Investigation of single, fixed-time artificial insemination in sows and gilts. Lima Freya Rodrigues. A Thesis Presented to The University of Guelph

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1 Investigation of single, fixed-time artificial insemination in sows and gilts by Lima Freya Rodrigues A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Population Medicine Guelph, Ontario, Canada Lima Rodrigues, January, 2018

2 ABSTRACT INVESTIGATION OF SINGLE, FIXED-TIME ARTIFICIAL INSEMINATION IN SOWS AND GILTS Lima Freya Rodrigues University of Guelph, 2018 Advisor: Dr. Robert Friendship The studies included in this thesis evaluated the use of two single, fixed-time artificial insemination (FTAI) techniques in both sows and gilts. One method involved IM injections of 600 IU of equine chorionic gonadotropin (ecg) followed by 5 mg of porcine luteinizing hormone (plh) and the other method was a 200 µg intravaginal dose of triptorelin acetate. Time of ovulation was monitored by ultrasound in a subset of animals. Both FTAI techniques resulted in sows farrowing within a short interval, leading to older and heavier pigs at weaning compared to controls. However, FTAI was associated with decreased farrowing rates, smaller litters, and fewer pigs produced per group in certain circumstances. The use of FTAI should be further investigated to determine why reproductive performance is sometimes reduced.

3 ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Robert Friendship. Thank you for introducing me to the swine industry and allowing me to gain a greater appreciation for agriculture. Thank you for giving me this opportunity and for all that I have experienced because of it. It has been a privilege learning all that I know from such a passionate mentor. You have always been a constant pillar of support and I could not be more grateful for that. To my committee member, Dr. Terri O Sullivan, thank you for your guidance and advice throughout my research. I appreciate all the knowledge you have shared with me. I would like to thank both Glen Cassar and Rocio Amezcua for the countless hours spent on farm collecting data, as well as your willingness to always help when I needed it. Additionally, I appreciate the staff at the Arkell Swine Research Station for their assistance. I would also like to acknowledge Ontario Pork and the Ontario Ministry of Agriculture, Food and Rural Affairs-University of Guelph Research Partnership for funding these projects. Lastly, I would like to thank my friends and family for their invaluable support and guidance throughout this learning experience. To my parents, thank you for always encouraging me to venture beyond what I expected for myself. Todd, words cannot express how grateful I am to have your unconditional support every step of the way. My time as a Master s student has been very enjoyable and this is something I will never forget. Thank you to everyone who made it a special experience for me. iii

4 CONTRIBUTIONS Lima Rodrigues contributed in the fieldwork, data management and analyses, interpretation of results, and was principal author of all chapters. Dr. Robert Friendship led the research projects, helped coordinate the fieldwork, interpreted results, and provided critical feedback on all chapters. Dr. Glen Cassar assisted with examination and interpretation of ultrasound results and data collection on Farm 2. Maria Amezcua helped with the field work and data collection on Farm 1. She also assisted with data management and analyses. Dr. Terri O Sullivan assisted with data analyses. Dr. David Pearl helped with data analyses when needed. Alison Jeffery assisted with data validation. Jordan Buchan helped with timing and recording the breeding of sows for the economic analysis. Elanco supplied triptorelin acetate for the research trials. Ontario Pork and the Ontario Ministry of Agriculture, Food and Rural Affairs-University of Guelph Research Partnership provided funding for these research projects. iv

5 TABLE OF CONTENTS LIST OF TABLES... vii LIST OF ABBREVIATIONS... viii CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW Introduction Female Porcine Reproductive Anatomy and Physiology Anatomy Estrous cycle Hormones Ovulation Artificial Insemination Synchronization Protocols Estrus Synchronization Ovulation Synchronization Gonadotrophin-releasing hormone (GnRH) and its analogues Human chorionic gonadotrophin (hcg) Porcine luteinizing hormone (plh) Conclusions/Thesis Objectives References CHAPTER 2: COMPARISON OF SINGLE, FIXED-TIME ARTIFICIAL INSEMINATION IN WEANED SOWS USING ecg-plh OR TRIPTORELIN ACETATE TO SYNCHRONIZE OVULATION ON TWO FARMS Introduction Materials and Methods Results Discussion Acknowledgements References CHAPTER THREE: COMPARISON OF SINGLE, FIXED-TIME ARTIFICIAL INSEMINATION IN GILTS USING ecg-plh OR TRIPTORELIN ACETATE TO SYNCHRONIZE OVULATION Introduction Materials and Methods Results Discussion Conclusions Acknowledgements References v

