. Theriogenology 65: Thatcher WW, Drost M, Savio JD, Macmillan KL, Schmitt EJ, Entwistle KW, De la Sota RL, Morris GR. (1993) New clinical uses of GnRH and its analogues in cattle. Anim Reprod Sci 33: Thatcher WW, Moreira F, Santos JEP et al (2001) Effects of hormonal treatments on reproductive performance and embryo production. Theriogenology 55:75 89 Toner JP, Seifer DB (2013) Why we may abandon basal follicle-stimulating hormone testing: a sea change in determining ovarian reserve using antimullerian hormone. Fertil Steril 99: Tribulo R, Balla E, Cutaia L et al (2005) Effect of treatment with GnRH or hcg at the time of embryo transfer on pregnancy rates in cows synchronized with progesterone vaginal devices, estradiol benzoate and ecg. Reprod Fertil Dev 17:234 (Abstract) Tríbulo A, Rogan D, Tribulo H et al (2011) Superstimulation of ovarian follicular development in beef cattle with a single intramuscular injection of Folltropin-V. Anim Reprod Sci 129:7 13 Tríbulo A, Rogan D, Tríbulo H et al (2012) Superovulation of beef cattle with a split-single intramuscular administration of Folltropin-V in two concentrations of hyaluronan. Theriogenology 77: van Wagtendonk AM, den Daas JHG, Rall WF. (1996) Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: Vitrification one-step dilution versus slow freezing and three-step dilution. Theriogenology 48:

28 Vieira LM, Rodrigues CA, Castro Netto et al (2014) Superstimulation prior to the ovum pick-up to improve in vitro embryo production in lactating and non-lactating Holstein cows. Theriogenology 82: Vieira LM, Rodrigues CA, Castro Netto A et al (2015) Efficacy of a single intramuscular injection of porcine FSH in hyaluronan prior to ovum pick-up in Holstein cattle. Theriogenology 84:1-10. Voelkel SA, Hu YX. (1992) Direct transfer of frozen-thawed bovine embryos. Theriogenology 37: Wiltbank MC. (1997) How information of hormonal regulation of the ovary has improved understanding of timed breeding programs. Proceedings of the Annual Meeting of the Society for Theriogenology pp Wallace LD, Breiner CA, Breiner RA et al (2011) Administration of human chorionic gonadotropin at embryo transfer induced ovulation of a first wave dominant follicle, and increased progesterone and transfer pregnancy rates. Theriogenology 75: Walsh JH, Mantovani R, Duby RT et al (1993) The effects of once or twice daily injections of p-fsh on superovulatory response in heifers. Theriogenology 40: Wehrman ME, Fike KE, Melvin EJ et al (1997) Development of a persistent ovarian follicle and associated elevated concentrations of 17β-estradiol preceding ovulation does not alter the pregnancy rate after embryo transfer in cattle. Theriogenology 47: Wilson JW, Jones AL, Moore K et al (1993) Superovulation of cattle with a recombinant-dna bovine follicle stimulating hormone. Anim Reprod Sci 33:71 82 Wock J, Lyle L, Hockett M (2008) Effect of gonadotropin-releasing hormone compared with estradiol-17β at the beginning of a superstimulation protocol on superovulatory response and embryo quality. Reprod Fertil Dev 20:228 (Abstract) Wrathall AE, Simmons HA, Bowles DJ, Jones S. (2004) Biosecurity strategies for conserving valuable livestock genetic resources. Reprod Fert Dev 16: Wright JM. (1981) Non-surgical embryo transfer in cattle. Theriogenology 15:

29 Oviductal and Early Uterine Effects on Pregnancy Success Mario Binelli a, Angela Maria Gonella-Diaza b, Thiago Martins b, Mariana Sponchiado b a. Department of Animal Sciences, University of Florida, Gainesville, FL, USA b. Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga-SP, Brazil Correspondence: Mario Binelli, Department of Animal Sciences, University of Florida, PO Box , Gainesville, FL 32611, USA, mario.binelli@ufl.edu 1. Synopsis Biochemically well-defined oviductal and uterine luminal environments are required for successful preimplantation embryo development in cattle. Developmental needs of the growing embryo/conceptus are expected to change over time. In this paper, we will discuss specific mechanisms that must be in place to execute the dynamic changes needed to generate and modify the luminal molecular compositions required to support early pregnancy events. 2. Introduction Ask any bovine embryo transfer practitioner if she/he would rather transfer a fresh, in vivo-produced, or a fresh, in vitro-produced embryo to obtain a successful gestation and the answer would unanimously favor the in vivo option. The simple difference between these two types of embryos is the exposure to the female reproductive tract. What does the reproductive tract provide to the embryo that influences its developmental capacity is the key research question for many groups including ours. Understanding reproductive tract functions to support early-embryo development is critical to advance the field of embryo production and embryo transfer. Preimplantation embryo development in vivo depends on successful interactions between the embryo and the reproductive tract. Initial development occurs in the oviductal lumen, and about 4-5 days after estrus, the embryo is transported to the uterine lumen to continue development through implantation, that occurs about 20 days after estrus (Figure 1). A key feature of these initial three weeks of development is that there are only loose connections between embryonic and maternal tissues. Thus, the mucous environment surrounding the embryo must possess the biophysical and biochemical characteristics that support its growth and development. Moreover, such characteristics are expected to change dynamically throughout the window of pre-implantation development, in order to attend to the changing needs of the developing embryo. Therefore, it is obvious that change in the composition of luminal components throughout early pregnancy is a regulated activity. Systemic regulators of such activity are the temporal fluctuations in the concentrations of sex steroids, estradiol (E2) and progesterone (P4). Sex steroids actions are through cognate receptors in the oviductal and endometrial cells, and are expected to induce tissue, cellular, and molecular changes that will ultimately affect biophysical and biochemical properties of the environment surrounding the embryo. Additional critical regulation of reproductive tract function is provided by the embryo itself. Indeed, embryo-derived micro- and macromolecules, such as prostanoids and proteins, target the oviduct and endometrium to regulate their activities. Collectively, pregnancy success depends on individual and dynamically-interacting properties of the reproductive tract and the developing embryo. The luminal compartment has the unique property of connecting the maternal and the embryonic units, providing the milieu for functional molecular exchanges. In this review, we will show information to support the importance of the luminal composition for pregnancy success and show evidence for sex-steroidal and embryonic control of reproductive tract function to support pregnancy in beef cattle. 27

30 Figure 1. Reproductive events occurring in the female reproductive tract and temporal changes in sex steroid concentrations from estrus to D7 of pregnancy in cattle. Each panel represents a specific day of the estrous cycle, the expected localization of gametes or the embryo and its association with oviductal or endometrial cells. On the right side of each panel, the bar graph shows relative concentrations of E2 and P4, represented as a percentage of the maximal concentration of each hormone achieved during the estrous cycle. Day 0 (standing estrus): during the pre-ovulatory phase, the preovulatory follicle produces maximal concentrations of E2. The ampulla develops morphological characteristics compatible with a high secretion, i.e.: tunica mucosa became more folded, the luminal epithelium is taller and, specifically, the secretory cells secrete macromolecules via secretory granules to the oviductal lumen. In the isthmus, the ciliated cells gain more cilia, and ciliary beating increases. In both regions, proliferation of secretory cells and vascularization is observed. After mating, spermatozoa migrate and accumulate in the caudal portion of the isthmus, as they bind to ciliated cells. Day 1 (ovulation): under decreasing E2 concentrations, the cumulus oocyte complex (COC) is transported from the infundibulum to the site of fertilization in the ampulla within min. At the site of fertilization, cumulus cells establish a strong connection with the oviductal epithelial cells, which pauses COC movement. Meanwhile, the sperm cells are released from the isthmus, resume their cranial migration, and begin hyperactivation and early acrosome reaction. 28

31 Figure 1 continued Day 2: as soon as a sperm cell penetrates the zona pellucida, the zygote detaches and continues its caudal migration. Embryonic cells undergo mitosis and the embryo grows. Decreasing concentrations of E2 and increasing concentrations of P4 decreases the speed of embryo transport. This allows exposure of the embryo to ampullary secretions that may affect its composition and development. Day 3: the oviductal secretions affect the development of the embryo and, in turn, the embryo modulates the oviductal transcriptome and secretome in a complex two-way communication. The oviductal fluid is the sole extraembryonic source of nutrients and growth factors that the embryo needs to continue developing. As the embryo transitions to the isthmus, it finds an increasing proportion of ciliated cells. Day 4: final transport of the embryo to the uterus occurs around 3.5 days after fertilization, under increasing concentrations of P4. The endometrium is preparing for embryo s arrival. Both, luminal and glandular epithelial cells are in active proliferation. Also, various types of metabolites are accumulating inside endometrial cells. Day 7: elevated luteal P4 inhibits oviductal secretory and transport activities. The oviduct prepares for the next estrous cycle. The embryo is already inside the uterine lumen and is exposed to secretions from endometrial cells (especially by endometrial glands). At this stage, the embryo is capable of sending signals to the surrounding endometrial cells. These signals, which may include interferon-tau, are very localized at this point, but they are able to stimulate the expression of specific genes and change cellular function. 3. Embryo effect vs. reproductive tract effect The fact that both the reproductive tract and the developing embryo play decisive roles in pregnancy outcome creates the scientific and practical challenge of determining which is at fault when pregnancy fails. Attempts have been made to study these two components individually. Embryonic response to a given uterine environment seems to be variable and unpredictable. For example, Betteridge et al. (1980) observed striking differences in the size of embryos recovered from super-ovulated donors 14 days after estrus (D14; mm) and on D16 ( mm). This was also true for an individual donor, that had conceptuses that varied in length from 4 to 40 mm on D14. Similarly, Garrett et al. (1988) verified that conceptuses recovered after natural mating from D14 uteri of P4-treated cows (37.3 ± 14.9 mm) were longer than those recovered from controls (3.8 ± 1.9 mm); however, they noticed a large variability within treatments (control: 1 13 mm; P4 treated: mm). Others studies also reported variability on the length of conceptus recovered on D14 to D16 post-estrus in cattle (Clemente et al., 2009) and sheep (Rowson e Moor, 1966). Further, competence of an individual embryo to develop, elongate and signal is intrinsically confounded with the ability of the oviduct and uterus to support embryonic development. It is well accepted that the ability of the uterus to support preimplantation embryo development is associated with the biophysical and biochemical properties of the luminal fluid, also termed histotroph (Spencer et al., 2004). To study the importance of histotroph composition on pregnancy outcome, we conducted uterine flushings as a mean to remove molecules that are critical for embryo development, generating an impoverished environment. Specifically, we flushed the uterine lumen of recipients 1, 4 or 7 days after estrus with 30 ml of PBS, or did not flush them, and transferred three fresh IVF embryos on D7.5 (Martins et al., 2018). Three embryos were transferred to minimize the random effect of a poor-quality, developmentally-incompetent embryo on the pregnancy outcome (Berg et al., 2010). Pregnancy rates measured on D25 were severely reduced when washings were performed on D4 (29.4% [5/17]) or D7 (37.5% [6/16]) compared to D1 or control (61.3% [19/31]). These data confirm that an artificially impoverished uterine luminal milieu is detrimental for pregnancy success. Furthermore, it was noteworthy that even when three embryos were transferred, approximately one-third of the recipients in the non-manipulated control group was unable to support embryo development. These findings suggest that mortality was due to poor uterine receptivity, since it would be unlikely that all three embryos transferred were developmentally incompetent. It was also remarkable that even when washing was conducted just 12 hours prior to embryo transfer, a proportion of recipients was still able to maintain their pregnancies. Altogether, our data provide clear indication that recipients have varying abilities to sustain pregnancies, and embryos have varying resiliencies to thrive in uterine environments of very distinct quality. In the field, tools are lacking to practitioners to manipulate embryo s ability to survive and maintain a gestation. In contrast, embryo recipients may be set up to maximize receptivity to the transferred embryo. Adequate E2 priming during proestrus and P4 exposure during early diestrus are associated positively with pregnancy success. 29

32 We manipulated the growth of the pre-ovulatory follicle to influence peri-ovulatory concentrations of sex-steroids. Reduced concentrations of E2 and P4, for example, verified when cows submitted to timed artificial insemination were induced to ovulate smaller follicles ( 11.0 mm) resulted in lower fertility (Perry et al., 2005; Bridges et al., 2013). In corroboration, we verified that cows ovulating smaller follicles (10.8 mm) presented lower pregnancy rate [41.5% (17/41)] than those ovulating larger follicles [13.2 mm; 55.6% (25/45); Pugliesi et al., 2016]. Therefore, reproductive tract ability to maintain pregnancy is programmable by sex-steroid hormones. We propose that such ability is through specific tissue, cellular and molecular changes in the oviductal and the uterine functions that support embryo development. We will next review information on sex-steroid regulation of oviductal and endometrial function. 4. Sex-steroid regulation of oviductal morphology, molecular function and luminal composition The oviduct plays a major role in sperm storage and capacitation, fertilization and early embryo development. It has different regions that fulfill specific functions (Hunter et al., 1983; Abe, 1996; Hunter, 1998). Based on its macro-anatomical characteristics, the oviduct can be divided into four regions: the infundibulum, the ampulla, the isthmus, and the oviductal portion of the utero-tubal junction (UTJ; Figure 1). The infundibulum contains fimbriae that, after ovulation, pick up the oocyte and takes it into the lumen of the ampulla. The ampulla possesses a remarkably secretory epithelium. There, the oocyte completes its nuclear and cytoplasmic maturation, and fertilization and first cell divisions of the embryo take place. The isthmus and UTJ play major roles prior to fertilization, initially by retaining and capacitating spermatozoa. After fertilization, they interact with the developing embryo until it is transported into the uterine lumen, at the stage of 8 16 cells in cattle (Hunter, 1998; 2012). In vivo, the oviductal tissue is exposed locally and systemically to drastic changes in the ovarian steroid profile. These hormonal changes promote significant morphological (Eriksen et al., 1994; Abe, 1996; Morita et al., 2001), biochemical (Bishop, 1956; Ayen et al., 2012), and physiological (Wijayagunawardane et al., 2005; Sostaric et al., 2008; Wijayagunawardane et al., 2009) changes in the oviduct during the estrous cycle. Many studies have demonstrated that appropriate timing and prominence of sex-steroid hormones is important to ensure maternal receptivity (Ashworth et al., 1989; Demetrio et al., 2007; Morris e Diskin, 2008). It has also been established that cows ovulating larger follicles can attain greater proestrus E2 plasma concentrations, form a larger corpus luteum (CL), and produce greater P4 concentrations during early diestrus (Vasconcelos et al., 2001; Demetrio et al., 2007; Peres et al., 2009; Mesquita et al., 2014). Using an animal model in which the growth of the pre-ovulatory follicle was manipulated, we created two contrasting groups: cows ovulating a large follicle and forming a large CL (LF-LCL group), and animals ovulating a small follicle and forming a small CL (SF-SCL group). The LF-LCL group presented greater E2 concentrations at proestrus and P4 concentrations at early diestrus (Mesquita et al., 2014), and was associated with a more-receptive uterine state (see section 5, below) and fertility (Pugliesi et al., 2016). Using these experimental groups, we conducted a series of experiments in order to explore the sex-steroid control of the oviductal biology and function. In these studies, animals were slaughtered on D4 and tissue samples of ampulla and isthmus, as well as oviductal luminal fluid samples, were collected. In a first study (Gonella-Diaza et al., 2015), ampulla and isthmus transcriptome was obtained by RNAseq and the protein distribution of Progesterone Receptor (PGR) and Estrogen Receptor alpha (ERα) was evaluated by immunohistochemistry. There was a greater abundance of PGR and ERα in the oviduct of LF-LCL animals, indicating a greater availability of receptors and possibly sex steroids-stimulated signaling in both regions. The transcriptomic profiles showed a series of genes associated with functional characteristics of the oviduct that are regulated by the periovulatory sex steroid milieu and that potentially affect oviductal receptivity and early embryo development. Enriched biological processes included tissue morphology changes [extracellular matrix (ECM) remodeling], cellular changes (proliferation), and secretion changes (growth factors, ions, and metal transporters). Transcripts associated to these processes were up-regulated in the LF-LCL group (Gonella-Diaza et al., 2015). Then, a series of morphological features were evaluated in order to establish whether there was evidence for active secretion in the ampulla region (Gonella-Diaza et al., 2017). The LF-LCL group presented a more folded tunica mucosa, which indicates a greater epithelial area. In addition, the LF-LCL group presented a greater number of secretory cells and cells undergoing mitosis (Ki-67 positive cells). Collectively, we propose that phenotypic changes agreed with the transcriptomic findings. Furthermore, transcriptomic evidence suggested that ECM remodeling was stimulated in the LF-LCL group. This is a very important biological process. The ECM is an arrangement of extracellular molecules that offer structural support and interact with the surrounding cells to regulate important processes such as migration, proliferation, 30

33 apoptosis, and/or cellular differentiation (Lu et al., 2011; Bonnans et al., 2014). Also, growth factors that are stored in the ECM, are normally released locally during the remodeling process through ECM cleavage. We evaluated the expression of nine proteins involved in the ECM remodeling process and we localized and quantified an ECM structural protein (Collagen type 1; Gonella-Diaza et al., 2018). The isthmus of the LF-LCL possesses not only more transcripts but also more proteins that are active part of the ECM remodeling process. This was related with fewer collagen fibers when compared with the SF-SCL animals. Additionally, LF-LCL animals had a greater abundance of growth factors transcripts. Next, quantitative mass spectrometry was used to determine the concentration of 21 amino acids (AA), 21 biogenic amines (BA), 40 acylcarnitines (AC), 76 phosphatidylcholines (PC), 14 lysophosphatidylcholines (LP), 15 sphingomyelins (SM), hexoses, and 17 prostaglandins and related compounds in the oviductal luminal Fluid (unpublished data). Multivariate analyses showed that the overall metabolite profiles of the LF-LCL and SF-SCL groups were significantly different and that samples from each group were divided clearly into two nonoverlapping clusters. The most influential variables to separate the two groups included AAs, PCs, LPs and arachidonic acid. These results were further confirmed by univariate statistical analyses. There were statistical differences in the concentration of 31 metabolites (P 0.05) between groups. We concluded that the composition of the oviductal luminal fluid is different between cows with contrasting receptivity and fertility status. In summary, LF-LCL animals possess more ERαa and PGR receptors and differential transcriptome in ampulla and isthmus, more secretory and proliferating cells in ampulla, an active ECM remodeling process in the isthmus and a specific composition of their oviductal fluid. All these data demonstrate that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates the oviductal receptivity. 5. Sex-steroid regulation of endometrial molecular function and luminal composition The bovine endometrium is a dynamic tissue that undergoes spatio-temporal functional changes orchestrated by the ovarian steroids E2 and P4. Regarding sex-steroid programming of endometrial function, manipulation of preovulatory follicle growth and associated changes in proestrus E2 and diestrus P4 concentrations regulate the endometrial transcriptome and function (Mesquita et al., 2015; Oliveira et al., 2015; Ramos et al., 2015), and fertility (Pugliesi et al., 2016). As mentioned above, our group described a model to manipulate preovulatory follicle growth to produce groups of cyclic beef cows with distinctly different preovulatory E2 concentrations and early diestrus P4 concentrations (Mesquita et al., 2014). Based on the contrasting ovarian and endocrine characteristics of these two groups of animals, we studied the endometrial transcriptomic profile by RNA sequencing and different candidate pathways involved in endometrial receptivity on day 4 and 7 after induction of ovulation. The results revealed differential enrichment of biological processes, as subsequently described for selected endometrial pathways. The periovulatory endocrine milieu affected the D7 endometrial molecular signature (Mesquita et al., 2015). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF-LCL endometrium, whereas SF-SCL endometrium transcriptome was biased toward cell proliferation (Figure 1, days 4 and 7). Data also suggested a reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF-SCL endometrium. LF-LCL endometrium showed an earlier onset of proliferative activity, whereas SF-SCL endometrium expressed a delayed increase in glandular epithelium proliferation. Collective interpretation of transcriptome and phenotypic data is suggestive of a shift from a proliferation-permissive phenotype to a more biosynthetic and metabolically active endometrium. Biosynthesis may be necessary to provide the endometrium-derived trophic factors toward the early developing embryo present in the endometrial lumen. In agreement, Scolari et al. (2016) verified a down-regulation of ECM-related transcripts in the LF-LCL group. This finding was consistent with a greater abundance of total collagen content in SF-SCL on D4 endometrium, indicating that specific endocrine profiles drive ECM related gene expression profiles and even phenotypic characteristics of the ECM in the tissue. Using the same animal model, Ramos et al. (2015) showed that cows from the SF-SCL group display decreased capacity to control its redox status. Consequently, there was an increased lipid peroxidation in the endometrium on D7. These data put forward that the redox environment found in the group with smaller ovulatory follicles might be one of the causes of the reduced fertility found in these animals, as described in the literature. In addition, Belaz et al. (2016) characterized the phospholipid profile of endometrial tissue collected on days 4 or 7 from animals treated to ovulate a SF or a LF. Data generated by MALDI-MS showed that global and specific phospholipid abundances were indeed modulated by periovulatory endocrine milieus. 31

34 Collectively, extensive data from our group have shown that timing and magnitude of pre-ovulatory follicular E2 and post-ovulatory P4 programs endometrial transcriptome and function. Moreover, we also have shown that the sex-steroid regulation of the endometrial function defines the biochemical quality of the uterine microenvironment. Using the same animal model mentioned above (Mesquita et al., 2014), França et al. (2017) showed that concentrations of amino acids in the uterine flushings on D4 were less for the LF-LCL group but on D7 concentrations were greater for that group. Our unpublished data on metabolomics of the luminal fluid show consistent results. Our interpretation is that on D4, the actively proliferating endometrium on LF-LCL cows uses metabolites and energy sources for growth, therefore sparing less of these compounds to the uterine lumen. On D7, there is an increase in the secretory activity of the endometrium that is reflected by an enrichment in the luminal abundance of specific metabolites. We speculate that such increased abundance is necessary to support embryo growth. 6. Early embryo-dependent regulation of oviductal and endometrial function Embryo-maternal interactions consist in a stepwise progression of dynamic events that are initiated and sustained by the developing embryo and the properly steroid-stimulated oviductal and uterine tissues to ensure establishment and maintenance of pregnancy. In the present review, after discussing the sex-steroid control of oviductal and endometrial functions, we now introduce the topic of embryo-dependent programming of the maternal tract function during the pre-implantational period. For many years, researches believed that the oviduct was merely a conduit that only served for the passage of the oocyte to the endometrium. Currently, it is well accepted that the oviductal environment plays a major role in the reproductive process and that oviductal cells interact intimately with gametes and the embryo as they transit through the oviductal lumen. For example, in mares, the oviduct has the ability to recognize unfertilized oocytes, which are retained at the UTJ, while viable embryos can reach the uterine lumen (Betteridge et al., 1979). In cattle, Wetscher et al. (2005) reported that bovine embryos of different qualities and developmental stages show distinct migration patterns after intra-tubal embryo transfer. Also, the porcine oviduct transcriptome is modulated differently in response to X- and Y-chromosome-bearing spermatozoa (Almiñana et al., 2014). When in vivo and in vitro embryo culture conditions are compared, the results of embryo implantation, conception rate, and survival rate after vitrification are superior for the in vivo conditions (Rizos, Dimitrios, Fair, Trudee, et al., 2002; Rizos, Dimitrios, Ward, Fabian, et al., 2002). Also, embryonic transcriptome (Rizos, D. et al., 2002; Nagatomo et al., 2015) and epigenome (Salilew-Wondim et al., 2015) are different between in vivo and in vitro produced embryos. Collectively, these data suggest that the oviduct has the capacity to establish a communication process with the embryo. This process, however, is challenging to study, because of its biochemical and molecular nature, the difficulty of obtaining oviductal samples and the fact that tissue responses may be restricted to limited portions of tissue. Many studies using non-ruminant models (mice, rabbits, pigs) demonstrate that oviductal gene expression was modulated in the presence of embryos. Maillo et al. (2015) showed that, using a model where multiple embryos (Up to 50) were transferred inside the oviductal lumen, the expression of a series of immune-related genes was downregulated in the oviduct. However, authors failed to detect changes in oviductal gene expression in response to a single embryo. Regarding early-embryo effects in the endometrium, we recently reported that the endometrial abundance of specific transcripts is altered by the presence of a day 7 embryo in a spatial-specific manner (Sponchiado et al., 2017) in vivo. The most effects were found in the cranial portion of the ipsilateral uterine horn, close to where the embryo is located on D7. This is probably due to the limited capacity of synthesis, secretion, and diffusion of signaling molecules from the early embryo. Transcripts that were affected by the embryo presence include classical interferon-stimulated genes and prostaglandin biosynthesis genes. In agreement, recent studies showed that bovine embryos are able to modulate co-cultured endometrial epithelial and peripheral blood mononuclear cells (PBMCs) in vitro from early stages of development (Talukder et al., 2017; Rashid et al., 2018). Gomez et al. (2018) reported that endometrial epithelial cells are able to recognize embryonic sex before day 8 in vitro. However, our understanding of how the embryo-maternal dialogue fine-tunes the uterine microenvironment and how the embryo can be in turn benefit from such signaling for optimal development is limited. Very recently, Talukder et al. (2018) detected interferon-tau protein expression in 16-cell stage embryos co-cultured with bovine oviductal epithelial cells, but not in embryos cultured in the absence of those cells. In addition, medium conditioned by the embryo was not able to modulate gene expression in cultured immune cells (PBMCs); whereas medium from a co-culture of embryos and bovine epithelial cells increased the expression of interferon-stimulated 32

35 genes, PTGES and TGFB1 in PBMCs. This finding indicates an interesting two-way interplay between the embryos and oviductal cells in vitro, where oviductal cells stimulate embryos to produce interferon-tau, which then acts on immune cells to promote an anti-inflammatory response in the oviduct. Studies over the past 20 years have indicated the existence of complex paracrine and endocrine in vivo communication between early embryo and the maternal tract in mammalians (Wolf et al., 2003). The functional relevance of the oviductal/uterine programming by the early embryo can be questioned due to the fact that in vitro produced bovine embryos can be transferred to the uterus as late as day 7 and are able to establish gestations successfully. However, we propose that exposure to the pre-hatching embryo may fine-tune oviductal and endometrial function to support subsequent pregnancy events. 7. Conclusions and perspectives It is widely accepted that the superior capacity of an in vivo produced embryo to maintain a pregnancy to term is due to its exposure to the reproductive tract in comparison to the current sub-optimal in vitro culture conditions. Indeed, there is increasing knowledge of reproductive tract functions, both from the oviduct and the uterus, that support embryo development. Importantly, some of these functions can be manipulated to further stimulate pregnancy outcome to artificial insemination and embryo transfer. Furthermore, there is growing evidence for previously unrecognized roles of the pre-hatching embryo to influence reproductive tract function to further support pregnancy success. Advances in embryo culture methodologies should incorporate the newly generated information on reproductive tract biology. 8. References Abe, H., The mammalian oviductal epithelium: regional variations in cytological and functional aspects of the oviductal secretory cells. Histology and histopathology 1996, 11 (3), Almiñana, C.; Caballero, I.; Heath, P. R.; Maleki-Dizaji, S.; Parrilla, I.; Cuello, C.; Gil, M. A.; Vazquez, J. L.; Vazquez, J. M.; Roca, J., The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa. BMC genomics 2014, 15 (1), 293. Ashworth, C. J.; Sales, D. I.; Wilmut, I., Evidence of an association between the survival of embryos and the periovulatory plasma progesterone concentration in the ewe. Journal of Reproduction and Fertility 1989, 87 (1), Ayen, E.; Shahrooz, R.; Kazemie, S., Histological and histomorphometrical changes of different regions of oviduct during follicular and luteal phases of estrus cycle in adult Azarbaijan buffalo. Iranian Journal of Veterinary Research 2012, 13 (1), Belaz, K. R. A.; Tata, A.; França, M. R.; Santos da Silva, M. I.; Vendramini, P. H.; Fernandes, A. M. A. P.; D'Alexandri, F. L.; Eberlin, M. N.; Binelli, M., Phospholipid profile and distribution in the receptive oviduct and uterus during early diestrus in cattle. Biology of reproduction 2016, 95 (6), Berg, D. K.; Van Leeuwen, J.; Beaumont, S.; Berg, M.; Pfeffer, P. L., Embryo loss in cattle between Days 7 and 16 of pregnancy. Theriogenology 2010, 73 (2), Betteridge, K. J.; Eaglesome, M. D.; Randall, G. C. B.; Mitchell, D., Collection, description and transfer of embryos from cattle days after oestrus. Journal of reproduction and fertility 1980, 59 (1), Betteridge, K. J.; Eaglesome, M. D.; Flood, P. F., Embryo transport through the mare's oviduct depends upon cleavage and is independent of the ipsilateral corpus luteum. Journal of reproduction and fertility. Supplement 1979, (27), Bishop, D. W., Active secretion in the rabbit oviduct. American Journal of Physiology-Legacy Content 1956, 187 (2), Bonnans, C.; Chou, J.; Werb, Z., Remodelling the extracellular matrix in development and disease. Nature reviews Molecular cell biology 2014, 15 (12), 786. Bridges, G. A.; Day, M. L.; Geary, T. W.; Cruppe, L. H., Triennial Reproduction Symposium: deficiencies in the uterine environment and failure to support embryonic development. Journal of animal science 2013, 91 (7), Clemente, M.; de La Fuente, J.; Fair, T.; Al Naib, A.; Gutierrez-Adan, A.; Roche, J. F.; Rizos, D.; Lonergan, P., Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium? Reproduction 2009, 138 (3),

