2005 JOINT CONVENTION PROCEEDINGS

Transcription

1 American Embryo Transfer Association Canadian Embryo Transfer Association 2005 JOINT CONVENTION PROCEEDINGS September 8 10, 2005 Minneapolis Marriott City Center Minneapolis, Minnesota

2 Table of Contents Equine ET101 Seminar Recipient Herd and Facility Management for Equine Embryo Transfer Programs Dr. Phil Matthews, Peterson and Smith Equine Hospital, Ocala, FL... 1 Superovulation in the Mare Dr. Ed Squires, Colorado State University, Ft. Collins, CO... 4 Embryo Freezing Dr. Ed Squires, Colorado State University, Ft. Collins, CO... 6 Efficiency of Programs that Control Follicular Development and Ovulation for the Donor Superovulation Without Estrus Detection Dr. Gabriel Bo, IRAC, Cordoba, Argentina Evaluation of the Postpartum Mare Dr. Phil Matthews, Peterson and Smith Equine Hospital, Ocala, FL Bovine Viral Diarrhea Virus Update, Syndromes, Approaches, and Control/Vaccinations Dr. Vic Cortese, Pfizer, Downington, PA Factors Affecting Pregnancy Rates in an IVF Embryo Transfer Program Dr. Cliff Lamb, University of Minnesota, Grand Rapids, MN Application of Fixed-Time Artificial Insemination and Embryo Transfer Programs in Beef Cattle Operations Dr. Gabriel Bo, IRAC, Cordoba, Argentina Designing Vaccination Programs that Incorporate Spirovac Leptospira Borgpetersenii Serovar Hardjo-bovis Dr. Vic Cortese, Pfizer, Downington, PA Pregnancy Loss in Cattle Dr. Juan Romano, University of Minnesota, St. Paul, MN Donor and Recipient Factors Affecting an Embryo Transfer Program Dr. Cliff Lamb, University of Minnesota, Grand Rapids, MN Sponsors and Exhibitors AETA Office 1111 N. Dunlap Savoy, IL Tel: Fax: aeta@assochq.org Web site: CETA/ACTE Office Karen McDermott, Secretary-Manager Box 2000, 595 County Road #44 Kemptville, Ontario Canada K0G 1J0 Tel: Fax: ceta@ebi.ca Web site:

3 Recipient Herd and Facility Management for Equine Embryo Transfer Programs Phil Matthews, DVM Peterson and Smith Equine Reproduction Center Facility Design Much of what can be achieved in the creation and maintenance of a recipient herd and facility is dependent on your geographic location and the logistics that it creates. Concerns and Difficulties Resourcing mares. Geographic location will determine breed availability. Does breed really matter? Size of mares Client concerns and prejudices Cost of mares Mare upkeep Facility Size Function Managing herd size Breeding season vs. off season HUGE COMMITMENT (Commitment in both capital and energy) Benefits Greatly expands your ET program Allows your facility to handle all facets of an embryo program Owners encouraged to use your mares = increase revenue Can guarantee availability of a recipient Can create a ship in embryo program Allows you complete control of potential recipient Management of your own mares should result in an increased success rate Ancillary Benefits of a Recipient Herd Population of mares for other activities Semen fertility testing Jump/tease mares Population of animals for wet labs Veterinarian instruction Veterinary seminars Research studies 1

4 Leasing vs. Selling Pregnant Recipients Benefits of leasing One time purchase of mare Increase herd quality, reproductively and otherwise Mares may be used consecutive years Fewer maiden mares in herd Benefits of selling No consideration of transport to return mare Geographic Factors Cost of land Cost of feed and labor Availability of pasture Cost and availability of mares in your region Clientele interested in embryo transfer Key Design Factors Minimize labor Safety Herd health concerns (especially capacity to quarantine new arrivals) Efficient control of herd movement Quick and efficient examination of mares Lighted area for conversion of anestral mares Separation of recipient herd from donor mares Electrobraid Fencing Less expensive to build and maintain Negates the need for lanes between pastures for more efficient use of land Minimizes injuries Adequate Pastures Depends on geography and cost of land Helps with herd health concerns Minimizes feed cost Lane System Lanes should connect all pastures and examination area Small crew can move a large number of mares Examination Area Should be separate from donor mare exam area Should allow for rapid and safe examination with minimal labor Staging area and chute system is very helpful Quick access holding pens to allow for separation of different category mares is helpful 2

5 Lighted Paddock(s) are Imperative A facility must have the ability to convert seasonally anestrus mares Should be tied in to chute and lane systems Reproductive Management of the Recipient Mare Selection of Mares for Purchase Adequately sized mares for your program Reasonably pasture sound Reasonable body condition score Maiden mares are acceptable (desirable?) A higher propensity for fetal loss Smaller foals Known reproductive status Ages 3 10 Reproductively normal, i.e. Two normal sized functioning ovaries with a normal sized uterus and normal cervix on both speculum and digital exam. (Digital exam probably not necessary with maiden mares.) Uterine fluid, adhesions to uterus, cervical tears, cervical adhesions, cervical discharge, urine pooling, poor perineal confirmation are all unacceptable Cribbers are unacceptable Reproductive Evaluations As the season progresses cycling mares should be grouped into an active status so that time isn t spent handling and examining noncycling or inactive mares Examination of mares is done as frequently as follicle size dictates. Mares with ovulatory sized follicles should be scanned daily. Ovulation should be pinpointed within a 24 hour period Five day checks are critical Speculum exam should reveal tight pale cervix Sonogram should confirm CL and complete absence of uterine or edema. Rectal exam should determine uterine tone to be reasonable and cervix to be tight Mares with uterine fluid on a five day check should not be used for transfer that cycle and should be considered for culling Consistent good scores do enhance a mares status Mares that receive an embryo but are not confirmed pregnant on early exams are given another chance before culling IF they do not have uterine fluid nor appear infected at time of exam Synchronization Window Still appears to be +1 to 3 Preferred is 0 to 2 3

6 Superovulation in the Mare E.L. Squires, N. Logan and P.M. McCue Animal Reproduction and Biotechnology Laboratory Colorado State University Fort Collins, CO USA Embryo recovery in a commercial program is approximately 40 to 50% per flush. The major expense in most embryo transfer programs is maintenance of recipients waiting on an embryo to be available for transfer. Thus, if one could improve embryo recovery, then recipient mares would be maintained fewer days and the cost of embryo transfer could be reduced. There are certain breeds of mares that spontaneously double ovulate, such as Warmbloods, Thoroughbreds and draft horses. These mares make excellent donors since embryo recovery is more than double in donors that spontaneously multiple ovulate. Although studies have been done on superovulation of mares since the early 1970 s, only in the past several years has an equine FSH product been available to the veterinarian for superovulation of mares. Equine FSH (efsh) is a purified pituitary extract available from Bioniche Animal Health (Bogart, GA). Several studies have been conducted in our laboratory over the past three breeding seasons to evaluate the efficacy of efsh for superovulation in mares. In the first study, Niswender et al. (2003) evaluated two doses of efsh: 12.5 mg and 25 mg twice per day. A dose of 25 mg twice per day resulted in a greater number of preovulatory follicles, however many of these follicles did not ovulate. The 12.5-mg dose of efsh given twice daily followed by hcg once mares had obtained preovulatory-sized follicles ( 35 mm) resulted in 3.4 ovulations per mare and, at 14 days after insemination, 1.8 pregnancies per mare compared to 0.6 pregnancies for untreated controls. Thus, the dose of 12.5 mg twice per day was selected for subsequent studies and commercial use. In Brazil, 16 mares were used during two cycles (Squires et al., 2003). The first cycle was considered a control cycle. In the second cycle, mares were given efsh (12 mg bid). Embryo recovery was performed seven days after ovulation. In the control cycle, mares ovulated one follicle per mare and embryo recovery was 0.5 embryos per mare. In contrast, mares given efsh had 3.6 ovulations and 1.9 embryos recovered per mare. In order to improve the success of using efsh for superovulation, a study was conducted in our laboratory in 2004 (Welch, 2005). The objective was to determine if once-daily injection of 25 mg of efsh would provide the same response as twice-daily injection of efsh (12.5 mg). The second objective was to determine if delaying hcg administration after the end of efsh treatment would enhance the ovulatory response. A third objective was to determine if efsh enriched with LH given during the last 3.5 days of efsh treatment would improve ovulation and embryo recovery. Forty cycling mares were used in this study on two consecutive cycles. Mares were treated for approximately six days with efsh. The number of preovulatory follicles after efsh treatment was higher for those given 12.5 mg bid and coasting of the follicles allowed before hcg administration. The number of ovulations was higher for mares given 12.5 mg of efsh twice a day and coasting compared to those given 25 mg once a day with coasting (4.1 vs 2.8). The percent of mares with greater than two ovulations averaged 85%. Overall embryo recovery was 2.1 embryos per flush and embryo recovery did not vary among the four groups. However, the highest embryo recovery numerically was in the group given 12.5 mg twice daily with follicle coasting (2.6). In 46 cycles, mares had 4

7 bilateral multiple ovulations with a mean embryo recovery rate of 2.4 embryos per flush. In contrast, 26 mares had unilateral multiple ovulation with a mean embryo recovery rate of 1.5. Although there is no way to predetermine if a superovulated mare will develop follicles unilaterally or bilaterally, if unilateral ovulations are detected, lower embryo recovery can be expected. Ninety-six percent of the embryos were graded 1 or 2 (excellent or good). Only 6 of the 148 (4% of the embryos recovered from superovulated mares) were graded fair or poor. Twenty-two of the mares were bred during a cycle immediately after the second efsh treatment cycle. Mares were evaluated by transrectal ultrasonography 12 to 16 days after ovulation to verify pregnancy. Pregnancy rates were detected in 21 of 22 (95%) mares. Thus, there does not appear to be any adverse effect in establishing pregnancy after mares have been treated with efsh for two consecutive cycles. One of the concerns of administering efsh to mares is the number of days that the mare must be treated and the cost involved. If mares are treated seven or eight days at 12.5 mg twice per day, then the cost for superovulation can be considerable. Therefore, studies have been conducted to determine if a shorter duration of treatment can be used and the same response obtained compared to a longer duration of treatment. In our laboratory, initiation of efsh treatment was delayed until mares had at least one follicle 20 to 25 mm in size. efsh was then given for three consecutive days and hcg delayed until the majority of follicles were 35 mm in size. This treatment was compared to initiating efsh once mares had a 20- to 25- mm follicle and continuing to administer efsh until the majority of follicles were >35 mm. The average number of days mares were treated with this approach was 4.5 days. The number of ovulations in both treatments was similar (3.7 and 5.5, respectively, for short-term and long-term treatment). The number of embryos tended to be higher for mares given an average of 4.5 days of treatment (2.1) compared to those given only 3 days of treatment (0.7). It would appear that administering efsh twice a day for only 3 days does not provide the same benefit as providing efsh for a slightly longer timeframe (4.5 days). In another study conducted in our laboratory, we attempted to answer the question as to whether mares can be administered efsh on several consecutive cycles. We compared this treatment with mares given efsh every other cycle with a non-treated control cycle between the efsh treatment cycles. The number of ovulations for both treatments was similar (5.5 and 4.1 for every other cycle and every cycle, respectively). Furthermore, the number of embryos collected from mares in each of these groups was 2.1 and 1.8 per flush for the two treatment groups, respectively. Thus, there did not appear to be any adverse effect of administering efsh for at least three consecutive cycles. Part of the variability in response to efsh treatment is due to the variable follicular population at the onset of treatment. In an attempt to control the follicular population at the onset of treatment, a study was performed in which mares were administered progesterone and estradiol (150 mg progesterone and 10 mg estradiol) for 10 days prior to efsh treatment. On day 10 of the progesterone+estradiol treatment, prostaglandin was administered and mares were given efsh once they had obtained 20- to 25-mm follicles on their ovaries. This treatment was compared to initiation of efsh 5 to 7 days after ovulation, regardless of the follicular population. The results of this study demonstrated that pretreatment of mares with progesterone+estradiol prior to efsh was of no value. The number of ovulations and the number of embryos were higher in mares given efsh without progesterone priming and the number of mares giving more than two embryos was higher in the efsh alone group, vs the progesterone+estradiol efsh group. Currently, studies are ongoing in our laboratory to determine if a lower dose of efsh can be used to induce modest superovulation in mares. Unlike the situation in cattle, where multiple embryos from a given donor-sire combination is accepted, in the horse industry most breeders only want one embryo from a given mare/sire combination. It is our intent to give a lower dose of efsh (6 mg twice per day) in order to induce 5

8 two or three ovulations and one to two embryos per embryo recovery attempt. The results of these studies are not yet available. The current recommendation for use of efsh for superovulation in mares is to begin examination of the mare with ultrasonography five to seven days post-ovulation. Once mares have obtained at least one follicle 20 to 25 mm in size, then efsh treatment can commence. Prostaglandin administration is given on day 2 of efsh treatment. Twice daily injections of efsh continue for approximately 3 to 5 days, until the majority of the cohort of follicles is 35 mm. The efsh treatment is then stopped and hcg is administered 36 hr later. If embryos are to be frozen, then the mare s uterus should be flushed 6.5 days after the majority of the ovulations or, alternatively, 8 days after hcg. If embryos are to be transferred fresh, then the mare s uterus should be flushed 7.5 days after the majority of the ovulations or 9 days after hcg. There are still many challenges to the use of efsh for superovulation. The variability in the number of ovulations and the embryo recovery in response to efsh is considerable. Ideally, one would like to have 3 to 5 ovulations, with ovulations occurring on both ovaries. In most cases, embryo recovery is approximately 50 to 60% per ovulation. On the average, approximately two embryos are collected per mare. However, similar to cattle, approximately 25% of the mares provide no embryo on a given recovery attempt. It appears that waiting until a mare has follicles in the range of 20 to 25 mm does shorten the duration of treatment from approximately 7 to 8 days down to 3 to 5 days. Further studies are needed to determine the best strategy for breeding mares that have been superovulated annd to identify factors affecting variability in embryo recovery. More than likely, with additional clinical use of efsh, the protocol for treatment will be fine-tuned such that the response of the mare becomes more predictable. References Niswender, K.D., M.A. Alvarenga, P.M. McCue, Q.P. Hardy and E.L. Squires Superovulation in cycling mares using equine follicle stimulating hormone (efsh). J. Equine Vet. Sci. 23: Squires, E.L., P.M. McCue, K. Niswender and M. Alvarenga A review on the use of efsh to enhance reproduction performance. Proc. 49th AAEP Annual Convention, pp Welch, S.A The use of equine follicle stimulating hormone to increase ovulation rate and embryo recovery in mares. M.S. Thesis, Colorado State University, Fort Collins, CO. 6

9 Embryo Freezing E.L. Squires, E.M. Carnevale and P.M. McCue Animal Reproduction and Biotechnology Laboratory Colorado State University Fort Collins, CO USA The number of equine embryos frozen is relatively small compared to the number of fresh embryos transferred. This is likely to change once the breed registries accept the use of this technology and allow foals to be registered from embryo recipients receiving frozen-thawed equine embryos. The largest equine breed, the American Quarter Horse Association, is in the process of deciding whether to accept foals born from frozen-thawed embryos. There appear to be three major reasons for freezing equine embryos: 1) import-export, 2) embryo banking, and 3) to reduce recipient herds. With the recent availability of equine FSH (efsh), superovulation and collection of multiple embryos from the mare provides extra embryos that can be frozen. It is likely that breed registries will change their attitudes about registering foals from frozen embryos and, with the simplified new procedures for vitrification, embryo freezing is likely to become more of a standard procedure used in equine embryo transfer. One major drawback to freezing equine embryos is that the embryo must be of a certain size and developmental stage to survive the freezing and thawing process. Based on numerous studies, only embryos <300 µm (morula or early blastocysts), with a zona pellucida, survive the freezing-thawing process. Whether one uses slow-cooling or vitrification procedures, embryos >300 µm result in very poor pregnancy rates after freezing, thawing and transfer (Slade et al., 1985; Squires et al., 1989; Maclellan et al. 2002). This means that the mare s uterus must be flushed for an embryo approximately 6.5 days after ovulation. Mares flushed later than this time provide embryos that have an acellular capsule formed underneath the zona. Apparently, this acellular capsule may, in fact, impeded the penetration of cryoprotectants into the inner cell mass and trophoblastic cells and explains why larger embryos freeze very poorly. There are two approaches to timing the flushing of the mare s uterus in order to obtain small embryos. One is to examine the mare by ultrasonography either once or several times per day in order to determine the exact time of ovulation and then flush the mare 6.5 days after ovulation. The other approach is to flush the mare s uterus 8 days after administration of hcg. Studies conducted in our laboratory have shown that the majority of mares flushed 8 days after hcg provide embryos that are <300 µm in size (Eldridge-Panuska et al., 2005). Generally, mares ovulate approximately 36 hr after hcg. Thus, when flushing the mare s uterus 8 days post-hcg, the embryo is approximately 6.5 days old. There are two different procedures used for freezing of equine embryos. One is a slow-cooling method similar to the protocol used for freezing bovine embryos. Several studies (Slade et al., 1985; Lascombes, 2002; Maclellan 2002) have reported excellent fertility of embryos slow-cooled, thawed and transferred into recipients. Typically, embryos are packaged in 0.5-ml plastic straws, cooled to 6 C at 4 C/ minute, seeded and held for 15 min, then cooled at 0.3 C/minute to 35 or 38ºC and then plunged into liquid nitrogen. The majority of studies have been done with the cryoprotectant glycerol, however ethylene glycol appears to be a good alternative to glycerol for equine embryos. The disadvantage of the slow-cool method is the time involved in freezing, as well as the need for a programmable cell freezer. 7

10 The alternative is a vitrification procedure. The vitrification procedure is much simpler and does not require a cell freezer. Several studies have been conducted in our laboratory in the last two years to evaluate the fertility of embryos that have been vitrified and thawed. In the initial study by Eldridge-Panuska et al. (2005), 39 lactating light horse mares from 3 to 15 years of age were used. Embryo collections were made 6 to 6.5 days after detection of ovulation or 8 days after administration of hcg. Upon identification, embryos were washed through 3 to 6 drops of holding medium (Vigro Holding Plus, AB Technology, Inc., Pullman, WA). Embryos were classified for developmental stage and measured. The vitrification solutions were: VS1 1.4 M glycerol for 5 min, then VS2 1.4 M glycerol M ethylene glycol for 5 min, then VS3 1.4 M glycerol M ethylene glycol for <1 min. The embryos were loaded into a straw and the straw was heatsealed and placed into a cooled plastic goblet surrounded by liquid nitrogen for 1 min. The entire goblet containing the straws was then plunged into liquid nitrogen. Embryos were transferred nonsurgically into the uteri of recipients. Twenty-six of 48 embryos transferred resulted in viable pregnancies (54%). In 2004 (Hudson et al., 2005), a study was done to determine if cooling embryos for 12 to 19 hr prior to vitrification would result in similar pregnancy rates to embryos vitrified immediately upon collection and to determine the viability of vitrified embryos from superovulated mares. Mares were administered 12.5 mg of efsh (Bioniche Animal Health, Bogart, GA) twice daily for 5 to 7 days. Embryos were flushed from the uterus 6.5 days after ovulation, if ovulations were asynchronous, or 8 days post-hcg for synchronous ovulations. Upon identification, each embryo was measured using an eyepiece micrometer and graded. After sizing and grading, each embryo was rinsed four times in Vigro Holding Solution (AB Technology, Inc., Pullman, WA). Embryos were either assigned to be vitrified immediately or to be cooled in a passive cooling device (Equitainer, Hamilton Thorne Biosciences, Beverly, MA) for 12 to 19 hr prior to vitrification. All embryos were vitrified as described previously by Eldridge-Panuska et al. (2005). Embryos were transferred during the month of August 2004 into synchronized recipients. Embryos were transferred into recipients that had ovulated 4 to 6 days previously to the transfer. Embryos were warmed by removing the straw from the liquid nitrogen tank and holding the straw in air at room temperature for 10 sec prior to plunging into a bath of 20 to 22ºC water for an additional 10 sec. The straws were removed from the water bath and flicked like a clinical thermometer 4 to 5 times to ensure mixing of the solutions. For transfer, the straws were loaded into a special Cassou gun designed for cut straws. Each embryo was transferred into a recipient within 8 min of removal of the straw from the liquid nitrogen tank. There were no differences in pregnancy rates between embryos vitrified immediately after collection (15 of 20, 75%) and embryos cooled for 12 to 19 hr prior to vitrification (13 of 20, 65%). This provides the flexibility of shipping an embryo from a farm or veterinary clinic to a centralized facility that has the expertise for vitrification. Alternatively, the embryos can be collected and vitrified immediately. In summary, pregnancy rates from transfer of vitrified equine embryos have been quite acceptable (50 to 70%). These rates are similar to what one would obtain from transfer of fresh embryos. Furthermore, the viability of embryos obtained from superovulated mares is similar to that from single-ovulating, nonsuperovulated mares. 8