6 CHAPTER 4: CONCLUSIONS Research Summary and Conclusions References APPENDIX I APPENDIX II vi

7 LIST OF TABLES Table 2.1: Production performance, represented by mean values ± standard deviation, of sows receiving a single, fixed-time artificial insemination (Group 1 and 2*) versus conventional double mating during a natural estrus (Group 3) on Farm 1 and Farm Table 2.2: The final model* illustrating the effect of FTAI (Groups 1 and 2) versus conventional (Group 3) breeding on litter size (total count) on Farm Table 2.3: The final model* illustrating the effect of FTAI (Groups 1 and 2) versus conventional (Group 3) breeding on weaning weight (kg) on Farm Table 2.4: The number of days it took for the last sow in each treatment group* to farrow, after the first sow + in each batch farrowed (Day 0) on Farm Table 2.5: Detailed ultrasound results of time of ovulation as experienced by 10 sows per treatment group* on Farm 2 beginning on Day 5 post-weaning Table 3.1: The number of days it took for the last gilt in each treatment group* to farrow, after the first gilt + in each batch farrowed (Day 0) and the range in breeding days for Group Table 3.2: The final model* illustrating the effect of FTAI (Groups 1 and 2) versus conventional (Group 3) breeding on the piglet weaning weight (kg) of gilts Table 3.3: Production performance, represented by mean values ± standard deviation, of gilts receiving a single, fixed-time artificial insemination (Group 1 and 2*) versus conventional double mating during a natural estrus (Group 3) Table 4.1: The cost breakdown ($ CAD) of FTAI protocols versus conventional double mating in a single sow vii

8 LIST OF ABBREVIATIONS AI artificial insemination DIUI deep intrauterine artificial insemination ecg equine chorionic gonadotrophin FSH follicle-stimulating hormone FTAI fixed-time artificial insemination GnRH gonadotrophin-releasing hormone hcg human chorionic gonadotrophin IM intramuscular(ly) IU International Unit IUI intrauterine artificial insemination LH luteinizing hormone plh porcine luteinizing hormone viii

9 CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction The Canadian swine industry is continuously improving reproductive technologies to meet higher production targets in order to gain an economic advantage in the world market. In a farrow-to-finish operation, the performance of the breeding herd determines the flow of pigs through the various production stages. A high level of production and consistent performance is important. If too many sows are bred at a particular time, then the consequences will be overcrowding in the nursery and grower stages as well as a problem of finding sufficient space to farrow the excess pregnant sows. If too few sows are successfully bred, then there will be a gap in later stages of production (an under-utilization of resources) resulting in a loss in potential returns. The two major factors that contribute to sufficient breeding numbers are sow longevity, so that young sows are not culled prematurely, and a reliable pool of gilts. Ensuring availability of service-ready gilts is necessary to manage breeding targets (Manjarín et al., 2015). The entry of gilts into the breeding herd contributes to non-productive days as they experience estrus unpredictably and it is difficult to manipulate their estrous cycle so that the right number of gilts is available to breed. This is especially true when a farm employs batch farrowing. Since swine producers replace 30 to 50% of sows with gilts annually (Estienne et al., 2010), it is of particular interest to the industry to develop and improve new reproductive technologies for gilts that will effectively optimize reproductive efficiency and production rate (Manjarín et al., 2015). Farms that utilize a batch farrowing management technique are required to breed females on a tight schedule, which may become a challenge with gilts and non-pregnant sows (Kauffold et al., 2007). By controlling the timing of the processes of estrus and ovulation through 1

10 pharmaceutical treatments (i.e. synchronization), producers may be able to overcome these difficult circumstances. Artificial insemination in pigs has been widely used as a commercial application since the 1980s but has since been further developed (Shinde & Gupta, 2016). Increased accuracy in the prediction of ovulation time could make it possible to implement a single insemination at a fixed-time with a similar probability of conception compared to the conventional method of breeding a sow twice or three times during an estrus. This is known as fixed-time artificial insemination (FTAI). The use of FTAI synchronizes breeding so that sows farrow within a narrower window of time, allowing better supervision at farrowing and during the first few days of neonatal care (Kraeling & Webel, 2015). This also creates a uniform group of similar aged pigs at farrowing and weaning. Uniform age at weaning is desired because it reduces the likelihood of pigs that are too young being moved to the nursery and mixed with older and larger animals. Using protocols such as FTAI will allow gilts and sows to be more easily integrated into a scheduled program such as a batch farrowing system, reducing the number of non-productive days (Kauffold et al., 2007). This literature review will be discussing the reproductive physiology of a female pig and synchronization protocols associated with artificial insemination. 1.2 Female Porcine Reproductive Anatomy and Physiology Anatomy In swine, major structures of the female reproductive tract include the vagina, cervix, uterus, oviducts and the ovaries (Senger, 2012). The tract may be envisioned as a series of interconnected tubes that begins with the vagina. It is the female copulatory organ that connects the vulva, an external structure, to the cervix. The cervix leads to the uterus and is made up of numerous cervical rings, which form interlocking projections. The uterus is a hollow, tubular 2