36 Demetrio, D. G. B.; Santos, R. M.; Demetrio, C. G. B.; Vasconcelos, J. L. M., Factors affecting conception rates following artificial insemination or embryo transfer in lactating Holstein cows. Journal of Dairy Science 2007, 90 (11), Eriksen, T.; Terkelsen, O.; Hyttel, P.; Greve, T., Ultrastructural features of secretory cells in the bovine oviduct epithelium. Anatomy and embryology 1994, 190 (6), Forde, N.; Carter, F.; Fair, T.; Crowe, M. A.; Evans, A. C. O.; Spencer, T. E.; Bazer, F. W.; McBride, R.; Boland, M. P.; O'Gaora, P., Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle. Biology of Reproduction 2009, 81 (4), Forde, N.; Spencer, T. E.; Bazer, F. W.; Song, G.; Roche, J. F.; Lonergan, P., Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium. Physiological genomics 2010, 41 (1), França, M. R.; da Silva, M. I. S.; Pugliesi, G.; Van Hoeck, V.; Binelli, M., Evidence of endometrial amino acid metabolism and transport modulation by peri-ovulatory endocrine profiles driving uterine receptivity. Journal of animal science and biotechnology 2017, 8 (1), 54. Garrett, J. E.; Geisert, R. D.; Zavy, M. T.; Gries, L. K.; Wettemann, R. P.; Buchanan, D. S., Effect of exogenous progesterone on prostaglandin F2α release and the interestrous interval in the bovine. Prostaglandins 1988, 36 (1), Gómez, E.; Sánchez-Calabuig, M. J.; Martin, D.; Carrocera, S.; Murillo, A.; Correia-Alvarez, E.; Herrero, P.; Canela, N.; Gutiérrez-Adán, A.; Ulbrich, S., In vitro cultured bovine endometrial cells recognize embryonic sex. Theriogenology 2018, 108, Gonella-Diaza, A. M.; da Silva Andrade, S. C.; Sponchiado, M.; Pugliesi, G.; Mesquita, F. S.; Van Hoeck, V.; de Francisco Strefezzi, R.; Gasparin, G. R.; Coutinho, L. L.; Binelli, M., Size of the ovulatory follicle dictates spatial differences in the oviductal transcriptome in cattle. PLoS One 2015, 10 (12), e Gonella-Diaza, A. M.; Mesquita, F. S.; da Silva, K. R.; de Carvalho Balieiro, J. C.; dos Santos, N. P.; Pugliesi, G.; de Francisco Strefezzi, R.; Binelli, M., Sex steroids modulate morphological and functional features of the bovine oviduct. Cell and tissue research 2017, 370 (2), Gonella-Diaza, A. M.; Mesquita, F. S.; Lopes, E.; da Silva, K. R.; Cogliati, B.; Strefezzi, R. D. F.; Binelli, M., Sex steroids drive the remodeling of oviductal extracellular matrix in cattle. Biology of reproduction Havlicek, V.; Wetscher, F.; Huber, T.; Brem, G.; Mueller, M.; Besenfelder, U., In vivo culture of IVM/IVF embryos in bovine oviducts by transvaginal endoscopy. Journal of Veterinary Medicine Series A 2005, 52 (2), Hunter, R. H. F., Components of oviduct physiology in eutherian mammals. Biological Reviews 2012, 87 (1), Hunter, R. H. F., Have the Fallopian tubes a vital role in promoting fertility? Acta obstetricia et gynecologica Scandinavica 1998, 77 (5), Hunter, R. H. F.; Cook, B.; Poyser, N. L., Regulation of oviduct function in pigs by local transfer of ovarian steroids and prostaglandins: a mechanism to influence sperm transport. European Journal of Obstetrics & Gynecology 1983, 14 (4), Leese, H. J., Metabolic control during preimplantation mammalian development. Human Reproduction Update 1995, 1 (1), Lu, P.; Takai, K.; Weaver, V. M.; Werb, Z., Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harbor perspectives in biology 2011, a Maillo, V.; Gaora, P. Ó.; Forde, N.; Besenfelder, U.; Havlicek, V.; Burns, G. W.; Spencer, T. E.; Gutierrez-Adan, A.; Lonergan, P.; Rizos, D., Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?1. Biology of Reproduction 2015, 92 (6), 144, , 1-8. Martins, T.; Pugliesi, G.; Sponchiado, M.; Maio, Gonella-Diaza, A. M.; Ojeda-Rojas, O. A.; Diaz, F.; Ramos, R. S.; Basso, A. C.; Binelli, M., Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle. Journal of Animal Science and Biotechnology, Accepted. Mesquita, F. S.; Pugliesi, G.; Scolari, S. C.; França, M. R.; Ramos, R. S.; Oliveira, M.; Papa, P. C.; Bressan, F. F.; Meirelles, F. V.; Silva, L. A., Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows. Theriogenology 2014, 81 (6), Mesquita, F. S.; Ramos, R. S.; Pugliesi, G.; Andrade, S. C. S.; Van Hoeck, V.; Langbeen, A.; Oliveira, M. L.; Gonella-Diaza, A. M.; Gasparin, G.; Fukumasu, H., The receptive endometrial transcriptomic signature indicates an earlier shift from proliferation to metabolism at early diestrus in the cow. Biology of reproduction 2015, biolreprod

37 Morita, M.; Miyamoto, H.; Sugimoto, M.; Sugimoto, N.; Manabe, N., Alterations in cell proliferation and morphology of ampullar epithelium of the mouse oviduct during the estrous cycle. Journal of Reproduction and Development 2001, 43 (3), Morris, D.; Diskin, M., Effect of progesterone on embryo survival. Animal 2008, 2 (8), Nagatomo, H.; Akizawa, H.; Sada, A.; Kishi, Y.; Yamanaka, K.-i.; Takuma, T.; Sasaki, K.; Yamauchi, N.; Yanagawa, Y.; Nagano, M., Comparing spatial expression dynamics of bovine blastocyst under three different procedures: in-vivo, invitro derived, and somatic cell nuclear transfer embryos. Japanese Journal of Veterinary Research 2015, 63 (4), Oliveira, M. L.; D'Alexandri, F. L.; Pugliesi, G.; Van Hoeck, V.; Mesquita, F. S.; Membrive, C. M. B.; Negrão, J. A.; Wheelock, C. E.; Binelli, M., Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus. Reproduction, Fertility and Development Peres, R. F. G.; Júnior, I. C.; Sá Filho, O. G.; Nogueira, G. d. P.; Vasconcelos, J. L. M., Strategies to improve fertility in Bos indicus postpubertal heifers and nonlactating cows submitted to fixed-time artificial insemination. Theriogenology 2009, 72 (5), Perry, G. A.; Smith, M. F.; Lucy, M. C.; Green, J. A.; Parks, T. E.; MacNeil, M. D.; Roberts, A. J.; Geary, T. W., Relationship between follicle size at insemination and pregnancy success. Proc Natl Acad Sci U S A 2005, 102 (14), Pugliesi, G.; Santos, F. B.; Lopes, E.; Nogueira, É.; Maio, J. R. G.; Binelli, M., Improved fertility in suckled beef cows ovulating large follicles or supplemented with long-acting progesterone after timed-ai. Theriogenology 2016, 85 (7), Ramos, R. S.; Oliveira, M. L.; Izaguirry, A. P.; Vargas, L. M.; Soares, M. B.; Mesquita, F. S.; Santos, F. W.; Binelli, M., The periovulatory endocrine milieu affects the uterine redox environment in beef cows. Reproductive Biology and Endocrinology 2015, 13 (1), 1. Rashid, M. B.; Talukder, A. K.; Kusama, K.; Haneda, S.; Takedomi, T.; Yoshino, H.; Moriyasu, S.; Matsui, M.; Shimada, M.; Imakawa, K., Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus. Biochemical and Biophysical Research Communications 2018, 500 (4), Rizos, D.; Fair, T.; Papadopoulos, S.; Boland, M. P.; Lonergan, P., Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Molecular Reproduction and Development: Incorporating Gamete Research 2002, 62 (3), Rizos, D.; Lonergan, P.; Boland, M. P.; Arroyo-Garcia, R.; Pintado, B.; Fuente, J. d. l.; Gutierrez-Adan, A., Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biology of reproduction 2002, 66 (3), Rizos, D.; Ward, F.; Duffy, P.; Boland, M. P.; Lonergan, P., Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Molecular reproduction and development 2002, 61 (2), Rowson, L. E.; Moor, R. M., Development of the sheep conceptus during the first fourteen days. Journal of anatomy 1966, 100 (Pt 4), 777. Scolari, S. C.; Pugliesi, G.; de Francisco Strefezzi, R.; Andrade, S. C.; Coutinho, L. L.; Binelli, M., Dynamic remodeling of endometrial extracellular matrix regulates embryo receptivity in cattle. Reproduction 2016, REP-16. Sostaric, E.; Dieleman, S. J.; Van De Lest, C. H. A.; Colenbrander, B.; Vos, P. L. A. M.; Garcia Gil, N.; Gadella, B. M., Sperm binding properties and secretory activity of the bovine oviduct immediately before and after ovulation. Molecular Reproduction and Development: Incorporating Gamete Research 2008, 75 (1), Spencer, T. E.; Johnson, G. A.; Bazer, F. W.; Burghardt, R. C., Implantation mechanisms: insights from the sheep. Reproduction 2004, 128 (6), Sponchiado, M.; Gomes, N. S.; Fontes, P. K.; Martins, T.; del Collado, M.; de Assumpção Pastore, A.; Pugliesi, G.; Nogueira, M. F. G.; Binelli, M., Pre-hatching embryo-dependent and-independent programming of endometrial function in cattle. PloS one 2017, 12 (4), e Talukder, A. K.; Rashid, M. B.; Yousef, M. S.; Kusama, K.; Shimizu, T.; Shimada, M.; Suarez, S. S.; Imakawa, K.; Miyamoto, A., Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells. Scientific reports 2018, 8 (1),

38 Talukder, A. K.; Yousef, M. S.; Rashid, M. B.; Awai, K.; Acosta, T. J.; Shimizu, T.; Okuda, K.; Shimada, M.; Imakawa, K.; Miyamoto, A., Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator. Journal of Reproduction and Development 2017, 63 (4), Vasconcelos, J. L. M.; Sartori, R.; Oliveira, H. N.; Guenther, J. G.; Wiltbank, M. C., Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate. Theriogenology 2001, 56 (2), Wijayagunawardane, M. P. B.; Kodithuwakku, S. P.; De Silva, N. T.; Miyamoto, A., Angiotensin II secretion by the bovine oviduct is stimulated by luteinizing hormone and ovarian steroids. Journal of Reproduction and Development 2009, 55 (5), Wijayagunawardane, M. P. B.; Kodithuwakku, S. P.; Yamamoto, D.; Miyamoto, A., Vascular endothelial growth factor system in the cow oviduct: a possible involvement in the regulation of oviductal motility and embryo transport. Molecular Reproduction and Development: Incorporating Gamete Research 2005, 72 (4), Wolf, E.; Arnold, G. J.; Bauersachs, S.; Beier, H. M.; Blum, H.; Einspanier, R.; Fröhlich, T.; Herrler, A.; Hiendleder, S.; Kölle, S., Embryo Maternal Communication in Bovine Strategies for Deciphering a Complex Cross Talk. Reproduction in Domestic Animals 2003, 38 (4),

39 Breed Associated Embryo Freezing Capacity Claude Robert PhD Université Laval, Département des sciences animales Centre de recherche en reproduction, développement et santé intergénérationnelle (CRDSI) Réseau québécois en reproduction (RQR) Institut sur la nutrition et les aliments fonctionnels (INAF) Summary Embryo cryopreservation is routinely done in the dairy industry allowing for short- or long-term storage of the embryos, their safe transportation over long distance and a better management of donors and recipients. It is commercially advantageous because genetics can be exchanged easily without exporting live animals. Although routinely done, damage to the embryo occurs frequently. Several factors affecting the embryonic capacity to survive cryopreservation have been highlighted and now a large focus is directed at intracellular lipid content. Genetics is known to impact lipid metabolism which can impact the surrounding microenvironment in which the embryo is submitted. Lipid accumulation can be influenced in vitro by the presence of lipid in the media. In vivo, reports show that the follicular fluid lipid content is partly correlated with serum levels suggesting that the embryonic lipid content could be influenced by maternal nutrition. There is also growing evidence that maternal nutrition and the overall maternal metabolic state is influencing oocyte quality and therefore the quality of the ensuing embryo. There is still a knowledge gap in the definition of the nutritional and metabolic conditions that are best suited for producing high quality embryos that will offer levels of intracellular lipids that will confer the best cryopreservation capacity. Cryotolerance In the field of embryo transfer, there is currently an increase in the demand for frozen rather than fresh embryos. However, not all embryos survive cryopreservation. During the freezing process, water crystals form and can cause structural damage to the embryonic cells to the extent of compromising cellular viability. The addition of protecting molecules such as ethylene glycol, propylene glycol or sucrose aims to reduce the formation and the size of water crystals. The traditional freezing procedure involves a controlled ramped decrease in temperature which is referred to as slow freezing. The second approach is a rapid freezing procedure called vitrification that aims to prevent the formation of large water crystals by inducing immediate freezing by dunking the embryos in liquid nitrogen. Rapid freezing produces more compact crystals that are less damageable to cell membranes. Both methods have been successfully used to freeze bovine embryos that were thawed and induced gestations to term. Since not all embryos are equal, not all embryos survive cryopreservation even when frozen in the same batch and coming from the same embryo collection. Identifying the determinants of what differ between an embryo that will survive freezing from another that will not has been challenging. Embryo morphology is not always predictive of the outcome following cryopreservation but better looking homogenous and compact embryos tend to be better than fragmented ones. However, it has been reported that lipid content plays an important role in the freezing potential since cell membranes are composed of lipids and that modifying their physical properties is crucial for the successful cryopreservation of bovine embryos (Sata et al. 1999; Kim et al. 2001). The colour of the embryo appears to be a predictor of embryo tolerance to cryopreservation (Massip 2001; Van Soom et al. 2003). Genetics/ breed effect Embryonic and sperm cryotolerance is known to be species specific. For instance, species such as pigs do not tolerate the freezing process very well. Therefore, there are intrinsic factors to each species that influence the capacity of cells to survive the stress of freezing. Furthermore, it has been shown that even within the same species, genetic subgroups can exhibit different tolerance to cryopreservation. In livestock, breeds are defined by their unique characteristics making individuals from the same breed distinguishable from the other breeds and animals from different breeds are inter-fertile. 37

40 In the dairy industry, cattle breeds differ considerably. The Holstein is recognised for providing the greatest volume of milk, whereas the Jersey is popular because of its relatively small body frame and the high milk composition in protein and butterfat percentage. Breeders regard the Jersey cow as robust, versatile and well suited to any production system. Compared to Holstein, Jerseys are more fertile when considering pregnancy rates from insemination during a natural cycle. However, it was found that Jerseys have a lower pregnancy rate after transferring cryopreserved-embryo than Holstein (Steel and Hasler 2004). However, it has also been observed that Jersey embryos do not tolerate freezing very well. Steel and Hasler (2004) showed that Jersey cryopreserved embryos produced significantly fewer gestations than did Holstein embryos. This problem is believed to be associated with the high lipid content of the Jersey embryo that is visually observed by being darker than Holstein embryos. Similarly, other studies have shown an impact of the breed on the color of the embryos. For example, Holstein embryos obtained in vivo were darker than Belgian Blue (Van Soom et al. 2003; Leroy et al. 2005). Moreover, Nellore embryos (Bos indicus) were found to be paler than Holstein (Visintin et al. 2002) or Simmental embryos (Sudano et al., 2012). In some breeds such as Jersey, results suggests that the gene pool leading to the high fat content of milk may involve differences in lipid metabolism (Beaulieu and Palmquist 1995; Bladoceda et al., 2015a). Lipids and embryo quality The roles played by lipids into embryogenesis is not well understood. While neutral lipids supply energy to embryos and are linked to improved developmental competence and early embryonic development (Kim et al. 2001; Aardema et al. 2011), several studies have clearly shown adverse effects of lipid accumulation on embryonic quality (Aardema et al. 2011) and on freezing tolerance of embryos (Van Soom et al. 2003). Therefore, lipids are essential as constituent of cellular membranes and as energetic substrate but this seems counterbalanced but the nature of the lipids that are accumulated. In vitro studies have shown that oocyte and early embryos are able to uptake lipids from their microenvironment. Early development was negatively affected when oocytes were cultured in the presence of palmitic (16:0) and stearic (18:0) acids (Aardema et al. 2011; Van Hoeck et al. 2011). During embryo culture, presence of high concentration of serum in the medium has been shown to induce the accumulation of intracellular cytoplasmic lipid droplets (Crosier et al., 2001). An inverse correlation between cytoplasmic lipid content and tolerance of freezing has been observed among embryos cultured in presence of serum (Abe et al. 1999, Yamashita et al. 1999; Hasler 2001; Reis et al. 2003). When comparing the lipid content of embryos of different breeds collected from animals submitted to a standardized nutritional regimen, Jersey embryos displayed more lipid droplets and the average droplet volume was smaller than in Holstein embryos. Deeper analysis of lipid composition showed that Jersey embryos were richer in palmitic (16:0) and linoleic (18:2) fatty acids compared to Holstein (Baldoceda et al., 2015a). Currently, palmitic acid was shown to exert some cytotoxic effect on oocytes and early embryos (Van Hoeck et al., 2011) whereas the role played by linoleic acid is still unclear whether it is positive or negative. These effects on early development seems to be dependent on concentration and to be reversible given change in the composition of the surrounding microenvironment. Analysis of embryonic gene expression between Jersey and Holstein showed differential expression of genes associated with lipid metabolism. Amongst these differentially expressed genes, some are known to be impacting lipid content by modulating their metabolism (adiponectin receptor) and the lipin protein family which are key effectors of triglyceride and phospholipid biosynthesis (Reue and Zhang 2008). Studies have shown that LPIN1 and LPIN2 modulate lipid droplet size, amount and fatty acid composition in mammalian cells (Sembongi et al. 2013). Another gene, Fatty Acid Elongase 5 (ELOVL5) is involved in lipid biogenesis which in turn appears to play an important role in modifying membrane fluidity by changing lipid content and fatty acid composition (Kim et al. 2001; Ferreira et al. 2010). This could explain the different embryonic sensitivity to cryopreservation observed between Jersey and Holstein (Steel and Hasler 2004). 38

41 Mitochondrial functions Mitochondria are intracellular organelles that play a multitude of key cellular functions such as energy production, control of apoptosis, lipid metabolism, steroidogenesis, and they produce metabolites that have important regulatory roles in various signaling pathways (Dumollard et al., 2007; Van Blerkom, 2009). In mammalian oocytes, a close spatial association and hence metabolic relationship between mitochondria and lipid droplets has been reported (Hyttel et al. 1986; Sturmey et al. 2006). Oocytes, have atypical mitochondrial contingent composed of immature organelles that have limited cristae resulting in a limited potential for the traditional ATP production via oxidative phosphorylation and glycolysis (Trimarchi et al., 2000). Since lipids can be metabolized by the mitochondria to produce energy, mitochondrial activity can potentially control intracellular lipid content. When Jersey embryos were compared to Holstein counterparts, mitochondrial activity was found to be lower in Jersey compared to Holstein (Baldoceda et al., 2015a). At the ultrastructure level, cells of Jersey embryos had a contingent of more immature mitochondria compared to Holstein (Baldoceda et al., 2015a,b). Similar observations were reported where an inverse relationship between the number of lipid droplets and the number of mitochondria in Holstein embryos compared with Nellore (Visintin et al. (2002). Combined, these observations suggest that the genetic background and mitochondrial activity can influence how lipids are managed therefore changing the nature and amount of intracellular lipids that can affect survival to cryopreservation. It is interesting that the darker cytoplasm observed in bovine embryos produced in vitro in several studies appears related to lipid uptake from the serum added to the culture medium and to be a consequence of impaired mitochondrial function (Abe et al. 1999; Reis et al. 2003; Plourde et al. 2012). Modulating the intracellular lipid content (in vitro and in vivo) To improve survival to cryopreservation, many possible solutions have been tested in attempts to reduce the intracellular lipid content, such as serum-free culture media (Abe et al. 2002; Rizos et al. 2003) or supplementation with different fatty acids (Aardema et al. 2011; Van Hoeck et al. 2011). Some positive experimental results have been obtained, but none of these approaches has met with notable success in commercial practice. Another tested approach was to induce lipid catabolism by helping mitochondrial functions. To do so, L-carnitine was added to culture media. This metabolic regulator could have the dual effects of regulating both lipid levels by transporting fatty acids to the mitochondria for catabolism and also protect the cells against accumulation of reactive oxygen species (ROS), thus improving development. Although the presence of L-carnitine significantly reduced lipid content, response to treatment was variable as some embryos were unaffected. Again, results highlighted a genetic contribution where the proportion of treatment-resistant embryos was significantly higher in Jersey than Holstein (Baldoceda et al. 2015b). Since such treatment is limited to in vitro embryo production, an in vivo treatment would have to account for lipid availability during oogenesis which takes place within the ovarian follicle and during embryogenesis which most of the pre-hatching development takes place in the oviduct. Recent observations are starting to elucidate the mechanism by which oocytes are uploaded with lipids found in the extracellular environment. It has been shown that the first layers of cumulus cells surrounding the oocyte have cellular projections extending across the zona pellucida and that these channels contain lipid carrying proteins (Del Collado et al., 2017). This suggests that the ovarian cells can upload lipids from the follicular fluid and transfer them to the oocyte. A potential solution to increase the production of freezing tolerant embryos could thus involve modifying the lipid composition of the follicular fluid. Since it has been shown that follicular fluid lipid content is partly correlated with serum levels (Leroy et al., 2005), maternal nutrition could potentially be used to control lipid content of the oocyte and ensuing embryo. Currently, the impact of maternal diet during oogenesis and early embryo development is not fully understood. The high energy or fat supplementation diets used in lactating cow to increase energy of the rations and limit the negative energy balance are not formulated for embryo production but to maximize milk production. However, the increase in energy or fatty acids content in the ration have a negative impact on quantity or quality of recovered oocytes and on the embryo yield. This effect is believed to be caused by the aberrant endocrine signaling caused by the negative energy balance (Wathes et al. 2007) that leads to the mobilisation of body fat reserves which releases non-esterified fatty acids (NEFAs) in the bloodstream. Palmitic acid and stearic acids are NEFAS that have been found to exert a toxic effect on the oocyte (Leroy et al. 2005, Van Hoeck et al. 2011). 39

42 Although it is clear that the lipid content of oocytes and early embryos is influenced by the surrounding microenvironment, there is still a knowledge gap in our understanding of the role played by maternal nutrition in modulating the nature and amount of cytoplasmic lipids to the extent of improving embryonic tolerance to cryopreservation. Concluding remarks Several factors contribute to the success of embryo transfer, including production (in vivo or in vitro), embryo quality, composition of the culture media and lipid content. Over the years, increasing productivity has led to improved management, nutrition and breeding programs. Anything impacting metabolism, including the genetic background, has the potential to influence the lipid content and composition of the microenvironment in which oogenesis and embryogenesis occur. Due to active uptake, lipid exposure can profoundly impact the embryonic lipid content and in turn impact freezing capacity. Aardema, H., Vos, P. L., Lolicato, F., Roelen, B. A., Knijn, H. M, Vaandrager, A. B., Helms, J. B., and Gadella, B. M. (2011). Oleic acid prevents detrimental effects of saturated fatty acids on bovine oocyte developmental competence. Biol. Reprod. 85, Abe, H., Yamashita, S., Itoh, T., Satoh, T., and Hoshi, H. (1999). Ultrastructure of bovine embryos developed from in vitromatured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Mol. Reprod. Dev. 53, Abe, H., Yamashita, S., Satoh, T., and Hoshi, H. (2002). Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Mol. Reprod. Dev. 61, Baldoceda L, Gilbert I, Gagné D, Vigneault C, Blondin P, Ferreira CR, Robert C. (2015a) Breed-specific factors influence embryonic lipid composition: comparison between Jersey and Holstein. Reprod Fertil Dev. doi: /RD Baldoceda L, Gagné D, Ferreira CR, Robert C. (2015b) Genetic influence on the reduction in bovine embryo lipid content by l-carnitine. Reprod Fertil Dev. doi: /RD Beaulieu, A., and Palmquist, D. (1995). Differential effects of high fat diets on fatty acid composition in milk of Jersey and Holstein cows. J. Dairy Sci. 78, Crosier, A., Farin, P., Dykstra, M., Alexander, J., and Farin, C. (2001). Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol. Reprod. 64, Del Collado M, da Silveira JC, Sangalli JR, Andrade GM, Sousa LRDS, Silva LA, Meirelles FV, Perecin F. (2017) Fatty Acid Binding Protein 3 And Transzonal Projections Are Involved In Lipid Accumulation During In Vitro Maturation Of Bovine Oocytes. Sci Rep. 7(1):2645. Dumollard, R., Duchen, M., and Carroll, J. (2007). The role of mitochondrial function in the oocyte and embryo. Curr. Top. Dev. Biol. 77, Ferreira, C. R., Saraiva, S. A., Catharino, R. R., Garcia, J. S., Gozzo, F. C., Sanvido, G. B., Santos, L. F., Lo Turco, E. G., Pontes, J. H., Basso, A. C., and Bertolla, R. P., Sartori, R., Guardieiro, M. M., Perecin, F., Meirelles, F. V., Sangalli, J. R., and Eberlin, M. N. (2010). Single embryo and oocyte lipid fingerprinting by mass spectrometry. J. Lipid Res. 51, Hasler, J. (2001). Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology. 56, Hyttel, P., Callesen, H., and Greve, T. (1986). Ultrastructural features of preovulatory oocyte maturation in superovulated cattle. J. Repro. Fert. 76, Kim, J. Y., Kinoshita, M., Ohnishi, M., and Fukui, Y. (2001). Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes. Reproduction 122, Leroy, J. L., Opsomer, G., De Vliegher, S., Vanholder, T., Goossens, L., Geldhof, A., Bols, P. E., de Kruif, A., and Van Soom, A. (2005). Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows. Theriogenology 64,

43 Massip, A. (2001). Cryopreservation of embryos of farm animals. Reprod. Domest. Anim. 36, Plourde, D., Vigneault, C., Lemay, A., Breton. L., Gagné, D., Laflamme, I., Blondin, P., and Robert C. (2012). Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts. Theriogenology 78, Reis, A., Rooke, J., McCallum, G., Staines, M., Ewen, M., Lomax, M., and McEvoy, T. (2003). Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro. Reprod. Fertil. Dev. 15, Reue, K., and Zhang, P. (2008). The lipin protein family: dual roles in lipid biosynthesis and gene expression. FEBS Lett. 582, Rizos D., Gutiérrez-Adán, A., Pérez-Garnelo, S., de la Fuente, J., Boland, M., and Lonergan, P. (2003). Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol. Reprod. 68, Sata, R., Tsuji, H., Abe, H., Yamashita, S., and Hoshi, H. (1999). Fatty acid composition of bovine embryos cultured in serum-free and serum-containing medium during early embryonic development. J. Repro. Dev. 45, Sembongi, H., Miranda, M., Han, G., Fakas, S., Grimsey, N., Vendrell, J., Carman, G., and Siniossoglou, S. (2013). Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells. J. Biol. Chem. 288, Steel, R., and Hasler, J. (2004). Pregnancy rates resulting from transfer of fresh and frozen Holstein and Jersey embryos. Reprod. Fert. 16, 182. [Abstract]. Sturmey, R. G., O'Toole, P. J., and Leese, H. J. (2006). Fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte. Reproduction 132, Sudano, M. J., Santos, V. G., Tata, A., Ferreira, C. R., Paschoal, D. M., Machado, R., Buratini, J., Eberlin, M. N., and Landim- Alvarenga, F. D. (2012). Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Biol. Reprod. 87, 130. Trimarchi JR, Liu L, Porterfield DM, Smith PJ, Keefe DL. (2000) Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos. Biol Reprod 62: Van Hoeck, V., Sturmey, R. G., Bermejo-Alvarez, P., Rizos, D., Gutierrez-Adan, A., Leese, H. J., Bols, P. E., Leroy, J. L. (2011). The effect of elevated NEFA during bovine oocyte maturation on early embryo physiology. PLoS ONE 6, e Van Soom, A., Mateusen, B., Leroy, J., and de Kruif, A. (2003). Assessment of mammalian embryo quality: what can we learn from embryo morphology?. Rep. Biomed. Online 7, Visintin, J. A., Martins, J. F., Bevilacqua, E. M., Mello, M. R., Nicácio, A. C., and Assumpção, M. E. (2002). Cryopreservation of Bos taurus vs Bos indicus embryos: are they really different? Theriogenology 57, Wathes DC, Fenwick M, Cheng Z, Bourne N, Llewellyn S, Morris DG, Kenny D, Murphy J, Fitzpatrick R. (2007) Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow. Theriogenology. 68 Suppl 1:S Yamashita, S., Abe, H., Itoh, T., Satoh, T., and Hoshi, H. (1999). A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing. Cytotechnology. 31,