11 References Eldridge-Panuska, W.D., V. Caracciolo di Brienza, G.E. Seidel, Jr., E.L. Squires and E.M. Carnevale Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos. Theriogenology 63: Hudson, J., P.M. McCue, E.M. Carnevale, S. Welch and E.L. Squires The effects of cooling and vitrification of embryos from efsh-treated mares on pregnancy rates after nonsurgical transfer. J. Equine Vet. Sci. Submitted. Lascombes, F.A. and R.L. Pashen Results from embryo freezing and postovulation Cryopreservation of equine embryos using glycerol and 1,2 propanediol as cryoprotectants. Equine Vet. J., Suppl 15: Maclellan, L.J., E.M. Carnevale, M.A. Coutinho da Silva, P.M. McCue, G.E. Seidel, Jr. and E.L. Squires Cryopreservation of small and large equine embryos pre-treated with cytochalasin-b and/or trypsin. Theriogenology 58: Slade, N.P., T. Takeda, E.L. Squires, R.P. Elsden and G.E. Seidel, Jr A new procedure for the cryopreservation of equine embryos. Theriogenology 24: Squires, E.L., G.E. Seidel, Jr. and A.O. McKinnon Transfer of cryopreserved equine embryos to progestin-treated ovariectomized mares. Equine Vet. J., Suppl. 8:

12 Efficiency of Programs that Control Follicular Development and Ovulation for the Donor Superovulation Without Estrus Detection G.A. Bó 1, P. Chesta 1, L.F. Nasser 2 and P.S. Baruselli 2 1 Instituto de Reproducción Animal Córdoba (IRAC), J.L. de Cabrera 106, X5000GVD Córdoba, Argentina; 2 Departamento de Reprodução Animal, FMVZ-USP, São Paulo, Brazil, INTRODUCTION Although embryo transfer techniques are widely used around the world, with more than 500,000 embryos being transferred each year, variability in response to the superstimulatory treatments remains an important limitation (13). The better understanding of ovarian function, acquired in recent years through the use of ultrasonography, has provided possibilities for a greater capability of controlling follicular development and ovulation. Recent protocols, designed to control both luteal and follicular function, permit the initiation of superstimulatory treatments and the synchronization of recipients at a self-appointed time. The intention of this manuscript is to review these protocols and discuss how they may impact on the effectiveness and application of bovine embryo transfer programs. The conventional protocol of initiating ovarian superstimulation during mid-cycle was originally based on anecdotal and experimental information in which a greater superovulatory response was reported when superstimulatory treatments were initiated 8 to 12 d after estrus (reviewed in 10). However, none of these early studies specifically evaluated follicular status of the animals when superstimulation treatments were initiated. Monitoring follicular development ultrasonically in cattle was, in most cases, in the early stages of development and not available in many laboratories. Through information generated by ultrasonography, it is now known that 8 to 12 d after estrus (equivalent to Days 7 to 11 after ovulation) would be the approximate time of emergence of the second follicular wave in two- or three-wave cycles (19) and a cohort of growing follicles would be present around that time. However, the day of emergence of the second follicular wave has been shown to differ between two-wave cycles and three-wave cycles (1 or 2 d earlier in three-wave cycles), as well as between individual animals (19). In this regard, it has been clearly shown that superovulatory response was higher when superstimulatory treatments were initiated at the time of follicular wave emergence rather than later (1, 26). Starting gonadotropin treatments as little as 1 d after wave emergence significantly reduced the superovulatory response compared to initiating treatments on the day of wave emergence (1, 26). Based on duration of the developmental phases of dominant follicles in two-wave and three-wave interovulatory intervals, the probability at any given time that the dominant follicle is not functionally dominant (late-static or regressing phases) is approximately 30% (6 of 20 d) for two-wave heifers and 35% (8 of 23 d) for three-wave heifers. More importantly, only approximately 20% (4 or 5 d) of the estrous cycle is available for initiating superstimulatory treatments at the time of follicular wave emergence. Therefore, 80% of the estrous cycle is not conducive to an optimal superovulatory response. The necessity of waiting until mid-cycle to initiate superstimulatory treatments implies monitoring estrus and an obligatory delay. To obviate these problems, an alternative approach is to initiate superstimulation treatments subsequent to the exogenous control of follicular wave emergence. 10

13 One approach involves transvaginal ultrasound-guided follicle ablation of all follicles 5 mm to synchronized wave emergence at random stages of the estrous cycle, followed by FSH (Folltropin-V, Bioniche Animal Health, Canada) treatment 1 d after ablation and PGF 48 h later (9, Table 1). It was shown that the timing of estrus could be more accurately controlled when a progesterone/progestogen implant was inserted for the period of superstimulation and 2 injections of PGF were administered on the day of implant removal. Nonablated control animals were given FSH 8 to 12 d after estrus and PGF 48 h later. Combined over 2 experiments, there was no difference in the superovulatory response between the ablated and non-ablated control groups. Transvaginal ultrasound-guided follicle ablation of all follicles (20) or just the dominant follicle (14, 20, 21) 2 d prior to superstimulation during mid-diestrous, has been shown to result in a higher superovulatory response than when the dominant follicle was not ablated (Table 1). Conversely, in a retrospective analysis of superovulatory responses of lactating dairy cows, follicle ablation resulted in a significantly higher number of ova/embryos collected, but a comparable number of transferable embryos than when cows were superstimulated 7 to 13 d after estrus (29; Table 1). In a more recent study, ablation of the 2 largest follicles at random stages of the estrous cycle was as efficacious in synchronizing follicular wave emergence for superstimulation as ablating all follicles 5 mm, eliminating the need to determine which follicle is the dominant follicle (2). Table 1. Mean (± SEM) number of transferable embryos in cows superstimulated 1 or 2 d after follicle ablation or 8 to 12 d after estrus Author Ablation No Ablations P value n Transf. Emb. n Transf. Emb. Bergfelt et al., 1996 (9) ± ± Bungarts and Niemann, ± (dom. fol.) 1.0 ±0.1 < (14) 14 (no dom. fol.) 10.1± Hill & Kuehner, 1996 (20) ± ±0.6 <0.001 Shaw and Good, 2000 (29) ± ± Shaw and Good, 2000 (29) ± ± Kim et al., 2001 (21) ± ±0.8 <0.05 Follicular wave emergence has also been synchronized in estrus synchronization protocols through the use of GnRH or porcine LH (plh). However, the reported asynchrony in follicular wave emergence (from 3 d before to 5 d after treatment) suggests that this approach may not be feasible for superstimulation (24). In spite of the wide range in day of follicular wave emergence that occurred in that study (24), the new follicular wave emerged within 3 d of treatment (many by chance because of the stage of the estrous cycle) in 44 of 54 (81 %) of heifers. Indeed, in one study involving 3 different experiments (18), GnRH or plh treatments consistently resulted in a lower number of embryos collected (and thus, transferable embryos) than when follicular wave emergence was synchronized with estradiol or by follicular ablation. Therefore, we do not recommend this approach to the synchronization of follicular wave emergence for the purpose of superstimulation in cattle. Our preferred approach to the synchronization of follicular wave emergence for superstimulation involves the treatment with estradiol-17â (E-17β) and progesterone (P4) by intramuscular injection at the time of progestogen/progesterone device insertion, followed by FSH beginning 4 d later (11). Data from experiments and commercial superovulation programs (Table 2; reviewed in 10,12,28) have shown that superovulatory response of donors treated with E-17β and P4 at unknown stages of the estrous cycle was comparable or better to that of donors superstimulated beginning 8 to 12 d after observed estrus (13,28). 11

14 Table 2. Superovulatory response in beef and dairy cattle superstimulated 8 to 12 d after estrus (traditional) or 4 d after treatment with estradiol-17β, progesterone and a progestogen/progesterone releasing device (P4 + E-17β; adapted from 10, 12 and 28). Beef Cattle Dairy Cattle Treatment n Total Transferable n Total Transferable ova/embryos embryos ova/embryos embryos Traditional ± ± ± ± 0.3 E-17β + P ± ± ± ± 0.4 Means did not differ (P>0.2). Estradiol-17β is not readily available for commercial use in many countries. Therefore, we investigated the possibility of using other commercially available estrogen esters such as estradiol benzoate (EB) or estradiol valerate (EV). Treatment with 2.5 mg EB and 50 mg P4 given at the time of CIDR-B insertion resulted in synchronous emergence of a new follicular wave 3 to 4 d later (15). Superstimulatory treatments initiated 4 d after 2.5 mg EB and 50 mg P4 resulted in superovulatory responses comparable to those initiated 4 d after treatment with 5 mg E-17β and 50 mg P4 (16) or 2.5 mg E-17β and 50 mg P4 (17, Figure 1 and Table 3) or those initiated 8 to 12 d after estrus (25). Treatment with 5 mg EV and 3 mg norgestomet resulted in less synchronous emergence of a follicular wave and a lower superovulatory response than 5 mg E-17β and 100 mg P4 in cows with two SMB implants (22). However, lower dosages of a dose of 1.0 or 2.0 mg EV resulted in follicular wave emergence in 3-4 d, with little variability (23). These results suggest that lower doses of estradiol esters may be useful in the synchronization of follicular wave emergence for superstimulation. Superstimulatory experiments are needed to confirm these observations. 2.5 mg EB +50 mg P4 FSH (AM and PM) AI Embryo Collection Progesterone device Treatment days PGF (AM and PM) Figure 3. Treatment protocol for superstimulation of donor cattle with progesterone (P4) releasing devices and estradiol benzoate (EB). Treatment consists of insertion of progesterone releasing device and 2.5 mg EB plus 50 or 100 mg P4 im on Day 0. Superstimulatory treatments are initiated on Day 4, with FSH in twice daily im injections over 4 d. Donors receive PGF treatment in the morning and evening of Day 6 and progesterone devices are removed with the second PGF treatment. Donors are inseminated 12 and 24 h after observed estrus or 48 and 60 h after progesterone device removal. Ova/embryos are collected non-surgically on Day 15 12

15 Table 3. Superovulatory response of beef cows treated with progesterone releasing devices and estradiol 17b (E-17b) or estradiol benzoate (EB) on Day 0 and Folltropin-V beginning on Day 4 (adapted from17). 2.5 mg E-17β + 50 mg P4 2.5 mg EB + 50 mg P4 N Total ova/embryos 12.7± ±1.0 Fertilized ova 8.1± ±0.9 Grade 1 4.4± ±0.6 Grade 2 and 3 2.4± ±0.3 Means did not differ (P>0.1). Collectively, these studies demonstrated that exogenous control of follicle wave emergence offers the advantage of initiating superstimulatory treatments at a time that is optimal for follicle recruitment, regardless of the stage of the estrous cycle. The treatment is practical, easy to follow by farm personnel and, more importantly, the need for detecting estrus or ovulation and waiting 8 to 12 d to initiate gonadotropin treatments is eliminated. Synchronization of follicular wave emergence by follicle ablation or estradiol/progesterone treatments has resulted in comparable superovulatory response (2, 7, 10). It is noteworthy that in studies involving superstimulation coincident with follicular wave emergence, the response to a single bolus injection of FSH was not different from that in response to a multiple injection scheme (9, 11). The nadir between FSH surges is responsible for preventing the emergence of a new follicular wave (8); provision of exogenous FSH during the period of the nadir in FSH may have resulted in break through growth of small follicles prior to the time of expected new wave emergence (i.e., effects of dominant follicle suppression were overcome by exogenous FSH). This may explain how large doses of exogenous FSH in conventional superstimulation schemes can overwhelm the endogenous rhythm and mask the wave effect on ovarian response. If a superstimulatory treatment is given for a long enough period of time, follicle recruitment will become apparent, regardless of follicular wave status at the time of gonadotropin treatment. However, asynchronous recruitment may result in more variability in ovarian follicular response and in the quality of embryos collected (11, 28). SUPEROVULATION OF THE FIRST FOLLICULAR WAVE Initiation of treatments at the time of emergence of the first follicular wave may be another option for using a synchronized follicle wave for superovulation. It has been demonstrated that optimal superovulatory responses can be obtained when treatments were initiated on the day of ovulation (26). Furthermore, Adams et al. (1) have shown that there were no differences in superovulatory response when superstimulatory treatments were initiated at the time of emergence of the first or second follicular wave. Although these studies were done over ten years ago, the industry has not taken advantage of natures way of synchronizing follicle wave emergence i.e., ovulation, probably because methods of synchronizing ovulation were not known or understood at that time, and the fear that PGF will not be able to induce luteolysis on Days 4 or 5 of the estrous cycle. We have recently revisited this idea, by combining a treatment that synchronizes ovulation with the superovulation regime (27). The purpose of this study was to evaluate the responsiveness of Bos indicus donors to superstimulatory treatments administered at the time of emergence of the first follicular wave. Eighteen Nelore donor cows were randomly allocated to 1 of 3 treatment groups. Cows in Groups 1 and 2 were superstimulated during the first follicular wave and cows in the Group 3 were superstimulated after synchronized follicular wave emergence using estradiol and progesterone. In this experiment, cows in Groups 1 and 2 were synchronized with a CIDR- 13

16 B plus EB + P4 protocol followed by injection of 12.5 mg plh (Lutropin-V; Bioniche Animal Health) 24 h after CIDR-B removal to synchronize ovulation. Superstimulatory treatments were initiated at the expected time of ovulation; cows in Group 2 also received a new CIDR-B device at the time of the first FSH injection. Cows in Group 3 (controls) were superstimulated starting on 4 d (expected time of wave emergence) after insertion of a CIDR-B device and treatment with EB + P4. All cows in the 3 groups were superstimulated with a total dose of 133 mg NIH-FSH-P1 of Folltropin-V (FSH, Bioniche Animal Health) divided into twice daily injections of 13.3 mg FSH in 1 ml of diluent over 5 d. On the last day of FSH treatment, all animals received PGF after each FSH injection and cows in Groups 2 and 3 had their CIDR-B removed at the time of the last FSH injection. All cows received 25 mg of plh 24 h after the last FSH treatment and were inseminated 12 and 24 h later. Ova/embryo collection and evaluation was done 7 d after plh treatment by the same embryo transfer practitioner. Results, summarized in Table 4, indicate that there were no differences in the superovulatory response, as measured by numbers of ovulations, among groups. In addition, there was no difference in the numbers of transferable embryos in CIDR-B-treated donor cows whether superstimulatory treatments were initiated at the time of emergence of either the first follicular wave (Group 2) or following synchronization of follicular wave emergence with EB+P4 (Group 3), but both were greater than when superstimulatory treatments were initiated at the time of emergence of the first follicular wave without the use of a CIDR-B device (P<0.05). Table 4. Superovulatory response and embryo quality in Nelore cows treated with CIDR-B plus 2.5 mg EB and 50 mg P4 on Day 0 and Folltropin-V on Day 4 (controls) or superstimulated at the beginning of the first follicular wave with or without the addition of a CIDR-B device during the superstimulatory treatment. Group 1 Group 2 Group3 First Wave First Wave EB+P4 without CIDR-B with CIDR-B (control) n Follicles > 8 mm on the day of AI 21.5± ± ±3.7 CL 15.3 ± 4, ± ± 2.5 Total ova/embryos 8.3 ± ± ±2.3 Fertilized ova 1.3±3.3 a 9.2±5.0 b 6.7±4.8 ab Grade 1 embryos 0.0 ± 0.0 a 5.8 ± 1.4 b 5.1±1.7 b Grade 1 and 2 embryos 0.2 ± 0.2 a 8.0 ± 1.8 b 6.6±2.0 b ab Means within rows with different superscripts differ (P < 0.05) Although the response based on the number of ovulations was expected, the poor embryo quality in the donors treated at the time of emergence of the first wave without a CIDR-B was not expected. Therefore, a second experiment was performed to confirm the results of the first experiment. Treatments were done exactly as for Groups 1 and 2 in the first experiment, and the control group was dropped. Therefore, all cows were superstimulated during the first follicular wave with or without the insertion of a CIDR-B during the treatment. In this case, cows were also bled to measure plasma progesterone concentrations during the superstimulatory treatment. Results are shown in Table 5 and Figure 4. 14

17 Table 5. Superovulatory response and embryo quality in Nelore cows treated with Foltropin-V at the beginning of the first follicular wave with or without the addition of a CIDR-B device during the superstimulatory treatment. Group 1 Group 2 First Wave First Wave P value without CIDR-B with CIDR-B n Follicles >8mm at the time of plh treatment 19.9 ± ± CL 5.4 ± ± Total Ova/embryos 4.0 ± ± Fertilized ova 1.5 ± ± Grade 1 embryos 1.2 ± ± Grade 1 and 2 embryos 1.3 ± ± P4 concentration during treatment (ng/ml) 1.3 ± ± P4 (ng/ml) * * * * Days of treatment With CIDR-B Without CIDR-B Figure 4. Mean plasma progesterone (P4) concentrations in Nelore cows superstimulated during the first follicular wave with or without the insertion of a CIDR-B device during treatment. * means differ (P<0.05). Results suggest that in Nelore donor cows superstimulated during the first follicular wave, the addition of a progesterone during the period of FSH treatments results in improved embryo quality. Whether the same applies to Bos taurus breeds of cattle needs to be determined, but this could have important implications in superstimulatory protocols. Furthermore, this treatment may be an alternative superstimulation protocol in countries where estradiol is not licensed or when on farm conditions make follicle aspiration impractical. Obviously, further studies with a larger number of donors are needed to confirm these results in Bos taurus and Bos indicus cattle. 15

18 ATTEMPTS TO SYNCHRONIZE OVULATION IN DONOR COWS Recent studies, mainly performed in Brazil, have been directe8d toward the development of a superstimulation protocol that allows for fixed-time AI in Nelore cattle (3,4,5). The treatments explored were similar to those previously described, involving a progesterone-releasing vaginal insert and injection of estradiol and progesterone on day 0 and FSH treatments initiated at the expected time of follicle wave emergence on Day 4. However, in these studies progesterone devices were removed in the morning or afternoon of Day 7 (Day 7 AM and Day 7 PM; Table 3; 30) instead of the afternoon of Day 6. All donors also receive 25 mg of plh (Lutropin-V, Bioniche Animal Health) or a dose of GnRH in the morning of Day 8 and are inseminated 12 and 24 h later. This treatment has been named depending on the time from the first PGF injection to the time of removal of the progesterone releasing device. Therefore when the progesterone device is removed on Day 7 AM, it is called P24 and when it is removed on Day 7 PM, the treatment is called P36. Although no differences were found in Nelore cattle when progesterone devices were removed on Day 7 AM or 7 PM (Table 6), the P36 protocol is preferred for fixed-time AI in Nelore cattle (Figure 5). Table 6. Effect of the time of progesterone device removal on superovulatory response of Nelore cows treated with progesterone releasing devices, estradiol benzoate (EB) and 130 mg NIH-FSH-P1 Folltropin-V. Progesterone devices were removed 24 h after the first PGF injection (Day 7 AM; P24) or with the last Folltropin-V injection (Day 7 PM; P36) and all cows received plh in the morning of Day 8 with AI 12 and 24 h later (adapted from 30). Day 7 AM (P24) Day 7 PM (P36) N Total ova/embryos 21.2± ±3.7 Fertilized ova 16.0± ±3.8 Viable embryos 9.3± ±1.9 Means did not differ (P>0.05). 2.5 mg EB +50 mg P4 FSH GnRH or plh AI Embryo Collection Progesterone device Treatment days PGF (AM and PM) Figure 5. Treatment protocol for fixed-time AI of superstimulated Nelore cattle. Treatment consists of insertion of progesterone releasing device and estradiol benzoate (EB) plus progesterone (P4) im on Day 0. Superstimulatory treatments are initiated on Day 4, with FSH given in twice daily im injections over 4 d. Donors receive PGF treatment in the morning and afternoon of Day 6 and progesterone devices are removed with the last FSH, in the afternoon of Day 7. Donors also receive plh or GnRH in the morning of Day 8 and are inseminated without estrus detection 12 and 24 h after GnRH/pLH. Ova/ embryos are collected non-surgically on Day