11 organ that links the cervix to the oviducts. In swine, this is characterized by having two highly developed uterine horns, up to 1.5 m in length, and a small uterine body located at the junction of the two uterine horns (Senger, 2012). The site at the end of the uterine horn, which attaches to the oviduct, is known as the uterotubal junction. The oviducts are small, convoluted tubes joining the uterus and ovaries, which are located at the end of each oviduct. Multiple ova, or eggs, are released from the follicles on the ovaries during ovulation. Ova are fertilized in the oviducts and the resulting embryos develop into fetuses in the uterus (Youngquist, 1997) Estrous cycle The estrous cycle in the female pig consists of a follicular phase of 5 to 7 days and a luteal phase of 13 to 15 days. During the follicular phase, small antral follicles develop into large, pre-ovulatory follicles. The pig may ovulate from 15 to 30 follicles. During the luteal phase, follicle development is less pronounced due to progesterone inhibition of gonadotrophic hormones. Nevertheless, formation of the early antral follicle pool probably has a major impact on follicle dynamics in the follicular phase in terms of number and quality of follicles. After farrowing, sows experience a lactational anoestrus period until they wean their offspring and the follicular phase is initiated once again, resulting in estrus and ovulation 4 to 7 days after weaning. Estrus detection is of vital importance in ensuring good timing of insemination and improved reproductive performance. Estrus, also known as standing heat, is the period of the estrous cycle when the female is fertile and sexually receptive. The onset of estrus is variable among sows and unpredictable in gilts. Signs of estrus include seeking of the boar, swelling and reddening of the vulva, erect ears in breeds where that is possible, decreased appetite, and a locked-up position in which the muscles tense up to prepare her to be mounted by the boar. If a female displays lordosis when presented with a boar, she is in standing heat and will permit 3

12 breeding. Lordosis is the characteristic mating posture of females in estrus; it is when she arches her back in anticipation (Senger, 2012). During estrus detection, a boar is moved down the aisle in front of the females and a stockperson observes for signs of estrus. To determine if she is ready to be bred, the stockperson applies pressure on her back. If she moves around, vocalizes, or walks forward she is not in standing heat. However, if she seems locked into place then she is in standing heat and breeding may proceed. This is a very time-consuming process that requires patience and stockmanship skills. The duration of estrus can range from 1 to 2.5 days and ovulation typically occurs at about 70% of the way through estrus (Wongkaweewit et al., 2012). With artificial insemination, it is crucial that estrus detection is properly identified to ensure breeding is carried out during the optimal period to increase the likelihood of fertilization. Although it can be labour intensive, the apparent onset of estrus and its duration is influenced by the frequency of estrus detection, intensity of the female s behaviour, and the stockman s observational skills (De Rensis & Kirkwood, 2016) Hormones The hormonal interactions of the hypothalamo-pituitary-ovarian axis regulate the onset of estrus and ovulation. The hormonal cascade begins with the hypothalamus releasing gonadotrophin-releasing hormone (GnRH), which stimulates the anterior pituitary gland to release follicle-stimulating hormone (FSH) (Turner et al., 2005). FSH promotes the ovarian follicle to grow and produce estrogen (Turner et al., 2005). The rise in estrogen is relayed back to the pituitary gland stopping the production of FSH and promoting the release of luteinizing hormone (LH) (Turner et al., 2005). The surge of LH induces matured eggs, or ova, to be released from the ovary; this process is known as ovulation (Turner et al., 2005). FSH, estrogen, 4

13 and LH return to their basal levels 3 to 35 hours after the surge and once ovulation is complete (Brinkley, 1981). Presented below is a brief overview of some of the hormones commonly used in synchronization protocols. Altrenogest, commercially available as Regu-Mate, is an orally active progestogen that inhibits gonadotrophin release, thereby, inhibiting follicular growth (Kauffold et al., 2007). It has been used to synchronize estrous cycles by blocking the growth of medium-sized follicles and initiating the cycle once altrenogest is withdrawn (Kauffold et al., 2007; De Rensis & Kirkwood, 2016). Equine chorionic gonadotrophin (ecg) is a hormone that has FSH-like activity when administered to swine; therefore, it is involved in the onset of estrus by stimulating follicle development (Brüssow et al., 2009; Senger, 2012). Human chorionic gonadotrophin (hcg) is a hormone that has LH-activity when administered to swine. This is used in combination with ecg to promote follicle growth and is also involved in inducing ovulation (De Rensis & Kirkwood, 2016; Ulguim et al., 2016). PG600 is a combination of ecg and hcg, that causes follicular development and ovulation; therefore, it is often used to induce puberty in gilts (Kraeling & Webel, 2015). The dosage rate of PG600 that is typically used is 400 IU of ecg in combination with 200 IU of hcg. Lastly, triptorelin acetate, commercially available as OvuGel, is a GnRH agonist that stimulates the release of FSH and LH, inducing and advancing ovulation (Kraeling & Webel, 2015). It has been formulated for intravaginal delivery in a gel form (Knox et al., 2017) Ovulation Ovulation is the process by which oocytes (eggs) of a female pig are released from the ovary into the reproductive tract during estrus for fertilization by sperm to reproduce. The onset of ovulation requires the presence of mature follicles on the ovary that are able to respond to a 5