44 Damage Prevention at Storage and Transportation of Frozen Bovine Embryos Angelika E. Stock Services vétérinaires mobiles de thériogénologie, Chambly-Saint-Hyacinthe, QC, Canada INTRODUCTION The damage of frozen semen and embryos due to careless handling, storage and transfer is an underestimated cause of pregnancy failure in an embryo transfer program Most of the points lined out in this presentation may well be known by the experienced embryo transfer practitioner, however I find they cannot be repeated often enough. This paper is intended to help educate the young and therefore less experienced veterinarian, veterinary student, technician, sales personnel as well as farmer and his employees who may handle embryos. Professionals who freeze embryos, know how crucial it is to place the embryo in a cryoprotectant solution and to perform the so called seeding technique on each single straw before the freezing process is started. Cryoprotectant and seeding allows for the embryo to be slowly dehydrated before it is frozen and for small ice crystals to be formed that spread throughout the solution avoiding the sudden formation of large crystals which may harm the embryonic cells. If we allow, however, temperature to rise in a straw with a frozen embryo for only a few seconds, thawing of some ice crystals may occur and then water recrystallizes when the straw is placed back into the liquid nitrogen. It is this recrystallization that can damage the cellular component of the embryo. If we deal with a straw of 0.5 cc and 20 million sperm, we may still have enough semen to accomplish fertilization. For a 0.25 cc straw with 2 million of sex sorted semen, fertility may be more easily compromised, because its surface to volume ratio is larger, i.e. the smaller diameter straw heats up faster. If a straw with one single embryo is damaged, the chance for a pregnancy is slim. STORAGE OF EMBRYOS AND SEMEN IN A LIQUID NITROGEN TANK The maintenance of very low temperature in the inner chamber of the nitrogen tank is due to its high- quality solid insulation material and vacuum in the outer chamber. The inner chamber is connected to the outer chamber via the neck tube. Jarring or excessive swinging could therefore result in a crack in that neck tube and subsequently cause loss of vacuum, increase the temperature in the inner chamber and cause a more rapid evaporation of liquid nitrogen. Appearance of frost at the top of the tank is a typical sign for a failing nitrogen tank. For all these reasons, it is advised to store the nitrogen tank in a place with acceptable light, where the tank is easily visible and people in charge pass daily. Ideally the tanks are protected from possible injury i.e. placed behind a fenced area or under a counter top. The nitrogen level should be monitored regularly and nitrogen loss per week recorded. The tank should be placed on an elevated area to avoid any corrosion due to wet or poorly ventilated areas (as often noticed on farms). Keeping them on a wooden or metal support with wheels helps for a smooth, injury-free manipulation of the tank. Ideally the tank should be filled up 3 /4 with liquid nitrogen and weekly measurements can help adjust the level so that the embryo and the semen in the upper goblet is covered with liquid nitrogen. It has been shown that even only 5 cm liquid nitrogen will keep the content of a tank safe. However, when nitrogen levels are low, one needs to keep in mind, that the temperature within the tank rises much faster when we open it, so the danger of injury to frozen straws is much greater. For tanks, that are discovered dried up, all is wasted. Each nitrogen tank should have the appropriate paper work with it to avoid damage to embryos and semen. First there should be a sheet with weekly measured and added nitrogen, initialed by the person who did the checking. Secondly, there should be a clear inventory of embryos and semen to avoid unnecessary searching and canister lifting to find the right specimen. Keep these records close to each tank or on the wall above the tanks. Well kept 42

45 paperwork may not only prevent damage to the embryos or the semen but may also become important for insurance claims if there is a sudden problem with a tank. THE TRANSFER OF EMBRYOS OR SEMEN FROM ONE TANK TO THE OTHER This process should be coordinated and rapid. It is recommended to put the two tanks close to each other and ask a second person to help. It is recommended to fill both tanks with nitrogen before handling the canes. An available inventory will avoid to unnecessarily lift canisters and thus prevent to harm all straws in a Canister each time you need to lift it. Often the use of a flashlight helps to look for the right cane tab leaving the whole canister as far down the neck as possible. I find using a flashlight especially helpful when dealing with canes where the top tab of the cane is hard to read or had been bent down to avoid cracking a straw during removal from a full goblet, a situation that is often found with the tightly packed sex sorted 0.25 cc straws or when there are too many embryo straws in one goblet. Straws should not be removed with the fingers, especially if the straw is not sealed with a plastic ID-plug, but is powder- or heatsealed, because the temperature in the straw will heat up much faster than when pre-cooled tweezers are used. Early Investigators have shown that frozen semen can be damaged any time the temperature rises above -130 C. The rate of warming can be influenced by many factors, such as ambient temperature, length of exposure, wind velocity and intensity of solar radiation, therefore even for one specific tank, rates will vary. It is also important to know that tanks differ in the diameter of their neck and insulation properties. Whereas the vapor of liquid nitrogen just above the liquid is quite close or below C, the temperature 1 inch below the top of the tank maybe only a few degrees colder than the ambient temperature (Fig 1). It was shown that it takes only 10 to 12 seconds for the internal temperature of a straw to reach -100 C in the neck of a medium sized tank that was half filled with liquid nitrogen. Therefore, the so-called 8 second rule was established for retrieving frozen semen or embryos from a tank: this rule states that canes and canisters should not be exposed any longer than 8 seconds in the neck of a Dewar and not being lifted above the frostline of a tank. This rule is not only important for the embryo we want to thaw and transfer, but important for all the remaining samples in the same goblet and canister. As shown in Fig. 2 many years ago for frozen semen, the damage from exposure to elevated temperatures is additive. Again, it is 43

46 important to realize that it is less dangerous for a straw to be lifted, when the tank is well filled with liquid nitrogen. Also, lifting straws with an embryo or semen in a goblet that is filled with liquid nitrogen and not only vapor is much safer for all the straws in that goblet. SAFE TRANSPORT OF NITROGEN TANKS It is obvious, that nitrogen tanks are considered dangerous and hazardous, because of the heavy burn injuries that can be inflicted on people and animals through accidental spilling. In general, people transporting liquid nitrogen need a specific certification or license to keep them in their vehicle and are demanded to wear protective gloves, aprons, shoes and eyewear. The secure attachment of a tank in a vehicle is paramount. DRYSHIPPER A Dryshipper is a vapor nitrogen tank that holds semen and embryos frozen in vapor only, at about -140 C. Therefore, these shippers are IATA approved and classified as non-dangerous and non-hazardous for travel. A dry shipper consists of an outer metal jacket and an inner shell, with the space between filled with insulation and vacuum sealed. The interior of the packaging contains a cylindrical void, which holds the material requiring refrigeration in one canister, surrounded by absorbent material. The absorbent material is saturated with the liquid nitrogen and releases nitrogen constantly into the void. These shippers have become very useful for transporting frozen material, including semen and embryos over the world. Depending on the model they can hold the temperature for a few days only to several weeks. Crucial to the use of a Dryshipper for safe transport of valuable material is a diligent preparation as well as critical reception and transfer of straws to the wet shipper. Ideally one should follow the manufacturer s instructions for filling, i.e. wearing insulated gloves made for handling liquid nitrogen and a face shield. Liquid nitrogen should be added slowly since a significant volume of nitrogen gas will form as the cold liquid contacts the warm surfaces. When the liquid level reaches ¾ of the void, filling should be stopped and the Dryshipper set aside for the period specified by the manufacturer to allow the liquid nitrogen to saturate the absorbent. Seeing the liquid nitrogen bubbling inside, tells us that the absorption process in still ongoing. Filling should be repeated until the liquid level no longer drops on standing. Filling a Dryshipper that is new or was not used for some time may require as many as 10 filling repetitions over a day. Very important is to consider that filling a warm Dryshipper too fast may cause cracks in the shell and compromise its usefulness in the future. We may not always have a thermocouple, an electrical device with a probe to measure the actual temperature inside a Dryshipper. It is therefore quite useful to have the manufacturers empty and full weights for their dry shippers at hand and the amount of nitrogen that is usually evaporated per day in that specific tank. Dry shippers that will not achieve their full weight or loose more weight per day than the manufacturer s manual describes, may indicate a problem with the absorbent s ability to hold the nitrogen. This may prevent maintaining liquid nitrogen temperature during shipment and may damage the samples. It is very unwise to rush to prepare a Dryshipper. If a Dryshipper is not shipped with a full weight, straws may be damaged, especially when an international transport takes longer than anticipated. Each of my own Dryshippers has the empty and full weight written on the tank, so the receiver has some way to know if there was still enough nitrogen absorbed at arrival. Sometimes all semen or embryo straws cannot be found in a Dryshipper, that we open. A goblet may contain only one instead of the expected three straws. In such a case, we might detect those missing straws on the bottom of the Canister, i.e. they fell out of the goblet during transport. In this situation, I think it is best to prepare a styrofoam dish with 2 cm of nitrogen. Long, precooled forceps can then be used to retrieve the straws. Personally, I simply take out the whole canister and empty it quickly upside down over the prepared liquid nitrogen dish. Once the straws are submerged in the liquid nitrogen, one can easily and securely identify them under the liquid nitrogen, from where they can be transferred safely in their new storage tank. Last, but not least important is to consider a possible damage to the frozen embryos during transport in the transfer gun, i.e. when loading and carrying the straw to the recipient. After proper thawing, damage by water infiltration should be avoided by wiping the straw dry before cutting and insertion in the gun. Cold shock by cold outside temperature can be avoided by warming the gun before use or by using batterie operated gun warmers. Another important detail to teach the beginners is to pay attention to cut off the straws squarely, i.e. at a clean 90 degree- angle. This ensures a snug fit with the transfer sheath and avoids that the embryo gets squeezed and lost backwards in the sheath instead of being pushed out into the uterine horn. 44

47 The take home message of this presentation is to emphasise that a successful embryo transfer program implies not only attention to detail and meticulous working habits when preparing donors and collecting embryos, but also thereafter during storage of frozen embryos and semen used to inseminate the donor cows. Every person who has the responsibility for the storage and transport of frozen semen and embryos should be well trained and knowledgeable about the facts mentioned above to avoid major economic losses. REFERENCES Farin PW, K Moore and M Drost: Assisted Reproductive Technologies in Cattle. In: Current therapy in large animal theriogenology, , 2007 Picket BW and WE Berndtson: Procedures for handling bovine semen in the field. IN: Current therapy in Theriogenology, D.A. Morrow, , 1980: Diagnosis, treatment and prevention of reproductive diseases in animals, 1980 Saacke RG, Lineweaver JA, Aalseth EP: Procedures for handling frozen semen. Proceedings of the 12 th conference on AI of Beef cattle, NAAB: 46-61, 1978 Stroud B: Consequences of mishandling frozen semen and embryos IN: Proceedings, Applied Reproductive Strategies in Beef Cattle, Sioux Fall, SD, ,

48 Delivering IVF Calves: Challenges and Opportunities Gilles Fecteau, DMV, DACVIM Saint-Hyacinthe, Québec INTRODUCTION AND BACKGROUND Assisted reproductive techniques are commonly used in bovine practice. Due to client concerns and the high risks associated with some pregnancies, food animal practitioners are commonly asked to provide advices and expertise in specific situations. This, sometimes push us outside our comfort zone and we may be reluctant to step forward. The objective of this paper is to summarize our experience delivering 26 cloned calves ( ) and eventually helps with hundreds of IVF calves over the last 10 years. The challenges that these calves represent should be seen as an opportunity for the veterinary profession and the diversity of the services offered by your clinic. STANDARDIZING THE MANAGEMENT OF DELIVERY AND MONITORING OF THE NEWBORN Providing an adequate level of care begins by standard management (operating) procedures (SOPs). It is crucial that procedures are performed in a very precise manner so we can assess efficiency and make the appropriate corrections over time. If each case is unique and approach differently, no conclusion could be drawn and over time, the expertise cannot be expand. Several aspects are important to discuss: monitoring appropriately the late gestation, parturition induction or natural delivery (protocols and due dates), continuous surveillance during delivery, immediate assistance and the minimal data information necessary to assess the situations. Calves should be examined promptly after birth to assess respiratory function. Nasal and pharyngeal secretions should be removed by suction. The cardiorespiratory function should be assessed with particular attention paid to respiratory effort, heart rate and rhythm. Apnea, persistent bradycardia, bradyarrhythmia or bradypnea would justify interventional support. This means that the veterinarians or an assistant is on site? Is it possible for all animals? Orotracheal intubation, followed by manual or mechanical ventilation should be performed when considered necessary. Complete blood count (CBC), serum biochemistry profile (SBP) and thoracic radiographs or ultrasound (first 24 hours) and arterial and/or venous blood gas analysis could help the overall assessment if they are available. Oxygen administration by intranasal route can be beneficial. A soft plastic cannula is inserted in a nostril, up to the level of the medial canthus, and fixed on a modified small halter. The oxygen should be humidified and at room temperature. The maximal oxygen rate in one cannula is 15 L/min. If greater concentration is needed, a second cannula could be placed in the other nostril. In our experience, placement of an auricular arterial catheter (medial branch) is extremely useful. When successfully placed, the catheter is then flushed every hour with 1 ml of diluted heparin solution (10 IU/mL). Catheters are left in place as long as there is no local irritation or until no longer required. If respiratory failure is diagnosed (persistent hypoxemia combined with hypercapnia) then assisted mechanical ventilation should be initiated using an Esprit ventilator (Respironics Inc.). This requires the necessary equipment and an expert team to monitor and be successful. The technique used is described in Buczinski et al. Umbilical care included disinfection with a 5% iodine tincture solution. Frozen colostrum from healthy donor cows (or fresh good quality) should be offered to the calves within 2 h of birth. A colostrum with a known status against important infectious diseases (BVD, BLV, paratuberculosis and IBR) is the best. Colostrum quality (IgG concentration) should be verified. A sample of colostrum can be submitted for bacteriological culture at the moment of freezing and only low bacterial count colostrum should be kept. The feeding schedule after colostrum feeding should be closely discussed with the client to ensure to meet the needs. Orogastric intubation is a useful tool if the calf failed to nurse on its own. 46

49 POTENTIAL MEDICAL CHALLENGES ENCOUNTERED Ante Partum The heifers carrying IVF embryos should be monitored closely by the owner. While most gestation are normal, in our experience 2 major problems could be faced during pregnancy; hydrops and hepatic lipidosis. Progressive and variable abdominal distension caused by uterine fluid accumulation is a possible observation in the last trimester. It could be subtle in some cases, while it becomes a reason for veterinary consultation and intervention in other cases. The precise incidence remains unknown. Also of importance is the possible ante partum hepatic lipidosis (pregnancy toxemia). These animals are anorectic and severely ketotic. Some animals cannot stand. If the disease is recognised early some treatment may be efficient while animals responded poorly in advanced stages. Our hypothesis is that the large fluid filled uterus combined with the overweight of some foetus occupy most of the abdominal space, thus preventing the dam from obtaining its nutritional requirements. A particular and specific nutrition plan should be adjusted, particularly in the last trimester. To characterize the anomalies developing during the pregnancy, ultrasound monitoring can be used. Transabdominal ultrasonography can also be a valuable technique for the evaluation of the fetuses and the fetal adnexa in the late pregnancy. In general, the examination technique was easy to perform and well tolerated by the dams. The information obtained may be limited by the size of the fetus and the abdominal depth of the dam, however fetal integrity, morphology of the placentomes and uterine fluids can be assessed. Ultrasonography allowed the early identification of fetal anasarca, which indicates the potential of this technique for early detection of fetal abnormalities. At Birth The newborn clones were extremely challenging. IVF calves are more often completely normal. However, medical problems are diverse. One common problem in clone calves that is not as frequent in IVF is excessive birth weight. Heavy newborns cannot stand on their own, and seem to loose considerable weight in the first few days of life. A second problem is the enlarged umbilical vessels and concurrent excessive umbilical haemorrhage. The umbilical vessels could be grossly abnormal in size. When delivered by C-section, the natural stretching and subsequent rupture of umbilical vessels does not take place. Spontaneous rupture is frequently close to the body wall leaving a very short umbilicus. As a consequence, persistent bleeding is possible and subsequent infection occurred frequently. After Birth Even if cardiorespiratory adaptation occurs uneventfully, IVF newborns require constant attentive vigilance. Hypoxemia without hypercapnia could developed. It is our impression that while this hypoxemia is common and marginal, it is associated with clinical signs of weakness and lost of suckling reflex (loss of interest in nursing). We observed clinical improvement when O2 supplementation is provided. The precise aetiology of respiratory dysfunction remains unclear and further investigations are in progress. Chest radiographs revealed abnormal findings of various severity and nature in animals in which radiographs were taken. Respiratory difficulties were the most prevalent but not the only clinical anomalies identified in cloned calves examined (20/26). While IVF are different than cloned calves, we feel that it is one of the common post partum problem. Associated with respiratory problems, some neonates becomes extremely weak and unable to stand. The generalised weakness is so severe that some calves could not elevate their heads. In association with the generalised weakness, the nursing ability is often impaired. If nursing ability is impaired, providing required nutrient via esophageal feedings is crucial. However, the weakness makes the procedure more at risk since false deglutition may occurs. As a consequence, this problem can be approached with a combination of partial parenteral nutrition combined with esophageal feedings. The parenteral nutrition, even if partial, allowed a reduction in the quantity of milk administered per day while still meeting nutritional requirements and preventing a malnourished state from developing. A very common and important problem is the umbilical infection and related complications. Umbilical vessels are sometimes large and normal physiological rupture does not occur during c-section delivery. The external structures becomes infected despite careful management. In our experience, the infection are rarely limited to the external umbilical structures but also involved the umbilical vein, arteries or the urachus (or a combination). Antimicrobial therapy combined with surgical resection is often necessary. 47

50 CONCLUSION Although IVF calves sometimes represent a challenge, they are also great opportunities for veterinary clinics to offer a higher level of care. Multiple techniques and material adapted or developed for IVF calves will also served your regular clients. It is a very good moment to initiate some SOP (standard operating procedures) on farm. Some of those procedures apply both to high value animal as well as regular replacement heifers. Suggested readings Brisville, AC, G. Fecteau, S. Boysen, A. Desrochers, P. Dorval, S. Buczinski, R. Lefebvre, P. Hélie, P. Blondin, LC Smith. Neonatal morbidity and mortality of 31 calves derived from somatic cloning. Journal of Veterinary Internal Medicine, Buczinski, S., G. Fecteau, RC Lefebvre and LC Smith. Assessment of fetal well-being in cattle by ultrasonography in normal, high-risk, and cloned pregnancies. Canadian Veterinary Journal, 52(2): , Brisville, AC, G. Fecteau, S. Boysen, P. Dorval, S. Buczinski, P. Blondin and LC Smith. Respiratory disease in neonatal cloned calves. Journal of Veterinary Internal Medicine, 25(2): , Buczinski, S., G. Fecteau, RC Lefebvre, LC, Smith. Fetal well-being assessment in bovine near-term gestations : Current knowledge and future perspectives arising from comparative medicine. Canadian Veterinary Journal. 48(2): , Buczinski, S. S. Boysen, G. Fecteau. Mechanical ventilation of a cloned calf in respiratory failure. Journal of Veterinary Emergency and Critical Care. Blackwell Publishing, Oxford, UK : : 2, ref. 48

51 What is Genomics and Epigenomics? Claude Robert PhD Université Laval, Département des sciences animales Centre de recherche en reproduction, développement et santé intergénérationnelle (CRDSI) Réseau québécois en reproduction (RQR) Institut sur la nutrition et les aliments fonctionnels (INAF) Summary Dairy cattle has been at the forefront of selective breeding for decades. Complex selection indices with high reliability estimates have been in place since the mid-1990 s and have been evolving ever since. Through the introduction of new traits that are weighted in accordance to their importance in productivity and profitability, comprehensive selection indices have been developed and are widely used by breeding companies and dairy producers. The dairy industry has also been the first to implement genomic based selection. The basis of this progressive attitude is partly explained by some determinants of the bovine reproductive physiology as well as the presence of strong producer associations who have invested in putting together national evaluation programs. The following aims at brushing a general portrait of the selective breeding program currently in place including the genomic-assisted genetic evaluations. It will also bring into perspective the next emerging wave of interest that is epigenomics where the environmental conditions can program for some extent of time how the genome is expressed. The favourable bovine reproductive physiology Because reproductive technologies such as artificial insemination and embryo transfer are routinely performed, it is often taken for granted that the bovine species is somewhat unique. Indeed, for most species, many reproductive technologies involving the manipulation of gametes and embryos are still very challenging and can only be achieved using complex procedures. For instance, the capacity of the bovine spermatozoon to survive well cryopreservation is instrumental to the dissemination of the genetic of high merit bulls. Distribution of bovine semen can be done worldwide and since it can remain frozen for decades while keeping its fertility, it allows for genetic banking very efficiently. However, for example, this capacity to survive freezing is not shared in pig where most spermatozoa do not regain their mobility after thawing (Blanch et al., 2014; Lee et al., 2015). As a consequence, commercialization of boar semen is done using fresh cooled semen thus limiting the timeframe and geographical range at which genetics can be efficiently distributed. The general perspective is similar on the female side where oocytes and early embryos can be more efficiently recovered in cow compared to many other livestock species. Embryo collection from hormonally controlled multiple ovulations followed by artificial insemination and sample collection by uttering flush is routinely practiced directly on farm whereas embryo collection and transfer generally require surgical procedures in other species such as in pig (Martinez et al., 2004; Wieczorek et al., 2015). This capacity to access the genetic pool from the female side is also instrumental in the dissemination of elite genetics and for the production of the next generations of bulls that will enter service for artificial insemination. Nowadays, the use of assisted reproductive technologies is closely intertwined with breeding schemes. The most cutting edge technologies such as ovum pick up followed by in vitro fertilization and embryo culture can be applied in cattle (Blondin et al., 2002; Nivet et al., 2011) whereas in other livestock species several technical challenges are still limiting their routine application. This capacity to manipulate the bovine gametes makes it extremely powerful when combined with precise methods to estimate the genetic merit of animals. Indeed, the ranking of animals based on their genetic merit estimates allows to identify which breeders should be considered for the application of assisted reproductive technologies. 49

52 Estimation of genetic merit At first, the genetic merit was determined based on productivity that was measured by the amount of milk produced per lactation. Phenotypic estimates have since been broaden to include many other traits that are directly and indirectly associated with productivity and profitability (Egger-Danner et al., 2015). These traits can be boldly classified in the three main categories that are: 1) production itself accounting for milk yield as well as milk protein and fat contents; 2) conformation traits that account for physical attributes. Some of them are known to be directly associated with milk production while others are important to account for the capacity of the animal to withstand the extreme physical stress associated with high productivity; 3) functional traits associated with maintenance costs including fertility parameters, disease resistance and estimation of potential to remain productive in the herd for several lactations. To enable the estimation of the genetic merit for all these traits individually, phenotypic recording must be in place. This is generally done through the breed associations who then channelized resources to put together national genetic evaluation programs through the establishment of standardized measurement procedures, automatic data recording, establishment of databases and calculation matrixes to extract the genetic contribution from the recorded phenotypes. To accomplish this with some degree of reliability, several measurements per animal must be accounted for. This requirement for minimal precision makes it difficult to generate precise genetic merit estimates for cows based on their own phenotypic records since they will generally have less than four lactations (Haine et al., 2017). In turn, sires in service do not have milk production records nor conformation traits records themselves but will have numerous daughters and each of their phenotypic records can be used to estimate the genetic merit of the bull. By assuming that bulls are randomly mated in the pool of females, the maternal contribution is expected to be around the breed average for all cohorts of dams mated to every available sires. In such model, the maternal contribution becomes a similar variable for all tested males. Therefore, if the daughters of a particular sire perform on average better than the breed average, this increased performance is assumed to be attributable to the quality of the sire. Following this simplified overview of how genetic merit is estimated, it is noteworthy to mention that these estimates only account for a portion of the entire genetic contribution. Indeed, the genetic makeup of any animal (here the discussion will be restricted to mammals that are diploid thus having one set of chromosomes originating from the sire and another being passed by the dam) is represented by the summation of the interactions between the genetic copies received from both parents. To illustrate this, three types of genetic interactions can be described. First, sometimes, both copies will act jointly in an additive manner where each copy with be expressed independently of the nature of the other. This will be termed as additive interactions (also termed codominance). These are the ones that are estimated and the sum of all additive interaction are reported as the genetic merit of an animal (Kennedy et al., 1991). However, other types of interactions exist where parental copies can influence one another depending on their respective sequence. These are referred to as dominance interactions and can be found in all shades in regard to the extent of their mutual influence. In some extreme cases, one copy can completely mask or inhibit the expression of the other copy. This is what is observed in the cases of dominant/recessive traits where animals carrying different sequences on both copies are phenotypically identical to animals carrying two copies of the dominant form of the gene. Such complete dominance/ recessive interaction is often the hallmark of genetic defects (e.g. bovine leucocyte adhesion deficiency, complex vertebral malformation, etc) or single gene traits such as the traditional black and white vs. red and white Holstein. The third type of interaction involves complex interactions between different genes or traits where one region on the genome will influence the expression of another located somewhere else. These complex interactions between distant sequences are referred to as epistatic and are very difficult to predict thus very difficult to estimate. Because non-additive interactions are very difficult to estimate with precision and most often require to know the genetic composition of the partner s to which the genome will be paired, genetic merit estimates generally account for the additive interactions only. The following question is then to appreciate how much of the trait of interest is actually driven by additive interactions. Part of the answer is that the proportion of this additive contribution in the overall genetic contribution to the phenotype is trait specific. It can be estimated from trait heritability which can be summarized as the capacity to pass on the trait of interest to the next generation. When a higher proportion of the overall genetic contribution is due to additive interactions, heritability estimates are higher. Conversely, when complex interactions (dominance and epistatic) are at the basis of the expression of a trait, it is more difficult to pass on the 50

53 right genetic combination to the next generation and heritability estimates are lower. Typically, reproductive and disease resistance traits have low heritability estimates which is perceived as involving a large number of genes with low individual contribution but also with potentially involving a high degree of complex interactions. Furthermore, low heritability traits are most often highly influenced by the environmental conditions to which the animal is subjected. Taken together, it is harder to estimate the genetic merit for low heritability traits and it is harder to transmit the trait to the offspring. Thus when facing a low heritability trait, phenotypic improvement of the population will take many more generations than for highly heritable traits. Where genomics fits in Dairy cattle was the first livestock species to implement genomic selection on a population-wide scale. Genomics is currently the cutting edge method for estimating genetic merit (Hayes et al., 2009; Wiggans et al., 2017). It does so through in a very different manner than the traditional phenotypic based estimation described above. In fact, a crude definition of genomics could be no more than the knowledge of the DNA sequence composing the genome. This information is not very useful unless variances in the DNA sequences are associated with the expression of a phenotype. These sequences then become genetic markers. This is often exemplified using genetic defect where a mutation leads to a deleterious phenotype. However, dairy production, conformation and functional traits are not single gene phenotypes and therefore the search for the milk production gene seems naïve. For complex traits, the situation is different where thousands of genes are involved. Individually, each gene has little effect but the combination of thousands of small effects can lead to the expression of an advantageous phenotype. The challenge in exploiting the information of the genome when facing complex traits is to adopt a genome-wide approach. This became available following the decryption of the bovine genome which provided a reference genome assembly for cattle. This was first completed in It allowed to survey where differences in the DNA sequence would rely in the genome and to put together a catalog of varying (polymorphic) regions. All sort of DNA sequence variations exists including deletion, insertion (additional sequence) and substitutions where one DNA nucleotide is replaced by a different one. This latter sequence discrepancy is referred to as a single nucleotide polymorphism (SNP) and it is known to be highly abundant in all genomes. On average, there is a SNP every 300 bases. Given that large mammals have a genome comprised of about three billion bases, anyone could expect that the bovine SNP catalog could be comprised of 10 million SNPs. Moving alongside the decryption of genomes, genotyping platforms were developed and commercially offered. Currently, genotyping platforms that are commonly used for livestock species interrogate about 50,000 to 80,000 SNPs per sample. These SNPs are selected across the entire genome and provide an equidistant coverage of about 60,000 to 37,750 nucleotides. This means that amongst all the known SNPs in the bovine genome, some were selected at regular interval to cover the entire genome. The panel of SNPs interrogated by the platforms was not selected for their know association with a phenotype but rather to be spaced apart at an equidistant interval. These SNPs are referred to as anonymous since they are not known to be associated with the expression of a phenotype. It is such genotyping platforms that have been the workhorse of genomic selection in all livestock species. To use the genotyping information in the context of complex traits to estimate genetic merit, the common approach is to define a signature of the animals expressing the trait of interest. In other words, the information is not used to identify a subset of SNPs that would be associated with the expression of the trait of interest but rather to identify the most common bases found in animals expressing the trait of interest. This provides an estimation of a SNP effect for every base interrogated by the genotyping platform. It is the summation of these small SNP effects that provides the estimation of the genetic merit. This genomic-based estimation is thus based on the genotypic information but it also requires to have a reference population of the best performing animals which is still based on phenotypic records. Therefore, genomic evaluations are not independent of phenotype recording. One of the main benefit of genomic evaluation resides in the capacity to provide an estimate of genetic merit as long as DNA is available for genotyping. This can be done readily at birth and even prior to embryo transfer from an embryonic biopsy. Determining the genetic value of the animals at an early stage of life avoids the need for progeny testing that is costly financially and timewise. To have good reliability, the database used to for the calculation must have tens of thousands and even hundreds of thousands of animals with both phenotypic and 51