19 The use of these protocols has also been recently investigated in high producing Holstein cows (6). In this experiment, 40 Holstein cows were treated with 1 or 2 Crestar implants plus 3 mg EB and 50 mg P4 im on Day 0; FSH treatments were initiated on Day 4 and PGF was given in the morning and afternoon of Day 6. Crestar implants were removed in the afternoon of Day 7 and GnRH was given in the morning of Day 8 (12 h) or in the afternoon of Day 8 (24 h) with AI 12 and 24 h later. So far, no differences have been detected between 1 or 2 Crestar. However, treatment with GnRH 24 h after Crestar removal resulted in fewer (P<0.05) number of unovulated follicles (1.7±0.3) than when GnRH was given 12 h after Crestar removal (3.5±0.5). This was not reflected in a significant increase in the number of transferable embryos, but numbers favor treatment with GnRH 24 h after Crestar removal (GnRH 24 h: 4.2±1.3 vs GnRH 12 h: 2.7±0.8). More cows are required in order to make statistically valid conclusions and recommendations. Nevertheless, results from the current studies in Brazil and Argentina suggest that alternatives may be available in Bos taurus cattle: 1) remove progestogen/progesterone devices on Day 6 PM and detect estrus and AI 12 and 24 h after estrus or 48 and 60 h after device removal (Figure 3); 2) remove the progestogen/progesterone device in the morning of Day 7, give GnRH or plh in the morning of Day 8 and AI 12 and 24 h later; or 3) remove the devices in the afternoon of Day 7, give GnRH or plh in the afternoon of Day 8 and AI 12 and 24 h later. Embryo collection should be done later in the third alternative, to avoid the collection of early morulae which do not survive freezing and thawing procedures as well as more advanced embryos. Obviously, more experiments are needed to determine optimum treatment protocol for fixed-time AI of Bos taurus donor cattle. SUMMARY AND CONCLUSIONS Incorporation of techniques designed to control follicular wave dynamics, like those discussed herein, will reduce the variability caused by superstimulating donor cows at different stages of the follicular wave. However, studies done to date have still not provided a way of minimizing the variability in ovarian response to superstimulation, but protocols involving synchronization of follicular wave emergence do offer the convenience of being able to initiate treatments quickly and at a self-appointed time, without the necessity of estrus detection and without sacrificing results. Furthermore, evidence is mounting that these protocols can be incorporated into protocols that involve fixed-time AI of donor cows, without sacrificing ova/embryo quality. REFERENCES 1. Adams GP, Nasser LF, Bo GA, Mapletoft RJ, Garcia A, Del Campo MR. Superstimulatory response of ovarian follicles of wave 1 versus wave 2 in heifers. Theriogenology 1994; 42: Baracaldo MI, Martinez M, Adams GP, Mapletoft RJ. Superovulatory response following transvaginal follicle ablation in cattle. Theriogenology 2000; 53: Barros CM, Nogueira MFG. Embryo transfer in Bos indicus cattle. Theriogenology 2001; 56: Baruselli PS, Marquez MO. Ultimos avances en superovulacion de donantes de razas cebuinas. Resúmenes del IV Seminario Internacional de Reproducción en Grandes Animales. CGR, Biotecnología Reproductiva E.U. División Capacitación. Bogotá 25 al 27 de septiembre; Medellín 27 y 28 de septiembre 2003; Baruselli PS, Marquez MO, Reis EL, Nasser LF, Silva RCP, Menegatti JA, Valentin R, Santos ICC. Adequacao da dose de FSH (Folltropin-V) em protocolos de superovulacao de vacas nelore (Bos taurus indicus) com inseminacao artificial em tempo fixo. XVII Reuniao Anual da Sociedade Brasileira de Technologia de Embrioes, Fortaleza; Acta Scientae Veterinarie 2003; Baruselli PS, Sa Filho M, Martins C, Reis EL, Nasser LF, Bo G.A. Novos Avancos nos tratamentos de superovulacao em doadoras de embriones bovinos. VI Simposio internacional de Reproduccion Animal, Cordoba, Argentina, 2005;

20 7. Beal WB. Practical application of ultrasound in bovine embryo transfer. 18th Annual Convention AETA, Colorado Springs, CO, USA, 1999; Bergfelt DR, Plata-Madrid H, Ginther OJ. Counteraction of inhibitory effect of follicular fluid by administration of FSH in heifers. Can J Anim Sci 1994; 74: Bergfelt DR, Bo GA, Mapletoft RJ, Adams GP. Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle. Anim Reprod Sci 1997; 49: Bo GA, Adams GP, Pierson RA, Mapletoft RJ. Exogenous control of follicular wave emergence in cattle. Theriogenology 1995; 43: Bo GA, Adams GP, Pierson RA, Mapletoft RJ. Effect of progestogen plus E-17β treatment on superovulatory response in beef cattle. Theriogenology 1996; 45: Bo GA, Mapletoft RJ. Control del desarrollo folicular y su aplicación en programas de superovulacion de donantes de embriones. Taurus 1999; 4: Bo GA, Baruselli PS, Moreno D, Cutaia L, Caccia M, Tríbulo R, Tríbulo H, Mapletoft RJ. The control of follicular wave development for self-appointed embryo transfer programs in cattle. Theriogenology 2002; 57: Bungartz L, Niemann H. Assessment of the presence of a dominant follicle and selection of dairy cows suitable for superovulation by a single ultrasound examination. J Reprod Fert 1994; 101: Caccia M, Bo GA. Follicle wave emergence following treatment of CIDR-B implanted beef heifers with estradiol benzoate and progesterone. Theriogenology 1998; 49:341 abstr. 16. Caccia M, Tribulo R, Tríbulo H, Bo GA. Effect of different estrogen and progesterone treatments on superovulatory response in beef (Bos taurus) cattle. Arq Fac Vet UFRGS, Porto Alegre, Brazil, 1998; 26 (Suppl): 211 abstr. 17. Caccia M, Tribulo R, Tribulo H. Superovulatory response of beef cows treated with progesterone devices and estradiol-17β or estradiol benzoate. Theriogenology 2002; 57:762 abstr. 18. Deyo CD, Colazo MG, Martinez MF, Mapletoft RJ. The use of GnRH or LH to synchronize follicular wave emergence for superstimulation in cattle. Theriogenology 2001; 55:513 abstr. 19. Ginther OJ, Knopf L, Kastelic JP. Temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J Reprod Fert 1989; 87: Hill BR, Kuehner LF. Follicle aspiration prior to superovulation in cattle. Theriogenology 1996; 43:324 abstr. 21. Kim HI, Son DS, Yeon H, Choi SH, Park SB, Ryu IS, Suh GH, Lee DW, Lee CS, Lee HJ, Yoon JT. Effect of dominant follicle removal before superstimulation on follicular growth, ovulation and embryo production in Holstein cows. Theriogenology 2001; 55: Mapletoft RJ, Martinez MF, Adams GP, Kastelic J, Burnley CA. The effect of estradiol preparation on follicular wave emergence and superovulatory response in norgestomet-implanted cattle. Theriogenology 1999; 51:411 abstr. 23. Mapletoft RJ, Colazo MG, Small JA, Ward DR, Kastelic J P. Effect of dose of estradiol valerate on ovarian follicular dynamics in CIDR-treated beef cows. Reprod Fert Dev 2004; 16:130 abstr. 24. Martinez MF, Adams GP, Bergfelt D, Kastelic JP, Mapletoft RJ. Effect of LH or GnRH on the dominant follicle of the first follicular wave in heifers. Anim Reprod Sci 1999; 57: Meyer JA, Wideman DJr, Looney CR, Long CR, Bo GA, Day ML, Anderson JC, Forrest DW. Embryo production rates of cattle superovulated with and without the presence of an intravaginal progesteronereleasing device. Theriogenology 2000; 53: 504 abstr. 26. Nasser L, Adams GP, Bo GA, Mapletoft RJ. Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 1993; 40:

21 27. Nasser LF, Reis EL, Oliveira AM, Bo GA, Baruselli PS. Effect of time of ecg pretreatment on pregnancy rates in Bos indicus x Bos taurus recipients synchronized with progesterone vaginal devices and transferred without estrus detection. Reproduction Fertility and Development 2004; 212 abstr. 28. Nigro M, Burry E, Villata ML, Bo GA. Effect of different estrogen and progestogen treatments on superovulatory response in beef and dairy cattle. Theriogenology 2002; 57:769 abstr. 29. Shaw DW, Good TE. Recovery rates and embryo quality following dominant follicle ablation in superovulated cattle. Theriogenology 2000; 53; Zanenga CA, Marquez MO, Santos ICC, Valentin R, Baruselli PS. Comparacao entre dois protocolos de superovulacao com inseminacao artificial em tempo fixo en vacas Nelore. XVII Reuniao Anual da Sociedade Brasileira de Technologia de Embrioes, Fortaleza; Acta Scientae Veterinarie 2003;

22 Evaluation of the Postpartum Mare Phil Matthews, DVM Peterson and Smith Equine Reproduction Center When examining a mare and foal for the first time I usually assess the mare first. If there is a severe problem existing or suspected with the foal it has already been examined earlier as a priority. However, in the routine first exam the foal can usually be found sleeping or nursing and not require immediate attention and my initial attention will be focused on the mare. Foaling facilities that are staffed with knowledgeable, experienced staff compliment the efforts of any veterinarian tremendously and these people should be relied upon to alert you to problems that may be occurring with either the mare or foal. Certainly breed differences should be taken into account in interpreting your findings. An example of this is the Quarter Horse halter mare. These mares certainly have a much higher propensity to become laminitic with postpartum complications such as retained placenta, and because of this require more prompt and aggressive treatment. General Assessment of the Mare The general demeanor of the mare should be assessed. Is she alert, eating, and carrying herself normally? Or is she depressed, off feed, or painful? Is she attentive to the foal? Mares may certainly exhibit fatigue or even exhaustion after foaling but they will still act alert when people approach and are usually still attentive to the foal. The mare with her head down and ears flattened out that is not eating well and obviously not very alert is probably more than simply tired, and a very thorough examine is indicated. The early parts of the exam then are making these general observations about the mare s attitude as you enter the stall. Approaching the mare s head to assess the look in her eye can be very helpful. A quick look at her gums to determine that her mucous membranes are neither hyperemic nor pale. A visual exam and palpation of the udder while working caudally to finally look under her tail. If the groom/owner has not taken the mare s temperature then that should be done. While making these assessments, it is a great opportunity to ask some questions regarding the mare. Is she passing stool and eating normally? Has she had a discharge and if so what has the amount and character of that discharge been? Has she been painful or even colicy acting? If all of my findings and the answers to these questions indicate that everything is normal my examination is complete. Although this examination is carried out quickly several aspects of it are key. Obviously if either the exam findings or the history indicates the mare is not normal than a complete physical exam is in order which may include blood work. Examination of the Udder The udder should be examined to determine several things. This examination should not only be visual but the udder should be palpated. Several observations should be made: 1) Is the mare s udder development sufficient to produce enough milk? Obviously a subjective call but one that should be attempted. Maiden mares certainly have smaller udders and less milk production as a general rule. They also may have more tendency to come in to their milk a little later than multiparous mares. Is feed supplementation or medication (such as domperidon) indicated for the mare? What about production of colostrum and does it appear that the foal should be supplemented from another source? 2) Are her legs covered with milk like a mare that has been running milk before foaling which may have resulted in depleting her colostrum. (This is a good time to remember to ask the client about this same issue.) Palpating the udder as well as visualizing it is very useful. Is the bag soft and pliant or is it firm and even painful. Most often if the udder is tight and painful it is the result of edema occurring at term which 20

23 should subside in a day or two. This condition is far more prevalent in the maiden mare. It s significance lies mostly in the fact that the mare (especially the maiden mare) is reluctant to let the foal nurse. Administering flunixin meglumine (50mgs/100lbs) and an azium/diuretic combination (such as Naquazone) will help these mares get through this and allow the foals to nurse more completely and earlier. A cause of a swollen and painful udder can be mastitis although it is rare in the post-foaling mare. Usually mastitic udders are warm to the touch and often result in a mild fever. Stripping a few drops of milk and observing it can often help confirm or refute this condition. Mastitis requires antibiotic as well as anti-infammatory therapy. 3) Is the entire udder functional? Often times no one has paid good attention to the udder until foaling. Has neoplasm or infection (such as nocardiosis) rendered a portion of the udder nonfunctional? 4) Shape and size of the teats can be assessed. Sometimes in older mares the teats can become distorted in shape and very large. Clients may ask about the foal s ability to nurse these but it never seems to be an issue. The foals will suckle them successfully. 5) One side of the udder being nursed and not the other can be a client concern. Certainly some foals will start out this way but all will eventually suckle both sides masterfully. 6) Evaluation of the colostrum is usually best done by farm staff so that early supplementation with another colostrum source can be given in time. Examination of the Perineal Area Lifting the tail to observe the vulva and perineum is the next order of business. Obviously one aim is to assess the degree of trauma if any is evident. The results of trauma will fit in to three categories; generalized swelling (bruising and edema), hematomas and lacerations. Lacerations can be vulvar, vaginal or vestibular, cervical, uterine, or rectovaginal. (An experienced foaling attendant can help to prevent these problems or may alert you to look for some such as a rectovaginal perforation.) The findings upon examining the perineal area for trauma should lead you to some conclusions and some possible treatments. Maiden mares are more likely to be traumatized due to poorer relaxation, but certainly any mare can have generalized swelling and possibly hematomas in this area. As a result these mares are also more likely to be more painful after foaling. Any mare that is painful and or has evidence of trauma will be less likely to defecate normally. It is helpful to oil these mares in order to help ensure that they continue to pass stool and do not become constipated/impacted. Flunixin meglumine is my analgesic choice for these mares, which I will administer as often as twice daily (50mgs/100lbs) until they appear comfortable. Hematomas in this area always seem to resolve themselves and not require drainage. Lacerations require different approaches dependent where in the reproductive track they occur. The most unfortunate (but maybe the most common) are vulvar tears that result from a caslicks not being opened prior to foaling. Unfortunate because they were preventable. If lucky, the caslicks only will tear out, but many times the tear will affect the peirvulvar tissue and can be quite extensive. Obviously tears in to the perivulvar tissue can occur even if the caslicks was opened. Swelling and bruising to the tissue always accompany such tears or lacerations. Because of this I never attempt to repair these lacerations immediately but elect to wait. I want the swelling to totally subside as well as the granulation tissue that forms at the wound edges. I firmly believe that trying to repair these wounds in the face of this bruising or granulation tissue will lead to dehiscence of your initial repair and often a worse situation due to a supporative reaction and further tissue loss. This may be a controversial opinion in that some people believe it is paramount to close the wound in order to protect the mare s reproductive track from contamination. However, I have not realized this to be an issue of practical concern in these post foaling mares and believe that making a single, good closure is of paramount importance. If there is no evidence of perineal trauma I will not do a vaginal exam at this time. (An exception to this is if a dystocia had occurred which required fetal manipulation or fetotomy.) If the evidence of external trauma is significant a vaginal exam is in order. Vaginal and vestibular lacerations can be extensive. These 21

24 lacerations require a speculum and a manual vaginal exam to determine their full extent. Surgical intervention is inappropriate and treatment consists of initial anti-inflammatories and broad-spectrum antibiotics until healing has taken place. Cervical tears can be very difficult to assess until some initial involution of the cervix has occurred. Cervical tears can be disastrous if the full length of the cervical tunnel is compromised. Sometimes this can be realized quickly or in a matter of days post foaling. However, many cervical tears are incomplete and time is necessary to make this determination. Only several millimeters of competent cervical tunnel are necessary to maintain pregnancy. If the entire tunnel is compromised cervical repair will be required, but unfortunately not always successful depending on the severity of the trauma. Uterine tears will most often occur in a horn and will most likely not be evident for hours or even days. The general health of the mare will alert you to pursuing this diagnosis, as signs of peritonitis become evident. Surgical intervention to repair the tear or extensive medical treatment to support the mare while healing takes place are the treatment options. Rectal vaginal lacerations are evident when the tear is externalized. Broad-spectrum antibiotics are indicated until a healthy bed of granulation tissue is formed. Fluinixine meglumine and oiling is also indicated. Rectal vaginal perforations are often suspected by the foaling attendant because of a foal s foot either protruding from the rectum or at least being forced up into the rectum. A vaginal and rectal exam will confirm or refute the presence of a fistula. Initially the treatment is the same. These mares can be bred on a thirty-day heat if caution is used in the mating or if AI is appropriate. A high percentage of these fistulas will repair with time. If healing does not occur sufficient to repair the fistula the mare can be surgically repaired once in foal or at the conclusion of the breeding season. Obviously surgery can be performed earlier if desired but this will delay breeding longer. In addition to looking for evidence of trauma the presence of placental membranes should be noted. How much time elapses before considering a placenta to be retained is subjective and seems to range between one and eight hours. Most mares will deliver the placenta in less than two hours therefore I feel that anything past two hours is abnormal. Besides your observations, questions need to be asked of the staff to ascertain if the placenta has been dropped yet. If they affirm it has then the judgement can be made if you should examine it yourself. Knowledgeable, experienced personnel can be fully capable of examining the placenta and bringing any concerns to your attention.. A vaginal exam is indicated if the placenta has not been found or if it is suspected to be torn or missing a piece. It can be difficult to reach the tips of the uterine horns in a mare that has only recently foaled but an attempt should be made if suspicion exists that some placenta remains in the mare. As a general rule I do not enter the uterus unless I feel there is justification to do so. The endometrium is very friable at this stage and bleeds very easily. If the choice is made to manually enter the uterus covering your tracks with an infusion of broad-spectrum antibiotics is indicated. In the face of a retained placenta attempting manual removal is called for if done judiciously. Gentle traction on the placenta (or partial placenta), twisting it, or gently working your fingers between the placenta and endometruim are all techniques that may free the placenta. If the placenta is tightly adhered you will quickly know this and should stop your efforts. If placenta is left behind after this initial attempt an infusion of antibiotics is indicated and systemic antibiotics and anti-inflammatories should be considered. Administration of oxytocin is indicated with a retained placenta as well. Repeated doses every 4 hours of 20 to 40 units given IM will often cause release of the retained placenta. A single dose of 100 units oxytocin added to a liter of saline given IV can have dramatic results almost always causing rapid release of a retained placenta. The use of oxytocin prior to attempting manual delivery of the placenta is probably desirable in most instances and the IM doses of oxytocin can be administered by farm personnel. 22

25 Non-Reproductive Considerations Mares with prior history of laminitis can suffer recurrent laminitis with seemingly little provocation other than parturition. However with signs of laminitis occurring a thorough examination should be conducted to try to ascertain the cause, such as retained placenta, severe endometritis or metritis. Certainly there are breed predilections for laminitis and in these mares a retained placenta can be thought of as an emergency. These laminitic mares must be treated for the laminitis as well as for the inciting cause if one can be ascertained. Certainly the post foaling mare can be painful and must be differentiated from other sources of pain. The mare that is only painful most often will have a normal or only slightly elevated heart rate, normal motility and normal mucous membrane color and refill time. These mares are usually quite responsive to flunixin meglumine as well. Other sources of pain can be uterine artery bleeding, colic and peritonitis. Mares that are bleeding from a uterine artery rupture will be painful and will usually exhibit a noticeably anxious behavior. Keeping these mares as quiet as possible, in their familiar surroundings and treating them medically is indicated. Medical treatment can consist of a combination of naloxone (12mgs IV), aminocarpoic acid (3 vials in I liter fluids) and one liter of a 5% formalin solution given slowly IV. The post foaling mare can suffer from routine types of colic that can be monitored and treated medically. These mares should certainly be monitored closely and body temperature, response to treatment and a CBC are important parameters in assessing the postpartum colic. Generalities can be applied to these mares that can be quite accurate. One should be suspicious of a ruptured bowel in the violent or severe colic that occurs soon after foaling. The mare that deteriorates over 24 hours (or even days), possibly exhibiting colic symptoms as well as depression and fever should be suspected as having a uterine perforation. Obviously there is no substitution for a complete physical exam that should include blood work and an abdominocentesis. Uterine Discharge Vulvar discharge from the post foaling mare needs to assessed not only for the character but the volume of that discharge. The presence of other clinical signs such as fever, malaise or even laminitis are also an important part of this assessment and when present are often indicative of the need for systemic treatment of a minimum of antibiotics and anti-inflammatories. Lochia A discharge of normal post foaling lochia can be brought to the attention of the veterinarian occasionally especially when it is voluminous. If determined that it is lochia and not purulent, systemic oxytocin will be all that is necessary to aid in its expulsion. Hemorrhagic Discharge At times the post foaling mare may exhibit a discharge consisting of frank blood. Upon further evaluation the uterus may be found to be filled with blood which continues to spill through the vulvar lips. The source of this bleeding is the endometrium and these mares don t seem to be in danger of life threatening blood loss. With time, and as uterine involution occurs the bleeding stops and all is evacuated from the uterus. Administration of oxytocin will help with this process. Although the frank blood would appear to be a wonderful media for bacterial pathogens, uterine infection is not prevalent in these mares. More commonly a heommorhagic discharge has the character of old blood with a dark, tarry appearance. This may be indicative of more endometrial hemorrhage than normal or slower clearance than normal, but treatment with systemic oxytocin is sufficient for these mares. 23