14 preovulatory LH surge (Knox, 2015). The LH surge is the distinct and substantial rise in the levels of LH before ovulation, which is induced after a period of time following estrogen exposure (Knox, 2015). In fact, administration of estrogen has been shown to induce the LH surge (Knox, 2015). Exposure of the follicle to the LH surge initiates the process of ovulation and it rapidly becomes independent of subsequent gonadotropin control (Knox, 2015). The presence and levels of gonadotrophins and the process of follicular development prior to ovulation determine the rate of ovulation (Soede et al., 2011). It has been established that LH is responsible for the selection and maturation of follicles while FSH also plays a supporting role in follicle development (Soede et al., 2011). It has been well established that maximum fertility, including fertilization rate, farrowing rate, and litter size, is achieved by breeding females within the 24-hour period prior to ovulation (Cassar et al., 2005; Knox et al., 2017). This is influenced by the short survival time of the oocyte after ovulation and the lifespan of the sperm within the female reproductive tract (Knox et al., 2017). Therefore, if you inseminate too early relative to ovulation it will result in decreased fertility and inseminating too late has the same effect (Castagna et al., 2003; Rozeboom et al., 1997). If the time of ovulation could be determined, a single insemination per female could be sufficient in impregnating her, regardless of expression of estrus (Hühn et al., 1996). This is the basis of the FTAI program. There have been countless studies conducted for developing protocols that synchronize ovulation as there are many potential benefits. It would facilitate the introduction of gilts into the breeding herd, reduce labour and costs associated with estrus detection, and reduce the need for multiple inseminations, substantially increasing the economic and genetic merit of semen usage (Knox, 2015; Kraeling & Webel, 2015). Inducing ovulation in 6

15 sows post-weaning would improve the synchrony of ovulation among the group and allow for a FTAI program (Knox et al., 2017). 1.3 Artificial Insemination The use of artificial insemination (AI) as a viable technique to inseminate female pigs dates back to the early 1930s (Knox, 2016; Shinde & Gupta, 2016). However, it was not until the 1980s that its true development and commercial application was widely adopted around the globe. By the year 2000, numerous countries bred almost all pigs by AI (Knox, 2016; Shinde & Gupta, 2016). The acceptance and success of breeding by AI is most likely a result of further development of AI techniques and equipment as well as improvements in fertility, labour efficiency, genetics, and production (Knox, 2016). For example, through the use of AI, genes from superior boars can remain focused on desirable production traits such as feed conversion, growth performance, and carcass measures (Knox et al., 2017). The current standard breeding protocol used in North America for inseminating swine is checking for signs of estrus once or twice per day, using boar exposure as an aid. Once the sow is found to be in standing heat, best indicated by the standing reflex, she is inseminated once. She is inseminated a second time 12 to 24 hours later only if she is still displaying signs of estrus. Each insemination comprises of 2.5 to 3.0 billion motile sperm per dose of semen resulting in an industry average of 84% farrowing rate with 12 total piglets born per litter (Knox, 2016; Knox et al., 2017). The vulva is cleaned to remove urine and feces and to reduce the introduction of bacteria into the reproductive tract (Knox, 2016). Using a new or clean AI foam tip catheter, the rod is lubricated and inserted into the cervix. Once fixed into place, the semen dose is attached to the catheter and is allowed to flow into the cervix by gravity and suction from the sow over a 3 to 4 minute period while pressure is applied to the back to mimic the mounting of a boar (Knox, 7

16 2016). The onset of estrus determines the time of insemination in this standard protocol, however, the duration of estrus and the interval from estrus to ovulation is variable and influenced by many factors (Knox et al., 2017). There are at least two other methods, based on location, for depositing sperm into the reproductive tract of the female pig. Intrauterine artificial insemination (IUI) is a method where an inner rod is passed through the outer rod that is locked into the cervix, so sperm is released into the uterine body (Knox, 2016). A second method is deep intrauterine artificial insemination (DIUI) where a flexible device is moved into the uterine horn in a similar way as IUI but as deep as possible so the sperm is released near the uterotubal junction (Roca et al., 2003). Each procedure has its benefits and limitations, however, the deposition of sperm into the cervical channel remains the most practiced method (Roca et al., 2003). Researchers have developed protocols that allow the time of ovulation to be predictable and therefore, insemination can be performed at a predetermined time independent of estrus detection for successful breeding (Kauffold et al., 2007). Due to increasing implementation of batch farrowing management, FTAI programs have become increasingly important. With reproductive performance goals and expectations constantly rising, priorities have shifted towards making advancements by using fewer inseminations and smaller doses of sperm in the window of time with the highest potential for fertilization (i.e. 24 hours before ovulation) (Knox, 2016; De Rensis & Kirkwood, 2016). FTAI is one way to facilitate this. 1.4 Synchronization Protocols Estrus Synchronization Due to the unpredictability of gilts, the estrous cycle must be synchronized into the breeding schedule of a batch farrowing system when they are introduced into a breeding herd 8