54 genotypic records. Without such resources, genomics does not provide better reliability than the traditional evaluation of the genetic merit. And now epigenomics Because the use of genomics for genetic merit evaluation is still relatively recent it is still difficult to fully appreciate its impacts on the landscape of the dairy cattle genetic pool. Nonetheless, the reduction of the generation interval (since calves can receive a breeding value at birth) has increased the rate of genetic improvement (Wiggans et al., 2017). Although genomics has not been fully digested yet, a new aspect of genetic-related consideration is emerging namely epigenetics. It has been known for a long time that phenotypes/performances results from the effects of genetics and of the environmental conditions in which the animals live. More recently, it has been shown from epidemiological studies and in animal models that the environmental conditions can leave some sort of gene expression memory or long term effects on gene expression even after the animal has been removed from the environment (Reik et al., 2003; Dunford and Sangster, 2017; Lucas and Watkins, 2017). In other words, some environmental cues can program the genome for some extent of time that will last even after the cues are no longer there. The way such environmental genetic programing is done is now known to be through epigenetic programing. The epigenome is different from the genome (the DNA sequence itself) and can be illustrated as a coat of molecules over the DNA that can modulate the access to the DNA sequence and thus influence gene expression (Handy et al., 2011). It is now known that environmental conditions influencing metabolism can profoundly influence the epigenome and leave a long term effects that are now termed as genome by environment interactions (or G x E). Epigenomics is only in its infancy and it is still difficult to grasp how much of the observed phenotypes can be explained by the epigenome. One differing aspect from genomics is that the epigenomics is not static and can be modulated trough life. Also, it can be independent of the DNA sequence which could explain some of the differences observed between identical twins or clones in the case of animals. This upcoming wave named epigenetics is still emerging and it is not known how it will be used to complement the information provided by the DNA sequence. Concluding remarks Selective breeding using genomics is currently dramatically modifying the landscape of the dairy genetic industry. The race to generate the next top ranked elite bulls has ever been tougher given that all genetic pools are now compared through a common ground that is sequence based. The use of DNA to estimate genetic merit has almost been adopted as a mystical and infallible method. It is undeniable that with sufficient phenotypic and genotypic information, more reliable estimates of SNP effects can be calculated. The comparison of these genomic signatures is still aimed at capturing the portion of the genetic effect that is additive. Therefore, in addition to the genetic merit estimates, environmental conditions must be accounted for in order to maximize productivity and profitability. Even in presence of genomic evaluations, low heritability traits still offer a challenge. Upcoming perspectives are forecasting that the environment interacts with the genome to the extent that it can program gene expression on the long term independently of the DNA sequence. This is now referred to as epigenetic programing. Some developmental widows such as during gametogenesis and fetal development or during the pre-pubertal growth phase could be prone to genomic programing by environmental cues. Lastly, the capacity to specifically and surgically edit the DNA sequence using molecular scissors has now open the opportunity to tailor the genome almost at will. These new perspectives will complement the current effort to benefit from the genomic information. Blanch E, Tomás C, Hernández M, Roca J, Martínez EA, Vázquez JM, Mocé E. (2014) Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin. J Reprod Dev. 60(2): Blondin P, Bousquet D, Twagiramungu H, Barnes F, Sirard MA. (2002) Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol Reprod. 66(1):

55 Dunford AR, Sangster JM. (2017) Maternal and paternal periconceptional nutrition as an indicator of offspring metabolic syndrome risk in later life through epigenetic imprinting: A systematic review. Diabetes Metab Syndr. Suppl 2:S655- S662. Egger-Danner C, Cole JB, Pryce JE, Gengler N, Heringstad B, Bradley A, Stock KF. (2015) Invited review: overview of new traits and phenotyping strategies in dairy cattle with a focus on functional traits. Animal. 9(2): Haine D, Delgado H, Cue R, Sewalem A, Wade K, Lacroix R, Lefebvre D, Arsenault J, Bouchard É, Dubuc J. (2017) Culling from the herd's perspective-exploring herd-level management factors and culling rates in Québec dairy herds. Prev Vet Med. 147: Handy DE, Castro R, Loscalzo J. (2011) Epigenetic modifications: basic mechanisms and role in cardiovascular disease. Circulation. 123(19): Hayes BJ, Bowman PJ, Chamberlain AJ, Goddard ME. (2009) Invited review: Genomic selection in dairy cattle: progress and challenges. J Dairy Sci. 92(2): Kennedy BW. (1991) C. R. Henderson: the unfinished legacy. J Dairy Sci. 74(11): Lee YS, Lee S, Lee SH, Yang BK, Park CK. (2015) Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation. Anim Reprod Sci. 159: Lucas ES, Watkins AJ. (2017) The Long-Term Effects of the Periconceptional Period on Embryo Epigenetic Profile and Phenotype; The Paternal Role and His Contribution, and How Males Can Affect Offspring's Phenotype/Epigenetic Profile. Adv Exp Med Biol. 1014: Martinez EA, Caamaño JN, Gil MA, Rieke A, McCauley TC, Cantley TC, Vazquez JM, Roca J, Vazquez JL, Didion BA, Murphy CN, Prather RS, Day BN. (2004) Successful nonsurgical deep uterine embryo transfer in pigs. Theriogenology. 61(1): Nivet AL, Bunel A, Labrecque R, Belanger J, Vigneault C, Blondin P, Sirard MA. (2011) FSH withdrawal improves developmental competence of oocytes in the bovine model. Reproduction. 143(2): Reik W, Santos F, Dean W. (2003) Mammalian epigenomics: reprogramming the genome for development and therapy. Theriogenology. 59(1): Wieczorek J, Koseniuk J, Mandryk I, Poniedziałek-Kempny K. (2015) Piglets born after intrauterine laparoscopic embryo transfer. Pol J Vet Sci. 18(2): Wiggans GR, Cole JB, Hubbard SM, Sonstegard TS. (2017) Genomic Selection in Dairy Cattle: The USDA Experience. Annu Rev Anim Biosci. 5:

56 Sexed Semen Utilization in IVF and ET Daniela C. Pereira 1, Alfredo Castro 2, Eduardo Benedetti 3 STgenetics 1 Vienna IVF Production Lab Manager, 2 Diretor de Mercado Internacional, 3 ET Lab, Navasota, Texas Sexed semen is used by the dairy and beef industry as a very interesting tool for the animal production system, because the option to select the next generation s gender contributes to genetic improvement and production financial gain. The objective of this is to demonstrate the efficiency of the new sexed semen technology, SexedULTRA, on AI of dairy heifers and milking cows, on the super ovulated donors and In Vitro embryo production of dairy and beef females. However, it is hard to comment on the impact of the new technology on artificial insemination and embryo production without explaining the effects that occurred in the sexed semen production system. For several years Sexing Technologies has put a lot of effort into searching for the exact cause of the fertility difference between the OLD frozen sexed semen (Legacy) compared with conventional frozen semen. To make this research easier, the production system was divided in steps and each step was evaluated individually. The ST researchers concluded, after intensive study, that the major problem was related to the sorting steps; therefore adjustments were required on all steps in order to result in a less stressed sorted sperm cell. Changes in the steps of the production SOP, new equipment, and adjusted software were gradually introduced to the system and the effects and results were evaluated through analyzing the semen physical parameters, DNA fragmentation, IVF fertilization, blastocyst production, and blastocyst eclosion rates compared between OLD sexed semen, Conventional Semen and NEW sexed semen. As a conclusion, a new production system was developed and was named as SexedULTRA, which consists of: 1. Equipment modernization 2. Temperature control 3. Stanning Optimization 4. Media and extender improvement 5. Ph control Buffers 6. Oxidation reduction 7. Dead cell separation The new product, SexedULTRA, showed a significant thaw gain on sexed semen physical parameters and on IVF embryo productions rates and quality. The average Day 8 blastocyst production was 30.10% and 51.7% for the old sexed semen and SexedULTRA respectively (Goncales, C, 2013). An important point in the IVF embryo production rate is that there was a great reduction on the performance differences between bulls and batches of the same bull, indicating that the deviation has been reduced. This new product was intensively tested in AI field trials performed by ST Genetics and third party companies and showed similar results as the ones observed on the IVF trials. Trials in France, Germany and Scotland demonstrated that SexedULTRA had 90 to 95% of the conventional semen conception rate. During these trials we learned that with SexedULTRA semen conception rates were positively affected by the concentration of sperm cells in the straw. In an experiment in Germany, different concentrations of sexed semen were compared with conventional semen, showing that 4 Million sperm-cells straws had similar conception rate to conventional semen. This is why SexedULTRA 4M TM was born. 54

57 Something else that was learned in this extensive SexedULTRA trial is that the sexed sperm has a different physiological behavior compared with conventional semen. It gets ready a little earlier than conventional semen, which is a sign that it needs to be AI earlier than conventional semen. 55

58 The technical evolution of the SexedULTRA 4M TM Conventional/Sexed Semen Cows and Heifers can be easily shown in the Table.3 USA AI results SexedULTRA 4M TM made possible the extensive use of sexed semen on milking cows with very consistent conception rate results around 95% of the conventional semen. On the embryo production side the changes were even better because the very poor results with OLD Sexed semen. Sexed ULTRA 4M made it possible to flush using sexed semen with very similar results to conventional. TABLE 2. Embryo Production - Holstein Heifers ST Genetics Navasota Texas. Spring Semem Doadoras Estr/don. Viav./don Sex. XY 5.0MI straws 20M SexedULTRA straws 12M The results above have been observed in several sites around the world shown in details during the presentation. Higher embryo production was possible with less semen used. Evaluating the results on In Vitro embryo production, the results with SexedULTRA 4M TM were better as well, with a lower deviation between bulls and a higher blastocyst rate compared with OLD sexed semen and conventional semen. Table 2. Blastocyst rate (XY/ULTRA) ST Genetics Vienna Farms - Multibovine Semen Oocytes Embryos % Blast Sex. XY 1/ Conventional Semen SexedUltra The new product SexedULTRA 4M TM improved the embryo quality and production rate per donor. The majority of the donors were Holstein heifers of 10 to 18 months. In conclusion, the new product SexedULTRA 4M TM allows us to achieve similar results compared to conventional semen in AI programs, conventional flush and In Vitro fertilization. 56

59 Ultrasonography and Endocrine Parameters in Recipients with Successful Embryo Transfer Angelika E. Stock Services vétérinaires mobiles de thériogénologie, Chambly-Saint-Hyacinthe, QC, Canada Introduction Many factors qualify a heifer or cow as a good recipient. Post-partum interval, nutrition, body condition, age, weight and previous fertility are all important. This presentation will focus on factors that the embryo practitioner can evaluate by rectal palpation and ultrasonography at his pre-transfer exam of the recipient. Are there any insights from the current research that may help the practitioner to decide for or against transfer? Also, can a heifer or cow be prepared through hormonal interventions to become a better recipient? Synchrony/Cycle stage of recipient In herds with a small embryo transfer program (as we have many in Québec) observation of heat or met-oestrus bleeding are a very important component of the success of embryo transfer. Ideally, the recipient should be at Day 7 after oestrus for embryo transfer. If only met-oestrus bleeding was observed, then transfer should be performed 5 days afterwards. Any deviation of more than 24 h of Day 7 of midcycle may decrease the pregnancy results. Usually the recipient should not be less than on Day 6.5 and not more than on Day 8.5 of the oestrus cycle at time of transfer (Hasler, 2012). Therefore, in contrast to embryo transfer recipients under a synchronization schedule, i.e. a fixed time embryo transfer (FTET) in very large herds, it is better to verify the date of heat or met-oestrus bleeding with the owner. It is always a good idea to ask if the recipient has regular estrous cycles or had been inseminated or received an embryo before. Depending on record keeping practices of the farm I have personally found recipients pregnant 24- days when performing the pre-transfer exam via ultrasound, or cows on Day 14 of the cycle that may have shown signs of mid cycle oestrus 7 days ago, as described in a study from India by Sood et al, Obviously in those situations embryo transfer has to be declined. In rare instances a small accumulation of clear fluid and increased uterine tone and thickness may not be favorable for a transfer. Site of transfer Small or large embryo transfer program, a rectal examination before each transfer needs to be performed to verify that the recipient has a corpus luteum (CL) compatible with Day 7 of the estrous cycle and to know if the corpus luteum resides on the left or right ovary. It is paramount to transfer the embryo into the ipsilateral uterine horn, i.e. the horn leading to the ovary bearing the corpus luteum. It is quite common for the embryo transfer practitioner to mark all the acceptable recipients on their specific side for transfer after the pre-exam, so that no mistake will be made when the actual transfer is performed. Embryo transfer ipsilateral to the side of the corpus luteum ensures the local message of the developing embryo, via its interferon tau production, to the uterus. This way luteolysis is deferred and pregnancy maintained through the critical window of maternal recognition at Day Interferon tau does not simply inhibit uterine PGF production by the uterus, but, as we know now, seems to influence the prostaglandin regulatory enzyme responsible for the molecular switch from PGF2alpha to PGE2, the latter being luteotrophic. (Brooks et al., 2014). This may also explain why giving meloxicam, an inhibitor of this PG regulatory enzyme to cows on Day 15 after artificial insemination (AI) did not rescue but substantially decrease pregnancy rate (Erdem et Guzeloglu, 2010). To administer this drug at the time of transfer is reported to improve transfer results when the recipient is hard to be kept quiet and/or a poorer-grade embryo is transferred (Lamb and Mercadante,2015). However, Purcell et al., 2005 observed no benefit to give meloxicam at the time of transfer. Thus, the embryo transfer practitioner may not support establishment of pregnancy by giving prostaglandin inhibiting drugs to the recipient at time of transfer or later during the critical window. Transfer of embryos in the contralateral horn may occur mistakenly. Interestingly, pregnancies have also been reported contralateral to the CL in animals that were inseminated. Pregnancies that establish contralateral to the CL bearing ovary, if after insemination or transfer, have little chance to be maintained until birth, but may be saved 57

60 with progesterone supplementation and the induction of accessory corpora lutea. Accessory luteal structures on the ipsilateral ovary seem to have the highest success rate to maintain the pregnancy to the end, as reported in one study (Zbylut and Czeladko, 2009) Size of the corpus luteum at time of transfer Ultrasonography Since ultrasonography has become a daily routine to the bovine practitioner, it is more and more used to examine the corpus luteum of the recipient, especially if there are any doubts after rectal palpation. In the situation, where the embryo is extremely valuable, the practitioner may be inclined to use additional tools to feel more confident in their decision. Numerous papers have investigated the possible importance of corpus luteum diameter on day of transfer for maintenance of pregnancy. For example, an American research group using 526 recipients, receiving fresh and frozen in vivo collected embryos, concludes from their data that there is no association of CL diameter, luteal volume and progesterone value on Day of transfer with pregnancy rate (Lamb, 2005, refer to Table 3 above, adapted from his article). Yet, a study with in vitro embryo transfers performed in Columbia, describes higher pregnancy rates (39.7%) when the CL was larger than 24 mm in diameter, whereas lower pregnancy rates (24.2%) were observed at a diameter of less than 14 mm (Gonella-Diaza et al, 2013). The difference between these two studies is that the researchers from Minnesota chose the 526 out of 763 synchronized recipients that showed heat (heat observation 4 x daily) whereas the Columbian study was based on a synchronized recipient herd with fixed time ETs (FTET). In a FTET program, usually applied in very big herds, where individual estrus observation is not possible, follicular wave and ovulation are heavily manipulated with different hormonal regimens (see Bo et al, 2012, Lamb and Mercadante, 2015 for review on FTET). Therefore, a small CL size after a fixed time ET may reflect an ansynchronous CL, i.e. a corpus luteum that has not truly the age and maturity of a Day 7 corpus luteum and may therefore be less competent to keep the pregnancy maintained. A different study from Brazil with lactating beef cows using a fixed time insemination trial (FTAI), did not only measure the size of the CL on Day 7 after insemination but also the size of the pre-ovulatory follicle at time of insemination prior to CL formation (Pfeifer et al., 2012). 58

61 Interestingly, they found, that the best pregnancy rates in these cows were associated with a specific size category of the ovulating follicle at insemination, namely follicles that measured mm (refer to Fig 2 above, as adapted from Pfeifer et al, 2012). The remaining cows with smaller than 13 and larger than 15 mm ovulatory follicles resulting in a corresponding smaller or larger CL had a lower pregnancy rate. In this study, a smaller follicle may have been too immature to result in a competent CL maintaining a pregnancy, as well as a large and possible persistent follicle with prolonged growth may not ensure adequate CL formation. It was shown that prolonged dominant follicles may still ovulate but not result in pregnancy (Stock and Fortune, 1994). Unfortunately, in practice, measuring the size of the follicle 7 days before the transfer is unpractical. In one of the textbooks, a minimal diameter of a solid CL at transfer has been set at 12mm (Farin et al, 2007) Compact corpus luteum, corpus luteum with cavity and luteal cyst at time of transfer Before the routine application of ultra-sound in clinical diagnosis in the early 80 s, researchers were intrigued by the frequent appearance of bovine corpora lutea with cavity found in bovine slaughterhouse reproductive tracts and talked about so called corpus luteum cysts. Luteal cell cultures developed from slaughter house corpora lutea of early and midcycle revealed no difference in progesterone secretion or stimulation by gonadotropins in vitro (Stock,1984). Thus, the cavity in the CL did not seem to be of pathologic significance. Soon thereafter Kahn et al,1986, from the same group at University of Munich Germany showed by daily ultrasound in cycling heifers that the cavity becomes smaller at the end of the cycle (see Fig below adapted from Kahn 1986). Pregnant heifers on Day 28 no longer bear the cavity in their CL. 59

62 The volumes of corpora lutea with and without cavity during early pregnancy in heifers adapted from: W. Kahn. 1986, Munich, Germany These observations were confirmed by many studies that followed. This means that a recipient bearing a corpus luteum with a fluid filled cavity at time of transfer should not be excluded from transfer. A very big fluid filled structure with a tiny wall of luteal tissue showing higher echo-density, however, may most likely be a luteal cyst, i.e. a follicle that did not ovulate but luteinized (see images A, B and C, adapted from Ginther 1998). Whereas image A is a typical follicular cyst, the 2 different luteal structures (B and C) may not always be easy to distinguish, however the cavity of a Cl is often formed irregularly and the ovulation papilla palpable or visible on ultrasound, whereas luteal cysts are often round and big structures (image C). Luteal cysts may produce very little progesterone and do not have the typical ovulation papilla. If a luteal cyst is suspected, transfer should be declined. adapted from Ginther: Ultrasonic Imaging and animal reproduction: Cattle, Book 3, 1998 Uterus Concerning the uterus, the pre-transfer ultrasound examination may be a useful tool to detect early pregnancy, uterine infections, and other pathological conditions such as freemartin or segmental aplasia of the uterus. Uteri with slight edema and/or uterine fluid as well as increased tone may be suspect for a recipient presented at late diestrus/early prooetrus rather than early diestrus. 60

63 Endocrine parameters Progesterone A tremendous amount of scientific papers studied the relationship of early diestrus progesterone levels and fertility after AI or at time of embryo transfer (for example: Inskeep 2003, Lonergan 2013, Wiltbank et al 2014). Generally, it is believed that there should be a minimal value on Day of transfer or Day 7 after AI to ensure maintenance of pregnancy. It is also postulated that a fast rise in progesterone after ovulation is beneficial to fertility. Like studies on the significance of the size of the CL, the review of some of the published data on the importance of progesterone offers quite controversial results. Some researchers found that progesterone values did not differ at time of transfer between heifers that became pregnant (1.5 +/-1.05 ng/ml) versus those diagnosed non-pregnant (1.31 +/ ng/ml) (Nogueira et al, 2012). In lactating dairy cows the mechanism involved in the relationship between circulating P4 concentrations and fertility seems quite complex, which may be part of the lower success of embryo transfer if a lactating dairy cow is the recipient. In this context data analyses by Wiltbank et al, 2014, revealed intriguing results: cows with high genetic merits for fertility seem to have 34% greater circulating progesterone levels than cows with lower genetic value for fertility. For a long time it was thought that increased progesterone during early gestation, i.e. on day 7 of the estrus cycle, reduces the incidence of early embryonic mortality. Therefore, already 18 years ago researchers looked for methods to increase progesterone in early diestrus. For example, an experiment with a small number of cows, reported that treatment of cows on day 7 after oestrus with 1000 I.U. hcg raised diestrus plasma progesterone and increased pregnancy rate due to the formation of an accessory corpus luteum (Rajamahendran and Sianangama, 1992). This method is still advocated in recently published textbook chapters (Lamb and Mercadante 2015). Many studies report to increase or supplement progesterone during early and later stages of diestrus. Usually hcg or GnRH is used to induce ovulation of the dominant follicle of the first wave or progesterone supplementation using PRID s, CIDR s or injectable progesterone are given. Yet, the increase in fertility was not significant in most of the reports. It was shown that increasing progesterone during early diestrus increases the growth rate of the conceptus and therefore seemed to be beneficial for embryo development at first (Carter et al., 2008). Then, researchers working in Spain, USA and Ireland report paradoxical results using supplemental progesterone from Day 3-7: although progesterone speeded up the growth and elongation of the embryo, supplementing progesterone early in diestrus inhibited CL development and lowered its endogenous progesterone production (Bo et al., 2012, O Hara et al, 2014). Therefore, supplementing progesterone in early diestrus has a potentially negative effect on CL life span. Similar results were reported where PMSG was used to increase the number of ovulating follicles and thereby preparing recipients with several corpora lutea. When embryos were transferred in these recipients with PMSG induced corpora lutea, pregnancy rate was not improved, confirming again that artificially high progesterone in early diestrus may be rather detrimental for the maintenance of pregnancy (Nogueira et al., 2004). Inserting a CIDR device later, at Day of transfer until Day 20 (i.e. CIDR in place for 13 days from Day 7 to Day 20, i.e. during mid and end of diestrus) was also not favorable to increase pregnancy rate after transfer, suggesting that some antiluteolytic strategies to improve fertility, as reviewed by Binelli et al, 2001, may not apply. The only positive observation using the 13day CIDR insertion in this study was the improved synchrony of the return to oestrus for the non-pregnant recipients (Purcell et al, 2005). Interestingly a Turkish study reported that in repeat breeder cows the insertion of a PRID device resulted in 40 % pregnant animals when a PRID was inserted from Day 11 to 18 of the cycle compared to 26 % when the PRID was inserted from Day 4 to 11 of the cycle (O. Ergene, 2012). In general, researchers point to the importance of a fast but specific rise of progesterone after ovulation. This specific rise will occur when the CL is competent after ovulation to ensure a spatial and temporal interrelationship with the uterus and to modulate and prepare the uterine environment and the secretion of histiotroph, which will have its effect long before the embryo arrives at the critical period of maternal recognition. In one report researchers conclude from their data that not absolute values, but a timely increase, i.e. a 2.7 fold increase of peripheral progesterone from day 0 to Day 7 and a 1.48 fold increase from Day 7 to 14 was the most obvious finding associated with pregnancy confirmed on Day 63 (Kenyon et al., 2013) Thus, as several authors conclude, there exists no minimal or specific value of peripheral progesterone on the day of transfer that can be correlated with successful establishment of pregnancy. 61

64 Estrogens: Estrogens are thought to have a negative effect on pregnancy because they may help to promote the luteolytic cascade, during the critical period of maternal recognition. Investigators in Japan have shown that Japanese Black beef cows having peripheral progesterone levels higher or equal to 2.5 ng/ml and estrogen levels lower than 1.5pg/ml on day 6 post oestrus, i.e. one day before embryo transfer had the best result in pregnancy rate. According to them a E2/P4 ratio lower than 1 around transfer is associated with a higher pregnancy rate (Nishigai et al., 2000). During pre-examination of recipients we often observe the presence of follicles, which are part of the first follicular wave. If a specific follicle and his estrogenic activity around the time of transfer is beneficial or inhibiting to the establishment of pregnancy is not clear. Like progesterone, it may be the amount of circulating estrogen produced at the right time before, during or after transfer that allows for specific signals to the uterus to ensure embryo survival. Madsen et al from South Dakota, USA presented interesting data in 2015 concerning the role of estrogens on embryo survival and establishment of pregnancy. She used ovariectomized beef cows as recipients, primed with CIDR implants 9 days before GnRH. GNRH was used with or without (control) an injection of ECP (-36 h) or EB (-12h) prior to GNRH injection to result in a LH surge. Early diestrus progesterone increase was mimicked by increasing injections of progesterone from Day 3 to 6 after GnRH. On Day 7 embryos were transferred and then progesterone was supplemented by inserting a CIDR on Day 8. The CIDR was replaced every 6 days until Day 30. Interferon production by the embryo as well as pregnancy specific protein B were measured to confirm normal development and attachment by the embryo. Ultasonography on Day 29 and 32 confirmed a positive, embryonic heartbeat. The importance of periovulatory estrogen was clearly shown in this elegant study with ovariectomized cows. Control cows, i.e. cows that had only received GnRH to induce the LH surge had a much lower pregnancy rate (4%) than the estrogen treated cows (25%), confirming that a single increase of periovulatory estrogenic rise in the recipient is very important in preparing the uterus for embryo survival and attachment for the next 30 days, and maybe longer. Taken together, ultrasonography is a valuable tool to help a practitioner in the decision to accept a recipient for transfer. Basing that decision on the size of the corpus luteum remains controversial. In some textbooks a minimal diameter of mm at transfer is considered as a requirement to increase chances for pregnancy. Luteal cysts and cavitary CLs need to be distinguished. A cavity in a CL should not be a reason to deny transfer. The importance of peripheral progesterone levels at the time of transfer have been studied for a long time intensively but this parameter seems to lose the attention of scientists because absolute progesterone levels seem to vary depending on age, breed and most often on the individual cow, i.e. genetics. Progesterone values at the time of transfer are no longer considered to be predictive of pregnancy rate after transfer. In addition, studies that tried to improve pregnancy rates by increasing circulating levels of progesterone during early diestrus, as well as during and/or after transfer of embryos report somewhat ambiguous results and are, as the title of one paper calls it, paradoxical. Therefore, the embryo practitioner should be careful to increase progesterone artificially to attain better results. Any artificial methods to increase progesterone at time of transfer are most likely not worth the effort or cost. Results of the reviewed literature make us understand that preovulatory events, such as preceding luteal phase, periovulatory estrogen and a timely rise in progesterone induce a very tight sequential interaction between ovary and uterus, which prepares for maintenance of pregnancy long before the critical period of maternal recognition (Bridges et al, 2014). The effect of a healthy steroidogenic capacity of the ovulatory follicle for maintenance of pregnancy after AI or ET has been shown and was actually reported by Inskeep and co-workers, 15 years ago. Interestingly the possible effect of estrogen secreting follicles during diestrus on embryo survival is far less studied, although a possible interplay with progesterone and an effect on the uterus may be possible. The importance of the size of the ovulatory follicle, and its estrogenic activity are still subject of extensive research in large herds with a FTET program, where follicular wave emergence and ovulation are controlled for synchronizing recipients, but where quality of the ovulatory follicle and the subsequent CL still represent a challenge. For Lamb and Mercadante, 2015, FTET, allows, despite these challenges, for a higher proportion of recipients to receive an embryo and therefore increase the overall pregnancy rate in a big embryo transfer program compared to transfers based on estrus detection of recipients. 62