26 Purulent Discharge A purulent discharge can vary in character and certainly in volume. Fever and malaise will often accompany these especially if the volume is large. Manual evacuation of the uterus is indicated along with flushing and infusion with antibiotics. Oxytocin administration is helpful. Systemic antibiotics and antipyretics are also indicated. Treatment should be continued each day until the discharge has been resolved. Although breeding the foal heat will be compromised, most of these mares will do well and be ready to breed by the thirty day heat or sooner. The incidence of purulent discharge is going to be greater in mares that have compromised uterine clearance. Mares that have ambulatory difficulties inhibiting their ability to be normally active will also have a greater tendency to acquire such a uterine discharge. Mares that are confined to the stall post foaling due to weather or difficulties with the foal are at higher risk to develop purulent uterine discharge. It should be noted that not all purulent discharges have their origin in the uterus. Mares that have suffered vaginal or vestibular lacerations will often times discharge pus as well as necrotic tissue tags. Broad spectrum systemic antibiotics as well as possibly analgesics and anti-inflammatories are indicated in these mares. General Management of the Post Foaling Mare As mentioned above, turnout for these mares is very important and should be practiced whenever possible. Uterine clearance is greatly enhanced when the mare is able to run and is free to get up and down and roll. Daily oxytocin administration to all post foaling mares will reduce the incidence of uterine discharge. Many factors can influence the decision to breed a mare on foal heat. Artificial insemination vs natural cover, the stallion s behavior when breeding natural cover, whether the semen is fresh, frozen or shipped, the fertility of the semen, the time of the year, etc. These are management decisions as well as veterinary ones. However how appropriate the mare is as a candidate for foal heat breeding should ultimately be determined by a reproductive exam. Mares that experienced problems foaling or since foaling are poor candidates for foal heat breeding. The lead time for booking the mare or ordering the semen will determine when the mare should first be checked on foal heat, but often day six or seven is most appropriate. Earlier can result in mares getting unfairly rejected and later can result in missing mares due to ovulation. The perineal area should be evaluated for evidence of trauma and discharge. A speculum exam should be performed on all of these mares to determine if evidence of cervical, vaginal or vestibular trauma exists and the presence or absence of a uterine discharge. A rectal exam will reveal the presence of an internal hematoma that may have occurred during foaling. The uterus can be assessed manually for size and tone, although these are subjective evaluations and their importance will differ according to the person performing the exam. An ultrasound exam is very helpful, not only to determine when to breed the mare, but to determine if there is fluid present in the uterus, which would render her a poor foal heat candidate. Management decisions can influence whether the borderline candidate is bred or not but to be fairly discriminate about which mares are bred on foal heat can be a useful rule of thumb. Even if the criteria are fairly stringent on which mares are good foal heat candidates, most mares will probably make the cut. 24

27 Bovine Viral Diarrhea Virus Update, Syndromes, Approaches, and Control/Vaccinations VICTOR S. CORTESE, D.V.M., PH.D., DIPLOMAT, ABVP (DAIRY PRACTICE) Director Cattle Business Unit Technical Services Pfizer Animal Health, Exton, PA INTRODUCTION Our knowledge of the bovine virus diarrhea virus is still changing, even though we have known about the virus for many years. One of the things we have learned is that the virus has the ability to mutate readily. The majority of BVD infections are mild or subclinical when they attack the cow or calf. The virulence of the strain and the susceptibility of the host determine the severity of the infection. The infection may be completely in apparent as is often seen in adult cattle or may cause a severe disease bordering on the appearance of mucosal disease. The one constant that appears with these infections is an immune suppression. Again the severity and duration of the immune suppression appears to be tied into the strain infecting the animal. In most of these infections if the animal is unexposed to other disease agents while undergoing the immune suppression it will recover, however if there is another disease agent present the mortality and morbidity rates can be greatly elevated. The respiratory syndrome looks very similar to IBR. The predominant signs are in the upper respiratory tract, primarily the trachea. It can involve the front section of the lungs but usually does not cause much pneumonia by itself. The newest syndrome is the thrombocytopenic syndrome. It is also caused the bleeder or hemorrhagic syndrome. In this syndrome the BVD virus attaches to platelets causing an increased destruction of the thrombocytes. These animals may start with a mild diarrhea or anorexia with a slight fever. The first sign is often bleeding into the conjunctiva. If the owner injects them the calf will often bleed from the injection site for several hours. On post mortem examination you will find hemorrhaging into the intestinal cavity or internal organs, you may find bleeding in the thoracic cavity or in large muscles. This is caused by a noncytopathic strain and these animals are not persistently infected. It appears to be primarily a disease of the Holstein breed. In order to understand the remaining syndromes it is necessary to review noncytopathic and cytopathic BVD. There are many different strains of BVD. These can be differentiated using monoclonal antibodies and then grouped into families based on common antigens. All BVD strains are divided into cytopathic (CP) and noncytopathic (NCP) strains. It appears that some of these strains have the ability to mutate from NCP to CP. Cytopathic BVD is rare in the cattle population. CYTOPATHIC VERSUS NONCYTOPATHIC STRAINS The CP/NCP differentiation is solely laboratory determined. When a CP strain is grown on a cell culture, the virus kills the cells, whereas a NCP strain does not. The NCP/CP designation does not relate to the virulence of the strain. Some of our most virulent strains in vivo at this time are NCP in vitro. Clinically you can t tell whether a NCP or CP strain is going through a herd. 25

28 REPRODUCTIVE SYNDROME These differences are still important, however, because of what it may indicate in the herd. The CP and NCP strains react most differently in the non-immune pregnant cow. If a non-immune cow is exposed to a NCP strain while in the first trimester of gestation, early embryonic death, abortion, mummification or persistently infected calves can result. If exposure occurs during the second trimester, birth defects, primarily involving nervous tissue, or occasionally persistent infection, are found. Infection during the last trimester usually has no effect on the fetus and the calf will be born with antibodies against BVD. Rarely, there is an overwhelming exposure that causes a late abortion. TYPE 1 VERSUS TYPE 2 Antigenic differences have been found on both the surface (strain variation) and internally on the GP 53 epitope. It is now felt that the pestiviruses can be split into four distinct groups that each shares approximately 65% homology with each other. These include type 1 BVD, type 2 BVD, true border disease in sheep, and hog cholera. These internal variations also contain surface variations that appear to be associated with each group. The type 1 versus type 2 designation also does not correlate with virulence. There can be severe death loss with either division depending on the strain. The only group specific disease is the thrombocytopenic form which is seen with only several type strains. The type two strains are not new but the classification is. PERSISTENT INFECTION When BVD infection occurs before the immune system has fully developed, the calf learns to recognize the virus as part of itself and never mounts an immune response against that particular strain of BVD virus. Persistently infected calves can be born normal and constantly shed the virus or they can be born weak and die. Persistently infected calves that appear normal can reach adulthood, breed and have persistently infected calves. They are also a constant source of viral shedding to the rest of herd. The current persistent infection rate in the United States among cattle under one year of age, is estimated at 1 ½ - 2%. This is similar to the death loss from mucosal disease seen in many feedlots. In some herds, 10-50% of the calves may be a carrier. In cow-calf and dairy operations reproductive failure is often the only sign of BVD exposure. Once an animal is persistently infected, nothing can eliminate the virus or stop its shed. MUCOSAL DISEASE In order for mucosal disease to occur a specific set of circumstances are required. First, the animal must be persistently infected. Then the animal must be exposed to another BVD virus that is cytopathic. Furthermore, new research indicates that this strain must be closely related to the noncytopathic strain causing the persistent infection to consistently cause mucosal disease. More antigenically distinct cytopathic BVD strains can cause this fatal disease but not as consistently. This exposure may be from additions to the herd or from a persistently infected animal spontaneously reverting to a cytopathic strain. This is the typical textbook BVD with which we are familiar with explosive diarrhea and ulcers through the digestive tract. Mortality rates with syndrome are 95% to 100%. 26

29 CHRONIC DISEASE In this form of BVD again persistent infection is a prerequisite. The animal again needs to be exposed to a cytopathic strain. It appears that if the strain is an intermediate in its antigenic relationship to the persistently infecting strain then a three to five month incubation period occurs that allows a recombination of the two strains and the chronic form to appear. These animals may begin with lameness involving multiple feet or with a mild, non-responsive diarrhea. It often appears like a Johnes case when it begins. The course of the disease is from one to two months and the mortality rate is very high with this complex also. There is nothing we can currently do to prevent mucosal disease or chronic BVD with vaccination or management except to minimize exposure if possible. DIAGNOSIS Diagnosis of BVD can be simple or difficult depending on the syndrome under investigation. Mucosal disease and Chronic BVD are fairly easy to get viral isolation from the blood if the lab is good. Also submission of ulcerated areas or Peyers patches will usually yield a positive FA. The newly developed microplate isolation and skin biopsy techniques are making the identification of persistently infected cattle easier, more rapid and more economical. Certainly BVDV eradication is possible in herds today. The diagnosis of subclinical BVD causing herd problems can be frustrating. The history will often give some clue. The most common history is that the herd has been experiencing a slow increase in reproductive problems manifested as early embryonic death with a few mummified calves and/or abortions. In some herds the first signs are higher than expected number of weak and stunted calves or increased calf morbidity and mortality from no one specific etiologic agent. Serology can be difficult to interpret in these herds. Often by the time a reproductive problem is diagnosed the exposure has already occurred. The other problem is that if the strain is antigenically different from the reference strain(s) used by the diagnostic laboratory you may get a false negative result. The level of a single sample titer can be useful to determine if more investigation is needed. Virus isolation can be very accurate if the timing of the sample corresponds to exposure and the laboratory performing the isolation is good. Confusion exists regarding vaccination programs because of the increasing number of BVD problemherds being diagnosed. Some of these herds have been on a killed BVD vaccination program, but have still seen a slow increase is reproductive problems over a two to three year period. There are probably several reasons. One has to do with our ability to better diagnose these infections in the laboratory, particularly the noncytopathic strains. In addition more samples are being submitted for testing. These samples include whole herd testing to find persistently infected animals. Last, we are seeing an increase in the number of persistently infected cattle on farms in the United States, probably due to strain variation, the virus s ability to mutate and correspondingly shortened duration of immunity. Our knowledge about ability of the different vaccines to protect against BVD infection is increasing rapidly. Recent studies have shown that the duration of immunity afforded by the killed vaccines is dependent on the antigenic similarity between the vaccine strain and the wild type virus to which the cow is exposed: If there are few common proteins, this protection can be as short as four months; if there are many common antigenic sites, it may last a year. Unfortunately, many persistently infected cows have strains that are antigenically distant (i.e. type 2) to our vaccine strains (i.e. majority are type 1). Cross protection between type 1 vaccines and type 2 strains has been demonstrated. Thus, these cows are a constant source of infection against which herdmates, if vaccinated with a killed vaccine annually, have little protection. The same holes in protection are found with modified live virus (MLV) vaccines but they do not become apparent until 12+ months after vaccination, closer to the time of annual revaccination and past the time when the virus can cause problems in early pregnancy. 27

30 These limitations primarily affect the cow-calf and dairy practitioners; the majority of feedlot veterinarians already use MLV BVD vaccines and these shortcomings are seen if exposure occurs during pregnancy. A total of four reproductive BVD studies have been performed using inactivated BVDV vaccines. Two studies have used modified live BVDV vaccines. Only the last studies gave protection at an acceptable level. If you have an open herd or a herd that has had diagnosed BVD problems, you have three options to maximize protection: 1. Increase the frequency of vaccination with the killed vaccines to three times a year. Rotation is probably not necessary with this vaccination schedule. 2. Give a MLV BVD vaccine to the open cow three weeks before breeding or on an annual basis. 3. Institute a virus isolation and cull program along with a vaccination program that includes an MLV BVD to all replacement animals. I start with the young stock and work backwards up the herd to minimize the farmers costs of testing. If killed vaccines are only to be given annually, vaccines containing multiple strains will provide broader protection. This protection may still be of insufficient duration to protect against reproductive problems. VACCINATION AND MUCOSAL DISEASE Mucosal disease is seen when an animal that is persistently infected is exposed to another closely related cytopathic strain of BVD. Theoretically, an animal can also have a spontaneous mutation of the noncytopathic BVD strain involved to a cytopathic strain, thereby causing mucosal disease without any subsequent exposure. High stress and immune depression may be involved in this reversion. One of the major concerns of using MLV vaccines is whether they have the ability to cause mucosal disease. I have never seen this in all the doses I have used in dairy animals. Many of you are also using them without difficulty. Dr. Bolin tried and failed to cause mucosal disease in persistently infected calves by vaccinating with MLV BVD vaccines. It appears that in order for mucosal disease to occur, the CP strain in the MLV vaccine must be closely related to the NCP strain in the persistently infected animal. With the degree of attenuation of the MLV vaccines today, a second set of circumstances is needed. The second predisposing factor is the background of the animal being vaccinated. If the animal is nutritionally deficient, persistently infected and severely stressed, the likelihood of inducing mucosal disease with vaccine may be higher. All of this doesn t mean that the MLV vaccines can t cause mucosal disease, but it does suggest that specific set of circumstances is required and that disease production, if it occurs, is rare. You do have options when it comes to BVD vaccination, and you need to realize the limitations of each approach. If a quick and accurate test for BVD is devised, we may be able to start an eradication program. In a small herd, a program of virus isolation, culling and annual vaccination is an attractive option for handling BVD problems. In large herds, the cost of testing may be prohibitive. In such cases, we must assume carriers are present and vaccinate accordingly. SUMMARY Bovine viral diarrhea virus infection is being diagnosed with increasing frequency. The number of herds containing persistently infected carriers is also on the rise. Recent research has shown that killed bovine virus diarrhea vaccines have a duration of immunity as short as four months following vaccination. This may partly account for the increase in infected herds. In order to maximize protection against bovine viral diarrhea virus killed vaccines must be given three times a year or a modified-live bovine virus diarrhea vaccine can be given annually to non-pregnant cattle. REFERENCES 28

31 REFERENCES 1. Bolin, S.R., Littledike, E.T., Ridpath, J.F., Serologic Detection and Practical Consequences of Antigenic Diversity Among Bovine Viral Diarrhea Viruses In a Vaccinated Herd. Am J Vet Res, 52(7): Corapi, W.V., Donis, R.O., Dubovi, E.J., Characterization of a Panel of Monoclonal Antibodies and Their Use In the Study of the Antigenic Diversity of Bovine Viral Diarrhea Virus. Am J Vet Res, Vol 51(9): Corapi, W.V., et. al., Thrombocytopenia and Hemorrhages In Veal Calves Infected with Bovine Viral Diarrhea Virus. JAVMA Vol 196(4): Perdrizet, J.A., et. al., Bovine Virus Diarrhea-Clinical Syndromes In Dairy Herds. Cornell Vet., 77: Bolin, S.R., The Current Understanding About the Pathogenesis and Clinical Forms of BVD. Vet. Med., Symposium on Bovine Viral Diarrhea, October 1990: Bolin, S.R., and Ridpath, J.F., Specificity of Neutralizing and Precipitating Antibodies Induced In Healthy Calves By Monovalent Modified-live Bovine Viral Diarrhea Virus Vaccines. Am J Vet Res, Vol 50(6): Ames, T.R., and Baker, J.C., Management Practices and Vaccination Programs That Help Control BVD Virus Infections. Vet. Med., Symposium on Bovine Viral Diarrhea, October 1990: Moennig, V., et.al., Reproduction of Mucosal Disease With a Cytopathogenic Bovine Viral Diarrhea Virus Selected In Vitro. Veterinary Record, 127: Bolin, S.R., et. al., Response of Cattle Persistently Infected WithNoncytopathic Bovine Viral Diarrhea Virus to Vaccination for Bovine Viral Diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea Virus. Am J Vet Res, Vol 46(12): Cravens, Ron and Bechtol, David, Clinical Response of Feeder Calves Under Direct IBR and BVD Virus Challenge: A Comparison of Two Vaccines and Negative Control, The Bovine Practitioner, No Brownlie,J, The pathogenesis of bovine virus diarrhea infections, Rev. sci. tech. Off. int. Epiz., 1990, 9 (1), Edwards, S., The diagnosis of bovine virus diarrhoea-mucosal disease in cattle, Rev. sci. tech. Off. int. Epiz., 1990, 9 (1), Kelling, Clayton, et. al., Investigation of bovine viral diarrhea virus infections in a range beef cattle herd, JAVMA, Vol 197, No. 5, September 1, Paton, D.J., Brockman. S. and L. Wood, Insemination of susceptibleand preimmunized cattle with bovine viral diarrhea virus infected semen,br. vet. J., (1990), Tarry, D.W., Bernal, L. and Edwards, S., Transmission of bovine virus diarrhoea by blood feeding flies, Veterinary Record(1991) 128, Bolin, S.R. and Ridpath, J.F., Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves, Am J Vet Res, Vol 53, No. 11, November Radostits, O.M. and Townsend, H.G., The Controversy Surrounding the role of the Bovine Virus Diarrhea Virus (B.V.D.V.) in the Pathogenesis of Pneumonic Pasteurellosis in Cattle, The Bovine Proceedings, No

32 18. Bolin, Steven R., BVD: What s The Latest, XVII World Buiatrics Congress, XXV American Association Of Bovine Practioners Conference Proceedings, 1992, Vol Vickers, Mary, BVD: Getting A Positive Diagnosis, XVII World Buiatrics Congress, XXV American Association Of Bovine Practitioners Conference Proceedings, 1992, Vol Mayling A, Rensholt L, Dalsgaard K, Jensen AM, Experimental exposure of vaccinated and nonvaccinated pregnant cattle to isolates of Bovine Viral Diarrhea Virus, McClurkin, AW, Coria, MF and Smith, RL, Evaluation of Acetylethyleneimine-killed bovine virak diarrhea-mucousal disease virus vaccine for prevention of BVD infection of the fetus. Proc US An Hlth, 79, Kaeberle, ML, Maxwell, D, Johnson, E, Efficacy of Inactivated Bovine Viral Diarrhea Virus vaccines in a Cow Herd. A.S Leaflet R Harkness, JW, et.al. The Efficacy of an Experimental Inactivated BVD-MD Vaccine, Frey, HR and Eicken, K, Undersuchungen uber die Wirksamkeit einer inaktivierten BVD-Vakzine zur Erhohung der Sicherheit einer BVD-Lebendvakzine, Tierarztl, Umschan, 50, (1995) 25. Cortese, V.S., Grooms, D.L., Ellis, J.A., Bolin, S.R., Ridpath, J.F., and Brock, K.V. Protection of pregnant cattle and their fetuses against infection with bovine viral diarrhea virus type 1 by use of modified live virus vaccine. Am J Vet Res 59(11): , Cortese, V.S., West, K.H., Hassard, L.E., Carmen, S.A., and Ellis, J.A. Clinical and immunologic responses of vaccinated and unvaccinated calves to infection with a virulent type-ii isoalte of bovine viral diarrhea virus. J.A.V.M.A 213(9): , Cortese, V.S., Whittaker, R., Ellis, J.A., Ridpath, J.F., and Bolin, S.R. Specificity and duration of neutralizing antibodies induced in healthy cattle after administration of a modified live virus vaccine against bovine viral diarrhea. Am J Vet Res 59(7): ,

33 Factors Affecting Pregnancy Rates in an IVF Embryo Transfer Program Cliff Lamb, Jamie Larson, Guilherme Marquenzini, Jose Vasconcelos University of Minnesota, Grand Rapids, MN, University of Sao Paulo, Botucatu, Brazil Summary The use of in vitro fertilized (IVF) embryos in embryo transfer programs is fast becoming an integral part of the industry. In recent years there have been many significant improvements in the development of new procedures that enhances viability and success of IVF embryos on a commercial basis. In the past, IVF was not a viable option for many producers because the rates of pregnancy success were significantly poorer than their in vivo produced counterparts. Therefore our goals were to assess factors that may or may not alter pregnancy rates in a large scale commercial IVF and MOET transfer facility. During the period from February 2002 to March 2004 data was collected on 13,485 transfers at several IVF facilities in central Brazil. Pregnancy rates to IVF transferred embryos were 35.9% compared to 32.4% for in vivo produced embryos. In addition to donor, recipient, and sire differences further factors that were assessed included corpus luteum (CL) score, right or left ovary transfer, embryo development stage, season of transfer, and synchrony between estrus and embryo transfer. Embryonic survival in from initial pregnancy diagnosis at 27 to 38 days of gestation until 90 days of gestation was 90%. Oocyte donor and technician had the greatest (P < 0.001) effect on overall pregnancy rates, but season of transfer and embryo stage also affected pregnancy rates significantly. Therefore, producers should be aware of the various factors affecting transfer of IVF embryos transferred to recipients on a commercial basis. Materials and Methods Between February 2002 and March 2004 data was collected from several IVF facilities in central Brazil. Twenty three experienced embryo transfer technicians transferred a total of 13,485 embryos in recipients that were detected in heat. Transfers were recorded for in vitro produced and in vivo produced embryos generated from 906 donors (1 to 215 transfers per donor), owned by 209 different owners. In addition, recipients were located at 34 separate locations. At the time of embryo transfer a quality score was applied to the CL of each recipient after palpation per rectum. Each single experienced embryo transfer technician used the following criteria to assign each CL a quality score: 1) the CL had a palpable diameter of >10 mm and firm or moderately firm consistency (excellent/good); or 2) the CL had palpable diameter of 10 mm, or the CL had a soft texture (poor). All recipients with any evidence of luteal tissue were designated to receive an embryo. All potential recipients were not estrous synchronized but were observed daily for signs of estrus. Each recipient deemed suitable for embryo transfer based on palpation per rectum received a single fresh in vitro (13,244 embryos) produced or in vivo (222 embryos) produced embryo using a standard embryo transfer technique in accord with the International Embryo Transfer Society (Savoy, IL). Oocytes or embryos had been collected from 906 donors. Embryos were transferred 5 to 9 days after detection of estrus. All embryos were assigned a developmental stage and quality grade according to standards set forth by the International Embryo Transfer Society (Savoy, IL). Developmental stage codes were: 3 = early morula; 4 = morula; 5 = early blastocyst; and, 6 = blastocyst. Embryos were transferred randomly to recipients without 31