17 (Brüssow et al., 2009). It is also usually necessary to synchronize estrus before inducing ovulation in gilts for FTAI protocols (Ulguim et al., 2016). Although sows typically come back into heat post-weaning, a proportion of weaned sows may not have adequate follicle development to predictably return to standing heat post-weaning. In addition, those diagnosed as non-pregnant may not correspond with the breeding schedule once they are rebred, causing a problem for managing the farrowing rooms. (Kauffold et al., 2007; Schlegel et al., 1978). For this reason, there has been extensive research on advancing the control of estrus, ovulation, and subsequent fixed-time artificial insemination in females. It has been shown that estrus can be best controlled and synchronized by using altrenogest and/or ecg and hcg protocols (Kauffold et al., 2007). Altrenogest, commercially available as Regu-Mate, suppresses the release of gonadotrophin-releasing hormone which inhibits follicular growth (De Rensis & Kirkwood, 2016). Once withdrawn, the follicular phase resumes (De Rensis & Kirkwood, 2016). Administering 15 to 20 mg as a daily oral dose for 14 to 18 days results in synchronized estrus in approximately 85% of females, 5 to 7 days post-drug withdrawal (Kraeling & Webel, 2015; De Rensis & Kirkwood, 2016). Since this occurs over the span of a week, gonadotrophin hormones may also be administered after altrenogest withdrawal or to sows after weaning to achieve better synchronization by stimulating follicle development (Brüssow et al., 2009). Analogous to FSH, ecg administered 24 hours after altrenogest withdrawal (Hühn et al., 1996) is shown to be effective at promoting and synchronizing follicle development at doses of 500 to 1,000 IU and 500 to 750 IU in gilts and sows, respectively (De Rensis & Kirkwood, 2016). A single dose of ecg has been demonstrated to stimulate estrus within 4 to 5 days (Knox, 2015). Hühn et al. (1996) states that accurate timing of ecg administration, post-altrenogest withdrawal, can ensure 9

18 the onset of estrus in 85 to 90% of gilts. As shown by a number of studies, the number of piglets born alive increased by 0.5 piglets at subsequent farrowing when ecg was routinely used (Hühn et al., 1996). As an alternative to ecg alone, combinations of gonadotrophins have also been observed to promote follicle growth (Brüssow et al., 2009). PG600, a commercially available product, is a commonly used protocol for the induction of estrus by the injection of a combination of 400 IU ecg and 200 IU hcg (Kraeling & Webel, 2015; Manjarín et al., 2015). Numerous studies have established that estrus occurs within 5 days of an injection of PG600 in gilts and sows at weaning (Kraeling & Webel, 2015). Although, at higher doses its use has been associated with an increased risk of ovarian cysts likely due to the increased LH-activity of hcg (Brüssow et al., 2009; Ulguim et al., 2016). In prepubertal gilts, the ecg and hcg ratio mentioned above is optimal for synchronizing estrus, increasing ovulation rate, and reducing cystic follicles, as confirmed by past studies (Estienne et al., 2010; Manjarín et al., 2015). In fact, when the ratio of ecg and hcg was increased from 400:200 to 700:350, there was a significant increase in ovarian cysts (88% versus 36%) in gilts (Brüssow et al., 2009). On the other hand, only 4% developed cysts when 1,000 IU ecg was administered to gilts (Schlegel et al., 1978). This is likely why PG600 seems to be used more often to induce first heat, or puberty, in prepubertal gilts and is the least preferred method for the synchronization of ovulation (Brüssow & Wähner, 2011; Estienne et al., 2010). Although these protocols may be beneficial for estrus induction, the variability in time of ovulation is too great to allow for implementation of a fixed-time artificial insemination program (Brüssow et al., 2009). 10