65 REFERENCES Binelli M, WW Thatcher, R Mattos and PS Baruselli: Antiluteolytic strategies to improve fertility in cattle. Theriogenology 56: , 2001 Bo GA, PS Baruselli and RJ Mapletoft: Increasing pregnancies following synchronization of bovine recipients. Anim. Reprod. 9: , 2012 Bridges GA, Day NL, Geary TW and LH Cruppe: Deficiencies in the uterine environment and failure to support embryonic development. J. Anim. Sci: 91, , 2013 Brooks k, G Burns and TE Spencer: Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol. Journal of Animal Science and Biotechnology 5: 53-65, 2014 Carter F, N Forde, P Duffy, M Wade, T Fair, MA Crowe, ACO Evans, DA Kenny, JF Roche and P Lonergan: Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in heifers. Reproduction, Fertility and Development: 20, , 2008 Erdem H et A Guzeloglu: Effect of meloxicam treatment during early pregnancy in Holstein heifers. Reprod.Domest.Anim.: , 2010 Ergene O: Progesterone concentration and pregnancy rates of repeat breeder cows following postinsemination PRID and GnRH treatments: Turk.J.Vet. Anim.Sci 36: , 2012 Farin,PW, K Moore and M Drost: Assisted reproductive Technologies in Cattle. In: RS Youngquist and WR Threlfall, Current Therapy in Large animal Theriogenology, 2nd ed., , 2007 Ginther OJ: Ultrasonic imaging and animal reproduction: Cattle, Book 3, 1998 Gonella-Diaza AM, G Holguin, D Montana and D Valbuena: Corpus luteum diameter and embryo developmental stage are associated with pregnancy rate: Data analysis from embryo transfers from a commercial in vitro bovine embryo production program. Anim. Reprod.: , 2013 Hasler JF: Bovine embryo transfer: Are efficiencies improving? Proceedings, Applied Reproductive Strategies in Beef cattle, Sioux Falls, SD: , 2012 Inskeep EK: Preovulatory, postovulatory, and postmaternal recognition effects of concentrations of progesterone on embryonic survival in the cow. J.Anim. Sci. 82 (Suppl), E24-E29, 2003 Jinks EM, MF Smith, JA Atkins, KG Pohler, GA Perry, MD MacNeill, AJ Roberts, RC Waterman, LJ Alexander and TW Geary: Preovulatory estradiol and the establishment and maintenance of pregnancy in suckled beef cows. J. Anim. Sci: 91, , 2012 Kahn W: Vorkommen und Wachstumsdynamik von Gelbkorpern mit Hohlraum wahrend des Ovarialzyklus bei Rindern und deren Hormonprofile. Dtsch. tierarztl. Wschr. 93: , 1986 Kahn W: Veterinary Reproductive Ultrasonography, Schlutersche Verlagsgesellschaft GmBH Germany 1994, Special edition, (Reprint), 2004 Kenyon AG, LGD Mendonca, G Lopes Jr, JR Lima, JEP Santos and RC Chebel: Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows. Health Advance 136: , 2013 Lamb C: Factors affecting an embryo transfer program. Proceedings, Applied Reproductive Strategies in Beef cattle, Reno Nevada: , 2005 Lamb GC and VRG Mercadante: Selection and Management of the Embryo Recipient Herd for Embryo Transfer. In: RM Hopper, Bovine Reproduction, 1 st ed., 2015 Lonergan P, L O Hara and N Forde: Role of diestrus progesterone on endometrial function and conceptus development in cattle. Anim. Reprod. 10: , 2013 Lopez-Gatius F, P Santolaria, JL Yaniz and RH Hunter: Progesterone supplementation during the early fetal period reduces pregnancy loss in high-yielding dairy cattle. Theriogenology: 62, , 2004 Madsen CA, GA Perry, CL Mogck, RF Daly, MD MacNeill and TW Geary: Effects of preovulatory estradiol on embryo survival and pregnancy establishment in beef cows. Animal Reproduction Science 158: ,

66 Nishigai M, H Kamomae, T Tanaka and Y Kaneda: The relationship of blood progesterone and estrogen concentrations on the day before and the day of frozen-thawed embryo transfer to pregnancy rate in Japanese Black beef cattle. J. Reprod. Dev 24: , 2000 Nogueira E, G Saravi Cardoso, HR Marques Junior, A Menezes Dias, LC Vinhas Itavo and J Correa Borges: Effect of breed and corpus luteum on pregnancy rate of bovine embryo recipients. R. Bras. Zootec, 41: , 2012 Nogueira MF, DS Melo, LM Carvalho, EJ Fuck, LA Trinca and CM Barros: Do high progesterone concentrations decrease pregnancy rates in embryo recipients synchronized with PGF2alpha and ecg? Theriogenology, 61: , 2004 O Hara L, N Forde, F Carter, D Rizos, V Maillo, AD Ealy, AK Kelly, P Rodriguez, N Isaka, ACO Evans and P Lonergan: Paradoxical effect of supplementary progesterone between Day 3 and Day 7 on corpus luteum function and conceptus development in cattle. Reproduction, fertility and Development: 26, , 2014 Pfeiffer LFM, del Carmen Bonilla de Souza Leal S, A Schneider, E Schmitt and MN Correa: Effect of the ovulatory follicle diameter and progesterone concentration on the pregnancy rate of fixed-time inseminated lactating beef cows. R. Bras. de Zootec: 41, , 2012 Purcell SH, WE Beal KR Gray: Effect of a CIDR insert and flunixin meglumine, administered at the time of embryo transfer, on pregnancy rate and resynchronization of estrus in beef cows. Theriogenology 64: , 2005 Rajamahendran R and PC Sianangama: Effect of human chorionic gonadotropin on dominant follicles in cows: formation of accessory corpora lutea, progesterone production and pregnancy rate. J. reprod. Fert. 95: ,1992 Sood P, NK Vasishta, M Singh and N Pathania: Prevalence and certain characteristics of mid-cycle estrus in crossbred cows. Veterinarski Arhiv 79: , 2009 Stock AE: Luteal cell culture as a model to study progesterone production of corpora lutea with or without cavity. Thesis, Fac vet. Med, Munich, 1984 Stock AE and JE Fortune: Ovarian follicular dominance in cattle: relationship between prolonged growth of the ovulatory follicle and endocrine parameters. Endocrinology 132: , 1993 Wiltbank MC, AH Souza, PD Carvalho, AP Cunha, JO Giordano, PM Fricke, GM Baez and MG Diskin: Physiological and practical effects of progesterone on reproduction in dairy cattle. Animal: 8, 70-81, 2014 Zbylut,J and J Czeladko: Contralateral pregnancies in cows. Medycyna weterynaryjna 65: ,

67 Sanitary Considerations at Collection, Search, Freezing and Transfer of Bovine Embryos Angelika E. Stock Services mobiles de thériogénologie, Chambly-Saint-Hyacinthe, QC, Canada Introduction Considering disease risk, the trade with embryos is certainly much safer than moving animals or semen across countries and continents. Still, the risk of transmission is possible. Therefore, the International Society of Embryo transfer (IETS) has developed practical and scientifically verified protocols, which consist mainly of preventive measures such as sanitary embryo handling at all time. For information about the epidemiology and pathogenesis of diseases of concern and embryo pathogen interaction from different species the reader is referred to more detailed reviews (Stringfellow and Givens, Julie Gard, and several chapters of the Manual of the IETS, see references). This presentation will focus on the techniques of sanitary handling of bovine in-vivo embryos to emphasise the importance of preventive measures against disease transmission by the embryo transfer practitioners and his team and to initiate the reader of this presentation to consult the corresponding chapters of the Manual of the International Embryo Transfer Society for a more in-depth knowledge on the subject. (IETS members have free access to the Manual online). Embryo collection Embryos can be collected on farm, in a clinic or in a special unit. Disease status of the farm should be known and the collaboration with the farm veterinarian is suggested to gather all possible information. Ideally collection is performed in a squeeze chute, yet the restraint of donors should not be stress full. The veterinary team and their employees should be well trained and not only diligent in technical procedures but also in sanitary aspects applying their techniques. Attention to detail is essential. Working clothes, car and equipment should be cleaned routinely. Clothes should be changed between farms and the designated place for the search and handling of the embryos. It is very important to know ahead of time if embryos are destined for export and if so, which country is importing the embryos. For example, export of embryos may not be possible if there was a clinical case of leukosis in the herd over the last 3 years. Semen used for the donor should have been collected in approved centers where the bulls and their semen have been proven pathogen free. A certificate for the bull semen is generally required if the embryos will leave for export. This does not mean, however, that one should be less careful when embryos will be transferred on the farm or remain in the same country. Prior to collection the rectum should be emptied and the tail region well scrubbed with soap and water to prepare for the epidural anesthesia. After epidural anesthesia the tail is to be tied towards the front of the donor and the vulvar region should be scrubbed and cleaned with soap, well rinsed thereafter and well dried. Disinfection of the clean and dried vulvar area with alcohol is the last step before catheter insertion. It is often mentioned in the literature to avoid soap and disinfectants to prepare the donor, because both are embryotoxic. If chemicals are used to clean, it is of course important to rinse and dry the region well and not to carry any of these substances into the uterus. The contamination of embryos by the uterine catherization of the donor should not be overlooked. In 1978, a study from Ontario, Canada found positive cultures from bovine vulvar swabs, in cows with vulvitis, but also in supposedly healthy cows. Among the infectious agents were Mycoplasma bovi-genitalis, Ureaplasma, Haemophilus somnus, Streptococci and E. coli. Interestingly, repeated vulvar culture from these cows revealed that Ureaplasma had persisted in some of them for at least 3 months (Ruhnke et al., 1978). Recently Ureaplasma has regained more attention since this agent was isolated on several occasions from aborted fetuses after embryo transfer. Contamination of embryos through the vulvovaginal route during catherization of the donor may be a possible route. Surprisingly we always use a sanitary sleeve for the transfer of embryos, whereas a simple sanitary plastic sleeve slipped over the collection catheter seems not to be used routinely. This could easily prevent contamination of uterus, flushing media and embryos coming from the vulvo-vestibular area and the vagina. 65

68 Material and equipment Any equipment should be clean, sterilized or new and disposable and this not only between farms, but also between animals on the same farm. Once the embryos are collected, the practitioner team should change clothes and shoes and clean their arms and hands well before entering the place where the search, washing and freezing of embryos will occur. The only material from the barn that should enter the place for searching, washing and freezing embryos should be a clean filter containing the embryos. Any equipment that was in contact with animal secretions and the flushing media needs to be sterilized before the use on the next animal. Sterilisation can be done in an autoclave or thoroughly washed and rinsed equipment and catheters may be submerged in boiling distilled water for 15 min. Disposable material should be used as much as possible, since its use is fast and clean. Nowadays, most media, plastic ware and pipettes for the embryo search, washing, freezing and transfer can be bought commercially, which safes the time for cleaning and the preparation and filtering solutions. The laboratory workspace should be clean, possibly wiped with liquid alcohol before. Any form of spray disinfectant, insecticides as well as heavy metals, detergents, lubricants, tobacco smoke or fuel fumes should be avoided, as well as any food or pets. Although the flushing media may contain antibiotics, embryos should be searched as soon as possible and then held in specific holding media. Unless using the microscope, prolonged exposure to bright light should be avoided since it may be harmful to the embryo. Clear identification of each dish with the appropriate donor is not only important to avoid inadvertent embryo mix-up but may also be prudent when embryos or medium of one of the donors is contaminated. Commercial holding and freezing media are sterile solutions that need to remain sterile for the next use. They should not be handled in the barn. Depending on the number of donors it is wise to prepare sterile syringes with attached needles that contain the anticipated amount of media at room temperature and to keep the reminder in a refrigerated environment to avoid bacterial or fungal growth. Searching and washing The washing procedure of embryos is clearly outlined in the IETS manual, see below Table 1. Grading and inspection (intact zona and adhering debris on the zona) of embryos should be done under magnification of at least 50x. Treating embryos with the enzyme trypsin is often recommended and required for the export of embryos. It is successful in cleaning the zona pellucida from adherent debris and from sticky pathogens such as the BHV-1 virus. Since trypsin is a protein enzyme, it is sensitive to its environment and easily inactivated (Thibier,2011). Commercially available are pathogen free trypsin solutions, that need to be frozen or, once thawed to be used. Each team member, using trypsin needs to be aware that trypsinization does not replace sanitary techniques. Table 2 categorizes different diseases and agents into categories. As we can see, category 4 includes several infectious agents such as bovine herpes virus, mycoplasma and ureaplasma, who may or may not be removed from the zona with the current methods of washing. Table 1. Essential requirements for proper washing of embryos (Chapter 6, IETS Manual, 3 rd ed, 1998) Only embryos from a single donor washed together (confirm before washing). Ten or fewer embryos washed at one time (count before washing). Only zona pellucida-intact embryos washed (confirm both before washing).* Only embryos free of adherent material washed (clean when necessary before washing).* Minimum of ten washes (allow sufficient time for thorough, gentle mixing in each wash). Use a new sterile micropipet each time embryos are moved from one wash to the next. Regulate volumes so that each was is at least 100-fold dilution of previous wash. *To confirm intactness of the zona pellucida and freedom from adherent material, embryos must be observed over all surfaces at a minimum magnification of 50X 66

69 Table 2. Diseases or infectious agents in cattle listed in 4 categories by IETS according to the risk for their transmission via in vivo derived embryos (OIE, 2012), adapted from Ponsart C and N Pozzi, 2013 Category 1: Sufficient evidence has accrued to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer according to the IETS Manual. Disease agent: Bluetongue, Bovine spongiform encephalopathy, Brucella abortus, Enzootic bovine leukosis, Foot and mouth disease, Infectious bovine rhinotracheitis: trypsin treatment required Category 2: Substantial evidence has accrued to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer according to the IETS Manual, but for which additional transfers are required to verify existing data. Disease agent: None Category 3: Preliminary evidence indicates that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer according to the IETS Manual, but for which additional in vitro and in vivo experimental data are required to substantiate the preliminary findings. Disease agents: Bovine immunodeficiency virus, Bovine viral diarrhea virus, Rinderpest virus Campylobacter fetus (subs.veneralis), Haemophilus somnus, Mycobacterium paratuberculosis, Neospora caninum Category 4: No conclusions are yet possible with regard to the level of transmission risk, or the risk of transmission via embryo transfer might not be negligible even if the embryos are properly handled according to the IETS Manual between collection and transfer. Disease agent: Akabane, Bovine anaplasmosis, Bovine herpesvirus-4, Enterovirus Lumpy skin disease, Vesicular stomatitis, Chlamydia psittaci, Escherichia coli 09: K99, Leptospira borgpetersenii serovar hardjobovis, Mycobacterium bovis Parainfluenza-3 virus Trichomonas foetus, Ureaplasma and Mycoplasma spp In vitro produced embryos Although the intact zona pellucida from IVF embryos is an effective barrier that is rarely penetrated, the steps recommended for washing in vivo embryos seem not as effective for the in-vitro embryos since several pathogens, such as BTV, BHV-1, foot and mouth disease virus and BVD virus, adhere more readily to the zona of IVF embryos (refer to Table 2 for in vivo embryos). The presence of virus reduces the rate of maturation, fertilization and development in vitro. In the IVF procedure, raw materials are often collected from the slaughterhouse, which poses an increased risk for contamination. Even though procedures like those used to cleanse in vivo embryos may reduce the level of contamination in IVF embryos, it seems that certain strains of some viruses, such as the BVD virus have a greater affinity to adhere to the IVF embryos than others. The use of antibiotics, antifungals and antiviral substances in the culture medium are investigated to minimize pathogens in IVP embryos (refer to Chapter 7, IETS manual for a detailed review on this subject) Freezing embryos When approaching the step of freezing of embryos, environmental contamination needs to be avoided when filling the straw with the embryos. Pre-sterilized straws and new and clean plugs for freezing need to be used each time a straw is prepared for freezing. Only unused media should be used to fill straws. It is also essential that the straw be filled and closed correctly, by wetting the cotton plugs in the straw, so that liquid nitrogen is not able to enter the straw during freezing, otherwise straws may crack or explode upon thawing. Depending on the model, the freezer compartment containing methanol or liquid nitrogen should be cleaned, disinfected and rinsed. The Styrofoam box (LN freezer) containing the nitrogen should be replaced periodically. People who work with liquid nitrogen for bovine embryo work may not be aware that liquid nitrogen itself can contaminate embryos or semen. This is more and more reported in human medicine, where frozen bone marrow samples have been contaminated with the Hepatitis B virus (Bielanski et al, 2000). Viral and bacterial agents easily survive in cryoprotectants and liquid nitrogen and may lead to cross -contamination, especially when structural defects of straws or faulty sealing is present. Direct contact of liquid nitrogen and embryo takes place when vitrification is used and puts therefore this technique more at risk for cross contamination. The fact that liquid nitrogen can be contaminated with viruses and bacteria from the donor but also environmental and waterborne bacterial and fungal agents, should make the professional rethink to handle liquid nitrogen wisely. Easy preventive measures may be to empty and clean wet shippers and liquid nitrogen storage tanks periodically, not to mix used liquid nitrogen with unused nitrogen as 67

70 well as filling tanks using a mesh filter to avoid accumulation of particles and debris on the bottom of the tank. Bielanski, 2005 has published recommendations on successful disinfection methods of Dryshippers, the main transporter vessel for frozen specimens all over the world. The same author states in another report from 2012, that the present cryopreservation technology seems to be sanitary sound if biocontainment measures as recommended by IETS standards and the World Organisation of Animal Health are strictly adhered to. Embryo transfer Prior to transfer, a standard thawing procedure for frozen embryos should be respected, such as a clean working area, clean water (30 degrees C) and a clean thermos to thaw the embryo. Transfer sheets should be sterile and the transfer gun cleaned after each use. As described for the donor, the recipient should be prepared for catherization of the cervix. Restraint without major stress, the epidural anesthesia is performed after emptying the rectum and cleaning of the vulvar area. Essential is the use of a protective sheath over the embryo transfer gun to protect the transfer gun from contamination before it enters cervix and uterus. Ideally a second person helps to slightly open the vulvar lips to insert the transfer gun quickly. The transfer gun should be sliding into the uterine horn smoothly, so that no trauma is caused. Recipients should be tested for certain pathogens, such as the bovine leukemia virus, known for its vertical transmission from mother to the fetus. The many first transfers that were performed without knowledge of the benefit of current sanitary procedures, show that embryo transfer has a certain innate safety considering disease transmission. It is important to maintain the awareness that technologies will change and new challenges may arise, as we have seen when we compare invitro produced embryos with the ones collected in-vivo. Hopefully, technical and ethical excellence will go hand in hand. References Bielanski A, S Nadin-Davis, T Sappand C Lutze-Wallace: Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 40: , 2000 Bielanski A: Experimental microbial contamination and disinfection of dry (vapour) shipperdewars designed for short - term storage and transportation of cryopreserved germplasm and other biological specimens. Theriogenology 63: , 2005 Bielanski A: A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contaminating during banking to prevent disease transmission in ET practices. Theriogenology 77: , 2012 Gard, J: Control of Embryo-borne Pathogens. In: RM Hopper, Bovine Reproduction, 1 st ed, 2015 Mapletoft RJ and GA Bo: General sanitary procedures and considerations associated with in vivo derived bovine embryos, Chapter 4, 5 th ed. Manual of the International Embryo Transfer Society, 50-56, 2017 Ponsart C and N. Pozzi: Sanitory requirements for bovine gametes and embryos in international trade. Anim. Reprod.10: , 2013 Ruhnke HL,PA Doig, AL MacKay, A Gagnon and M Kierstead: Isolation of Ureaplasma from bovine granular vulvitis. Can J Comp. Med 42: , 1978 StringfellowDA and MD Givens: Preventing disease transmission through the transfer of in-vivo derived bovine embryos. Livestock Production Science 62: , 2000 Thibier M: Embryo transfer: a comparative biosecurity advantage in international movements of germplasm. Rev.Sci tech.off.int.epiz. 30: ,

71 Starting an Oocyte Collection Lab and Incorporating IVF into Regular ET Practice Dr. Rob Stables Bow Valley Genetics Ltd., Brooks, Alberta Adding IVF as a service in our practice has been a huge practice builder. The hard work and frustration of 3 years while getting off the ground has paid off in We have been running at full capacity for the last 8 months which is about 15 donors per week. In % of our total donor collections were on IVF cycles. All donors have been beef or rodeo stock thus far. Learning OPU technique is easy in theory but in practice getting consistently good recovery rates can be challenging and frustrating. Having good mentorship and training is paramount. If at all possible work with an experienced practitioner to get you started. You will have questions and ideas that you need to bounce off others. Equipment is important and getting the experience of those who have gone through these challenges before is invaluable. Getting clients has not been an issue. There is a lot of interest from breeders and they have often acquired at least some minimal knowledge of the procedure from talking to other breeders or via Dr. Google. In beef practice the primary benefits of IVF are related to the seasonality of the breeding season in Canada. Majority of calves are born from January to the end of March but we are seeing herds move later into April and May. With IVF we can collect yearling heifers between puberty and first breeding to get embryos for early implant, and then collect again after breeding. Breeders are just beginning to realise the potential advantages of decreasing the generation interval. Traditionally beef cattle breeders prefer to wait until the donor is a proven producer, and the majority still have this opinion. However, some are realising that the potential benefits outweigh the risk of collecting what may turn out to be a less than stellar cow once proven. Increased risk but increased reward as well. For cows the major advantage of IVF is doing them while pregnant vs. keeping them open for a year for conventional flushing. The value of that calf should cover much of the added cost of IVF over conventional ET and it is better for the cow s longevity to be in calf rather than open and dry. For cows that have been extensively flushed successfully but now have some age and decreased viable embryo production, IVF can often bring those donors back into sustainable production. We have had a few of these type of donors in the past year and have done IVF cycles every 2 weeks for 6-8 months and produced more viable embryos per collection and in total than they had been doing on conventional ET just prior to switching. Some clients may come to you with the perception that IVF is a silver bullet for their poor producing donor. Often these donors will work better on IVF but not if they are low follicle count cows. I tell them it all comes down to a numbers game. We need to start off with enough follicles to generate the oocytes and we lose numbers at each step through the procedure. The clients also get excited to know they can split collections to different bulls. We don t do this unless they have a minimum of 16 oocytes total. Boviteq finds reduced development rates if less than 8 oocytes per dish. The average number of freezable embryos as a percentage of oocytes collected is about 33%. Once you freeze and transfer you may be looking at only calf or two per mating unless you happen to get above average development rates or higher preg rates. Generally, I don t recommend splitting collections unless there are 30 oocytes or more. There are extra costs from the lab associated with running two dishes that increase the cost. The client is generally further ahead by doing two separate collections. At the meeting I will present some data on the use of once per day FSH injections for both IVF and ET, our use of a 5 day co-synch recip protocol and the addition of GnRH at time of transfer. 69

72 Conception of a Bovine Ovum Pick-Up Mobile Unit Lab Dr. Jonathan Lehouiller Clinique Vétérinaire Centre-du-Québec inc., Bon Conseil, Québec Over the past 10 years, all over the world, in-vitro produced (IVP) embryos have been quickly increasing. Lots of research is done in commercial laboratories as well as universities to continually improve the efficacy of culture media and to try to mimic as much as possible the in-vivo process of oocyte maturation, fertilization and embryo culture. Research is also done on donor selection, preparation of the donor and super-ovulation to try to improve the numbers and the quality of collected oocytes. Ultrasound guided ovum pick up (OPU) is now being performed at multiple collections centers and universities. For years now, the need to bring donor cows to facilities for conventional embryo transfer has become obsolete. A similar change may be about to happen with regards to Ovum pick up. Ovum pick-up is an advanced procedure requiring optimal conditions in order to succeed and achieve a good blastocyst rate. Oocytes are known to be a lot more fragile than embryos when considering temperature and ph variations, air quality and other environmental factors. Two years ago, our team in Clinique Vétérinaire Centre-du- Québec inc. had an idea to develop a mobile unit that would allow us to perform OPU on farm with the objective of obtaining the same success rate that many achieve in a designated collection centers. The following is a list of optimal conditions that my team and I had determined as necessary factors for success: 2 separate rooms: a clean room being the laboratory for oocyte processing and a room for the cow Easy and safe entryway for the donor cow with good restraint A controlled temperature range of degrees Celsius for performing OPU Washable surfaces for cleaning and disinfection Air quality control/system for air purification/air filters/ventilated areas A system enabling both left-handed or right-handed veterinarians to perform OPU A manner to achieve a consistent aspiration rate when performing OPU With all those conditions in mind, and with some restrictions about length, weight and width (Quebec laws are strict), we started developing a mobile unit that would meet the criteria. Here is the best description of the finished product: Mobile unit made up of 3 areas: a restraint area for the cow (48 inches wide by 14 feet long), an area for the laboratory (42 inches wide by 10 feet long) and a dirty-to-clean changing/washing area at the front of the laboratory (42 inches width by 4 feet long) 2 hydraulic gates, one at the rear for the cow s entrance, and one in the front for the cow s exit Cow restraint system: no lateral or forward movement is possible, with a special set-up allowing for heifer restraint 2 separate heating systems, electrical or propane, to individually heat both the laboratory and cow areas to their desired temperature. (Heating in cow area is maintained at 26 C, if outside temperature is -20 C it takes about 5 minutes to reach this after closing the doors) A fan in the cow area to ensure adequate air flow Air conditioning system for hot summer days 70

73 Stabilizing system: Allowing one person to process oocytes in the lab area while someone else is performing the next OPU in the cow area. Cleaning and disinfection system and washable surfaces: Biosecurity is very important! Box for OPU ultrasound and pump/tube heater: Installed on a track to easily adapt for left or right-handed manipulators Window from cow area to lab area: allows for passage of the collection tube and prevents contamination of the lab Storage and proper ties to stop the equipment from moving around during travel An incubator for pre-heating solutions on the road There are a lot of advantages to being able to perform OPU on farm, the trailer ensure the work is being done always in the same conditions. Our aspiration system is always the same and is fixed ensuring a constant aspiration rate, the only variable is the cow s height. We actually can control that too, because we included a step in the restraint area. Producers are pleased by the reduced cow s stress level. By staying on farm, she climbs in the mobile unit for as little as 20 minutes instead of leaving the farm for a day. There is no milk loss for that reason, and biosecurity is optimal. The mobile unit is cleaned and disinfected at the end of each farm OPUs. The mobile unit also allows smaller farms to have access to OPU-IVF technologies without having to transport their cows, given that those farms are not doing enough OPU to build a collection room on their own site. The main disadvantage of this mobile unit is the limited number of different farms that can be done in one day. Travel is a limiting factor, and as we can only send oocytes to our IVF lab once a week, we sometimes need to delay OPUs. From our perspective though, we are certain that some OPUs performed in the past 2 years would not have been done if the producers had to bring his cow to a collection center. Our clientele includes a lot of producers who are interested in this technology and want to perform OPUs only once or twice per year. We do not have many producers doing large number of OPUs. This mobile unit is very convenient for our type of clientele and it certainly gives them more access to the IVF technology. This bovine OPU mobile unit is currently Patent Pending for Canada and United States. Please do not hesitate to contact me for more information. 71

74 IVP Embryo Evaluation for the Receiving Practitioner Patti Anderson and Dr. Jeff Anderson Trans Ova Genetics, Richland, WA, USA In 2017, over 200,000 in vitro produced (IVP) embryos were produced from Trans Ova Genetics across five regional labs in the United States. More than one-third were shipped fresh and transferred by ET practitioners in 35 states. Helping practitioners succeed with our embryos is a priority and warrants a review of day 6 embryo predictions, how that translates into day 7 results as well as receiving, unloading and evaluating IVP embryos from Trans Ova Genetics. Embryos evaluated on day 6 of development are assigned into quality, average and marginal categories. This evaluation is done by assessing stage of development, viable embryo mass, cell symmetry, and lipid content. A quality embryo typically develops into a freezable grade 1 or grade 2 embryo on day 7. An average embryo typically develops into a transferrable but non-freezable embryo on day 7. Approximately 25% of the total embryos in the marginal category will develop into transferrable, but non-freezable embryo on day 7. It is important to keep in mind that these are estimations of a biological system and actual embryo results on day 7 may vary from the criteria outlined above. After embryo selection has been determined on day 6, viable embryos from individual matings are packaged into tubes and placed in a heated shipping incubator. Embryo shipments can be couriered or shipped on Day 6 and transferred on Day 6.5 or Day 7. Upon receipt and prior to unloading embryos in a petri dish it is important to warm medias and disposable supplies needed for evaluation and transfer to avoid embryo shock. Using a stereo microscope with adequate magnification and light source is critical. The use of warm stages and slide warmers are important in environments where temperature control is challenging. Abrupt temperature changes, osmolarity changes in medias, and mechanical injury are the most common insults that IVP embryos will encounter and care should be taken to avoid these drastic changes in environment. Using aseptic techniques, embryos can be reloaded into shipping tubes if needed, to be transferred or frozen later. There has been a lot of discussion over the years in developing a separate grading system for IVP embryos. The current standards set by the International Embryo Transfer Society (IETS Manual, 4 th Ed) work well for in vivo derived embryos but do not address all the morphological differences seen with IVP embryos. Differences in stage of development, perivitelline space, extruded cells, cell compaction, cell shape, density, lipids, and vacuoles are all well-known and contribute to the difficulty of grading IVP embryos (Barfield 2015). The IETS Manual does address evaluating the stage of development, the percentage of extruded material in the perivitelline space, symmetry of the embryo mass, cell size, color, and density. Lipids and vacuoles are difficult to evaluate and are not mentioned specifically in the current IETS manual. The presence of lipids can vary appearing granular or iridescent, can be localized or diffuse, exist in large or small droplets, or present within or outside the embryo mass. Embryos with lipids are generally considered of lesser quality (grade 2 or 3) and are preferred to be transferred versus frozen when possible to optimize conception rates. If transferring the embryo is not an option, selecting embryos that have the least amount of lipids present will increase survivability post thaw. Receiving practitioner feedback about fresh IVP embryos transferred on Day 7 will improve the accuracy of day 6 predictions and is valuable for improving processes. Creating an avenue that is easy for practitioners to share this information should be discussed. Despite improvements in IVP embryo quality and conception rates, evaluating IVP embryos remains challenging. Utilizing the current standards to evaluate IVP embryos should continue but further research is needed on practical methods to evaluate lipid content in IVP embryos. The desire to develop IVP systems that create embryos that look, freeze, and make pregnancies similar to ET embryos is a continued effort. Barfield, J Evaluation of in vitro-produced bovine embryos CETA/ACTE & AETA Joint Convention. Niagara Falls, ON. Stringfellow, DA and Givens MD Manual of the International Embryo Transfer Society, 4 th Ed. 72