34 regard to donor-recipient synchrony or the structure and integrity of the corpus luteum. Embryos were loaded individually into straws and one embryo transferred to each recipient. Initial pregnancy diagnosis occurred at between d 27 and 47 of gestation by transrectal ultrasonography, using a real-time, B-mode ultrasound scanner equipped with a 5 MHz linear-array transducer. The CATMOD procedure of SAS was used to analyze all data involving categorical pregnancy data. The effects of using in vitro versus in vivo derived embryos, stage of embryo development, embryo age and embryo-recipient synchrony (embryo factors) and all two-way interactions on pregnancy rate were determined. Because embryo type (in vitro vs. in vivo) was the only significant embryo factor, differences in pregnancy rates among recipients due to CL score was tested individually using the CATMOD procedure with embryo type as a co-main effect in each analysis. Figure 1. Effects of transfer technician on pregnancy rates of recipients receiving IVF embryos at first pregnancy diagnosis. Results and Discussion Major advance have been made in the last 30 years in embryo transfer and related biotechnologies related to the cattle industry throughout the world. Most recently the cattle industry has noted an exponential increase in the use of in vitro fertilization and transfer of embryos to recipient females. However, in the United States the largest reason for the failure of this technology to be adapted has been the poorer pregnancy rates compared to in vivo produced embryos transfer. The cost of maintaining cows that are not pregnant in a United States cattle operation tends to be the most expensive portion of the reproductive management program. However, with improvements in maturation and fertilization media, embryo transfer techniques, and with an overall more complete understanding of IVF results are within the range of in vivo embryo transfer. Therefore, establishing characteristics of IVF embryos and recipients that may alter pregnancy rates or assist in allowing the technology to be more accepted were the goals of this large-scale study. Overall pregnancy rates were similar between in vivo (32.4%) and IVF (35.9%) derived embryos. In a subset of 4,459 females that were diagnosed pregnant at between 27 and 38 days of gestation, 464 had lost their pregnancies between 85 and 100 days of gestation. Therefore, embryo/fetus loss was 10%. In addition, when pregnancy loss was assessed at various seasons embryo survival did not differ among seasons. The greatest source of variation (P < 0.001) was due to the embryo/oocyte donor and embryo transfer technician. As could be expected the range in pregnancy rates from each donor ranged from zero pregnancies to 100% 32

35 pregnancy rates in the 906 donors. Of the 23 embryo transfer technicians, the greatest pregnancy rates were 48% compared to the poorest pregnancy rates of 0.4% in 251 transfers (Figure 1)! Speculation has occurred that embryos transferred into the uterine horn located on one side may have a greater chance of resulting in a pregnancy than embryos transferred in the uterine horn located on the other side. Our results indicate that pregnancy rates were similar for embryos transferred into the left uterine horn (35%) compared to the right horn (36%; Figure 2). Figure 2. Effect of transfer location (left or right horn) on pregnancy rates for IVF embryos. Many embryologists use a scoring system to assess the competence of the corpus luteum, similar to the criteria used by the technicians in this evaluation. However, the results indicate that no difference exists based on CL score (Figure 3). In our previous report we determined that ultrasound evaluation of the CL revealed that 79% of the corpra lutea possessed a measurable fluid-filled lumen (Spell et al., 2001) and pregnancy rates did not differ among recipients with a CL containing fluid-filled lumen and those recipients without a lumen. Further, concentrations of circulating progesterone in recipients with a fluid-filled lumen were similar to those recipients without a visible lumen. These results indicate that the presence of a fluidfilled lumen does not represent a decrease in the possibility of a recipient accepting a transferred embryo. Although an earlier report (Remsen and Roussel, 1982) failed to indicate any correlation between CL size and pregnancy rate of recipients, they used palpation per rectum to determine CL size. 33

36 Figure 3. Significance of CL score on pregnancy rates of recipients receiving IVF embryos. Embryo stage had a significant effect on pregnancy rates. Younger embryos such as morulas (30%) and early blastocysts (28%) had porrer overall pregnancy rates that blastocysts (35%) and expanded blastocysts (39%; Figure 4). Synchrony between the embryo development and recipient demonstrated that pregnancy rates for younger embryos was greater when transferred into recipients sooner after ovulation, whereas blastocysts and expanded blastocysts had greater pregnancy rates when the interval from estrus to embryo transfer was at least 7 days. Perhaps an 8 day interval is more desirable for expanded blastocysts (Figure 5). Season of embryo transfer is a significant contributor to the success of an embryo transfer program. When separating the transfer information into various seasons, a marked difference is noted. During the 2002 and 2003 a similar trend was demonstrated. The poorest pregnancy rates were experienced when embryos were transferred during the winter months of April to September (Figure 6). During this period of time in Brazil there is little precipitation and cattle undergo a significant decrease in nutrients available as forage. Therefore, cattle tend to be maintained on a declining nutrient balance. The result is that cattle are losing weight and pregnancy rates are poor. The greatest pregnancy rates were noted during the late summer months, when rainfall has replenshied pastures and cattle have regained their body condition. Ultimately, the result is that the transfer of IVF embryos was more successful during this period of time. Figure 4. Effect of embryo development stage on pregnancy rates of IVF embryos. 34

37 Figure 5. Differences in pregnancy rates based on embryo and recipient synchrony by embryo development stage, with 0 being a recipient receiving a day 7 embryo 7 days after estrus. Figure 6. Effects of season on IVF embryo pregnancy rates during two successive years (2002, black bars; 2003, open bars). 35

38 Conclusion This summary of data reflects the common variations found in the current IVF transfer industry. From our analyses it is evident that every effort should be made to transfer more developed embryos into recipients that were detected in estrus at least 7 days prior to transfer. However, allowing asynchrony of 24 hours between the recipient and embryo does not appear to significantly decrease conception rates. In addition, the best gauge of the suitability of a potential embryo transfer recipient is an observed estrus and a palpable corpus luteum, regardless of size or quality. However, special consideration should be related to the season in which transfers occur and the obvious evidence that technicians effects will alter overall success of the IVF embryo transfer program. Literature Cited Spell, A.R., W.E. Beal, L.R. Corah, and G.C. Lamb Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle. Theriogenology 56: Remsen, L.G. and J.D. Roussel Pregnancy rates relating to plasma progesterone levels in recipient heifers at day of transfer. Theriogenology 18:

39 Application of Fixed-Time Artificial Insemination and Embryo Transfer Programs in Beef Cattle Operations G. A. Bó 12, L. Cutaia 12, P. Chesta 13, E. Balla 123, D. Picinato 13, L. Peres 13, D. Maraña 13, D. Moreno 1, G. Veneranda 5, R.J. Mapletoft 6, P.S. Baruselli 4 1 Instituto de Reproducción Animal Córdoba (IRAC), 2 Universidad Católica de Córdoba, 3 Universdad Nacional de Córdoba, 4 Departamento de Reprodução Animal, FMVZ-USP, Brazil, 5 Los Lazos S.A., Argentina, 6 Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. gabrielbo@iracbiogen.com.ar INTRODUCTION The main objective of implementing artificial insemination (AI) and embryo transfer (ET) technologies in beef operations is to produce a genetic progress in the herd. However, a small percentage of the beef animals are inseminated or receive an embryo. In Argentina for example, only 4.5% of the beef breeding females are AI and 80% of those inseminated are heifers (52). Among the main factors that affect the extensive use of these technologies are those related to nutrition, management and inefficient estrus detection. Probably the most useful alternative to significantly increase the number of animals involved in AI and ET programs is the use of protocols that allow for AI or ET without the need for estrus detection, usually called fixed-time AI (FTAI) and fixed-time ET (FTET) protocols. Also, the development of protocols for suckled cows will allow the inclusion of a significantly larger population of animals, and not just limit the application of these technologies to heifers. The intention of this manuscript is to review the currently available protocols that synchronize ovulation and discuss how they may impact on the effectiveness and application of bovine ET and AI programs, paying particular attention to those currently applied in South America. SYNCHRONIZATION OF ESTRUS AND OVULATION IN RECIPIENTS Prostaglandin F2α (PGF) has been the most commonly used treatment for synchronization of estrus in cattle (reviewed in 61). Early studies showed that the maturity of the CL at the time of PGF treatment influenced the luteolytic response and that PGF did not effectively induce luteolysis during the first 5 or 6 d following estrus (60). Furthermore, in cattle in which luteolysis did occur the ensuing estrus was distributed over a 6-day period (43). Recent studies have shown that the interval from PGF treatment to expression of estrus and ovulation is determined by the stage of development of the dominant follicle at the time of treatment (35). If PGF is given when the dominant follicle of a wave is in the late growing or early static phase, ovulation will occur within 3 to 4 d. On the other hand, PGF treatment given when the dominant follicle is in the mid- to late-static phase (i.e., when it is no longer viable) will result in ovulation of the dominant follicle from the next follicular wave 5 to 7 d later (35). This interval is a reflection of the time required for the dominant follicle of the new wave to grow and develop to a preovulatory state, and emphasizes that both luteal and follicular control is required to obtain high pregnancy rates in FTAI or FTET programs that do not require estrus detection. The treatments generally used for synchronization of recipients consisted of the administration of PGF 11 to 14 d apart (24). If all the recipients are cycling, about 80% should show signs of estrus within 5 d of treatment. However, due to the low accuracy of estrus detection, about 50% of the treated recipients usually 37

40 have a CL and receive an embryo 7 d after estrus (16). This situation could be even worst if the recipients used are Bos indicus or Bos indicus crosses under pasture conditions. Table 1 exemplifies the rates of recipients transferred over those treated in a large commercial embryo transfer program in Brazil and Bolivia. The overall pregnancy rate was about 13% and was due largely to the low number of recipients seen in estrus and/or with a CL at the time of embryo transfer (Burry personal communication, cited in 21). This inefficiency greatly affects the feasibility of these programs in cattle kept under pasture conditions in South America. Table 1. Pregnancy rates in recipients receiving an embryo 6 to 8 d after an observed estrus following a synchronization treatment with PGF (adapted from 21). In estrus/ Transferred/ Pregnant/ Pregnant/ Treated (%) Treated (%) Transferred (%) Treated (%) Matto Grosso 384/ /854 89/226 89/854 (Brazil, 2000) (45.0%) (26.5%) (39.4%) (10.4%) Santa Cruz 479/ / / /700 (Bolivia, 2001) (68.4%) (31.9%) (49.8%) (15.9%) Total 863/ / / /1554 (55.5%) (28.9%) (44.5%) (12.9%) HORMONAL TREATMENTS THAT CONTROL FOLLICULAR WAVE EMERGENCE AND SYN- CHRONIZE OVULATION. GnRH It has been shown that GnRH will induce ovulation or luteinization of the largest follicle present at the time of treatment (44), and research efforts have been devoted to the development of treatment protocols that utilize GnRH and PGF for FTAI in beef and dairy cattle (62, 63, 68, 70, 73). This treatment protocol is known as the Ovsynch treatment (63) and consists of an injection of GnRH followed by PGF 7 d later, a second injection of GnRH 48 h after PGF treatment and fixed-time AI 16 h later. The rationale for the treatment is that the first injection of GnRH will induce LH release, resulting in ovulation of the dominant follicle and emergence of a new follicular wave within 2 d. The administration of PGF 7 d later is to induce luteolysis, and the second GnRH injection is to induce LH release and synchronize ovulation of the new dominant follicle. Others have used a similar protocol in beef cattle with an interval of 6 d between the first GnRH treatment and PGF (55, 79). The Ovsynch protocol has been more efficacious in cows than in heifers (63, 55). Results of a recent study confirmed that GnRH does not always result in ovulation or luteinization of the dominant follicle in heifers and the emergence of a new follicular wave was synchronized only when treatment caused ovulation (53). Hence, ovulation following the second GnRH treatment may be poorly synchronized if the first GnRH treatment does not synchronize the emergence of a new follicular wave. In addition, it has been reported that some heifers may show signs of estrus before the second GnRH treatment. Prevention of the early ovulations by addition of a CIDR-B (Pfizer Animal Health) device to a 7-day Ovsynch program has been shown to significantly improve pregnancy rates in heifers after FTAI (55). GnRH-based protocols have also been used to synchronize ovulation in recipients that received in-vivo- 38

41 (3, 34, 84) or in-vitro- (1) derived embryos. In two studies, Bos indicus x Bos taurus crossbred heifers (3, 84) and cows (84) were treated with a single dose of PGF and observed for estrus for 5 d or with an GnRHbased protocol without estrus detection. Seven days after estrus (PGF group) or after the second GnRH treatment (Ovsynch group), recipients with an ultrasonically detectable CL (3) or a palpable CL on rectal examination (84) were selected for embryo transfer. All selected recipients received frozen-thawed embryos by Direct Transfer. The overall pregnancy rate was higher in recipients treated with the Ovsynch protocol (Table 2); more recipients received embryos because the Ovsynch protocol was not dependent on the detection of estrus. It is also noteworthy that in one of the studies (3), 53.7 % of the heifers treated with PGF were observed in estrus, a reflection of the difficulty of estrus detection in Bos indicus-derived cattle (20, 25). Table 2. Pregnancy rates in recipients receiving embryos by Direct Transfer 7 d after a detected estrus following PGF treatment or following an Ovsynch treatment protocol without estrus detection (adapted from 3 and 84). Group n Transferred/Treated Pregnant/Transferred Pregnant/Treated (%) (%) (%) Heifers (6) PGF/estrus detect /177 (45.2 %) a 45/80 (56.3 %) a 45/177 (25.4%) a Ovsynch /168 (72.6 %) a 60/122 (49.2 %) a 60/168 (35.7 %) b Heifers (76) PGF/estrus detect 75 50/75 (66.6 %) 34/50 (68.0 %) 34/75 (45.3 %) Ovsynch 43 32/43 (74.4 %) 24/32 (75.0 %) 24/43 (55.8 %) Cows (76) PGF/estrus detect 58 30/58 (51.7 %) 18/30 (60.0 %) 18/58 (31.0 %) Ovsynch 55 35/55 (63.6 %) 20/35 (57.1 %) 20/55 (36.4 %) Overall PGF/estrus detect /310 (51.6 %) a 97/160 (60.0 %) 97/310 (31.3 %) a Ovsynch /266 (71.1 %) b 104/189 (65.0 %) 104/266 (39.1 %) b ab Proportions in columns, within experiments, or overall, with different superscripts differ significantly (P<0.05). In another study (9, 34), 499 lactating Bos taurus crossbred cows (28 to 92 d post-partum) were assigned to 1 of 3 treatment groups. Cows in the Control group were treated with GnRH on Day 0, PGF on Day 7 and detected for estrus using a Heat-Watch system (GnRH+PGF). Cows in the other 2 groups were treated with an Ovsynch protocol alone (Ovsynch) or an Ovsynch protocol plus a norgestomet (SMB, Syncro-Mate-B, Merial) implant for 7 d (Ovsynch+P) and were not observed for estrus. Six to 8 d after estrus (GnRH+PGF group) or 7 d after the second GnRH treatment (Ovsynch and Ovsynch+P groups), cows were examined by rectal palpation and those with a palpable CL received Grade 1 or 2 frozen-thawed embryos by Direct Transfer. Although the pregnancy rate of cows that received an embryo (recipients pregnant/recipients transferred) tended (P<0.07) to be higher in cows in the GnRH+PGF group, the overall pregnancy rates were not significantly different among groups, mainly because of the significantly larger (P<0.01) number of recipients selected for embryo transfer in the Ovsynch and Ovsynch+P groups (Table 3). The same investigators have also done field trials involving 1637 recipients treated with an GnRH-based treatment protocol plus a SMB ear implant or a CIDR-B device without estrus detection; overall pregnancy rate was 59.9 % (10). In summary, results of these studies indicate that acceptable pregnancy rates can be achieved when embryos are transferred to recipients that have received treatments that synchronize ovula- 39

42 tion, without the necessity of estrus detection. Figure 1 outlines the treatment protocol. Table 3. Pregnancy rates in recipients receiving an embryo by Direct Transfer 6 to 8 d after an observed estrus following GnRH on Day 0 and PGF on Day 7, or 7 d after the second GnRH injection in an Ovsynch protocol without (Ovsynch) or with a SMB implant (Ovsynch+P), without estrus detection (adapted from 9). Group n Transferred/treated Pregnant/transferred Pregnant/treated (%) (%) (%) GnRH+PGF /169 (63.9 %) a 67/108 (62.0 %) c 67/169 (39.6 %) (estrus detection) Ovsynch /165 (90.9 %) b 72/150 (48.0 %) d 72/165 (44.0 %) Ovsynch+P /165 (92.1 %) b 82/152 (53.9 %) d 82/165 (49.7 %) Proportions within columns with different superscripts differ ( ab P<0.01; cd P<0.07). GnRH PGF GnRH Fixed-time Embryo Transfer Progestogen implant or Progesterone device Treatment days Figure 1. Treatment protocol for fixed-time embryo transfer in cattle with an Ovsynch program or Ovsynch plus a progestogen/progesterone-releasing device (Ovsynch+P). The Ovsynch treatment consists of an injection of GnRH on Day 0, followed by PGF on Day 7 and a second injection of GnRH on Day 9. In the Ovsynch+P treatment a progestogen ear implant or a progesterone-releasing device is placed on Day 0 and removed on Day 7. Estrus is not observed and embryo transfer is performed on Day 16; recipients with a CL receive an embryo. Progesterone Studies performed to evaluate different progestogen/progesterone (P4) protocols have shown that treatments that are long enough to allow normal regression of the CL (i.e., 14 d), would induce synchronous estrus. However, these treatments were associated with the development of oversized (persistent) dominant follicles (27, 37, 69,72) and low fertility following AI (67, 82). The lowered fertility has been attributed to spontaneous maturation (i.e., germinal vesicle breakdown and cumulus expansion) of oocytes within the persistent dominant follicles (65). Treatments that induce regression of the persistent follicle and resulted in emergence of a new follicular wave improved pregnancy rates in AI programs (36, 41). Based on the results from one study, it was originally thought that persistent follicles did not affect the quality of the CL and pregnancy rates following ET (80). However, the overall pregnancy rates in this study were low (about 30% in all treatment groups) and results from a recent study have shown that the large CL derived from persistent follicles may be inadequate in some way and pregnancy rates were lower than normally ovulating control recipients (48). 40

43 Progesterone and Estradiol Estradiol and P4 treatments have been increasingly used over the past several years in estrus synchronization programs in beef and dairy cattle (47, 13, 26, 36, 55). Treatments consist of insertion of a P4 device and the administration of estradiol and P4 on Day 0 (to synchronize follicular wave emergence and avoid the development of persistent follicles), PGF at the time of device removal on Days 7, 8 or 9 (to ensure luteolysis), and the subsequent application of a lower dose of estradiol 24 h later (28) or GnRH/LH 48 to 54 h later (13, 55) to synchronize ovulation. Pregnancy rates to a single FTAI have been similar to those expected after detection of spontaneous estrus (36). A series of experiments were designed to evaluate the possibility of applying the estradiol and P4 protocols currently used for FTAI to synchronize recipients subjected to embryo transfer without estrus detection. In one experiment (76), cows in the estradiol/p4 group received a CIDR-B device combined with 2 mg estradiol benzoate (EB) and 50 mg injectable P4 im on Day 0, PGF at the time of CIDR-B removal on Day 7, and 1 mg EB on Day 8. Estrus was not observed and Day 9 was arbitrarily considered to be the day of estrus. Cows in the Control group were treated with 2 doses of PGF 14 d apart and observed for signs of estrus for 5 d. Seven days after observed estrus (PGF group) or the expected time of estrus (CIDR-B group), all cows with an apparently functional CL (>15 mm as estimated by rectal palpation) received frozen/thawed embryos by Direct Transfer. Overall pregnancy rates (recipients pregnant/recipients treated) did not differ between treatments (PGF: 32.0% vs CIDR-B: 37%; P>0.6). These results were further confirmed in two experiments in which no differences were detected between recipients treated with CIDR-B for 7 or 8 d (15) and DIB (Syntex, Argentina) devices for 8 d. (15). Although the use of estradiol and P4 has eliminated the need for estrus detection, overall pregnancy rates have remained 20 to 35%, requiring improvement (16). Therefore, two experiments were designed to determine if the time of PGF treatment in estradiol and P4 protocols would affect the number of recipients selected for embryo transfer. Cows received a P4 releasing device (DIB) and an injection of 2 mg EB and 50 mg P4 im on Day 0 and were randomly assigned to receive PGF on Day 4 (expected time of wave emergence) or on Day 8 (at the time of DIB removal). On Day 9, all cows received 1 mg EB im. Day 10 was considered the day of estrus; therefore, embryos were transferred on Day 17 in all recipients with a CL 12 mm in diameter. In the first experiment, in which follicular development was monitored by daily ultrasonography, giving PGF to initiate luteolysis at the time of wave emergence (Day 4) instead of the time of P4 device removal (Day 8), increased the diameter of the dominant follicle (13.2±0.2 mm vs. 11.5±0.2 mm; P<0.05); hastened the time of ovulation (66.6±0.4 h vs 70.8±2.3 h; P<0.01) and resulted higher plasma P4 concentrations at the time of embryo transfer (6.9±0.8 ng/ml vs 5.2±0.6 ng/ml; P=0.08; 57). In the second experiment, treatment with PGF on Day 4 resulted in an increased proportion of recipients selected for transfer (70.5% vs. 52.7%; P<0.02) and pregnancy rate (41.1% vs. 21.5%; P<0.004) compared to those that received PGF on Day 8. Furthermore, in a subgroup of recipients plasma P4 levels were higher in recipients treated with PGF on Day 4 (6.8±0.7 ng/ml; n=43) than in those treated with PGF on Day 8 (4.0±0.4 ng/ml; n=27; P=0.008). Treatments with ecg to increase circulating progesterone concentrations and pregnancy rates. There have been several studies investigating the relationship between circulating P4 and pregnancy rates in cattle (reviewed in 73). However, the use of supplementary P4 has resulted in inconsistent effects on pregnancy rates. Insertion of a CIDR-B device at the time of embryo transfer resulted in a 12.8% increase in pregnancy rates in cycling, lactating dairy recipients (46). However, we were unable to demonstrate any benefit of CIDR-B insertion in dairy cows, heifers or crossbred Bos indicus heifer recipients (75). Bousquet 41