19 1.4.2 Ovulation Synchronization Since optimal fertility is achieved by insemination during the 24-hour period prior to ovulation, timing is imperative (Cassar et al., 2005; Ulguim et al., 2016). Hormonal control of ovulation is a method that has been effective in limiting the variation of time of ovulation in swine. In pigs with mature follicles, ovulation can be induced by using gonadotrophin-releasing hormone (GnRH) or its analogues, human chorionic gonadotrophin (hcg), or porcine luteinizing hormone (plh) (Knox, 2015) Gonadotrophin-releasing hormone (GnRH) and its analogues Several GnRH analogues have been evaluated for ovulation induction in swine (Martinat- Botté et al., 2010). Past research has established that ovulation occurs in sows at approximately 38 hours after GnRH treatment (Wongkaweewit et al., 2012). Exogenous GnRH induces ovulation by acting at the anterior pituitary level in order to induce an endogenous preovulatory LH surge (De Rensis & Kirkwood, 2016; Ulguim et al., 2016). When a GnRH analogue was given to sows post-weaning, they experienced a mean time of ovulation at 39 hours after the onset of estrus (Martinat-Botté et al., 2010). In another study, when an analogue was given 72 hours subsequent to ecg, ovulation was induced in all prepubertal gilts that received treatment compared to only a few ovulations when gilts were not pretreated with ecg (Knox, 2015). Signifying that stimulating follicular development through the use of ecg may be advantageous when aiming for ovulation induction using a GnRH analogue. Involving ecg treatment at 24 hours after weaning, Rosales et al. (2008) showed that treatment with a GnRH analogue 56 hours after ecg administration successfully induced ovulation and additionally, improved farrowing rate and litter size compared to the controls. Martinat-Botté et al. (2010) applied a treatment using a GnRH analogue at 94 or 104 hours post-weaning and found that after 94 hours the 11

20 weaning to ovulation interval was condensed while maintaining fertility. Fertility was considerably decreased and the interval remained the same as the controls when the analogue was administered 104 hours post-weaning, indicating treatment may have been given too late (Martinat-Botté et al., 2010). Although the use of GnRH and its analogues has been shown to be successful in inducing ovulation while maintaining a high level of fertility and of fecundity in female gilts and sows, the timing and doses differ between studies (Brüssow et al., 1996; Martinat-Botté et al., 2010). GnRH agonists have been described as more effective than GnRH or hcg in advancing and synchronizing ovulation (Schlegel et al., 1978). Triptorelin acetate (OvuGel ) is a GnRH agonist. It stimulates the release of FSH and LH from the anterior pituitary, ultimately inducing ovulation (Kraeling & Webel, 2015). Currently, triptorelin acetate is only approved for use in sows and it is the first GnRH agonist product to be approved for synchronizing ovulation followed by a single FTAI program (Kraeling & Webel, 2015). Treatment with this product causes ovulation to occur 40 to 48 hours post-triptorelin administration (Kraeling & Webel, 2015). Hence, it is administered to sows as an intravaginal gel 96 hours post-weaning followed by a single FTAI performed 22 to 24 hours after triptorelin acetate administration to optimize fertility (Kraeling & Webel, 2015). It has been suggested that the transport of this gel across a membrane is augmented when the viscosity of the agent matches the viscosity of the cells in the membrane (Stewart et al., 2010). Treatment in gel form also reduces the stress induced by injection of other hormonal treatments and allows for ease of administration by technicians (Schlegel et al., 1978). This protocol occurs independent of the onset of estrus (Kraeling & Webel, 2015) and is reported to have similar fertility findings compared to controls given multiple inseminations (Knox et al., 2017). 12

21 Knox and colleagues (2017) evaluated the use of a single IUI using 1.5 or 2.5 billion sperm administered at either 22, 26, or 30 hours following triptorelin acetate treatment, given 96 hours post-weaning in sows. The average interval from triptorelin acetate to ovulation was 42.2 ± 0.4 hours and ovulation occurred within a 24 hour period in 88% of sows (Knox et al., 2017). The 2.5 billion sperm dose and inseminations at 22 and 26 hours following triptorelin acetate resulted in greater farrowing rate and litter size compared to 1.5 billion sperm and inseminations performed 30 hours after triptorelin acetate (Knox et al., 2017). The farrowing rate was also affected by other variables such as parity, estrus expression, and ovulation (Knox et al., 2017). Similarly, another study evaluated the effects of altering the dose and timing of intravaginal triptorelin gel in weaned sows (Knox et al., 2014). The intravaginal gel containing 0, 25, 100, or 200 µg of triptorelin was administered to sows 96 hours post-weaning, they were inseminated on each day they exhibited estrus (Knox et al., 2014). After 48 hours of receiving the treatment, more sows ovulated with the 200 µg of triptorelin gel compared to the 100 and 25 µg or Placebo groups (Knox et al., 2014). Using this dosage information, 200 µg was administered to sows at 72, 84, or 96 hours after weaning or they were left untreated (Knox et al., 2014). After 40 hours of receiving the treatment, more sows ovulated when treatment was received 72 and 84 hours post-weaning compared to 96 hours after or when left untreated, however, the farrowing rate was higher in the two latter groups (Knox et al., 2014). These results indicate that 200 µg of triptorelin gel administered intravaginally 96 hours post-weaning effectively synchronizes ovulation and results in fertility similar to the controls (Knox et al., 2014). Francisco et al. (2015) investigated the administration of triptorelin acetate followed by a single FTAI program in many commercial farm management conditions. Control sows were inseminated on the day they were detected in estrus and 24 hours later if still in standing heat 13