75 Impact of Early Life Nutrition on the Sexual Development and Fertility of Cattle David A. Kenny Animal and Bioscience Research Department, Teagasc Grange, Dunsany, Co. Meath, Ireland. Abstract The age at which cattle reach puberty and subsequent sexual maturation is fundamental to the reproductive and economic efficiency of both beef and dairy production systems worldwide. Sexual maturation is regulated by a complex network of neuroendocrine processes involving the biochemical interaction of key metabolic, neuroendocrine and reproductive tissues. While our understanding of the underlying biochemistry that conditions and eventually triggers the pubertal process has improved in recent years, much of the intricate mechanistic detail is yet to be elucidated. Management influences and principally, nutrition together with the inherent genetic makeup of the animal undoubtedly influence the timing of puberty in cattle. In particular, there is now overwhelming evidence to support the importance of early life nutrition in regulating the timing of puberty in both bulls and heifers. Indeed, there is growing evidence that improving the nutritional status of the calf in early life, significantly advances maturation of the hypothalamic-pituitary-gonadal axis; therefore, facilitating earlier sexual development. While advancing sexual maturation is a desirable goal, it is important that any strategy employed does not impinge upon normal gametogenesis or post-pubertal reproductive potential. With this in mind, the objective this review is to discuss the impact of early life nutritional management of male and female calves on (i) advancing the maturity of the hypothalamic-pituitary-gonadal axis and (ii) implications for subsequent fertility. A brief discussion of the influence of post pubertal nutritional management on aspects of both heifer and bull fertility is also included. Introduction The length of the generation interval has traditionally been an Achilles heel in terms of the rate of genetic improvement in cattle (Kasinathan et al. 2015). While the advent of genomic selection has contributed both to the earlier identification of potentially genetically elite animals, the potential of such technology is only truly realized when the timing of sexual maturation and ultimately breeding of cattle is optimized. This is true for both beef and dairy cattle and for both genders. Given the high rearing costs incurred, it is important that heifers become pregnant early and that bulls produce semen of adequate quantity and quality, in order to enable their effective recruitment as breeding animals within their first breeding season. This is particularly important, for both genders, within the context of seasonal based calving systems. Early onset of puberty and subsequent sexual maturation is thus fundamental to the timely recruitment of these young animals to the breeding herd and management practices must be appropriate to facilitate this (Kenny et al., 2018). Based on the published literature, the average age at puberty, is approximately 315 days of age (292 to 327 days; (Brito et al. 2007a; Brito et al. 2007c) for beef and 320 days (283 to 369 days; (Dance et al. 2015; Harstine et al. 2015; Byrne et al. 2018a) for dairy bred bulls. For heifers, the average age at which puberty is attained ranges from approximately 261 days (234 to 301 days; (Davis Rincker et al. 2011; Manthey et al. 2017) for dairy breeds to 366 days of age (276 to 438 days; (Rodriguez-Sanchez et al. 2015; Heslin et al. 2017; Zezeski et al. 2017) for heifers of beef breeds. There is, however, significant within breed variation for both genders (Wathes et al. 2014). Duittoz et al. (2016) recently described puberty as the best example of the interaction between genotype and environment and the age at which individual animals undergo puberty and their subsequent reproductive potential will be dictated by their particular genetic (breed type predominantly) and environmental (nutrition, health, season, sexual status of herdmates etc.) influence. 73

76 Measurement of puberty in cattle It is now widely accepted that the gonadotropin-releasing hormone (GnRH) neuronal network generates pulse and surge modes of gonadotropin secretion which is critical for puberty and fertility in mammals (Herbison, 2016). However, the establishment of the pubertal phenotype in cattle varies in its specificity and accuracy of establishment between the two genders. Indeed, caution is advised (Duittoz et al., 2016) in the interpretation of the factors that influence the age at puberty in cattle given the multitude of variables that have been cited as a measure of the phenotype for both heifers and bulls. For example, for heifers, traits measured range from readily available parameters including age at first service/calving/oestrus to more expensive and difficult to measure and invasive traits that involve regular ovarian ultrasonography and/or establishment of systemic concentrations of progesterone. For males, studies have monitored scrotal circumference, sperm quality (concentration, motility and morphology) as well as circulating blood concentrations of reproductive hormones. The widely accepted definition of puberty in the bull is as the ability to produce an ejaculate containing 50 million spermatozoa with 10% progressively linear motile spermatozoa (Wolf et al. 1965). The timing of the early transient rise in LH pulsatility is a critical factor in determining the age at which puberty is reached (Evans et al. 1995). This early rise has been reported for bulls to occur between weeks 10 and 20 of age (Rawlings and Evans 1995) and is thought to induce responsiveness of testicular Leydig cells to LH, leading to an increase in testosterone (TT) production (Amann and Walker 1983). This increase in TT is necessary for the differentiation of Sertoli cells and initiation of spermatogenesis (Amann and Walker 1983). The timing and magnitude of the early LH rise has been shown to be particularly sensitive to the prevailing metabolic status of the animal during calfhood (Dance et al. 2015; Byrne et al. 2017). In heifers, puberty is defined as ovulation, accompanied by visual evidence of oestrus and followed by the formation of a corpus luteum (CL) and an oestrous cycle of normal duration (Perry 2016). In most cattle studies, the timing of puberty has been assessed through the regular measurement of systemic concentrations of progesterone with two successive samples having a concentration of >1ng/mL, generally being accepted as primary evidence for first ovulation having occurred. The Gonadostat hypothesis describes the mechanism behind the initiation of puberty and the onset of the event, for heifers, is characterised by a progressive decrease in the sensitivity of the hypothalamic/anterior pituitary axis to the negative feedback of oestradiol and coincides with an incremental increase in uterine development, length of follicular waves, and size of the dominant follicle (Atkins et al. 2013; Wathes et al. 2014; Perry 2016). The endocrinological regulation of the hypothalamic-pituitarygonadal axis during the pre and peri-pubertal periods has been reviewed in detail for both heifers (Atkins et al. 2013; Duittoz et al. 2016) and bulls (Rawlings et al. 2008; Brito 2014) and a targeted discussion of factors that may influence the neuroendocrine control of this axis as well as gametogenesis will follow in later sections. Maternal nutritional and reproductive development of offspring Given the dynamic nature of foetal sexual differentiation and subsequent gonadal organogenesis, it is not implausible to expect that such processes may be affected by prevailing metabolic status of the dam during pregnancy, and particularly the first trimester. The ontogeny of mammalian sexual development commences during foetal life, and, it is important to acknowledge the potential influence that pre- as well as post-natal environment may exert on post-pubertal reproductive potential. In their review of pre-natal influences on the long-term health and productivity of animals, Reynolds and Caton (2012) propose that long-term effects of various insults during foetal or postnatal life may be induced by either (i) irreversible alterations in tissue and organ structure (i.e., a structural defect) or (ii) permanent changes in tissue function mediated through epigenetic perturbation (i.e., a permanent change in gene expression leading to a functional defect). While a detailed discussion is beyond the scope of this review, the reader is referred to a recent review by Kenny et al. (2018a) which summarises the development of bovine foetal gonads in utero as well as the current published literature on the effect of maternal nutritional status at various stages of pregnancy on reproductive outcomes for both male and female progeny. 74

77 Pre-pubertal nutritional status and sexual development in heifers and bulls Most authors now agree that nutritional augmentation has a greater effect on the rate of sexual maturation of both bulls and heifers, the earlier in life that it is implemented. While management regimen for replacement heifer and young bull rearing systems facilitate early intervention strategies in dairy herds, opportunities to manipulate the nutritional status of suckled calves in early life are limited unless calves are weaned prematurely (Gasser et al. 2006). For both genders, published studies have generally arbitrarily divided the pre-pubertal period (birth to puberty) into pre-weaning (zero to six months of age) and post-weaning (six months to puberty) periods. The effect of elevated pre-weaning plane of nutrition and growth rate on age at puberty onset for selected dairy and beef heifer studies is summarized in Table 1. In general, where sizeable differences in animal performance is achieved, commencing a nutritional improvement regimen for heifers in the first six months of life, typically hastens puberty onset by one to three months, compared to contemporaries reared to achieve a more moderate growth trajectory. When one examines the effect of improving the nutritional status of heifers after reaching the age of six months, more modest responses have been observed (Gasser 2013). Table 1. The effect of pre-weaning nutrition on age at puberty in heifers (adapted from Kenny et al., 2018) Study Animal Type Breed 1 Diet 2 n Age Duration ADG Age at Puberty Sig. Shamay et al., 2005 Dairy heifer HO WM MR 40 5D 55D <0.01 Meyer et al., 2006 Dairy heifer HO MR 29% CP MR 22% CP 78 10D 56D <0.01 Moallem et al., 2010 Dairy heifer HO WM MR 46 4D 56D D 320D <0.001 Rincker et al., 2011 Dairy heifer HO MR + CP conc MR + conc 80 2D 40D <0.01 Gasser et al., 2006 Beef heifer AAxSI 60% corn conc 30% corn conc D 70D <0.05 Guggeri et al., 2014 Beef heifer HE Dam + creep Dam only 46 75D 83D <0.10 Rodriguez- Sanchez et al., 2015 Beef heifer PMO Dam + creep Dam only D <0.01 Reis et al., 2015 Beef heifer AAxHE Dam + creep Dam only D D 323D ns Heslin et al., (2018) Beef heifer LMxHO AAxHO Grass silage + conc ad lib Grass silage kg conc Heslin et al. (unpublished) Beef heifer AAxHO Pasture + conc ad lib Pasture kg conc D 450D 1 Breed: HO = Holstein-Friesian; AA = Angus; SI = Simmental; PMO = Piedmontese; HE = Hereford; LM = Limousin 2 Diet: WM = whole milk; MR = milk replacer; CP = crude protein; conc = concentrate; ad lib = ad libitum 75

78 We have recently examined the effect of nutritional augmentation during various stages of the prepubertal period on aspects of the physiology of sexual development of beef heifers. In a study where 80 Angus x Friesian heifer calves were offered either a high compared or moderate plane of nutrition from 4.5 to 9 months of age puberty onset was advanced substantially in those heifers offered the high plane of nutrition, with 93 compared to 69% of heifers pubertal at 15 months of age for high (ADG: 1.15 kg/day) and moderate (ADG: 0.5 kg/day) planes of nutrition, respectively. Alternatively, in another recent study where we offered 309 crossbred beef heifers of varying ancestry (predominantly beef v beef x dairy genotypes were compared in the study) divergent planes of nutrition over a 13 week period commencing at eight months of age, a modest 15 day reduction in the age at onset of puberty, with no latent effects on post pubertal fertility, was observed. This was in spite of the fact that heifers on the high plane of nutrition (concentrate offered ad libitum) grew at twice the rate (1 kg v 0.5 kg/day) of their contemporaries offered a restricted diet (grass silage kg concentrate, daily; Heslin et al. unpublished). The results were consistent across genotype in that study. Similarly Friesian heifers achieving a moderate growth rate of 0.62 kg/day were three weeks younger (307 v 329 days old) at puberty that their feed restricted contemporaries growing at only 0.33 kg/day (Luna-Pinto et al. 2000). A study by Cardoso et al. (2014) showed that the growth trajectory of heifers can be altered from linear to a stairstep pattern and that this will not impact on age at puberty, so long as an adequate overall growth rate is achieved (0.8 to 0.9 kg/day). This supports the theory that there is some flexibility in how target weights are reached (Day and Nogueira, 2013) which, in turn, could be exploited to decrease the overall cost of rearing replacement heifers (Funston et al. 2012). Although most studies investigating the effect of nutrition on sexual development in bulls have focussed on the first six months of life (Brito et al. 2007a,b,c, Dance et al. 2015); there is a rapid increase in testicular growth after 24 weeks of age (Evans et al. 1996). It is likely, therefore, that nutrition and other environmental influences during this period may have an effect on the gonadal development and subsequent reproductive potential of the bull. In the studies mentioned above, diets were offered to dairy bulls from 2 to 31 weeks of age (~7.5 months) with a common diet offered thereafter (Dance et al. 2015). Beef bulls (Brito et al. 2007a, b, c) were offered their diets from 10 to 26 weeks (~6.5 months) with a control or enhanced diet offered subsequently, thus improving the plane of nutrition of half of the previously restricted animals. The same authors also offered beef bulls a high, medium or low diet from 10 to 70 weeks of age. There was one late peak in LH secretion at 39 weeks of age in beef bulls offered a low plane of nutrition from 10 to 70 weeks but LH was otherwise unaltered by plane of nutrition post-six months. FSH concentration was unaffected by plane of nutrition until 59 weeks of age when concentrations increased in animals on high and medium planes of nutrition until 70 weeks of age. Significant increases associated with age were detected for TT secretion, although the high and medium planes of nutrition had higher secretion at 30 weeks of age. Using Holstein-Friesian bulls, our research group has shown that effects of dietary restriction in early life on timing of puberty onset cannot be overcome by enhancing plane of nutrition after six months of age (Byrne et al., 2018a). Additionally, advantages in terms of hastening onset of puberty by offering young bulls a high plane of nutrition in the first six months of life will not be circumvented by imposing a more moderate diet subsequently. Together these data show that the plane of nutrition offered between six months of age and puberty has no little or no impact on the age at puberty in bulls (Byrne et al. 2018a). Neuroendocrinological control of puberty in cattle Sex differences in brain neuroanatomy and neurophysiology support considerable physiological and behavioural differences between females and males (Clarkson and Herbison, 2016). Within the hypothalamus, the arcuate (ARC) nucleus plays a central role in the regulation of both metabolic and reproductive functions through communicating metabolic signals from peripheral tissues to other regions of the brain (Schwartz et al. 2000; Filby et al. 2008; Allen et al. 2012). The ARC region consists of metabolic sensing and GnRH pulse regulator related neurons (Redmond et al. 2011), which are vital in co-ordinating reproductive function. In recent years, there has been much interest in the potential role of the neuropeptide, kisspeptin, as a mediator and even gatekeeper, signalling the metabolic status of the animal to the brain and, therefore, playing a pivotal role in puberty onset and reproductive function in mammals (Pinilla et al. 2012). The kisspeptin neurons in the ARC that innervate the projections of GnRH neurons in and around their neurosecretory zone are key components of the pulse generator in all mammals. However, Herbison (2016) points out that, by contrast, kisspeptin neurons located in the preoptic area project to GnRH neuron cell bodies and proximal dendrites and are involved in surge generation in female rodents (and possibly other species). Kisspeptin has also been shown to have leptin receptors which suggests that 76

79 this neuropeptide may mediate metabolic signalling between energy reserves and the hypothalamus (Sanchez- Garrido and Tena-Sempere 2013). Appetite promoting proteins such as neuro peptide Y (NPY) and appetite suppressing genes such as pro-opiomelanocortin (POMC) are expressed in the ARC and pre-optic area (POA) in close proximity to kisspeptin neurons in sheep (Backholer et al. 2010). The same authors also reported a possible interaction between these neuronal pathways following treatment with kisspeptin which resulted in increased NPY and decreased POMC. Data from a recent RNASeq study conducted by our research group shows that ghrelin receptor gene expression was upregulated in both ARC and anterior pituitary tissue of 18 week old Holstein- Friesian bull calves offered a low compared to high plane of nutrition from two weeks of age (English et al. unpublished). α-melanocortin (αmsh), a peptide product of the POMC gene, is involved in regulating appetite and reproductive processes (Amstalden et al. 2014). A larger population of αmsh neurons were found in the ARC when heifers were offered a high vs a low plane of nutrition (Cardoso et al. 2015), and it was hypothesized that this was due to the increased mrna expression of the polypeptide POMC. The same study reported that 20% of GnRH neurons were located in close proximity to αmsh containing fibres; however, there was no effect of prevailing metabolic status indicating the possibility of an indirect relationship. In contrast, there was a greater population of kisspeptin neurons in close proximity to αmsh containing fibres within the ARC of heifers gaining 1 kg compared to 0.5 kg bodyweight per day from four to eight months of age (Cardoso et al. 2015). An increase in LH secretion was observed following administration of the melanocortin agonist melanotan II to leptin treated ewes indicating a relationship between leptin, melanocortins and GnRH secretion (Backholer et al. 2010). Notwithstanding this, Naloxone, a melanocortin antagonist, was unsuccessful in stimulating LH release (Backholer et al. 2010) when administered to ovariectomised, leptin treated ewes as part of the same study; suggesting that the relationship between leptin and GnRH secretion may be indirect. As mentioned earlier, the presence of a population of kisspeptin neurons in close contact with POMC neurons was also demonstrated in that study, which indicates that kisspeptin may act as a mediator between melanocortins on GnRH secretion. Nesfatin is a polypeptide produced in the hypothalamus and other peripheral tissues including adipose and has roles in regulating feed intake and behaviour in mammals (Stengel et al. 2011). In pigs, intracerebral ventricular injection of nesfatin reduced feed intake and increased LH secretion on both pre and post pubertal pigs (Lents et al. 2013b). Hatef and Unniappan (2017), reporting the results of in vitro studies with murine brain tissue suggested that nesfatin may act directly, in concert with kisspeptin, on hypothalamic neurons and gonadotropes to elicit a generally positive influence on the endocrine milieu regulating reproduction. Thus, it is clear that complex neuroendocrinological processes within the hypothalamic-pituitary axis, influence the onset of puberty; however, despite data from extensive laboratory animal and in vitro models, the exact mechanisms that are modulated by prevailing plane of nutrition in cattle remain to be elucidated (Amstalden et al. 2014). In a recent study, Alves et al. (2017) reported that increased rates of body weight gain in heifers between 4.5 and 8.5 months of age altered the methylation pattern of genomic DNA obtained from the ARC region providing some further insight into how nutritional cues could influence the biochemical functionality of the hypothalamic-pituitary-gonadal (HPG) axis. Metabolic hormone influence on HPG function The most important period for nutritional manipulation of endocrinological profiles of bulls is during the proliferation phase of testicular development (8 20 weeks of age). Insulin-like growth factor-1 (IGF-1) and insulin are important for signalling metabolic status to the hypothalamus and studies have reported an increase in LH secretion between 12 and 18 weeks of age in bulls, associated with a concomitant increase in IGF-1 (Brito et al. 2007a; Dance et al. 2015). In beef bulls, endocrine profiles have been altered using three different planes of nutrition (Brito et al. 2007c); with a higher frequency of LH pulses detected when bulls were offered a high plane of nutrition (6-8 pulses/10 h vs pulses/10 h, high vs low plane of nutrition, respectively); this higher pulse frequency was detected monthly, from 14 to 26 weeks of age, during a 10 h intensive blood sampling. This rise in LH coincided with an increase in IGF-1 concentration which strengthens the case for nutritional modulation of the GnRH pulse generator. Similarly, data from our research show that a high plane of nutrition offered to Holstein- Friesian and Jersey bull calves, from two until 39 weeks of age, results in greater secretion of LH, in response to exogenous GnRH at 16, 24 and 32 weeks of age (Byrne et al. 2017). Many studies employing various mammalian species have reinforced and validated the paradigm of adipose tissue as an endocrine organ that impacts physiological mechanisms and whole body homeostasis (Lents et al. 2013a). Indeed adipokines, a group of over 600 bioactive molecules produced by adipose tissue that act as paracrine and 77

80 endocrine hormones and include amongst others, leptin, adiponectin, resistin and nesfatin. These molecules are important in the regulation of diverse processes including appetite and satiety, fat distribution, inflammation, blood pressure, hemostasis and endothelial function and signal to a host of metabolic and neuroendocrine tissues including the brain. The influence of adipose deposition and ancillary leptin concentrations on the timing of puberty remain unclear (Wathes et al., 2014) however, the concept that a threshold total body-fat level is required for the attainment of puberty and successful reproductive performance has been suggested (Perry et al., 2016). Leptin has been shown to increase LH secretion in the anterior pituitary and to increase GnRH in the hypothalamus of the pig (Barb and Kraeling 2004). In ruminants, leptin will only evoke a significant endocrinological response if the animal has been fasted or subjected to chronic negative energy balance (Amstalden et al. 2005). Studies in both beef and dairy bred bulls have reported no effect of plane of nutrition on systemic leptin prior to 31 weeks of age (Brito et al. 2007c; Dance et al. 2015), in agreement with data from our research group (Byrne et al. 2018b). In the latter study, despite augmenting plane of nutrition from six months of age leading to an increase in leptin concentrations, bulls which had undergone dietary restriction prior to six months of age were older at puberty than their contemporaries offered a high plane of nutrition during this period, again highlighting the importance of early life nutrition on age at puberty. As heifers approach puberty, their serum concentrations of leptin increase; however, changes in diet did not impact concentrations of leptin when percentage of total carcass fat was similar between treatments (Garcia et al. 2002). Reviewing the literature on the effect of exogenous leptin administration to both sheep and cattle offered various planes of nutrition, Lents et al. (2013a) concluded that metabolic state appears to be the primary determinant of hypothalamic-pituitary response to leptin in ruminants with LH secretion typically higher in underfed leptin treated animals. Several studies have shown that adiponectin, it s receptors (AdipoR1 and AdipoR2) and resistin are present in various reproductive tissues in both sexes of different species (Lents et al., 2013c). Adiponectin is not only present in the follicular fluid but the adipokine and its receptors are also detectable in ovarian cells of various species including granulosa cells, the corpus luteum (CL) as well as receptor expression in cumulus-oocyte complex but not in granulosa cells (Richards et al. 2012). In cattle, the physiological status of the ovary influences the expression pattern of adiponectin and its receptors in follicular and luteal cells (Tabandeh et al. 2010), most likely mediated by follicular E2 concentrations. In bulls, adiponectin and its receptors also play vital roles in the structural and functional sperm traits by regulating sperm capacitation (Kasimanickam et al. 2013). The adipokine resistin is linked with the modulation of adipogenesis (Bulcao et al. 2006). It is secreted by mature adipocytes and stimulated during adipocyte differentiation. It has been reported to be elevated in obese men and higher systemic concentrations have been linked with adverse effects on male reproduction (Shukla et al. 2014). Resistin is widely expressed in different-sized follicles (small <6 mm and large >6 mm) where it is localised in oocytes, cumulus, theca and granulosa cells as well as in the CL (Maillard et al. 2011). Many in vitro studies have shown that both adiponectin and resistin can regulate gonadal steroidogenesis and gametogenesis (Rak et al. 2017). Adiponectin can also exert effects on GnRH synthesis and the pituitary secretory functions that could then indirectly affect gonadal functions. However, the effects of resistin on GnRH and gonadotropin secretion are still unknown. Receptors for insulin have been identified in both oocytes and embryos up to blastocyst stage and it has been reported that excess exposure to insulin may be detrimental to oocyte and embryo development (Laskowski et al. 2016). Serum concentrations of insulin were reported to be positively correlated with nutrient intake and LH pulse frequency (Yelich et al. 1996). Insulin has been shown to stimulate ovarian function (Spicer et al. 1994) and increase follicular growth and development in vitro (Simpson et al. 1994). A more recent study by Allen et al. (2017) reported a younger age at puberty (54.5 v 60.2 weeks of age, respectively) and greater serum concentrations of insulin in heifers gaining 0.91kg/day and 0.45kg/day from 14 weeks of age, respectively. Caution must be advised when providing a high plane of nutrition to replacement heifers as excessive energy intakes in heifers have been reported to be negatively correlated with pregnancy success, possibly mediated through effects of elevated insulin on oocyte quality (Wathes et al. 2014). The initiation of puberty in heifers has been related to prevailing blood concentrations of IGF-1 which has also been reported to be an indicator of future reproductive success when measured in prepubertal calves (Kumar et al. 2015). Systemic concentrations of IGF-1 closely reflect nutritional status and growth rate in cattle and lower concentrations as a consequence of dietary restriction, prior to puberty have been reported to be associated with 78

81 a delay in pubertal onset (Wathes et al. 2014). A negative relationship (r=-0.54) between IGF-1 concentration at nine months of age and the age of puberty attainment has been reported for beef heifers (Rodriguez-Sanchez et al. 2015). Consistent with this immunisation against growth hormone releasing factor reduced follicular growth and also delayed puberty (Cooke et al. 2013). In the bull, stepwise regression models have shown that IGF-1 accounts for 69, 59, 72 and 67% of the variation in bodyweight, backfat, scrotal circumference and paired testes weight, respectively (Brito et al., 2007c). As the former two variables indicate metabolic status and the latter two, testicular development, these data suggest an important influence of IGF-1 in hastening age at puberty and also in defining testicular size in bulls. There is also evidence that IGF-1 has a positive effect on proliferation of Sertoli cells when cultured in combination with FSH (Dance et al., 2016a); suggesting that the metabolic status of the bull calf will impact on the number of Sertoli cells that are available to produce sperm cells in later life. Taken together, these studies indicate that IGF-1 and the somatotrophic axis is an important metabolic mediator involved in the onset of puberty in cattle, with evidence for direct action on the hypothalamic-pituitary-gonadal axis (Velasquez et al. 2008). Notwithstanding this, however, beef heifers selected for either high or low systemic concentrations of IGF-1 had similar ages at puberty (Yilmaz et al. 2006). Gonadotropin and steroid secretion Offering a high plane of nutrition has been shown to increase TT concentrations in pre-pubertal bull calves (Brito et al. 2007c; Byrne et al. 2017). Consistent with higher concentrations of IGF-1 and insulin (Brito et al. 2007b; Byrne et al. 2017b). Brito et al. (2007b) also observed a high plane of nutrition caused a greater number of LH pulses over a 10 h period compared to the control diet. The concentration of TT observed in bulls on the improved nutrition trended towards significance; however, the results show that TT concentrations in such bulls increase both earlier and at a faster rate than in the bulls on the low plane of nutrition. As previously mentioned, the initiation of puberty for heifers, is characterised by an amelioration in the initial inhibitory effect of oestradiol (E2) on GnRH secretion (Atkins et al. 2013). Reproductive development of replacement heifers has been categorised into four key periods namely; the infantile period (birth to two months of age), developmental period (two to six months of age), static phase (six to 10 months of age), and the peripubertal period (Day and Anderson, 1998, Day and Nogueira, 2013). Within the context of these developmental phases, Gasser et al. (2006) weaned beef heifers 73 days of age rather than the traditional six to eight months of age and implemented a nutritional programme from 99 to 286 days of age where heifers achieved a growth rate of 1.27kg/day in heifers when fed a 60% corn diet or 0.85kg/day for those heifers offered a 30% corn diet. As a result of this differential dietary regime, age at puberty was 262 and 368 days of age, respectively, with the faster growing heifers younger at puberty. As expected, frequency of LH pulses increased as animals grew older however; faster growing heifers experienced a greater number of LH pulses by 190 days of age and at blood samplings. Despite this, there was no difference in LH concentration between treatment groups. In a similar study, heifers that were fed to achieve either 0.9kg/day or 0.3kg/day were 372 and 435 days of age at puberty, while the slower growing heifers also experienced delayed ovarian follicular development (Bergfield et al. 1994). Despite six to 10 months of age being considered the static phase of development, Yelich et al. (1996) fed nine month old heifers to gain either 1.36kg/day or 0.23kg/day for 16 weeks followed by 1.36kg/day until puberty, with initial faster growing heifers 91 days younger at puberty (369 v. 460 days of age). Heifers on the high plane of nutrition exhibited greater serum concentrations of LH after 64 days and greater LH pulse frequency after 68 days of treatment but not when sampled within three weeks of puberty. Pharmalogical interventions, during the early life period can also be used to advance sexual development. The aim of many studies of pre-pubertal development in cattle has been focussed on manipulation of FSH, because of its proliferative effects on Sertoli cell proliferation in bulls (Orth 1984; Bagu et al. 2004) or ovarian follicular growth in heifers Day and Nogueira (2013). Bull calves treated every second day from four to eight weeks of age with exogenous FSH exhibited a significant increase in systemic FSH concentrations (Bagu et al. 2004) which hastened puberty. Histological evaluation of the testes at 56 weeks of age in that study (Bagu et al. 2004) revealed that FSHtreated bulls had a greater number of Sertoli cells, elongated spermatids, and spermatocytes. This potential for greater sperm production has also been associated with greater numbers of Sertoli cells and greater testicular weight (Berndtson et al. 1987). Some heifers may not have attained puberty by the beginning of the breeding season, and induction of puberty with the use of exogenous hormone treatment may be necessary. Normal 79