44 (personal communication) was only able to show a benefit of supplemental P4 after the transfer of poorer quality embryos, and Fuentes (31) reported higher pregnancy rates only when a PRID was inserted in heifers with lower quality (smaller size) CL. In a more recent study, the insertion of a CIDR-B from Days 7 to 20 of the estrous cycle did not result a significant increase in plasma P4 concentrations in heifers (49). An alternative strategy to increase circulating progesterone concentrations is to induce an accessory CL by inducing ovulation of the dominant follicle with hcg, or by insertion of Deslorelin implants on Day 5 of the estrous cycle (reviewed in 73). In Bos indicus recipients, treatment with hcg on Day 7 increased P4 concentrations (49) and in another study (50), treatment with GnRH, hcg, plh or a CIDR-B device increased pregnancy rates. However, in another study involving Bos Indicus x Bos taurus treated with P4 devices EB and ecg (78) pregnancy rates where not increased by treatment with hcg or GnRH at the time of FTET. Mapletoft (personal communication) was also unabled to improve pregnancy in Bos taurus recipients, treated with GnRH, or plh on Days 5 or 7 after estrus. Taking all studies into consideration, the beneficial effects of higher P4 seem to be evident only when pregnancy rates in controls (not treated) recipients were lower than expected, suggesting that body condition score, cyclicity and/or embryo quality may have been contributing factors.t Another approach to increasing circulating P4 concentrations in recipients is to induce multiple ovulations by an injection of ecg during the synchronization protocol. Fuentes and de la Fuente (32) first reported that treatment of Holstein heifers with 1000 IU of ecg on Day 4 of a 6.5-day treatment protocol with PRID devices and estradiol-17β (E-17β) resulted in multiple CL (2 to 5 CL per ovary), more (P<0.05) recipients selected for embryo transfer (89.7%) and a higher overall pregnancy rate (58.6%), as compared to heifers treated similarly but without ecg (49.1% and 22.2%, respectively), those treated with one PGF injection (44.8% and 19.0%, respectively) or those receiving an embryo 7 d after natural estrus (50% and 28.8%, respectively). In another study (4), Bos taurus x Bos indicus heifers were treated with a CIDR-B device for 7.5 d, combined with 2 mg EB and 50 mg P4 im on Day 0. Half of the heifers received 800 IU of ecg on Day 5 and all heifers received PGF on Day 7 and 1 mg EB im on Day 8. All animals were examined by ultrasonography 1 d prior to embryo transfer and a blood sample was taken for plasma P4 determination. Results, shown in Table 4, revealed that ecg treatment resulted in increased numbers of ovulations (CL numbers), plasma P4 concentrations and pregnancy rates. Table 4. Mean (± SEM) number of CL, plasma progesterone (P4) concentrations (1 d prior to embryo transfer) and pregnancy rates in recipients treated with CIDR-B devices and estradiol/p4 on Day 0, with or without 800 IU of ecg given on Day 5 (adapted from 4). Groups n CL numbers P4 Selected/treated Preg./transferred Preg./treated numbers (ng/ml) (%) (%) (%) Control ±0.5 a 1.3±0.8 a 17/50 (34.0) a 5/17 (29.4) 5/50 (10.0) a ecg ±2.9 b 4.2±3.7 b 42/50 (84.0) b 21/38 (55.3) 21/50 (42.0) b ab Means or percentages within columns with different superscripts differ significantly (P<0.05). It is noteworthy that, considering only the recipients with 1 CL, the area of the CL (P<0.01) and plasma P4 (P=0.1) concentrations were also greater in heifers treated with ecg (2.9 ± 0.6 cm 2 and 2.3±1.6 ng/ml; n=8), than in those not treated with ecg (2.2±0.5 cm 2 and 1.3±0.8 ng/ml; n=17). 42

45 A follow up study was designed to evaluate whether a lower dose of ecg would result in increased pregnancy rates in recipients following FTET. Bos taurus x Bos indicus crossbred cows were treated with a DIB device combined with 2 mg EB and 50 mg P4 im on Day 0. Half of the cows received 400 IU of ecg im and all received PGF on Day 5. DIB was removed on Day 8 and 1 mg EB im was given on Day 9. All cows were examined by ultrasonography 1 d prior to embryo transfer. Although this treatment did not result in as many multiple ovulations as in the previous study, ecg treatment resulted in increased CL diameters and pregnancy rates (Table 5). A subset of 55 recipients that were transferred was also bled for plasma P4 determination. Plasma P4 in recipients treated with ecg that had 2 or 3 CL (30.2±8.2 ng/ml; n=8) or only 1 CL at time of embryo transfer (7.5±0.7 ng/ml; n=18) was significantly higher (P<0.01) than in those recipients not treated with ecg (5.7±0.4 ng/ml; n=29). Table 5. Mean (± SEM) CL diameters (1 d prior to embryo transfer) and pregnancy rates in recipients treated with DIB devices and estradiol/progesterone on Day 0, with or without 400 IU of ecg given on Day 5 (adapted from 77). Group n CL a Transferred/treated Pregnant/transferred Pregnant/treated (mm) (%) (%) (%) Control ±0.4 b 127/156 (81.4%) b 53/127 (41.7%) b 53/156 (33.9%) b ecg ±0.4 c 132/156 (84.6%) b 76/132 (57.6%) c 76/156 (48.7%) c a CL diameters of the recipients transferred (CL 10 mm) as measured by ultrasonography. bc Means or percentages within columns with different superscripts differ significantly (P<0.02). In another experiment, we compared pregnancy rates following FTET in cows induced to ovulate with either EB or hcg (58). Bos taurus x Bos indicus crossbred beef cows with a body condition score between 2.5 to 3.5 (1 to 5 scale) were used. At the beginning of each replicate (Day 0), all cows received a DIB device and 2 mg EB plus 50 mg P4 im. All cows also received 400 IU of ecg im plus 500 µg cloprostenol on Day 5 and DIB devices were removed on Day 8. Cows were randomly divided to receive 1 mg EB im on Day 9 or 1500 IU hcg (Ovusyn, Syntex Argentina) on Day 10. As in the previous experiments, recipients were not observed for signs of estrus. On Day 17, all recipients were examined by ultrasonography and those with a CL >12 mm in diameter received fresh or frozen/thawed Direct Transfer embryos. Mean (± SEM) CL diameters on Day 17 were not different (P>0.1) between recipients induced to ovulate with EB (21.1±0.6 mm) or hcg (21.3±0.5 mm). There were no significant effects of fresh vs frozen embryos, embryo quality, or technician on pregnancy rates (P>0.4). Furthermore, pregnancy rates were not different between recipients induced to ovulate with EB or hcg (Table 6; P>0.9). It was concluded that the two treatments evaluated are equally efficacious to synchronize ovulation in Bos taurus x Bos indicus recipients. Table 6. Pregnancy rates in recipients treated with DIB devices for 8 d and estradiol/progesterone on Day 0, 400 IU of ecg on Day 5 and induced to ovulate with EB on Day 9 or hcg on Day 10. Embryos were transferred on Day 17 (adapted from 58). Group n Transferred/treated (%) Pregnant/transferred (%) Pregnant/treated (%) EB /106 (85.8) 52/91 (57.1) 52/106 (49.1) hcg /109 (82.5) 48/90 (53.3) 48/109 (44.0) Proportions did not differ (P>0.9). 43

46 Although the previous studies have shown the efficacy of this treatment protocol, it required that the cows be run through the chute at least four times for treatments. Therefore, a recent study was done to simplify the treatment protocol, by reducing the number of injections or the number of the days required for treatments. The first objective was to compare pregnancy rates in recipients treated with DIB devices and 400 IU of ecg given on Day 5 (1 d after wave emergence) or Day 8 (DIB removal, 59). A secondary objective was to determine the effect of injectable P4, given im at the time of insertion of the DIB device plus EB treatment, on the same end points. Crossbred Bos taurus x Bos indicus beef heifers were randomly assigned to 1 of 4 treatment groups in a 2 by 2 factorial design. All heifers received a DIB device plus 2 mg EB im on Day 0, with or without 50 mg of P4 given im at the same time. Heifers were further subdivided to receive 150 µg D (-) cloprostenol im and 400 IU of ecg im on Day 5 or on Day 8. In all heifers, DIB devices were removed on Day 8 and 1 mg EB im was administered on Day 9. On Day 17, all heifers were examined by ultrasonography to determine the number of CL and those with more than 1 CL or a single CL with a diameter >18 mm received an in vitro produced (IVP) embryo by non-surgical transfer performed by the same veterinarian. The proportion of recipients transferred/treated (P=0.15) and pregnant/transferred (P=0.23) were not different. However, the overall pregnancy rate (recipients pregnant/recipients treated) tended (P=0.1) to be higher in heifers treated with ecg on Day 5 than in those treated with ecg on Day 8. Conversely, there was no significant effect of injectable P4 in any of the end points evaluated (Table 7). Table 7. Mean (±SEM) CL number and pregnancy rates (%) in recipients treated with a progesteroneintravaginal device concurrently with 2 mg EB im with or without an injection of 50 mg of P4 im on Day 0 and 400 IU of ecg given on Day 5 or Day 8 (adapted from 59). Day 0 ecg N CL Transferred/ Pregnant/ Pregnant/ numbers treated (%) transferred (%) treated (%) EB Day ± 0.12 a 68/75 (90.7) 38/68 (55.9) 38/75 (50.7) Day ± 0.02 b 62/75 (82.7) 30/62 (48.4) 30/75 (40.0) EB + P4 Day ± 0.12 a 67/76 (88.2) 33/67 (49.3) 33/76 (43.4) Day ± 0.07 ab 65/75 (86.6) 31/65 (47.7) 31/75 (41.3) Main effects Day ± 0.08 e 135/151 (89.4) 71/135 (52.6) 71/151 (47.0) c Day ± 0.04 f 127/150 (84.7) 61/127 (48.0) 61/150 (40.7) d EB ± /150 (86.7) 68/130 (52.3) 68/150 (45.3) EB + P ± /151 (87.4) 64/132 (48.5) 64/151 (42.4) ab, ef Means within columns with different superscripts are different (P < 0.05). cd Percentages within columns with different superscripts are different (P = 0.1) A subset of 154 recipients were randomly selected across treatment groups and bled for plasma P4 determinations. Treatment with ecg on Day 5 resulted in a higher (P<0.05) number of CL (1.4±0.1 CL) and plasma P4 concentrations (2.4±0.3 ng/ml) on Day 17 (day of embryo transfer) than treatment with ecg on Day 8 (1.1±0.1 CL and 1.7±0.2 ng/ml, respectively). There was no significant effect of the 50 mg of P4 injection in any of the end points evaluated. 44

47 Results suggest that ecg treatment on Day 5 increases the number of CL, plasma P4 concentrations and tends to increase pregnancy rates in bovine embryo recipients synchronized with DIB devices and EB and transferred at a fixed-time. These results were further confirmed in a follow-up study designed to compare different dosages of ecg given on Day 5 or Day 8. There was no significant effect of ecg dosage (400 IU vs 500 IU vs 600 IU) on pregnancy rates. However, treatment with ecg on Day 5 resulted in significantly higher pregnancy rates than ecg treatment on Day 8 (Table 8). Table 8. Percentage transferred/treated and pregnancy rates in recipients treated with a DIB device and 2 mg EB im on Day 0 and 400 IU, 500 IU or 600 IU of ecg given on Day 5 or Day 8 (Adapted from 6). Group N Transferred/treated Pregnant/transferred Pregnant/treated (%) (%) (%) ecg Day /299 (87.0%) a 132/255 (51.8%) a 132/299 (44.1%) a ecg Day /295 (81.7%) b 108/240 (45.0%) b 108/295 (36.6%) b ab Percentages within columns with different superscripts differ significantly (P<0.05). We have recently designed an experiment to compare pregnancy rates in cows treated with DIB devices plus EB and ecg, and induced to ovulate with EB given at device removal or 24 h later. Non-lactating, cycling, crossbred Zebu cows (n=478), with a body condition score between 2.5 to 3.5 (1 to 5 scale) were used. Data were obtained in 5 replicates. All cows were treated with a DIB device and 2 mg EB im, on Day 0 and 400 IU of ecg im plus 150ìg D(+)cloprostenol im, on Day 5. On Day 8, DIB devices were removed and cows were randomly divided to receive 1 mg EB im at the time of DIB removal (EB0) or 24h later (Day 9; EB24). Recipients were observed for signs of estrus from Day 8 to 13. All recipients with >1 CL, or a single CL with an area >256mm 2 (16 mm in diameter), measured by ultrasonography, received fresh or frozen/thawed embryos by FTET on Day 16 (EB0) or Day 17 (EB24). The variables fresh or frozen embryos, technician, embryo stage and CL area did not affect pregnancy rates (P>0,05). However, embryo quality tended (P=0,06) to influence pregnancy rates (Grade 1: 152/253, 60.1%, Grade 2 32/65, 49.2%, Grade 3 11/33, 33.3%). The interval from DIB removal to estrus was shorter (P=0.0001) for the recipients in Group EB0 (26.5±0.6h) those in Group EB24 (39.8±0.8h). Nevertheless, pregnancy rates were not different (P=0.6) between recipients seen or not seen in estrus (Table 9). The rate of recipients transferred/treated was not different (P=0.5) among groups. Conversely, the rate of recipients pregnant/transferred and pregnant/ treated were higher (P=0.03) in Group EB0 than in Group EB24, respectively (Table 10). It was concluded that the use of EB at device removal could reduce the number of trips through the chute for treatments and possibly improve pregnancy rates in a FTET program. 45

48 Table 9. Pregnancy rates in relation to estrus observation in recipients treated with DIB devices for 8 d and EB on Day 0, 400 IU of ecg on Day 5 and induced to ovulate with EB on Day 8 (EB0) or on Day 9 (EB24h). EB 0 h EB 24 h In estrus (%) 137/241 (56.8 %) 117/237 (49.3 %) Pregnant/Observed in estrus (%) 83/137 (60.6 %) 60/117 (51.3 %) Pregnant/not observed in estrus (%) 27/43 (62.8 %) 25/54 (46.3 %) Percentages did not differ (P=0.6). Table 10. Pregnancy rates in recipients treated with DIB devices for 8 d and EB on Day 0, 400 IU of ecg on Day 5 and induced to ovulate with EB on Day 8 (EB0) or on Day 9 (EB24h). Embryos were transferred on Day 17. Group Transferred/treated (%) Pregnant/transferred (%) Pregnant/treated (%) EB0 180/241 (74.7%) 110/180 (61.1%) a 110/241 (45.6%) c EB24 171/237 (72.2%) 85/171 (49.7) b 85/237 (35.9%) d Pregnancy rates differ significantly (ab: P=0.03, cd: P=0.02) Although there have been many reports of satisfactory pregnancy rates in recipients synchronized with P4 devices (40,42,58), like those reported herein, there are those in which reduced pregnancy rates have been reported (38,74,81). The reasons for this discrepancy are not clear; however, it has been reported that the expression of estrus was higher in progestogen-treated animals than in PGF-treated animals (81). At least one study in lactating beef cows revealed that approximately 50% were in anestrous; therefore, the estradiol/p4 treatment may have induced estrus and ovulation in cows that did not have an adequate post-partum interval or were not in satisfactory body condition, accounting for the reduced pregnancy rates (74). This may also explain the differences between the control groups (no ecg) in the first experiments presented in this section. Although Bos taurus x Bos indicus crossbred cattle were used in both experiments, it is possible that the heifers used in the first experiment (4) were not in as good body condition as the cows used in the second experiment (77). These results further emphasize the importance of using cycling cows or heifers that are in good body condition and cows that are of an adequate post-partum interval, as embryo recipients. From 1542 recipients synchronized with estradiol/p4 and ecg in a commercial embryo transfer program, 1309 (84.9%) recipients received an embryo by non-surgical transfer and 692 (44.9%) were pregnant. Considering that one of the most costly items in an embryo transfer program is feeding recipients until they become pregnant (34), a protocol that results in 45% of the recipients becoming pregnant per synchronization treatment seems very cost-effective, especially considering that this treatment also avoids the necessity of estrus detection. Figure 2 outlines the recommended treatment protocol. 46

49 2 mg EB PGF IU ecg 1 mg EB Fixed-time Embryo Transfer Progesterone device Treatment days 2 mg EB PGF IU ecg 1 mg EB Fixed-time Embryo Transfer Progesterone device Treatment days Figure 2. Treatment protocols for fixed-time embryo transfer in cattle. Treatments consist of insertion of a P4 releasing device and 2 mg estradiol benzoate (EB) im on Day 0, PGF and 400 IU ecg on Day 5, device removal on Day 8, and EB on Day 9. Estrus is not observed and embryo transfer is performed on Day 17. Alternatively, the second EB treatment may be given on Day 8 and embryo transfer is performed on Day 16. APLICATION OF TREATMENTS THAT SYNCHRONIZE OVULATION IN FIXED-TIME ARTIFICIAL INSEMINATION PROGRAMS As with recipients, we can divide FTAI protocols into those using combinations of GnRH and PGF, called Ovsynch protocols (64) and those using P4 devices and estradiol (16,18,19). The Ovsynch protocol has resulted in acceptable pregnancy rates in dairy (23,64) and beef (55) cows in North America. However, results in beef herds under grazing conditions in South-America have not been satisfactory with low conception rates in anestrous cows (5). The addition of a P4 releasing device increased pregnancy rates in cows in anestrous and may be more applicable to cows in grazing conditions (39). The most commonly used treatment for FTAI using P4 releasing devices in beef cattle in South America consist in the administration of 2 mg of estradiol benzoate (EB) im upon insertion of the device (Day 0); on Day 7 or 8 the device is removed and PGF is administered im and 1 mg of EB im is given 24 h later (19). FTAI is done between 52 and 56 h after device removal. Data from 13,510 inseminations between December 2000 and December 2004 resulted in a mean pregnancy rate of 52.7% with a range from 27.8% to 75%. The factors that most affected pregnancy rates were body condition score (BCS) and cyclicity of the cows (30). 47