22 while treatment sows received triptorelin acetate according to protocol (Francisco et al., 2015). Farrowing rate was lower in the triptorelin acetate treatment group when the rate was calculated as the number farrowed of the number bred (Francisco et al., 2015). However, it is important to note that all sows in the treatment group were bred independent of estrus and only those in standing heat were bred in the control group (Francisco et al., 2015). When calculating the farrowing rate based on the number of sows in estrus at insemination on Day 5, it allows for a more accurate comparison between groups (Francisco et al., 2015). Using this method, there was no difference in farrowing rate between groups, although the treatment group achieved this with a single insemination (Francisco et al., 2015). The number of piglets born per litter was also comparable between groups, however, total born per semen dose was greater for the triptorelin acetate group (Francisco et al., 2015). By inseminating sows impartial to estrus, a proportion of sows which did not display estrus will become pregnant and farrow, increasing the number of pregnant sows on farm with the same sow inventory (Francisco et al., 2015). This would be a benefit to producers. Also evaluating the protocol on multiple farms, Allison (2013) compared triptorelin acetate-treated sows with controls and found no difference in farrowing rate between groups. This study revealed that 2.9 more doses of semen were used in control sows than in triptorelin acetate-treated sows (Allison, 2013). The value of saved labour was also highlighted when one of the farms reallocated an employee to attend farrowing sows overnight and discovered that the percentage of stillbirths significantly decreased from 7.7% to 1.9% with triptorelin acetate (Allison, 2013). Therefore, this protocol will permit labour to be redistributed to other high value farm responsibilities such as retaining the best technicians for artificial insemination, gilt management, and farrowing attendance for piglet care and to decrease pre-weaning mortality 14

23 (Francisco et al., 2015) Human chorionic gonadotrophin (hcg) Human chorionic gonadotrophin is a LH analogue, which works at the ovarian level (De Rensis & Kirkwood, 2016; Ulguim et al., 2016) and has an effective dose of 500 to 1,000 IU to induce ovulation (Knox, 2015). Past research has established that ovulation occurs in sows at approximately 42 hours post-hcg treatment (Cassar et al., 2005; Wongkaweewit et al., 2012). As recently reviewed by Brüssow et al. (2009), ecg was administered to females followed by hcg 80 hours later to induce estrus and ovulation, respectively. These females experienced ovulation between 42 to 53 hours post-hcg, whereas no control animals ovulated during this interval (Brüssow et al., 2009). A different study found that a combination of 300 IU hcg and 300 μg GnRH improved the synchronization of ovulation when compared to simply administering 500 IU hcg at first detection of estrus (Hühn et al., 1996). Conversely, a study comparing the administration of these hormones 72 hours post-weaning found that the administration of hcg resulted in development of smaller follicles and reduced estrus expression, while administration of GnRH resulted in follicle growth and greater cyst formation (Knox, 2015). Additionally, Manjarín and colleagues (2015) found that supplemental hcg, after ecg plus hcg treatment, did not increase the number of gilts ovulating, but it did increase the frequency of follicular cysts in a dose dependent fashion. This indicates that additional hcg may be associated with excess LH activity thus leading to increased instances of cyst development (Manjarín et al., 2015). In another occasion, gilts were treated with ecg to stimulate follicle development 24 hours after the last feeding of altrenogest, followed by either GnRH or hcg 80 hours later to induce ovulation (Brüssow et al., 1996). After GnRH or hcg, FTAI was used at 24 and 42 hours (Brüssow et al., 1996). Although this study lacked a negative control group, 15

24 minimal differences in farrowing rate and litter size between treatment groups were recorded (Brüssow et al., 1996) Porcine luteinizing hormone (plh) Comparable to hcg in its mechanism and effectiveness, exogenous porcine luteinizing hormone (plh) works at the ovarian level and induces ovulation within approximately 36 to 38 hours after injection in treated gilts and sows (Cassar et al., 2005; De Rensis & Kirkwood, 2016). In a previous study, Cassar et al. (2005) indicated that ecg-induced estrus combined 80 hours later with plh-induced ovulation, permitted predictable timing of ovulation and optimal timing of insemination relative to ovulation. Ovulation occurred at hours after plh treatment which allowed the use of a single FTAI (Cassar et al., 2005). In treated sows, farrowing rates were notably higher compared to control sows that were inseminated at least twice (Cassar et al., 2005). A study by Ulguim et al. (2016) also evaluated a single FTAI protocol with the use of plh at the onset of estrus in gilts and weaned sows, however, it was administered through the vulvar submucosal route rather than the typical injection. It was reported that the treatment enhanced time of ovulation in gilts but it had no effect in weaned sows (Ulguim et al., 2016). Degenstein et al. (2008) evaluated two single FTAI programs in a single study. Estrus was induced by ecg 24 hours after the last feeding of altrenogest and ovulation was induced by either hcg, plh, or saline 80 hours later allowing a single insemination to be performed 32 hours later (Degenstein et al., 2008). A reduced variation in the interval between treatment and ovulation was associated with the plh treatment compared to the hcg or control groups, which represents an important benefit of plh (Degenstein et al., 2008). This study demonstrated that the use of plh is an ideal treatment, allowing a single FTAI protocol within 30 to 40 hours after plh injection (Manjarín et al., 2015). To summarize, it is evident that the feeding of altrenogest 16