82 induction of puberty in heifers involves the use of progesterone either alone or in combination with E2, GnRH or equine chorionic gonadotropin (ecg). These hormonal regimens promote the necessary gonodotrophin activity to support final follicular growth and ovulation (Day and Nigeria 2013). In both Bos taurus and Bos indicus ovulation was stimulated in approximately 80% of pre pubertal heifers treated appropriately with progesterone (Rasby et al. 1998; Rodrigues et al. 2013). An important consideration when employing pharmalogical induction of puberty for heifers is that these interventions are most effective in heifers that are approaching the attainment of puberty and of appropriate metabolic status (Day and Nogueira 2013). Pre-pubertal nutrition and post-pubertal semen production and quality As the testes require a temperature of 2-6 C lower than body temperature for normal spermatogenesis (Kastelic 2014), it is important that offering bulls high plane of nutrition does not result in excessive fat accumulation in the scrotum, which may negatively affect thermoregulation. While feeding high energy diets, ad libitum, to young bulls may hasten onset of puberty; there is evidence that an increased dietary energy intake may reduce the amount of heat that can be radiated from the scrotal neck, thereby increasing the temperature of the testes and scrotum (Coulter et al. 1997). Although no negative effects on daily spermatozoa production were detected; bulls fed a high energy diet had a reduced percentage of morphologically normal sperm compared to bulls fed a moderate amount of energy thereby leading to ejaculates with reduced fertilizing capacity. While data from our research group indicate that offering Holstein-Friesian bulls ad libitum access to high energy, grain based diets for an extended period during pre-pubertal and early post-pubertal stages did not affect scrotal surface temperature or on any measurement of semen quality (Byrne et al. 2018a), caution should be used when applying similar nutritional management regimen to older animals where the potential for scrotal fat deposition may be greater. In a review of male reproductive physiology, Amann and Schanbacher (1983) noted that daily sperm production continues to increase for some time after puberty in the bull. This is likely to be highly variable, depending on health and nutrition. Sexual maturity is attained when a bull can produce an ejaculate with greater than 30% progressively motile spermatozoa and greater than 70% morphologically normal spermatozoa, as evaluated, using Bos indicus x Bos taurus cattle breeds (Brito et al. 2004). This definition of maturity was later examined in Bos taurus bulls exclusively where it was found that Bos taurus bulls met the criteria for the definition of maturity approximately 50 days after they had achieved puberty (Brito et al. 2012). A high plane of nutrition prior to 31 weeks of age has been reported to lead to a numerically younger age at sexual maturation in Holstein-Friesian bulls (Dance et al. 2016). This enhanced nutrition also increased the number of harvestable spermatozoa. Characteristics affecting fertility, such as post-thawing motility, IVF ability, live/dead ratios and spermatozoa proteins were unaffected by early life nutrition (Dance et al. 2016). This increase in number of harvestable sperm, following an enhanced early plane of nutrition was not found, however, to be consistent in Holstein-Friesian bulls (Harstine et al. 2015; Byrne et al. 2016a). In our study, while we estimated that the average number of straws produced per ejaculate was unaffected by the pre-pubertal plane of nutrition, once animals had reached puberty, animals offered a high plane of nutrition during early calf-hood yielded an overall greater quantity of saleable semen up to 18 months of age (Byrne et al., 2018a). Precocious puberty in heifers and subsequent fertility and longevity A common goal for both dairy and beef herds is to breed replacement heifers by 15 months of age so that they calve for the first time, on average, at 24 months (Diskin and Kenny 2014; Wathes et al. 2014). Indeed Wathes et al. (2014) concluded that age at first calving (AFC) of 23 to 25 months is optimal economically and does not have any adverse consequences as long as the heifers are of an adequate BW and stature. This is of particular relevance to seasonal calving herds where calving pattern is planned to coincide with maximal pasture growth. As a consequence, heifers invariably are either two or three years of age at first calving when managed under these systems. Even within less seasonally influenced systems, the range in AFC extends to 30 months for dairy herds in many countries (Wathes et al. 2014), while for beef herds it can extend well beyond this. Brickell et al. (2009) reporting on the results of a multi-herd UK based dairy farm study, cited differences in ADG for replacement heifers of between 0.5 to 1.0 kg/day during the first six months of life, while within herd heifer performance on these farms ranged from 0.45 to 1.15 kg/day. Similarly, Soberon et al. (2012) reported a range from 0.10 to 1.58 kg/day in dairy heifers over the pre-weaning period for US dairy herds. 80

83 The traditional recommended approach to rearing replacement beef heifers has been to meet or exceed a predetermined (and often arbitrary) target weight at breeding (proportion of estimated mature cow weight) to ensure puberty had occurred ahead of commencing the breeding season (Patterson et al. 1992). This philosophy was based, to some extent, on the findings of a study carried out by Byerley et al. (1987) who reported a 21 percentage point lower pregnancy rate in heifers bred on their first as opposed to third post pubertal oestrus. However, heifers bred on their third oestrus in that study were 375 days of age while heifers bred on their first oestrus were only 322 days of age, thus confounding the interpretation of the results (Endecott et al. 2013). Heifers developed to a lighter weight at breeding (51% v 57% of estimated mature BW) resulted in a lower pubertal rate at the start of breeding and more open heifers at the end of the breeding season and an extended calving pattern (Martin et al. 2008). In a recent study conducted by our own group, heifers (~8 months old at start) gained either 0.5 kg/day or 1kg/day over a 145-day differential winter feeding period which resulted in 9% and 31% of heifers pubertal at the onset of the breeding season, respectively. Despite this significant difference in pubertal status, there was no difference in either six (56% v 61%) or 12 week (91% v 87%) pregnancy rate (Heslin et al. unpublished). Buskirk et al. (1995) using regression analysis indicated that increasing both weaning-weight and post-weaning gain had a positive relationship with the probability for achieving puberty prior to the onset of the breeding season, while faster growing heifers prior to weaning have an increased probability of calving to first AI service. Quoting data from murine studies, Smith et al. (2014) suggested that first-wave follicles develop at approximately twice the rate of second-wave follicles, taking 23 days to reach the antral stage compared with 47 days for second wave follicles. The concept of rapidly growing follicles releasing immature oocytes during an animal s initial oestrous cycles may provide some insight to the issue of poor conception when heifers are bred at or shortly after puberty. Overall, notwithstanding how it was achieved, Cushman et al. (2014) have shown that conception early during their first breeding season and calving in the first three weeks of the calving season maximises lifetime productivity and longevity of replacement heifers. Thus the timing of puberty is critical to achieve early submission for breeding resulting in earlier calving and is greatly influenced by age, nutrition and breed type which should be appropriately harnessed in the design of management regimen for replacement heifers (Perry 2016). Post pubertal nutrition and fertility in heifers and bulls While pre-partum nutrition plays a key role in regulating the interval to resumption of postpartum ovulation in cows, mainly through its modulating effects on BCS, both concurrent plane of nutrition as well as dietary chemical composition during the breeding season, has been shown to affect conception and pregnancy rates. For example, in a study with beef heifers maintained on a high plane of nutrition at pasture, and subsequently offered either a high or sub maintenance plane of nutrition immediately post AI, Dunne et al. (2000) reported a close to 50% reduction in conception rate of the heifers offered the low post AI diet. Additionally, there was no evidence in that study that systemic concentrations of progesterone were implicated in the conception rates recorded. Consistent with this, Kruse et al. (2017) reported that nutrient restriction of beef heifers for 6 days immediately following AI resulted in poorer quality embryos that were delayed in stage of development, though neither systemic progesterone nor IGF-1 were affected by prevailing nutritional treatment. In a series of experiments, bulls were offered either a diet that met protein requirements (14% CP) or proteindeficient diets (8, 5 or ~1.35% CP) diets (Meacham et al., 1963). Testes, epididymis, seminiferous tubule diameter and epithelium thickness as well as seminal gland weights were markedly reduced in bulls fed protein deficient rations. However, diet had no negative effects on semen production. Interestingly, in that study, semen characteristics were not affected until CP was reduced to 1.35%; semen volume and total spermatozoa in the ejaculate were decreased, but sperm morphology and motility remained the same as for the other dietary treatments. Indeed the apparent robustness of the spermatogenic process was evident from the fact that, protein restriction was so severe in these studies that half of the protein restricted (1.35%) bulls died or were slaughtered before imminent death after losing ~40% of their initial BW. On the contrary, some authors have raised concern in the past over possible deleterious effects of high protein diets on reproductive efficiency of dairy cows and heifers in particular (Butler, 2000). While beef cows or heifers are generally not exposed to excessively high dietary levels of protein, or indeed its systemic metabolites, ammonia and urea, cows managed under temperate pasture based systems, may be grazing herbage with a high 81

84 rumen degradable protein (RDP) content leading to elevated concentrations of rumen ammonia and systemic ammonia and urea. In a series of experiments Kenny et al. (2001, 2002) conducted with beef heifers, examined the effect of artificially elevating the RDP content of forage diets both indoors and on pasture. Despite raising systemic concentrations of ammonia and urea beyond that previously reported to be associated with reduced fertility (Butler, 2000), no effect of diet was observed on conception rate in those studies. Similarly, Gath et al. (2012) recently no evidence of high CP or systemic ammonia/urea promoting diets on embryo development or survival in beef heifers offered high or low RDP diets. It is thus unlikely, that the range in protein intake typically experienced by beef females maintained on forage based production systems will appreciably affect reproductive efficiency. In addition to the quantity of feed offered, studies have examined the effect of altering the chemical composition of the diet on various reproductive outcomes in cattle. For example, there has been much interest over the past decade in the potential of dietary fat supplementation to improve reproductive performance of cattle. Strategies have included the use of fat supplements to augment energy intake but mainly to examine effects of their constituent fatty acids, on various aspects of the reproductive performance (Santos et al., 2008). Inconsistencies in fertility outcomes have often been suggested as being the result of differences in the fatty acid composition of the supplement (i.e. n-3 v n-6 fatty acids) or status of the animals employed (heifers v cows). While many studies have examined the effects of various fatty acid based supplements on aspects of the reproductive process in dairy cows, there have been few reports for beef cows. Scholljegerdes et al. (2009) recorded lower tissue concentrations of LH and IGF-1, follicle numbers, systemic oestradiol and overall conception rate in beef cows supplemented with highlinoleate safflower seeds compared with unsupplemented controls. SimiIarly, Martin et al., (2002) observed no advantage of supplementing heifers with soyabeans in either productive or reproductive efficiency. In a series of experiments conducted in our own laboratory, with beef heifers, Childs et al. (2008 a,b,c) failed to establish any positive effects of enriching diets with rumen bypass n-3 fatty acids on a range of reproductive variables including steroid concentrations, follicle size and on the quantity or quality of embryos, following superovulation. The lack of a positive effect of fat supplementation on beef females may be due, in part, to their overall general positive metabolic energy status in comparison to their dairy counterparts. There are a limited number of studies that have examined the potential of certain dietary fatty acid based supplements to augment reproductive potential in male ruminants (Gholami et al., 2010; Fair et al., 2014). Animals cannot synthesise omega-3 or omega-6 poly-unsaturated fatty acids (PUFA) de novo as they lack the appropriate fatty acid de-saturase enzymes and need to obtain these or their precursors from dietary sources. These PUFA are important components of animal cell membranes, and play a crucial role in oocyte fertilisation (Wathes et al., 2007). Indeed omega-3 and omega-6 PUFA cumulatively make up 30% to 40% of the lipid content in bovine spermatozoa cells (Byrne et al., 2017a). Offering Holstein-Friesian bulls a DHA-enriched nutraceutical for 9 weeks resulted in no difference in semen volume, concentration per ml or total spermatozoa number (Gholami et al., 2010). A subjective examination of motility in that study found a greater number of motile spermatozoa when bulls were fed the DHA-enriched diet; however, this was not substantiated when the same samples were analysed using computer-assisted semen analysis (CASA). The DHA-enriched diet led to a higher percentage of bulls displaying a positive hypo-osmotic swell test, suggesting an improvement in sperm cell membrane integrity in these animals. Unfortunately, cell FA compositional changes were not analysed in that study so it is impossible to gauge the level of cellular incorporation required to elicit the recorded response. In another study in bulls, employing alpha-linoleic acid (ALA) and palmitic acid (PA) supplements (Gürler et al., 2015), no difference in preliminary semen characteristics (volume, concentration, motility) was observed which agrees with the findings of Gholami et al. (2010). However, Bulls offered both the ALA and PA supplements had increased levels of plasma membrane and acrosome-intact cells post-thawing. Interestingly, while there was no difference in semen lipid peroxidation (LPO) levels, measured by BODIPY581/591, between treatments post-thawing; LPO was higher after a 3-h post-thaw incubation period in bulls offered the ALA supplement. While the quantity of spermatozoa produced has not been altered in bulls, dietary supplementation of rams with fish oil extract led to a higher semen concentration per ml; however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motility, ability to penetrate artificial mucus, or ability to resist lipid peroxidation in either fresh or liquid stored semen (Fair et al., 2014). Recent data from our research group (Byrne et al., 2017a) indicate that supplementing young postpubertal bulls (14 months) for 12 weeks with either an omega-6 (safflower oil) or a an omega-3 (distilled fish oil) enriched diet altered the PUFA composition of spermatozoal cells and seminal plasma but did not lead to any appreciable improvements to the quantity or quality of fresh semen. Many 82

85 of the reported improvements as a result of dietary PUFA supplementation are linked with post-thaw spermatozoa suggesting that PUFA are important for ensuring spermatozoa survive cryopreservation. Despite this, in our study we failed to observe any improvements in frozen-thawed semen analysed for a range of CASA motility or flow cytometry-based parameters (Byrne et al., 2017a). Trace elements play an important role in the health and performance of cattle and deficiencies are often suspected in cases of poor reproductive performance, though again, there are little data to substantiate this. The dietary mineral requirements of cattle have been reviewed by (Ledoux and Shannon, 2005) and for most processes a relatively large dietary range is evident. As part of a large scale on-farm study we measured the trace element status of lactating beef cows on 169 Irish spring-calving herds over two successive breeding seasons. Preliminary findings indicate that 15, 79 and 82% of cows are below limits considered acceptable for copper, iodine and selenium, respectively. Analyses are on-going to determine the association of these trace elements, if any, with various reproductive, health and animal performance traits. While not measured as part of this study, that same range in mineral status would be expected for natural service bulls running with these herds. In domestic farm species, zinc has been reported to play an important role in maintenance of testosterone production (Martin et al., 1994) and also synthesis of RNA and DNA polymerases, necessary for sperm function (Hidiroglou and Knipfel, 1984). Following copper and zinc supplementation to pre-pubertal beef bulls, Geary et al. (2016) reported that although a greater percentage of supplemented bulls reached puberty earlier; no effect on semen quality was evident. Using a large number (n =167) of yearling Angus bulls, Arthington et al. (2002) found that increasing the inclusion level (60 v. 40 ppm) of a combination of organic and inorganic minerals in a dietary supplement at a high (60 ppm) inclusion rate led to a reduction in the number of bulls failing pre-sale BBSE. Most improvements in semen quality following dietary mineral supplementation are likely observed where the base forage offered is of poor nutritional composition. Conclusions Limiting the generation interval in cattle is key to the speed of genetic improvement and economic sustainability of beef and dairy production and the advent of genomically assisted selection technologies has brought with it renewed interest in maximising the impact of genetically elite animals as early as possible. Onset of puberty and sexual maturation marks the commencement of the productive life of an animal within a herd and for heifers there is clear evidence that animals that are eligible for breeding and conceive early in the breeding season, lead longer and more productive and profitable lives. Such animals are also more environmentally and economically sustainable to maintain. Puberty is regulated by complex interaction between metabolic and neuroendocrine biochemistry and is influenced by genetic as well as environmental factors, the most notable of which is nutritional management. While the effect of developmental programming in utero is not clear with contrasting effects of maternal diet and timing of intervention, on the sexual development of male and female offspring, early post-natal life nutrition holds significant promise as a readily implementable management intervention to consistently advance puberty and sexual maturation in both heifers and bulls. The timing of intervention is important, particularly with bull calves, where unlike heifers, to a limited degree, there is little or no evidence that growth retardation in the first six months of life can be compensated for by improved nutrition thereafter. The precise biochemical regulatory mechanisms, controlling the influence of nutrition on the hypothalamic-pituitary-gonadal axis, are yet still to be fully elucidated but efforts thus far, have highlighted a number of metabolic signalling proteins and neuropeptides that may work in concert to modify gonadotrophin secretion and/or gonadal steroidogenesis. While improved early life nutritional status consistently advances puberty, concurrent or latent effects on gametogenesis are less clear. Certainly, independent of direct effects on post pubertal fertility, within the context of seasonal calving systems, earlier onset of sexual maturation is conducive to greater probability of dairy bulls being used through AI or dairy and beef heifers joining and being retained within the breeding herd. Ongoing advances in our understanding of the genomic control of reproductive traits together with genomically assisted selection technologies will facilitate greater future progress in advancing the sexual development and fertility of cattle. However, difficulties in the logistics of widespread accurate measurement of puberty in bulls and heifers will continue to hamper potential progress in the short to medium-term. Thus accurate and easily measured biomarkers for puberty amongst other key reproductive traits are urgently required if the true potential for progress in shortening the generation interval for cattle is to be fully realised. 83

86 References Allen, C., Tedeschi, L., Keisler, D., Cardoso, R., Alves, B., Amstalden, M., and Williams, G. (2017) Interaction of dietary energy source and body weight gain during the juvenile period on metabolic endocrine status and age at puberty in beef heifers. J Anim Sci 95(5), Allen, C.C., Alves, B.R., Li, X., Tedeschi, L.O., Zhou, H., Paschal, J.C., Riggs, P.K., Braga-Neto, U.M., Keisler, D.H., Williams, G.L., and Amstalden, M. (2012) Gene expression in the arcuate nucleus of heifers is affected by controlled intake of highand low-concentrate diets. J Anim Sci 90(7), Alves, B.R., Cardoso, R.C., Doan, R., Zhang, Y., Dindot, S.V., Williams, G.L., and Amstalden, M. (2017) Nutritional programming of accelerated puberty in heifers: alterations in DNA methylation in the arcuate nucleus. Biol Reprod 96(1), Amann, R.P., and Schanbacher, B.D. (1983) Physiology of male reproduction. J Anim Sci 57 Suppl 2, Amann, R.P., and Walker, O.A. (1983) Changes in the pituitary-gonadal axis associated with puberty in Holstein bulls. J Anim Sci 57(2), Amstalden, M., Cardoso, R., Alves, B., and Williams, G. (2014) REPRODUCTION SYMPOSIUM: Hypothalamic neuropeptides and the nutritional programming of puberty in heifers. Journal of animal science 92(8), Amstalden, M., Cardoso, R.C., Alves, B.R.C., and Williams, G.L. (2014b) Reproduction symposium: hypothalamic neuropeptides and the nutritional programming of puberty in heifers. J Anim Sci 92(8), Amstalden, M., Harms, P.G., Welsh, T.H., Jr., Randel, R.D., and Williams, G.L. (2005) Effects of leptin on gonadotropinreleasing hormone release from hypothalamic-infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin. Anim Reprod Sci 85(1-2), Arthington J, Corah L and Hill D The effects of dietary zinc concentration and source on yearling bull growth and fertility. The Professional Animal Scientist 18, Atkins, J.A., Pohler, K.G., and Smith, M.F. (2013) Physiology and endocrinology of puberty in heifers. Vet Clin North Am Food Anim Pract 29(3), Backholer, K., Smith, J.T., Rao, A., Pereira, A., Iqbal, J., Ogawa, S., Li, Q., and Clarke, I.J. (2010) Kisspeptin cells in the ewe brain respond to leptin and communicate with neuropeptide Y and proopiomelanocortin cells. Endocrinology 151(5), Bagu, E.T., Madgwick, S., Duggavathi, R., Bartlewski, P.M., Barrett, D.M., Huchkowsky, S., Cook, S.J., and Rawlings, N.C. (2004) Effects of treatment with LH or FSH from 4 to 8 weeks of age on the attainment of puberty in bull calves. Theriogenology 62(5), Barb, C.R., and Kraeling, R. (2004) Role of leptin in the regulation of gonadotropin secretion in farm animals. Anim Reprod Sci 82-83, Barth, A. (2014) Testicular Degeneration. In 'Bovine Reproduction.' pp (John Wiley & Sons, Inc) Bergfeld, E., Kojima, F., Cupp, A.S., Wehrman, M., Peters, K., Garcia-Winder, M., and Kinder, J.E. (1994) Ovarian follicular development in prepubertal heifers is influenced by level of dietary energy intake. Biol Reprod 51(5), Berndtson, W.E., Igboeli, G., and Pickett, B.W. (1987) Relationship of Absolute Numbers of Sertoli Cells to Testicular Size and Spermatogenesis in Young Beef Bulls1. J Anim Sci 64(1), Brickell, J., Bourne, N., McGowan, M., and Wathes, D. (2009) Effect of growth and development during the rearing period on the subsequent fertility of nulliparous Holstein-Friesian heifers. Theriogenology 72(3), Brito, L.F., Barth, A.D., Rawlings, N.C., Wilde, R.E., Crews, D.H., Jr., Boisclair, Y.R., Ehrhardt, R.A., and Kastelic, J.P. (2007a) Effect of feed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, testosterone, and on sexual development in bulls. Reproduction Suppl 134(1), Brito, L.F., Barth, A.D., Rawlings, N.C., Wilde, R.E., Crews, D.H., Jr., Mir, P.S., and Kastelic, J.P. (2007b) Effect of improved nutrition during calfhood on serum metabolic hormones, gonadotropins, and testosterone concentrations, and on testicular development in bulls. Domest Anim Endocrinol 33(4),

87 Brito, L.F., Barth, A.D., Rawlings, N.C., Wilde, R.E., Crews, D.H., Jr., Mir, P.S., and Kastelic, J.P. (2007c) Effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls. Domest Anim Endocrinol 33(1), 1-18 Brito, L.F.C., Silva, A.E.D.F., Unanian, M.M., Dode, M.A.N., Barbosa, R.T., and Kastelic, J.P. (2004) Sexual development in early- and late-maturing Bos indicus and Bos indicus Bos taurus crossbred bulls in Brazil. Theriogenology 62(7), Bulcao, C., Ferreira, S.R., Giuffrida, F.M., and Ribeiro-Filho, F.F. (2006) The new adipose tissue and adipocytokines. Curr Diabetes Rev 2(1), Buskirk, D., Faulkner, D., and Ireland, F. (1995) Increased postweaning gain of beef heifers enhances fertility and milk production. J Anim Sci 73(4), Butler WR Nutritional interactions with reproductive performance in dairy cattle. Animal Reproduction Science 60 61, Byrne CJ, Fair S, English AM, Holden SA, Dick JR, Lonergan P and Kenny DA 2017a. Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality. Theriogenology 90, Byrne, C.J., Fair, S., English, A.M., Urh, C., Sauerwein, H., Crowe, M.A., Lonergan, P., and Kenny, D.A. (2017) Effect of breed, plane of nutrition and age on growth, scrotal development, metabolite concentrations and on systemic gonadotropin and testosterone concentrations following a GnRH challenge in young dairy bulls. Theriogenology 96, Byrne CJ, Fair S, English AM, Urh C, Sauerwein H, Crowe MA, Lonergan P and Kenny DA. 2018a. Plane of nutrition pre and post-six months of age in Holstein-Friesian bulls: I. Effects on performance, body composition, age at puberty and postpubertal semen production. Journal of Dairy Science. 101(4): Byrne CJ, Fair S, English AM, Cirot M, Staub C, Lonergan P and Kenny DA. 2018b. Plane of nutrition during pre- and postsix months of age in Holstein-Friesian bulls: II. Effect on metabolic and reproductive endocrinology and physiological markers of puberty and sexual maturation. Journal of Dairy Science. 101(4): Cardoso, R.C., Alves, B.R., Sharpton, S.M., Williams, G.L., and Amstalden, M. (2015) Nutritional Programming of Accelerated Puberty in Heifers: Involvement of POMC Neurones in the Arcuate Nucleus. J Neuroendocrinol Cardoso, R.C., Alves, B.R.C., Prezotto, L.D., Thorson, J.F., Tedeschi, L.O., Keisler, D.H., Park, C.S., Amstalden, M., and Williams, G.L. (2014) Use of a stair-step compensatory gain nutritional regimen to program the onset of puberty in beef heifers1. J Anim Sci 92(7), Childs S, Carter F, Lynch CO, Sreenan JM, Lonergan P, Hennessy AA and Kenny DA 2008c. Embryo yield and quality following dietary supplementation of beef heifers with n-3 PUFA. Theriogenology 70, Childs S, Hennessy A, Sreenan JM, Wathes DC, Cheng Z, Stanton C, Diskin MG and Kenny DA 2008b. Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle. Theriogenology 70, Childs S, Lynch CO, Hennessy A, Stanton C, Wathes DC, Sreenan JM, Diskin MG and Kenny DA 2008a. Effect of dietary enrichment with either n-3 or n-6 fatty acids on systemic metabolite and hormone concentration and on ovarian function in cattle. Animal 2, Clarkson, J., and Herbison, A.E. (2016) Hypothalamic control of the male neonatal testosterone surge. Philos Trans R Soc Lond B Biol Sci 371(1688), Cooke, R., Arthington, J., Araujo, D., Lamb, G., and Ealy, A. (2008) Effects of supplementation frequency on performance, reproductive, and metabolic responses of Brahman-crossbred females. J Anim Sci 86(9), Cooke, R.F., Bohnert, D., Francisco, C., Marques, R., Mueller, C., and Keisler, D. (2013) Effects of bovine somatotropin administration on growth, physiological, and reproductive responses of replacement beef heifers. J Anim Sci 91(6), Coulter, G.H., Cook, R.B., and Kastelic, J.P. (1997) Effects of dietary energy on scrotal surface temperature, seminal quality, and sperm production in young beef bulls. J Anim Sci 75(4),

88 Cushman, R., McNeel, A.K., and Freetly, H.C. (2014) The impact of cow nutrient status during the second and third trimesters on age at puberty, antral follicle count, and fertility of daughters. Livest Sci 162, Dance, A., Thundathil, J., Blondin, P., and Kastelic, J. (2016) Enhanced early-life nutrition of Holstein bulls increases sperm production potential without decreasing postpubertal semen quality. Theriogenology 86(3), Dance, A., Thundathil, J., Wilde, R., Blondin, P., and Kastelic, J. (2015) Enhanced early-life nutrition promotes hormone production and reproductive development in Holstein bulls. J Dairy Sci 98(2), Davis Rincker, L.E., VandeHaar, M.J., Wolf, C.A., Liesman, J.S., Chapin, L.T., and Weber Nielsen, M.S. (2011) Effect of intensified feeding of heifer calves on growth, pubertal age, calving age, milk yield, and economics. J Dairy Sci 94(7), Day, M. and Anderson, L. (1998) Current concepts on the control of puberty in cattle. J Anim Sci 76(suppl_3), 1-15 Day ML and Nogueira GP Management of age at puberty in beef heifers to optimize efficiency of beef. Animal Frontiers 3, No 4, Diskin, M. and Kenny, D. (2014) Optimising reproductive performance of beef cows and replacement heifers. Animal 8(s1), Duittoz, A.H., Tillet, Y., Le Bourhis, D., and Schibler, L. The timing of Puberty (oocyte quality and management) In '32nd Meeting of the European Embryo Transfer Association (AETE)', 2016, Barcelona, Spain, pp Dunne LD, Diskin MG, Boland MP, O' Farrell KJ and Sreenan JM The effect of pre- and post-insemination plane of nutrition on embryo survival in beef heifers. Animal Science 69, Endecott, R., Funston, R., Mulliniks, J., and Roberts, A. (2013) Joint Alpharma-Beef Species Symposium: Implications of beef heifer development systems and lifetime productivity. J Anim Sci 91(3), Evans, A.C., Davies, F.J., Nasser, L.F., Bowman, P., and Rawlings, N.C. (1995) Differences in early patterns of gonadotrophin secretion between early and late maturing bulls, and changes in semen characteristics at puberty. Theriogenology 43(3), Evans, A.C., Pierson, R.A., Garcia, A., McDougall, L.M., Hrudka, F., and Rawlings, N.C. (1996) Changes in circulating hormone concentrations, testes histology and testes ultrasonography during sexual maturation in beef bulls. Theriogenology 46(2), Fair S, Doyle DN, Diskin MG, Hennessy AA and Kenny DA The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen. Theriogenology 81, Filby, A.L., van Aerle, R., Duitman, J., and Tyler, C.R. (2008) The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish. Biol Reprod 78(2), Funston, R.N., and Larson, D. (2011) Heifer development systems: Dry-lot feeding compared with grazing dormant winter forage. J Anim Sci 89(5), Garcia, M.R., Amstalden, M., Williams, S.W., Stanko, R.L., Morrison, C.D., Keisler, D.H., Nizielski, S.E., and Williams, G.L. (2002) Serum leptin and its adipose gene expression during pubertal development, the estrous cycle, and different seasons in cattle. J Anim Sci 80(8), Gasser, C. (2013) JOINT ALPHARMA-BEEF SPECIES SYMPOSIUM: Considerations on puberty in replacement beef heifers. J Anim Sci 91(3), Gasser, C.L., Bridges, G.A., Mussard, M.L., Grum, D.E., Kinder, J.E., and Day, M.L. (2006) Induction of precocious puberty in heifers III: hastened reduction of estradiol negative feedback on secretion of luteinizing hormone. J Anim Sci 84(8), Gath VP, Crowe MA, O Callaghan D, Boland MP, Duffy P, Lonergan P and Mulligan FJ Effects of diet type on establishment of pregnancy and embryo development in beef heifers. Animal Reproduction Science 133, Geary TW, Kelly WL, Spickard DS, Larson CK, Grings EE and Ansotegui RP Effect of supplemental trace mineral level and form on peripubertal bulls. Animal Reproduction Science 168, 1-9. Gholami H, Chamani M, Towhidi A and Fazeli MH Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls. Theriogenology 74,