50 Synchronization treatments for FTAI in Suckled-beef cows Under favorable conditions, a cow has the potential to produce one calf per year, with an interval of 12 months between calvings. However, cows in grazing conditions often present a high incidence of postpartum anestrus which extends the calving-conception interval and, consequently, negatively affects their reproductive performance. Therefore, techniques used to advance the resumption of cyclicity in the postpartum period have a great impact in beef production. A treatment that is commonly used for shortening postpartum anestrus is the insertion of subcutaneous norgestomet implants or intravaginal P4 devices. These treatments maintain subluteal plasma concentrations of P4, provoking an increase in the frequency of LH pulses, promoting follicular growth, maturation of the dominant follicle and its ovulatory capacity (7). It also sensitizes the genital system and prevents the formation of a short lifespan CL (66). The positive effect of these treatments has been reported by several authors. In one study, animals treated with P4 devices had an increased breeding rate during the first 45 d of the breeding season compared to controls (7). In another study, the calving to first estrus interval was reduced from d (control) to d (treated) without compromising conception rates in primiparous zebu-derived cows treated with norgestomet implants (71). When P4 and EB treatments were used for FTAI in postpartum anestrous cows, acceptable conception rates were obtained, increasing pregnancy rates (5). In another study with suckled beef cows that were 70 d postpartum, the use of a P4 device plus EB combined with temporary weaning (TW) from device removal to AI (50-52 h), improved pregnancy rates compared to cows that were only TW for 48 h and that received natural service for 60 d (2). Treatments with progesterone devices and ecg Progesterone devices combined with ecg have been widely used in postpartum anestrous cows. Treatment with ecg has been shown to increase pregnancy rates in suckled cows with a high incidence of anestrous (30). However, when these protocols were used in cows in good BCS, pregnancy rates were not higher than those obtained in the cows not treated with ecg. Follicular development in cows with good BCS was not compromised (18,30); therefore the stimulation of the growth of the dominant follicle was not needed to achieve acceptable pregnancy rates in these females. In a study by Cutaia et al. (30), the use of 400 IU of ecg at the time of P4 device removal resulted in increased pregnancy rates in cows with small follicles (< 8 mm) or with follicles > 8 mm but with no CL at the time of insertion of the P4 device. In another study (8), ecg treatment increased plasma P4 concentrations and pregnancy rates in suckled cows treated during postpartum anestrus. Therefore, ecg treatment may be an important tool for increasing pregnancy rates at FTAI, to reduce the postpartum period and to improve reproductive efficiency in post-partum beef cows (7). Figures 3 and 4 show data from 9,668 FTAI done by our group from December 2000 and December 2003, considering different factors such as BCS and cyclicity (22). As is shown in Figure 3, BCS is a critical factor affecting results following FTAI. Results presented herein and in other studies suggest that animals should have a minimum BCS of 2.5 (scale 1 to 5) or ideally 3, in order to obtain good pregnancy rates when using a FTAI protocol without adding ecg. within another study involving 678 cows treated with 400 IU ecg on Day 8 (DIB removal), the addition ecg allowed for pregnancy rates close to 50% in cows with a BCS of 2 (30). These results may be achieved only when cows are gaining weight during the breeding season. If drought conditions or lack of feed availability prevent the cattle from improving BCS during the breeding season, pregnancy rates will most probably be 35% or less, even with the administration of ecg (22,29). 48

51 Figure 3. Effect of BCS on pregnancy rates in cows treated with P4 releasing devices with (n=678) or without (n=9668) 400 IU ecg at device removal(p>0.1). Figure 4 shows the impact of ovarian status on pregnancy rates. Pregnancy rates were compared among cows that had a CL or follices at the time of P4 device insertion. Data showed that the addition of ecg did not improve pregnancy rates in cows with a CL, but significantly improved fertility in cows with only follicles, probably because many of those cows were not cycling at the time that treatments were initiated. Figure 4. Pregnancy rates in suckled cows that had a CL or only follicles at the insertion of a P4 releasing device and treated or not with 400 IU of ecg at device removal (P<0.01). Temporary Weaning and FTAI We have recently designed two experiments to compare the effects of ecg treatment and temporary weaning (TW) on ovulation and pregnancy rates in postpartum cows. In Experiment 1, 39 lactating multiparous crossbred Bos Indicus cows, 60 to 80 d postpartum with a BCS between 2.0 to 2.5 out of 5 were randomly allocated to 1 of 4 treatment groups, in a 2 by 2 factorial design. On Day 0, all cows received a DIB device and 2 mg EB im On Day 8, DIB devices were removed and all cows received PGF and were randomly divided to receive 400 IU ecg im at the same time or no further treatment. In addition, half of the cows in each treatment group had their calves TW for 56 h from the time of DIB removal; the other half 49

52 remained with their calves. All cows received 1 mg EB im on Day 9 and were examined every 8 h by ultrasonography, from the time of DIB removal until ovulation. The diameter of the dominant follicle on Day 8 [ecg, 7.8±0.5 mm vs no ecg, 8.2±0.4 and TW, 7.6±0.4 vs no TW, 8.4±0.5], the proportion of cows that ovulated [ecg, 12/20, (60%) vs no ecg, 9/19 (47%) and TW, 13/20 (65%) vs no TW, 8/19 (42%)], and the interval to ovulation [ecg, 72.0±1.4 h vs no ecg, 75.6±2.0 h and TW, 73.8±1.6 h vs no TW, 73.0±1.8 h] did not differ among groups (P>0.05). Although there was no effect of ecg treatment or ecg by TW interaction (P>0.3) on the size of the preovulatory follicle [ecg, 11.1±0.4 mm vs no ecg, 10.1±0.6 mm), it was smaller in TW cows (9.9±04), compared to those not TW (11.8±0.3; P<0.05). Nevertheless, the growth rate of the ovulatory follicle was greater (P<0.02) in cows treated with ecg (1.1±0.1 mm/d) than in those not treated with ecg (0.6±0.1 mm/d). Experiment 2 was conducted over 2 years; 769 Bos indicus cross-bred suckled cows (year n=393 and year n=376) with a BCS of 2 to 2.5 were used. All animals were examined by rectal palpation at the time of initiating the treatment to determine ovarian status. Based on presence of a CL (22.5%), follicles > 8 mm (30.0%) or ovaries with small follicles (<8 mm; 47.5%) cows were assigned to 4 treatment groups in a 2 by 2 factorial design (Control, ecg, TW and TW+eCG). Weaned calves were separated from their dams by approximately 1000 m, to prevent any kind of contact between cows and calves. All cows were FTAI between 52 and 56 h after DIB removal. Data were analyzed by logistic regression. As it is shown in Table 11, a lower overall pregnancy rate was obtained in 2005 compared to 2004 (P=0.01). Besides, the pregnancy rate was lower in cows that were not treated with ecg than in those that were treated with ecg (P=0.01). No differences were found between those that were TW and those that were not (P=0.7), or the TW x ecg interaction (P=0.7). Table 11. Effect of temporary weaning (TW) and ecg treatment on pregnancy rates in suckled cows that were treated with progesterone releasing devices and FTAI. Main factors YEAR 2004 YEAR 2005 TOTAL ecg 93/191 (48.7%) 61/186 (32.8%) 154/377 (40.8%) x No ecg 80/202 (39.6%) 48/190 (25.3%) 128/392 (32.6%) y TW 86/191 (45.0%) 55/188 (29.3%) 141/379 (37.2%) No TW 87/202 (43.0%) 54/188 (28.7%) 141/390 (36.1%) TOTAL 173/393 (44.0%) a 109/376 (29.0%) b Percentages within the same row (ab) or column (xy) with different superscripts differ (P=0.01). The use of ecg but not TW improved pregnancy rates following FTAI in postpartum Bos indicus cows. Results also suggest that the ecg-related increase in pregnancy rates may be due to the final growth rate of the ovulatory follicle (8,30). On the other hand, no or little effect of TW on pregnancy rates contrasts with data from other authors (reviewed in 8). IMPACT FIXED-TIME AI IN BEEF PRODUCTION SYSTEMS Undoubtedly, one of the main advantages of implementing FTAI programs in a beef herd is that heavier weaning weights may be obtained (30). Fifty percent of the cows could potentially become pregnant on the first day of the breeding season and result in a higher number of cows calving at the beginning of the breeding season. Therefore, their calves will be older and heavier at weaning. Besides, the use of genetically superior 50

53 bulls will also result in heavier calves at weaning (30). The impact of FTAI has proven to be equally efficient for different beef operations in Argentina and Brazil (17) and examples will be shown in the last part of this review. In 2002, the Estancia El Mangrullo (Lavalle, Santiago del Estero, Argentina) started implementing FTAI programs. This operation is located in the semiarid region of Argentina, with seasonal rainfalls of 600 mm per year from November-December to May-June (Summer and Fall). Animals are all zebu-derived and an absorbent cross-breeding program with Bonsmara has been implemented with the use of semen and embryos. Table 12 shows the evolution of the number of animals involved in FTAI programs and the results obtained. Table 12. Pregnancy rates following FTAI programs implemented in Estancia El Mangrullo in Lavalle, northeast of the Province of Santiago del Estero, Argentina. Category Year 2002/03 Year 2003/04 Year 2004/05 Total Heifers 148/ / / /2144 (50.7%) (55.1%) (45.7%) (49.1%) Dry cows 189/ /394 (47.9%) (47.9%) Suckled cows 156/ / / /2278 (54.0%) (43.7%) (37.5%) (41.7%) Total 304/ / / /4816 (52.3%) (48.5%) (41.7%) (45.5%) As it is shown in Table 12, an aggressive FTAI program was implemented with heifers and suckled cows which resulted in pregnancy rates between 40 and 50%. It is important to highlight that the summer of 2005 (i.e. the breeding season) was especially dry, with no rains between December and March which, undoubtedly affected the pregnancy rates. However, we can observe how an aggressive FTAI program may still result in acceptable pregnancy rates, even with the tough drought experienced that year. Probably, the main aspect of applying this system was its effect on calving distribution as shown on Figure 5. Initially, calvings were distributed over 6 months in 2002/03 (no FTAI) with a high number of cows calving from December to March (late calvers). In 2004/05, calvings occurred over 5 months, but began earlier and with a high proportion of cows calving earlier in the breeding season. In 2004, we also evaluated the impact of FTAI on weaning weights of the calves obtained through natural service compared to that of calves obtained through FTAI, similar to the comparison in a previous publication with Angus cows (30). Only one group of animals in which all calving data could be collected was used. The cows from the Natural Service Group were bred with 3% Bonsmara bulls for 90 d. Cows in the FTAI Group were inseminated at the beginning of the breeding season and exposed to 1.5% clean-up bulls. All cows were monitored during the calving season and calves born were identified with ear tags and weighted. Table 13 shows the weaning weights of calves produced through FTAI or natural service. Weight of the calves was adjusted at 180 d to determine what proportion of the weight difference between groups was due to the age of the calves and what proportion was due to a genetic improvement introduced with the bulls used for FTAI. 51

54 Figure 5. Distribution of calvings at El Mangrullo Santiago del Estero, Argentina. 52

55 Table 13. Weaning weights of zebu x Bonsmara calves produced through FTAI or Natural Service. Estancia El Mangrullo, Argentina, N Weaning weight (Kg) Adjusted 205 d-weight (Kg) (mean±ee) (Mean±EE) FTAI ±1.9 a 184.2±1.6 a Natural service ±1.5 b 173.8±1.4 b Difference ab Means with different superscripts in the same column differ (P= ) As shown in Table 13, calves from the FTAI group were heavier at weaning than calves in the Natural Service Group. Part of this difference (18.3 Kg) was attributed age, because the calves from the FTAI group were born earlier than those in the Natural Service Group. There was also a 10.4 Kg weight increase of the calves of due to genetic improvement. These data confirm previous results in Angus cattle (30) where differences in weaning weights were 34.6 Kg for calves produced through FTAI compared to those produced through natural service and show that it is possible to improve production in a beef herd with a FTAI program, at the beginning of the breeding season. Another program worth mentioning is the one applied at Estancia Santo Domingo, in Rio Ceballos, Córdoba. This operation is located in an area that receives more rainfall than El Mangrullo, about 800 to 1000 mm per year, in a seasonal fashion from October to June. This is a mixed operation (soybean and corn crops and beef cattle) with a purebred Brangus and Braford herd. FTAI was done in November and December in a group of 180 to 280 animals, including 22 to 26 month old heifers and suckled cows that were 45 to 70 d postpartum (approximately 100 heifers from 2001 to 2003 and 200 heifers in 2004). In this case, animals have always been in good BCS (2.5 to 3.5) at the beginning of the breeding season and the FTAI treatment used consist of a P4 device (Triu-B, Biogénesis, Argentina; DIB or CIDR-B) with 2 mg EB on Day 0, device removal and PGF on Day 7 or 8, 1 mg EB 24 h later and FTAI from 52 to 56 h after device removal. As the goal was to increase the number of offspring produced by AI, animals are re-synchronized with reinsertion of the P4 device on Day 13 after FTAI. EB (1 mg im) is also given to cows (not heifers) on Day 13. In this case, estrus is detected for 5 d after device removal on Day 20 and all animals are inseminated 8 to 12 h after onset of estrus. As it is shown in Table 14, pregnancy rates with FTAI are similar over the 4 years (P>0.88). Overall pregnancy rates with AI has decreased during the past year (P<0.05), compared to the two previous years, due to failures in estrus detection after the re-synchronization protocol, which demonstrates the sensitivity of systems that depend on estrus detection in beef cattle. However, pregnancy rates following FTAI were maintained more or less constant over the years in this type of system. 53

56 Table 14. Pregnancy rates with FTAI, estrus detection rate, conception and pregnancy rates at re-synchronization and overall pregnancy rates at Estancia Santo Domingo, Córdoba, Argentina. Years FTAI Re-synchronization Cumulative pregnancy rates Estrus detection Conception Pregnancy rate rate rate /189 44/82 ab 24/44 24/82 ab 131/189 ab (56.6%) (53.7%) (54.5%) (29.3%) (69.3%) /192 35/88 ab 35/49 35/88 b 139/192 b (51.2%) (55.7%) (71.4%) (39.7%) (72.4%) /228 71/100 b 36/71 36/100 b 164/228 b (56.1%) (71.0%) (50.7%) (36.0%) (71.9%) /279 50/130 a 25/50 25/130 a 174/279 a (53.4%) (38.4%) (50.0%) (19.2%) (62.4%) ab Proportions in the same column with different superscripts differ (P<0.05). A third example is the operation called Estancia Santa Dominga owned by Los Lazos S.A., in Olavaría, Buenos Aires, Argentina. This is a purebred Angus herd located in the best cattle country in Argentina, the Pampa Húmeda, with rainfalls of 1200 to 1500 mm evenly distributed throughout the year. In this farm, cows and heifers have always been in good BCS (2.5 to 3.5) at the beginning of the breeding season and have been treated with a CIDR-B protocol for 8 d, plus the administration of 2 mg EB on Day 0, PGF on Day 8 (upon CIDR-B removal), 1 mg EB on Day 9 and FTAI between 52 and 56 h after CIDR-B removal. All cows have been exposed to clean-up bulls for 90 d, beginning 15 d after FTAI. Pregnancy rates have been determined by ultrasonography 30 d after FTAI and then rectal palpation 60 d after the end of the breeding season. In an experiment done in 2003, it was shown that FTAI calves were 34.6 Kg heavier than those produced through natural service (30). Therefore, the use of FTAI was intensified and results of programs done in subsequent years are presented in Table 15. Table 15. Pregnancy rates following FTAI in cows and heifers at Estancia Santa Dominga, Olavarría, Buenos Aires. Year N Pregnant % Total Tables 16 and 17 show the pregnancy rates in the different herds in 2003 and 2004 breeding season, where the consistency of the pregnancy rates obtained in the different categories can also be observed. 54

57 Table 16. Pregnancy rates following FTAI in cows and heifers at Estancia Santa Dominga, Olavarría, Buenos Aires. (Year 2003). Herd Category N Pregnant % 1 Heifers Heifers Heifers Heifers Suckled Cows Suckled Cows Suckled Cows Suckled Cows Suckled Cows Overall Table 17. Pregnancy rates following FTAI in cows and heifers at Estancia Santa Dominga, Olavarría, Buenos Aires. (Year 2004). Herd Category N Pregnant % 1 Heifers Heifers Heifers Heifers Suckled Cows Suckled Cows Suckled Cows Suckled Cows Suckled Cows Suckled Cows Suckled Cows Overall SUMMARY AND CONCLUSIONS Reproductive endocrinology and estrous behavior of cattle have been studied for over 50 years; however, the relatively recent application of technologies such as real time ultrasonography and Heat-Watch systems has expanded our knowledge of the ovarian follicular wave dynamics during the estrous cycle and the time of ovulation. Duration of estrus is often short (<12 h) and very variable affecting the efficiency of reproductive programs in beef operations. The inefficiency of estrus detection has also limited the widespread application of AI and ET programs and emphasizes the need for treatments that control follicular development and ovulation. Therefore, the incorporation of techniques designed to control follicular wave dynamics and ovulation, like those discussed herein, will reduce the problem of estrus detection and provide possibilities for 55

58 the application of FTAI and FTET programs. Pregnancy rates to a single FTAI or FTET were similar or superior to those after detection of spontaneous estrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in cattle by offering the possibility of planning programs without the necessity of estrus detection and without sacrificing the overall results. Finally, the selection of the best program will depend on many management factors such as the availability of qualified personnel, facilities and the objectives of the breeding program. REFERENCES 1. Ambrose, J.D., Drost, R.L., Monson, R.L, Rutledge, J.J., Leibfried-Rutledge, M.L., Thatcher, M.J., Kassa, T., Binelli, M., Hansen, P.J., Chenoweth, P.J., Thatcher, W.W 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: Arteche, A.C., Rocha, D.C., Moreira, R., Cardozo, L.D., Borges J.B.S., Mattos R.C., Gregory, R.M Inseminación artificial a tiempo fijo de vacas tratadas con CIDR, benzoato de estradiol, asociado a ecg o destete temporal. V Simposio Internacional de Reproducción Animal, Huerta Grande, Córdoba; 378 abstr. 3. Baruselli, P.S., Marques, M.O., Carvalho, N.A.T., Valentim, R., Berber, R.C.A., Carvalho Filho, A.F., Madureira, E.H., Costa Neto, W.P Ovsynch protocol with fixed-time embryo transfer increasing pregnancy rates in bovine recipients. Arq Fac Vet UFRGS, Porto Alegre, Brazil; 28 (Suppl): 205 abstr. 4. Baruselli, P.S., Marques, M.O. Madureira, E.H.,, Costa Neto, W.P., Grandinetti, R.R., Bo, G.A Increased pregnancy rates in embryo recipients treated with CIDR-B devices and ecg. Theriogenology; 55:157 abstr 5. Baruselli, P.S., Madureira, E.H., Marques, M.O Programas de IA a tiempo fijo en Bos indicus. Resúmenes. Cuarto Simposio Internacional de Reproducción Animal, Huerta Grande, Córdoba; Baruselli, P.S., Reis, E.L Sincronización de receptoras cruza cebú en condiciones tropicales. Resúmenes del IV Seminario Internacional de Reproducción en Grandes Animales. CGR, Biotecnología Reproductiva E.U. División Capacitación. Bogotá 25 al 27 de septiembre; Medellín 27 y 28 de septiembre 2003; Baruselli, P.S., Marques, M.O., Reis, E.L., Bó, G.A Tratamientos hormonales para mejorar la performance reproductiva de vacas de cría en anestro en condiciones tropicales. Resúmenes V Simposio Internacional de Reproducción Animal. Huerta Grande, Córdoba Baruselli, P.S., Reis E.L., Marques M.O., Nasser L.F., Bo G.A The use of treatments to improve reproductive performance of anestrus beef cattle in tropical climates. Anim. Reprod. Sci Beal, W.B Streamlining embryo transfer. 18th Annual Convention AETA, Colorado Springs, CO, USA; Beal, W.E., Hinshaw, R.H Synchronization of estrus and ovulation in bovine embryo transfer recipients. Proceedings of the Advanced Embryo Transfer Seminar 12. Annual Meeting of the American Association of Bovine Practitioners, Vancouver, BC, Canada 11. Bó, G.A., Adams, G.P., Pierson, R.A., Mapletoft, R.J Exogenous control of follicular wave emergence in cattle. Theriogenology; 43: Bo, G.A, Caccia, M., Martinez, M., Mapletoft, R.J Follicular wave emergence after treatment with estradiol benzoate and CIDR-B vaginal devices in beef cattle. 13 th Int Congr Anim Reprod, Sydney, Australia; 7:22 abstr. 13. Bo, G.A., Medina, M., Tegli, J.C., Costamagna, A., Brogliatti, G.M Fixed-time artificial insemination in CIDR- B treated cows induced to ovulate with estradiol benzoate or GnRH. 14th International Congress on Animal Reproduction, Stockholm, Sweden; 2:45 abstr. 14. Bó, G.A., Cutaia, L., Brogliatti, G.M., Medina, M., Tríbulo, R., Tríbulo, H Programas de inseminacion artificial a tiempo fijo en ganado bovino utilizando progestágenos y estradiol. Resúmenes Cuarto Simposio Internacional de Reproducción Animal, Huerta Grande, Córdoba; Bo, G.A., Tribulo, H., Caccia, M., Tribulo, R Pregnancy rates in embryo recipients treated with progesterone vaginal devices and transferred without estrus detection. Theriogenology; 55:357 abstr. 16. Bo, G.A., Baruselli, P.S., Moreno, D., Cutaia, L., Caccia, M., Tríbulo, R., Tríbulo, H., Mapletoft, R.J The control of follicular wave development for self-appointed embryo transfer programs in cattle. Theriogenology; 57: Bó, G.A. y Baruselli, P.S Programas de Inseminación Artificial a Tiempo Fijo en el Ganado Bovino en Regiones Subtropicales y Tropicales. Capítulo XXXI. En: Avances en la Gandería doble propósito, C. Gonzalez- Stagnaro, Eleazar Soto Belloso y Lílido Ramírez Iglesia (Editores); Fundación Girarz, Maracaibo, Venezuela;

59 18. Bó, G.A., Cutaia, L., Tribulo, R Tratamientos hormonales para inseminación artificial a tiempo fijo en bovinos para carne: algunas experiencias realizadas en Argentina. Primera Parte. Taurus; 14: Bó, G.A., Cutaia, L., Tribulo, R Tratamientos hormonales para inseminación artificial a tiempo fijo en bovinos para carne: algunas experiencias realizadas en Argentina. Segunda Parte. Taurus; 15: Bó, G.A., Baruselli, P.S., Martinez, M.F Pattern and manipulation of follicular development in Bos indicus cattle. Anim. Repr. Sci. 78, Bó, G.A., Moreno, D., Cutaia, L., Caccia, M., Tribulo, R.J., Tribulo, H Transferencia de embriones a tiempo fijo: tratamientos y factores que afectan los índices de preñez. Taurus 2004; 21: Bo, G.A., Cutaia, L., Chesta, P., Balla, E., Picinato, D., Peres L., Maraña, D., Avilés, M., Menchaca, A. Veneranda, G., Baruselli, P.S Implementacion de programas de inseminación artificial en rodeos de cría de argentina. VI Simposio Internacional de Reproducción Animal, Córdoba, Argentina; Burke, J.M., de la Sota, R.L., Risco, C., Staples, C.R., Thatcher, W.W Evaluation of timed insemination using a gonadotropin-releasing hormone agonist in lactating dairy cows. J Dairy Sci; 79: Broadbent PJ, Stewart M, Dolmal DF Recipient management and embryo transfer. Theriogenology; 35: Cavalieri, J., Fitzpatrick, L.A Oestrus detection techniques and insemination strategies in Bos indicus heifers synchronized with norgestomet-oestradiol. Aust Vet J; 72: Colazo M.G., Bó, G.A., Illuminanti, H., Meglia, G., Schmidt, E.E., Bartolomé, J Fixed-time artificial insemination in beef cattle using CIDR-B devices, progesterone and estradiol benzoate. Theriogenology; 51:404 abstr. 27. Custer, E.E., Beal, W.E., Wilson, S.J., Meadows, A.W., Berardinelli, J.G., Adair, R Effect of melengestrol acetate (MGA) or progesterone-releasing intravaginal device (PRID) on follicular development, concentrations of estradiol-17b and progesterone, and LH release during an artificially lengthened bovine estrous cycle. J Anim Sci; 72: Cutaia, L., Moreno, D., Villata, M.L., Bo, G.A Synchrony of ovulation in beef cows treated with progesterone vaginal devices and estradiol benzoate administered at device removal or 24 hours later. Theriogenology; 55:244 abstr. 29. Cutaia, L., Tríbulo, R., Moreno, D., Bó, G.A Pregnancy rates in lactating beef cows treated with progesterone releasing devices, estradiol benzoate and equine chorionic gonadotropin (ecg). Theriogenology 59, 216 abstr 30. Cutaia, L, Veneranda, G, Tribulo, R, Baruselli, PS, Bó GA Programas de Inseminación Artificial a Tiempo Fijo en Rodeos de Cría: Factores que lo Afectan y Resultados Productivos. V Simposio Internacional de Reproducción Animal. Huerta Grande, Córdoba; Fuentes, S Utilizacion de tratamientos hormonales para la sincronizacion de receptoras de embriones. Producción Animal 160: Fuentes, S., De la Fuente, J Different synchronization treatments for direct embryo transfer to recipients heifers. Proc XIII Annual Meeting AETE, Lyon, France; 148 abstr. 33. Geary, T.W., Whittier, J.C., Downing, E.R., LeFever, D.G., Silcox, R.W., Holland, M.D., Nett, T.M., Niswender, G.D Pregnancy rates of post partum beef cows that were synchronized using Syncro-Mate B or Ovsynch protocol. J Anim Sci; 76: Hinshaw, R.H Formulating ET contracts. Annual Meeting Soc for Theriogenology, Nashville, USA; Kastelic, J.P., Ginther, O.J Factors affecting the origin of the ovulatory follicle in heifers with induced luteolysis. Anim Reprod Sci; 26: Kastelic, J.P., McCartney, D.H., Olson, W.O., Barth, A.D., Garcia, A., Mapletoft, R.J Estrus synchronization in cattle using estradiol, melengestrol acetate and PGF. Theriogenology; 46: Kinder, J.E., Kojima, F.N., Bergfeld, E.G.M., Wehrman, M.E., Fike, K.E Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci; 74: King, M.E., Odde, K.G., Lefever, D.G., Brown, L.N., Neubauer, C.J Synchronization of estrus of embryo transfer recipients receiving demi-embryos with Syncro-Mate-B or Estrumate. Theriogenology; 25:162 abstr. 39. Lamb, G.C., Stevenson, J.S., Kesler D.J., Garverick H. A., Brown, D.R., Salfen, B.E Inclusion of an intravaginal progesterone insert plus GnRH and prostaglandin F 2á ovulation control in postpartum suckled beef cows. J. Anim. Sci; 79: Looney, C.R., Roberts, J.W., Jones, M., Day, M.L., Anderson, J.C., Hafs, H.D., Forrest, D.W Synchrony and conception to insemination or embryo transfer in beef females treated with an intravaginal progesterone-releasing device with or without an injection of estradiol. Theriogenology; 51:266 abstr. 57

60 41. McDowell, C.M., Anderson, L.G., Kinder, J.E., Day, M.L Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle. J Anim Sci; 76: McGrath, A.B., Looney, C.R., Bluntzer, J.S., Odeon, A.J., Massey, J.M Comparison of norgestomet and prostaglandin F 2 a (PGF) for estrus synchronization of recipients nursing embryo transfer (ET) calves. Theriogenology; 23:207 abstr. 43. Macmillan, K.L., Henderson, H.V Analyses of the variation in the interval from an injection of prostaglandin F 2 a to estrus as a method of studying patterns of follicle development during diestrous in dairy cows. Anim Reprod Sci; 6: Macmillan, K.L., Thatcher, W.W Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol Reprod; 45: Macmillan, K.L., Peterson, A.J A new intravaginal progesterone releasing device for cattle (CIDR-B) for estrus synchronization, increasing pregnancy rates and the treatment of post-partum anestrus. Anim Reprod Sci; 33: Macmillan, K.L., Taufa, V.K., Hayman, D.L Pregnancy rates in lactating dairy cows used as recipients for frozen/thawed embryos and receiving supplemental progesterone. New Zealand Embryo Transfer Workshop, Hamilton, NZ; Macmillan, K.L., Burke, C.R Effects of oestrus cycle control on reproductive efficiency. Anim Reprod Sci; 42: Mantovani, A.P., Reis, E.L., Bó, G.A., Baruselli, P.S., Gacek, F Prolonged use of a progesterone-releasing intravaginal device (CIDR ) on the induction of persistent follicles in bovine embryo recipients. 15th International Congress on Animal Reproduction, Porto Seguro, Brazil; 133 abstr. 49. Marques, M.O., Madureira, E.H., Bo, G.A., Baruselli, P.S Ovarian ultrasonography and plasma progesterone concentration in Bos taurus x Bos indicus heifers administered different treatments on Day 7 of the estrous cycle. Theriogenology; 57: 548 abstr. 50. Marques, M.O., Nasser, L.F, Silva, R.C.P., Bo, G.A., Baruselli, P.S Increased pregnancy rates in bos taurus x bos indicus embryo recipients with treatments that increase plasma progesterone concentrations. Theriogenology; 59:369 abstr. 51. Maraña Peña, D., Cutaia, L., Borges Kruel, L.F., P.incinato, D., Peres Coelho L, Bó, G.A Efecto de la aplicación de ecg y destete temporario sobre la tasa de ovulación en vacas posparto tratadas con DIB y benzoato de estradiol. VI Simposio Internacional de Reproducción Animal, Córdoba, Argentina, abstr. 52. Marcantonio, S.A El mercado del semen bovino en Argentina. Taurus 19: Martinez, M.F., Adams, G.P., Bergfelt, D., Kastelic, J.P., Mapletoft, R.J Effect of LH or GnRH on the dominant follicle of the first follicular wave in heifers. Anim Reprod Sci; 57: Martínez MF, Kastelic JP, Adams GP, Janzen E, McCartney D, Mapletoft RJ Estrus synchronization and fertility in beef cattle given a CIDR and estradiol or GnRH. Can. Vet. J. 41, Martinez, M.F., Kastelic JP, Adams GP, Mapletoft RJ 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: Moreno, D., Cutaia, L., Villata, M.L., Ortisi, F., Bó, G.A Follicle wave emergence in beef cows treated with progesterone releasing devices, estradiol benzoate and progesterone. Theriogenology 55, 408 abstr. 57. Moreno, D., Cutaia, L., Villata, M.L., Caccia, M., Gatti, G., Tribulo, R., Tribulo, H., Bo, G.A Effect of the time of prostaglandin administration on pregnancy rates in embryo recipients treated with progesterone vaginal devices and transferred without estrus detection. Theriogenology; 57:552 abstr. 58. Moreno, D., Cutaia, L., Tribulo, R., Caccia, M., Tribulo, H., Chesta, P., Villata, M.L., Bo, G.A Fixed-Time embryo transfer in cows treated with progesterone vaginal devices and induced to ovulate with estradiol benzoate or hcg. Theriogenology; 59:307 abstr. 59. Nasser, L.F., Reis, E.L., Oliveira, A.M., Bo, G.A., Baruselli, P.S Effect of time of ecg pretreatment on pregnancy rates in Bos indicus x Bos taurus recipients synchronized with progesterone vaginal devices and transferred without estrus detection. Reproduction Fertility and Development; 212 abstr. 60. Momont, H.W., Seguin, B.E Influence of the day of estrous cycle on response to PGF 2 a products: Implications for AI programs for dairy cattle. 10th International Congress on Animal Reproduction; 3: Odde, K.G A review of synchronization of estrus in postpartum cattle. J Anim Sci; 68:

61 62. Pursley, J.R., Mee, M.O., Wiltbank, M.C Synchronization of ovulation in dairy cows using PGF2a and GnRH. Theriogenology; 44: Pursley, J.R., Kosorok, M.R., Wiltbank, M.C Reproductive management of lactating dairy cows using synchronization of ovulation. J Dairy Sci; 80: Pursley, J.R., Wiltbank, M.C., Stevenson, J.S., Ottobre, J.S., Garverick, H.A, Anderson, L.L Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J Dairy Sci; 80: Revah, I., Butler, W.R Prolonged dominance of follicles and reduced viability of bovine oocytes. J Reprod Fert; 106: Rivera G.M., Goñi C.G., Chaves M.A., Ferrero S.B., Bó G.A Orarian follicular wave synchronizatión and induction of ovulation in postpartum beef cows. Theriogenology 49: Roche, J.F Synchronization of oestrous in heifers with implants of progesterone. J Reprod Fertil; 41: Roy, G.L., Twagiramungu, H A fixed time AI program using the GnRH-PGF-GnRH method for beef females. J Anim Sci; 74 (Suppl 1): Savio, J.D., Thatcher, W.W., Morris, G.R., Entwistle, K., Drost, M., Mattiacci, M.R Effects of induction of low plasma progesterone concentrations with a progesterone-releasing intravaginal device on follicular turnover and fertility in cattle. J Reprod Fert; 98: Seguin, B Ovsynch: A method for breeding dairy cows without doing heat detection. The Bovine Practitioner; 31: Soto Belloso E., Portillo Martinez G., De Ondíz A., Rojas N., Soto Castillo G., Ramírez Iglesia L., Perea Ganchou Improvement of reproductive performance in crossbred zebu anestrus suckled primiparus cows by treatament with norgestomet implants or 96 h calf removal. Theriogenology 57, Stock, A.E., Fortune, J.E Ovarian follicular dominance in cattle: Relationship between prolonged growth of the ovulatory follicle and endocrine parameters. Endocrinology; 132: Thatcher, W.W., Moreira, F., Santos, J.E.P., Mattos, R.C., Lopez, F.L., Pancarci, S.M., Risco C.A Effects of hormonal treatments on reproductive performance and embryo production. Theriogenology; 55: Tribulo H, Bo GA, Kastelic JP, Pawlyshyn V, Barth AD, Mapletoft RJ Estrus synchronization in cattle with estradiol-17b and CIDR-B vaginal devices. Theriogenology; 43:340 abstr. 75. Tribulo R, Nigro M, Burry E, Caccia M, Tribulo H, Bo GA Pregnancy rates in recipients receiving CIDR-B devices immediately following embryo transfer. Theriogenology; 47:372 abstr. 76. Tribulo H, Bo GA, Gatti G, Tegli JC, Cutaia L, Moreno D, Brito M, Tribulo R Pregnancy rates in embryo recipients treated with estradiol benzoate and CIDR-B vaginal devices to eliminate the need for estrus detection. 14th International Congress on Animal Reproduction, Stockholm, Sweden; 2:115 abstr. 77. Tribulo H, Moreno D, Cutaia L, Gatti G, Tríbulo R, Caccia M, Bó GA Pregnancy rates in embryo recipients treated with progesterone vaginal devices and ecg and transferred without estrus detection. Theriogenology; 57:563 abstr. 78. Tribulo R., Balla, E., Cutaia, L., Bo, G.A., Baruselli, P.S., Peres, L Effect of treatment with GnRH at the time of embryo transfer on pregnancy rates in cows synchronized with progesterone vaginal devices, estradiol benzoate and ecg. Reproduction Fertility and Development; 17:234 abstr. 79. Twagiramungu H; Guilbault LA, Dufour JJ Synchronization of ovarian follicular waves with a gonadotropinreleasing hormone agonist to increase the precision of estrus in cattle: A review. J Anim Sci; 73: Wehrman ME, Fike KE, Melvin EJ, Kojima FN, Kinder JE Development of a persistent ovarian follicle and associated elevated concentrations of 17b-estradiol preceding ovulation does not alter the pregnancy rate after embryo transfer in cattle. Theriogenology; 47: Wenkoff, M.S The management of drug-induced manipulation of the estrous cycle in normal cows and heifers. Can Vet J; 28: Wiltbank, J.N., Zimmerman, D.R., Ingalls, J.E., Rowden, W.W Use of progestational compounds alone or in combination with estrogen for synchronization of estrus. J Anim Sci; 24: Yavas Y. and Walton J.S Postpartum acyclicity in suckled beef cows: a review. Theriogenology. 54: Zanenga, C.A., Pedroso, M.S., Lima, G.S., Santos, I.C.C Embryo transfer without estrus observation. Arq Fac Vet UFRGS, Porto Alegre, Brazil; 28(Suppl): 337 abstr. 59

62 Background Designing Vaccination Programs that Incorporate Spirovac Leptospira Borgpetersenii Serovar Hardjo-bovis Victor S. Cortese, D.V.M., Ph.D., Diplomat, ABVP (Dairy Practice) Associate Director Cattle Immunology Veterinary Speciality Group Pfizer Animal Health, Downingtown, PA Despite routine use of 5-way leptospiral vaccines in most U.S. dairy and cow-calf operations, bovine leptospirosis remains one of the most important infectious diseases affecting cattle reproduction The leading cause of disease is the gram-negative spirochete Leptospira borgpetersenii serovar hardjo bovis, formerly classified as Leptospira interrogans serovar hardjo-bovis. This is due to several factors; 1. Cattle are the maintenance host for hardjo bovis; the bacteria preferentially infects cattle. 2. The maintenance host infection is difficult to diagnosis and common diagnostic tests often give false negative results. 3. Maintenance host infections are characterized by subtle signs and long term infections and shedding of the bacteria. 4. The bacteria readily penetrates mucous membranes, making transmission to uninfected cattle fairly simple. 5. The hardjo-bovis contained in most current five way Leptospira vaccines is ineffective in preventing infection and transmission of the bacteria. They also are ineffective in preventing the bacteria s impact on reproduction. Bovine leptospirosis caused by hardjo bovis is characterized by: high incidence of infection inapparent clinical signs chronic disease infertility, reproductive failure, milk drop extended period of leptospiral shedding low or undetectable titers to the infecting organism Current studies have shown an incidence of infected dairy herds in the United States at with Leptospira sp. at 59%. In certain areas, housing and management practices have allowed the bacteria to infected 90% of the dairies. The infection rare in today s beef herds is unknown and studies are currently underway to determine the prevalence. If a herd is diagnosed as positively infected with hardjo-bovis, due to efficient trransmission, a large number of cattle will usually be infected in a herd. Very little of this description corresponds with the cattle industry s traditional understanding of leptospirosis as a disease associated with late-term abortion, which usually can be diagnosed by isolating and identifying a specific leptospiral agent from aborted fetuses. With hardjo bovis infection, the majority of pregnancy losses are reabsorptions in which the embryos are simply reabsorbed into the cow and typically go unnoticed and undiagnosed. 60

63 The widespread prevalence and insidious nature of hardjo bovis infection in U.S. dairies underscore the importance of obtaining an accurate diagnosis wherever cows thought to be pregnant are later found to be open and of initiating effective vaccination programs targeting L. borgpetersenii serovar hardjo bovis. Effect Upon Reproductive Efficiency A common measure of reproductive efficiency in contemporary dairies is the pregnancy rate (pregnancy efficiency), which is defined as the conception rate multiplied by the heat detection rate. Pregnancy rate also can be regarded as the proportion of available cows that become pregnant during 21-day windows from the voluntary waiting period. Most dairy herd management software programs are capable of calculating this important management number. Economic analysis has shown that optimal gross margin per cow is obtained when pregnancy rates exceed 30%. Most dairies in the U.S., though, have pregnancy rates ranging between 10% and 20%, a situation that represents a major loss of income for the industry. Adding to the losses is the fact that low pregnancy rates necessitate the purchase of replacements to maintain herd size and force managers to make culling decisions for reasons other than production. A variety of factors affect pregnancy rates. On one side of the pregnancy rate equation is heat detection, which is influenced by human management decisions related to time allocation, nutrition, and footing, among others. On the other side are conception rates. Under no physiological load (no milk production, metritis, stress, or infectious disease), cows can be expected to achieve conception rates of 65% to 75%, rates which have been reported by many heifer raising operations. Research has shown that one of the infectious diseases lowering conception rates is leptospirosis caused by L. borgpetersenii serovar hardjo bovis. Diagnostically Elusive As noted above, diagnosing hardjo bovis is relatively difficult. Source of the problem lies with the pathogenesis of the disease (Figure 1). Initially, hardjo bovis enters the body through mucous membranes of the nose, eyes, and mouth, or through breaks in the skin. The organism spreads immediately from the site of entry to the bloodstream where it begins replicating exponentially, with a doubling time of approximately 8 hours. During this period of leptospiremia (lasting anywhere from 3 to 20 days), the organism also invades and colonizes the liver, kidney, spleen, eye, and reproductive tract. Circulating antibodies are generated early in the course of infection and usually remove leptospires from the bloodstream and most tissues. However, in animals that become chronically infected, some leptospires survive by localizing in immunologically privileged sites such as the kidney tubules and genital tract where they are protected against circulating antibodies. Leptospiral growth continues in these privileged sites and eventually infectious leptospires pass out of the body in urine, a condition that causes more infection especially if the urine contaminates water sources frequented by susceptible animals. This chronic condition in which host animals show no clinical signs, antibody production is limited, and leptospires are shed from the kidneys in urine is known as the renal carrier state. Excretion may continue intermittently or continuously for periods lasting from several weeks, months, or years, to the lifetime of the animal. Such excretor animals play a pivotal role in disease transmission since they are regarded as the central distributors of leptospirosis within a given population. They also serve as a reservoir for zoonotic illness. 61

64 Figure 1 In chronically infected cows, leptospires survive in immunologically privileged sites where they are protected against circulating antibody to hardjo bovis. Until recently, no United States licensed vaccines were effective at controlling Leptospira borgpetersenii serovar hardjo bovis in cattle. Multiple studies have demonstrated Spirovac s ability to prevent colonization of the kidneys and uterus. This effectively stops the carrier stage of this infection and protects against the reproductive impacts of infection. Spirovac Leptospira borgpetersenii serovar hardjo bovis vaccine highlights include: 1. Two doses initial regimen ( booster dose administered 4-8 weeks after the first dose 2. Annual booster requirement 3. True year duration of immunity 4. No blockage by maternal antibody Instituting a Spirovac Vaccination program Several different programs can be used to begin an effective vacination program against Leptospira borgpetersenii serovar hardjo bovis utilizing Spirovac vaccine. The program chosen is determined by the severity of th eproblem being cuased by current infection and current management as well the herd s desire for rapid onset of protection. Beginning Spirovac vaccination may be done in one of the following manners: 1. Whole herd programs. This will give fastest herd control. 2. Dry/Open cows, springing and breeding age heifers. This may show the highest impact for vaccination dollars spent. 3. Young stock program, This will take the longest to see impact and is recommended only in low incidence/problem herds. 62