89 Gier, H.T., and Marion, G.B. (1969) Development of mammalian testes and genital ducts. Biol Reprod 1, Suppl 1:1-23 Gürler H, Calisici O, Calisici D and Bollwein H Effects of feeding omega-3-fatty acids on fatty acid composition and quality of bovine sperm and on antioxidative capacity of bovine seminal plasma. Animal Reproduction Science 160, Harstine, B.R., Maquivar, M., Helser, L.A., Utt, M.D., Premanandan, C., DeJarnette, J.M., and Day, M.L. (2015) Effects of dietary energy on sexual maturation and sperm production in Holstein bulls. J Anim Sci 93(6), Hatef, A., and Unniappan, S. (2017) Gonadotropin-releasing hormone, kisspeptin, and gonadal steroids directly modulate nucleobindin-2/nesfatin-1 in murine hypothalamic gonadotropin-releasing hormone neurons and gonadotropes. Biol Reprod 96(3), Herbison, A.E. (2016) Control of puberty onset and fertility by gonadotropin-releasing hormone neurons. Nat Rev Endocrinol 12(8), Heslin, J., Kenny, D.A., Kelly, A.K., and McGee, M. Age at puberty and pregnancy rate in beef heifer genotypes offered contrasting nutrition levels. In 'ASAS-CSAS Annual Meeting & Trade Show', 2017, Baltimore, MD, p. 235 Hidiroglou M and Knipfel J Zinc in mammalian sperm: a review. Journal of Dairy Science 67, Johnson, L., Varner, D.D., Roberts, M.E., Smith, T.L., Keillor, G.E., and Scrutchfield, W.L. (2000) Efficiency of spermatogenesis: a comparative approach. Anim Reprod Sci 60-61, Kasimanickam, V.R., Kasimanickam, R.K., Kastelic, J.P., and Stevenson, J.S. (2013) Associations of adiponectin and fertility estimates in Holstein bulls. Theriogenology 79(5), e1-3 Kastelic, J.P. (2014) Thermoregulation of the testes. In 'Bovine Reproduction.' pp (John Wiley & Sons, Inc) Kenny DA, Heslin J and Byrne CJ. (2018a). Early onset of puberty in cattle: implications for gamete quality and embryo survival. Reproduction, Fertility and Development, 2018, 30, Kenny DA and Byrne CJ. (2018b). The effect of nutrition on timing of pubertal onset and subsequent fertility in the bull. Animal. Mar 20:1-9. Kenny DA, Boland MP, Diskin MG and Sreenan JM Effect of pasture crude protein and fermentable energy supplementation on blood metabolite and progesterone concentrations and on embryo survival in heifers, Animal Science 73, Kenny DA, Boland MP, Diskin MG and Sreenan JM Effect of rumen degradable protein with or without fermentable carbohydrate supplementation on blood metabolites and embryo survival in cattle. Animal Science 74, Krause, A., Dias, F., Adams, G., Mapletoft, R., Huanca, W., Zwiefelhofer, E., and Singh, J. (2017) Antral Follicular Counts and Superstimulatory Response in Prepubertal Calves. Reprod Fertil Dev 29(1), Kruse SG, Bridges GA, Funnell BJ, Bird SL, Lake SL, Arias RP, Amundson OL, Larimore EL, Keisler DH and Perry GA Influence of post-insemination nutrition on embryonic development in beef heifers. Theriogenology. 90, Kumar, A., and Laxmi, N.A. (2015) Role of IGF 1 in male and female reproduction in bovines: a review. Asia Pac J Res Laskowski, D., Sjunnesson, Y., Humblot, P., Sirard, M.-A., Andersson, G., Gustafsson, H., and Båge, R. (2017) Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential. Reprod Fertil Dev 29(5), Ledoux DR and Shannon MC Bioavailability and antagonists of trace minerals in ruminant metabolism. In Porc. 16th Ann. Meet., Florida Ruminant Nutr. Symp., Gainesville, Florida, pp Lents, C., White, F., Ciccioli, N., Floyd-White, L., Rubio, I., Keisler, D., Spicer, L., and Wettemann, R. (2013a) Metabolic status, gonadotropin secretion, and ovarian function during acute nutrient restriction of beef heifers. J Anim Sci 91(9), Lents, C.A., Barb, C.R., and Hausman, G.J. (2013b) Role of Adipose Secreted Factors and Kisspeptin in the Metabolic Control of Gonadotropin Secretion and Puberty. In 'Gonadotropin.' (InTech) Lents, C.A., Barb, C.R., Hausman, G.J., Nonneman, D., Heidorn, N.L., Cisse, R.S., and Azain, M.J. (2013c) Effects of nesfatin-1 on food intake and LH secretion in prepubertal gilts and genomic association of the porcine NUCB2 gene with growth traits1. Domest Anim Endocrinol 45(2),

90 Luna-Pinto, G., and Cronje, P. (2000) The roles of the insulin-like growth factor system and leptin as possible mediators of the effects of nutritional restriction on age at puberty and compensatory growth in dairy heifers. S Afr J Anim Sci 30(2), Maillard, V., Froment, P., Rame, C., Uzbekova, S., Elis, S., and Dupont, J. (2011) Expression and effect of resistin on bovine and rat granulosa cell steroidogenesis and proliferation. Reproduction 141(4), Majerus, V., De Roover, R., Etienne, D., Kaidi, S., Massip, A., Dessy, F., and Donnay, I. (1999) Embryo production by ovum pick up in unstimulated calves before and after puberty. Theriogenology 52(7), Manthey, A.K., Anderson, J.L., Perry, G.A., and Keisler, D.H. (2017) Feeding distillers dried grains in replacement of forage in limit-fed dairy heifer rations: Effects on metabolic profile and onset of puberty. J Dairy Sci 100(4), Martin, J., Creighton, K., Musgrave, J.A., Klopfenstein, T.J., Clark, R., Adams, D.C., and Funston, R.N. (2008) Effect of prebreeding body weight or progestin exposure before breeding on beef heifer performance through the second breeding season. J Anim Sci 86(2), Meacham TN, Cunha TJ, Warnick AC, Hentges JF and Hargrove DD Influence of low protein rations on growth and semen characteristics of young beef bulls. Journal of Animal Science 22, Palmer, C.W. (2016) Management and Breeding Soundness of Mature Bulls. Vet Clin North Am Food Anim Pract Patterson, DJ, Perry, RC, Kiracofe, GH, Bellows, RA, Staigmiller, RB, and Corah, L. R. (1992). Management considerations in heifer development and puberty. Journal of Animal Science. 70, Perry, G.A. (2016) Factors affecting puberty in replacement beef heifers. Theriogenology 86(1), Pinilla, L., Aguilar, E., Dieguez, C., Millar, R.P., and Tena-Sempere, M. (2012) Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Rev 92(3), Rak, A., Mellouk, N., Froment, P., and Dupont, J. (2017) Adiponectin and resistin: potential metabolic signals affecting hypothalamo-pituitary gonadal axis in females and males of different species. Reproduction 153(6), R215-R226 Rasby, R.J., Day, M.L., Johnson, S.K., Kinder, J.E., Lynch, J.M., Short, R.E., Wettemann, R.P., and Hafs, H.D. (1998) Luteal function and estrus in peripubertal beef heifers treated with an intravaginal progesterone releasing device with or without a subsequent injection of estradiol. Theriogenology 50(1), Rawlings, N.C., and Evans, A.C.O. (1995) Androgen negative feedback during the early rise in LH-secretion in bull calves J Endocrinol 145(2), Redmond, J.S., Baez-Sandoval, G.M., Spell, K.M., Spencer, T.E., Lents, C.A., Williams, G.L., and Amstalden, M. (2011) Developmental changes in hypothalamic Kiss1 expression during activation of the pulsatile release of luteinising hormone in maturing ewe lambs. J Neuroendocrinol 23(9), Reynolds, L.P., and Caton, J.S. (2012) Role of the pre-and post-natal environment in developmental programming of health and productivity. Mol Cell Endocrinol 354(1), Richards, J.S., Liu, Z., Kawai, T., Tabata, K., Watanabe, H., Suresh, D., Kuo, F.T., Pisarska, M.D., and Shimada, M. (2012) Adiponectin and its receptors modulate granulosa cell and cumulus cell functions, fertility, and early embryo development in the mouse and human. Fertil Steril 98(2), e1 Rodrigues, A., Peres, R., Lemes, A., Martins, T., Pereira, M., Day, M., and Vasconcelos, J.L.M. (2013) Progesterone-based strategies to induce ovulation in prepubertal Nellore heifers. Theriogenology 79(1), Rodriguez-Sanchez, J.A., Sanz, A., Tamanini, C., and Casasus, I. (2015) Metabolic, endocrine, and reproductive responses of beef heifers submitted to different growth strategies during the lactation and rearing periods. J Anim Sci 93(8), Sanchez-Garrido, M.A., and Tena-Sempere, M. (2013) Metabolic control of puberty: roles of leptin and kisspeptins. Hormones and Behavior 64(2), Santos JE, Bilby TR, Thatcher WW, Staples CR and Silvestre FT Long chain fatty acids of diet as factors influencing reproduction in cattle. Reproduction in Domestic Animals 43 (Suppl 2), Scholljegerdes EJ, Hess BW, Grant MH, Lake SL, Alexander BM, Weston TR, Hixon DL, Van Kirk EA and Moss GE Effects of feeding high-linoleate safflower seeds on postpartum reproduction in beef cows. Journal of Animal Science 87,

91 Schwartz, M.W., Woods, S.C., Porte, D., Jr., Seeley, R.J., and Baskin, D.G. (2000) Central nervous system control of food intake. Nature 404(6778), Shukla, K.K., Chambial, S., Dwivedi, S., Misra, S., and Sharma, P. (2014) Recent scenario of obesity and male fertility. Andrology 2(6), Simpson, R., Chase, C., Spicer, L., Vernon, R., Hammond, A., and Rae, D. (1994) Effect of exogenous insulin on plasma and follicular insulin-like growth factor I, insulin-like growth factor binding protein activity, follicular oestradiol and progesterone, and follicular growth in superovulated Angus and Brahman cows. J Reprod Fertil 102(2), Soberon, F., Raffrenato, E., Everett, R. W., and Van Amburgh, M. E. (2012). Preweaning milk replacer intake and effects on long-term productivity of dairy calves. Journal of Dairy Science. 95, Spicer, L., Alpizar, E., and Vernon, R. (1994) Insulin-like growth factor-i receptors in ovarian granulosa cells: effect of follicle size and hormones. Mol Cell Endocrinol 102(1), Stengel, A., Goebel, M., and Taché, Y. (2011) Nesfatin-1: a novel inhibitory regulator of food intake and body weight. Obs Rev : an official journal of the International Association for the Study of Obesity 12(4), Tabandeh, M.R., Hosseini, A., Saeb, M., Kafi, M., and Saeb, S. (2010) Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development. Theriogenology 73(5), Velazquez, M., Spicer, L., and Wathes, D. (2008) The role of endocrine insulin-like growth factor-i (IGF-I) in female bovine reproduction. Domest Anim Endocrinol 35(4), Wathes DC, Abayasekara DR and Aitken RJ Polyunsaturated fatty acids in male and female reproduction. Biology of Reproduction 77, Wathes, D.C., Pollott, G.E., Johnson, K.F., Richardson, H., and Cooke, J.S. (2014) Heifer fertility and carry over consequences for life time production in dairy and beef cattle. Animal 8 Suppl 1, Wolf, F., Almquist, J., and Hale, E. (1965) Prepuberal behavior and puberal characteristics of beef bulls on high nutrient allowance. J Anim Sci 24(3), Wu, B., and Zan, L. (2012) Enhance beef cattle improvement by embryo biotechnologies. Reprod Domest Anim 47(5), Yelich, J., Wettemann, R.P., Marston, T., and Spicer, L. (1996) Luteinizing hormone, growth hormone, insulin-like growth factor-i, insulin and metabolites before puberty in heifers fed to gain at two rates. Domest Anim Endocrinol 13(4), Yilmaz, A., Davis, M., and Simmen, R. (2004) Estimation of (co) variance components for reproductive traits in Angus beef cattle divergently selected for blood serum IGF-I concentration. J Anim Sci 82(8), Zezeski, A.L., McCracken, V.L., Poole, R.K., Al Naib, A., Smith, J.K., McCann, M.A., and Rhoads, M.L. (2017) Metabolic and reproductive characteristics of replacement beef heifers subjected to an early-weaning regimen involving highconcentrate feeding. Animal 11(5),

92 The Au Courant Developments in Sex Sorted Semen and Application in Livestock Improvement Programs R (Vish) Vishwanath and J F Moreno STgenetics, State Hwy 6 South, Navasota, Texas Introduction The world s population is expected to grow to approximately 10 billion by Population growth, combined with trends of increased urbanization and per capita increases in income, is expected to increase food demand by 50% from 2012 levels. Satisfying increased food demands with existing production practices will result in more intense competition for natural resources, increased greenhouse gas emissions, and further deforestation and land degradation (Wu et al., 2014; FAO, 2017). The sustainability, even the very existence, of the world s cattle industry relies on strategies and initiatives to meet the protein needs of 10 billion people in a way that is economical, healthy, and good for the environment. Production efficiency (productivity per animal unit and land unit) relates to sustainability through its effects on economics and environmental impacts. Use of artificial insemination enabled large scale genetic selection programs in cattle and those have been the major contributors to increases in animal productivity, efficiency, product quality, and environmental and economic advancements observed in the last half century. As an example, milk production in the United States increased by 59% with 64% fewer cows in 2007 when compared to Production of the same volume of milk produced in 1944 required only 21% of the cows, 23% of the feedstuff, 10% of the land, and 35% of the water in As a consequence, greenhouse gas production also decreased 41% (National Research Council, 2015). In the last decade, the cattle industry has been revolutionized by the development and adoption of new genetics and breeding technologies, namely the use of genomics for animal selection and the commercial use of sexed semen for artificial insemination. Genomic selection has reduced generation intervals and accelerated the rate of genetic gain in extraordinary fashion in dairy cattle and new programs in beef cattle offer promising results. Although use of sexed semen used for artificial insemination is rightfully considered a reproductive biotechnology, it could be argued that it should also be considered genetic selection, since gender is a genetic trait. Most genetic traits can be manipulated through selection, but before sexed semen was available, producers had to accept the probability that 51% of all births would result in a male calf. Due to impact that gender has on animal production systems, it has been described as the most important genetic trait (Seidel., 2003). As such, sexed semen will continue to be one of the main drivers of cattle production efficiency and sustainability. Sexed semen production has greatly improved since the beginning of commercial application, but still continues to evolve rapidly. Incorporation of the most recent advancements into the production of sexed semen resulted in a differentiated product, SexedULTRA 4M TM, that now allows producers to obtain fertility rates comparable to those obtained with conventional semen. Brief history of sexed semen technology Sexed semen technology was initially developed at US government research centers. Studies started at the Lawrence Livermore National Laboratory in the 1970 s, where scientists studying the health effects of radiation using mouse sperm as a model to indicate damage to the germ-line developed flow cytometer techniques that allowed precise measurement of sperm DNA content that lead to the breakthrough demonstration of the potential use of the techniques to identify X- and Y-sperm populations based on DNA content differences (Garner & Seidel, 2008). Further development of the technology occurred at the USDA Beltsville Agricultural Research Center in the 1980 s and 1990 s, when changes to sperm staining methods and further advancements in flow-cytometry not only lead to the major breakthrough of live births of rabbits produced with sexed semen (Johnson et al., 1989), but also supported the potential commercial application of the technology. 90

93 After encouraging results using low dose insemination with fresh semen in cattle, in the mid 1990 s the USDA granted a license to XY Inc., a company funded by the Colorado State University Research Foundation, Cytomation Inc., and private investors to commercialize the Beltsville sexed semen technology for non-human mammalian sperm (Garner & Seidel, 2008). Further developments on rapid-speed flowcytometry lead to a leap in production from a few hundred sperm/second to ~3,000 sperm/second at approximately 90% accuracy (Johnson & Welch, 1999). Development of methods for sexed semen cryopreservation (Schenk et al., 1999) and demonstration of acceptable pregnancy rates obtained with frozen sexed semen (Seidel et al., 1999) further opened the doors for commercial application of the technology. Commercial licenses were granted to bull studs in the early 2000 s and commercial tests started being conducted around the world. Development of the technology changed when Sexing Technologies (ST) secured a sorting license in 2004 and started to establish a small number of sorting labs. In 2007, ST acquired XY Inc. and refocused the commercial approach to allow bull studs access to larger and consistently growing amounts of sexed semen of consistent quality at reasonable costs (Gilligan, 2014). Today all of the world s largest bull studs use ST technology to offer sexed semen from a diverse group of top bulls as an essential and important portion of their product portfolios. Overview of sexed semen production Sexed semen production is based on the difference in DNA content between X- and Y-sperm, resulting from the difference in size between X- and Y- chromosomes. On average, the difference in DNA content between bull X- and Y-sperm is approximately 4%, although subtle differences occur among breeds (4.22% in Jerseys, 4.07% in Angus, 4.01% in Holstein, and 3.7% in Brahman; Garner, 2006). Hoechst (H33342) is a dye that permeates the intact cell membrane and binds selectively to A/T base pairs along the minor groove of dsdna. Hoechst exhibits a relatively large Stokes shift (excitation/emission maxima of about 350/460 nm), making it very useful in assessing precise amounts of DNA in living cells (Seidel & Garner, 2002). A flow cytometer is used to quantify sperm DNA content. Briefly, H33342 dye DNA-bound molecules are excited by a laser as sperm pass two fluorescence detectors that measure the intensity of fluorescence. The strength of the fluorescence signals depends on the number of fluorescing molecules bound to DNA, thus allowing differentiation of X- and Y-sperm. After collection and evaluation, semen must be prepared for sorting. This involves extension with appropriate buffers and adjustment of cell concentration to optimal range. The sample is then incubated with optimal concentrations of H33342 for a predetermined period of time. Stained sperm are pumped in a stream in front of a laser beam when the illuminated sperm emit a very bright blue fluorescence. This fluorescence is measured as the sperm flow single-file in front of a photomultiplier tube (PMT). Specialized software is used to analyze the relative fluorescence of the X- and Y-sperm populations and select the population(s) to be captured (Figure 1). A crystal vibrator is used to break the fluid stream into individual droplets containing a single spermatozoon. Sperm are then sorted by placing opposite electrical charges on droplets containing X-sperm from those containing Y-sperm. The droplets fall past positive and negative electrical fields that separate the droplets into two streams for collection; a third stream of uncharged droplets is discarded (Johnson 2000; Seidel & Garner, 2002). Sorted sperm are collected into tubes containing appropriate buffers to protect cells during the sorting and cooling processes. After sorting, tubes are slowly cooled to 5 o C, additional extenders are added, and tubes are processed to obtain concentrated sperm pellets. After a period of equilibration, semen is loaded into straws and frozen in a programable freezer using the optimal freezing curve (Johnson 2000; Seidel & Garner, 2002). Post-thaw quality control involves evaluation of sperm motility and acrosome integrity after 3 hours of incubation at 35 o C, analysis of purity, concentration, and bacteriology. Altogether, sexed semen production involves over 20 sub-processes. 91

94 Figure 1. Flow cytometry histograms used to analyze the relative fluorescence of the X- and Y-sperm populations and select the sorted population. In histograms (1), the dead and properly oriented sperm populations can be differentiated and gated. The degree of difference (peak-to-valley ratio or PVR) in fluorescence intensity between the X- and Y-sperm in the oriented population can be visualized in histograms (2), whereas the population of interest (desired gender) is gated in histograms (3). High-Productivity sorting (A) results in the maximum number of straws produced per allotted time and requires a high event rate to obtain a high sorting rate; in this example, >40,000 sperm/second going through the sorter and >9,300 sperm/second actually sorted. High-Efficiency (B) sorting results in the maximum number of straws produced from the allotted amount of ejaculate and requires adjusting the event rate to maximize the proportion of sperm sorted from the overall sperm population; in this example, over 29% of available sperm are sorted for the desired gender (>5,000 sperm/second sorted for ~17,000 sperm/second going through the sorter). Different sorting modes allow bull studs to strategically plan production according to bull age, availability of semen, and demand to adequately fulfill their customers needs for sexed semen (Gilligan, 2014). SexedULTRA TM technology Continuous Research & Development investment in sexed semen production technology have resulted in significant improvements in semen quality and fertility, so much so that a new product label was created. Although the SexedULTRA TM label was officially launched in 2013, it is important to understand that the product is a culmination of a series of innovations that combined to create a product significantly different from that produced using XY Inc. legacy technology. Some of these innovations included optimization of flow cytometry media (sheath fluid) and extenders, large scale media and extender production for global distribution, optimization of staining conditions, and worldwide adoption of modern, standard equipment. Innovations also include advances in flow cytometry technology. The original equipment used for sexed semen production were adapted from medical research. These were expensive, bulky, difficult to operate, and with low throughput. Equipment optimization included reduction of fluidic instability and pressure, laser noise, electronic and photodetector noise, and acoustic vibration, while improving sperm orientation, light collection efficiency, resolution, and signal processing. Current state-of-the-art instruments at ST facilities use a solid-state laser for UV excitation, dual orthogonal detectors (at 0 o and 90 o to the laser), an orienting nozzle, and digital electronics to provide sorted subpopulations of X- or Y- sperm at rates of approximately 8,000 sperm/second with approximately 90% purity when operating at an input event rate of 40,000 sperm/second (Sharpe & Evans, 2009; Evans, 2010). Cytonome is a new company in the ST group specialized and dedicated to flow cytometry technology. The most recent advances made at Cytonome resulted in the Genesis TM sexed semen sorters. The new equipment is compact and easy to operate with integrated fluidics and all digital controls; this technology marvel is ushering a new era of large scale, industrial sexed semen production (Figure 2). 92

95 Figure 2. Genesis TM, the new generation of sexed semen flow cytometry sorters by Cytonome/ST, LLC. Figure 3. Effect of SexedULTRA TM technology on in vitro semen quality tests. Sperm motility and progressive motility were determined using computer-assisted semen analysis (CASA) and percentage intact acrosome (PIA) was determined using DIC microscopy (n = 12 bulls). **Bars with superscripts differ (P < 0.001). From Gonzalez-Marin et al., Initial laboratory evaluations indicated that results from in vitro semen quality tests, including sperm motility and acrosome integrity, were superior when semen was processed using SexedULTRA TM technology when compared XY legacy technology (Figure 3). In addition, use of SexedULTRA TM semen for in vitro fertilization resulted in greater production of blastocysts and greater proportion of freezable embryos (Table 1; Gonzalez-Marin et al., 2016). In an initial field trial with a small number of inseminations involving industry partners, Holstein and Jersey heifer conception rates were 7.4% greater when SexedULTRA TM was compared with XY legacy technology (Table 2). A larger field trial in collaboration with Select Sires in 41 Holstein commercial herds in the US indicated that heifer conception rates were 4.5% greater when SexedULTRA TM technology was used (Table 2; Vishwanath, 2014). 93

96 Table 1. Effect of SexedULTRA TM technology on in vitro embryo production. From Gonzalez-Marin et al., 2016 No. of oocytes Cleavage rate Blastocyst rate Freezable embryos* XY legacy 5, % 18.4% a 9.2% a SexedULTRA TM 5, % 22.3% b 13.2% b *Grades 1 and 2. a,b Rows with different superscripts differ (P < 0.05). Table 2. Effect of SexedULTRA TM technology on heifer conception rates. Select Sires trial results from Vishwanath, No. of inseminations Conception rate Sexing Technologies trial XY legacy 1, % a SexedULTRA TM % b CR improvement 7.4% Select Sires trial XY legacy 3, % a SexedULTRA TM 3, % b CR improvement 4.5% a,b Rows with different superscripts differ (P < 0.01) within trial. Data compiled by researchers from the USDA on sexed semen usage for Holstein females in the United States demonstrated the positive effects of SexedULTRA TM technology on conception rates. Data on sexed semen inseminations in heifers and cows between 2007 and 2015 showed a consistent reduction in conceptional rate differences between sexed and conventional semen coinciding with global introduction of SexedULTRA TM in 2013 (Figure 4; Hutchison & Bickhart, 2016). Interestingly, sexed semen utilization rate in heifers increased from 22.5% of the total number of insemination in 2013 to 30.7% in Although sexed semen utilization is still low in cows, rates increased from 0.5% in 2013 to 1% in The changes in utilization, especially in cows, are likely associated with the realization that comparable conception rates could be obtained with sexed semen. Data obtained through ST partnering Holstein and Jersey herds between 2012 and 2016 have shown very similar results to those compiled by the USDA. The proportion of inseminations using sexed semen has increased over the years, but the rate of sexed semen utilization increase was particularly more pronounced after 2013 and the introduction of SexedULTRA TM (Heuer et al., 2017). Sexed semen use is becoming more common in Holstein cows, especially in first and second lactation cows, and has virtually displaced the use of conventional semen in Jersey heifers and cows. When conception rates were evaluated using a subset of the data (lactation 0-2, service 1-3), means conception rates for sexed semen have increased and the differences from conventional semen have decreased consistently after the introduction of SexedULTRA TM in all female categories in both Holstein and Jersey cattle. Conception rates of 85-90% of that obtained with conventional semen can be obtained with SexedULTRA TM semen. 94

97 Figure 4. Conception rates in Holstein females in the United States. Only inseminations from 2007 through 2015 with confirmed outcomes were included: 5,963,876 heifer inseminations (1,323,721 to sexed semen) and 42,232,502 cow inseminations (253,586 to sexed semen). Mean conception rates for heifer sexed semen inseminations has recently increased due to improved technology (42% in 2007 compared to 49% in 2015). Comparable conception rates for heifer conventional inseminations were 56, and 59% for 2007, and 2015, respectively. Conception rates for sexed-semen inseminations to cows were 26% in 2007, and 30% in 2015 compared to 30, and 32% for conventional inseminations during the same years. Adapted from Hutchison & Bickhart, SexedULTRA 4M TM Successful sexed semen production must address the susceptibilities of sperm to staining, laser exposure, high dilution, elevated pressure, and resistance to the several changes in media composition that occur during the process. Historically, the compounding deleterious effects of these factors resulted in what could be described as uncompensable changes to sperm, since increasing insemination dosage from the 2.1 million sperm used as the industry standard resulted in little to no significant gain in conception rates. Although some sire-by-dosage interactions were observed, across sires sexed semen dosages of 2.1, 3.5 or 5 million sperm had no effect on conception rates in Holstein heifers and cows (DeJarnette et al., 2008; 2010). In another study comparing sexed and conventional semen dosages of 2.1 and 10 million sperm, sexed semen resulted in a decrease in conception rates by an almost identical magnitude within both sperm dosages. Although sexed semen conception rates were improved by the 10 million sperm dosage, conception rates were not comparable to either dosage of conventional semen (DeJarnette et al., 2011). One of the most interesting observations since the implementation of SexedULTRA TM technology is that not only have deleterious effects of semen processing been minimized, but also that resulting adverse biological changes to sperm became compensable. In a study conducted in collaboration with German Genetics International, ejaculates from 5 bulls were split four ways and processed using XY legacy technology with 2.1 million sperm dosage or using SexedULTRA TM technology with 2.1, 3, and 4 million sperm dosages; contemporaneously produced conventional semen with 15 million sperm dosage served as control. Fifty-six days non-return rates were evaluated after insemination of 7,855 heifers with sexed semen and 62,398 heifers with conventional semen. As expected, XY 2.1 million resulted in lower conception rates when compared to both SexedULTRA TM and conventional treatments. Although SexedULTRA TM 2.1 and 3 million sperm dosages produced results lower than conventional semen, increasing the dosage to 4 million sperm resulted in conception rates comparable to conventional semen (Lenz et 95

98 al., 2016). These results were the first to demonstrate (i) consistently improved conception rates with increased sexed semen dosage and (ii) conception rates equivalent to conventional semen with SexedULTRA 4M TM sexed semen. Encouraged by the early results obtained in the German trial, STgenetics adopted SexedULTRA 4M TM as its standard product in Data obtained through ST partnering Holstein and Jersey herds between 2012 and 2016 on the use of STgenetics sexed semen revealed that introduction of SexedULTRA 4M TM provided an additional increase in conception rates, especially for Holstein heifers. STgenetics officially launched SexedULTRA 4M TM in Some bull studs also conducted internal trails and have recently announced the release of similar products (see SELECTed SexedULTRA 4M from Select Sires and GenChoice 4M from Genex). Several other bull studs are currently conducting trial and the expectation is that SexedULTRA 4M TM will soon become the new industry standard for sexed semen. Figure 5. Effect of SexedULTRA 4M TM on 56 days non-return rates (NRR) recorded at German Genetics International (n = 5 bulls). a,b Bars with different superscripts differ (P < 0.001). Adapted from Lenz et al., Performance of sex sorted sperm in IVF programs Historically, it was always considered that the most economical method to use sex sorted sperm in breeding programs would be through IVF methods where a relatively small number of spermatozoa are required. This was until low dose inseminations with sex sorted sperm became feasible (Seidel and Garner 2002). Combined with ovum pickup (OPU), quite a large number of in vitro fertilized embryos are currently generated for breeding companies through the use of both X and Y sorted sperm ( These companies also provide a service using previously frozen sperm which are then thawed and subjected to the sorting process (reverse sorting). While, there are many studies that show similar rates of cleavage and blastocyst formation from embryos generated from sexed and non-sexed sperm (Xu et al., 2009) others report some reduction in blastocyst yield (Bermejo-Alvarez et al., 2010). However the conclusion at this time is that the overall calving rate following transfer of in vitro produced embryos with non-sorted or sex-sorted sperm is similar (Rasmussen et al., 2013) 96