25 followed by ecg regulates ovarian follicular growth and that the subsequent administration of GnRH, hcg, or plh controls time of ovulation without affecting farrowing rate and litter size, permitting successful FTAI programs in female swine (De Rensis & Kirkwood, 2016). 1.5 Conclusions/Thesis Objectives While the majority of sows return to estrus 4 to 7 days post-weaning, there is still considerable variation between the intervals of estrus to ovulation (Schlegel et al., 1978). Furthermore, although estrus is a reliable indicator of fertility, it is still an inaccurate predictor for time of ovulation due to this variation (Schlegel et al., 1978). With gilts, in addition to experiencing unpredictable estrus, external factors such as environmental influence and genetic background can affect the onset of puberty (Hühn et al., 1996). For these reasons, the development of methods for the control of puberty, estrus, and ovulation have become essential. Especially with the production of large batches of uniformly developed healthy pigs, batch farrowing management greatly benefits from the use of synchronization protocols (Brüssow & Wähner, 2011). However, since the hormones as well as numerous farm-related factors can impact the effectiveness and outcome of these protocols, it is important to adapt this technology to each individual farm (Brüssow & Wähner, 2011). It is also necessary to compare hormonal protocols before applying such treatments on farm in order to ensure production, safety, cost, effectiveness, and fertility (Knox, 2015). The control of ovulation in gilts and sows is possible by administration of a GnRH agonist (triptorelin acetate) or with ecg followed by plh. The success of these protocols is thought to depend on the dose and hormone used, the stage of follicle development, and ensuring that the treatment occurs before the LH surge (Knox, 2015). This level of synchronization of ovulation permits the opportunity to apply an FTAI program. Typically, multiple inseminations 17

26 are required to ensure that at least one occurs close to ovulation with the existing variability between estrus and ovulation (Knox et al., 2017). However, controlling the time of ovulation permits one insemination at an appropriate time, or the insemination of fewer sperm, to be sufficient for conception to occur (De Rensis & Kirkwood, 2016). Further potential benefits include a narrower window of farrowing dates, increased uniformity between piglets at weaning, cost savings associated with less labour, and more widespread use of superior quality boar semen resulting in higher genetic merit (Brüssow & Wähner, 2011). In the following thesis, a comparison of two synchronization protocols conducted in sows and gilts will be evaluated, one with a protocol using ecg followed by plh and a second using triptorelin acetate, a GnRH agonist. As previously demonstrated, both methods have been shown to be successful, however, they have not been compared within a single study. Furthermore, triptorelin acetate (OvuGel ) does not have a label claim for gilts. The objective will be to determine the best method of synchronizing the breeding of sows and gilts that will result in optimal fertility and a narrow conception period to promote uniformity of litters, particularly in a batch farrowing system. In addition, the economics of using the FTAI procedures will be evaluated. 18

27 1.6 References Allison G. Single fixed-time insemination of swine using OvuGel (Triptorelin acetate). Am Assoc Swine Veterinarians, Proceedings. 2013: Brinkley HJ. Endocrine signaling and female reproduction. Biol Reprod. 1981;24: Brüssow KP, Jöchle W, Hühn U. Control of ovulation with a GnRH analog in gilts and sows. Theriogenol. 1996;46(6): Brüssow KP, Schneider F, Kanitz W, Rátky J, Kauffold J, Wähner M. Review: Studies on fixedtime ovulation induction in the pig. Soc Reprod Fertil Suppl. 2009;66: Brüssow KP, Wähner M. Biological and technological background of estrus synchronization and fixed-time ovulation induction in the pig. Biotechnol Anim Husb. 2011;27(3): Cassar G, Kirkwood RN, Poljak Z, Bennett-Steward K, Friendship RM. Effect of single or double insemination on fertility of sows bred at an induced estrus and ovulation. J Swine Health Prod. 2005;13(5): Castagna CD, Peixoto CH, Bortolozzo FP, Wentz I, Ruschel F, Neto G. The effect of postovulatory artificial insemination on sow reproductive performance. Reprod Domest Anim. 2003;38(5): Degenstein KL, O Donoghue R, Patterson JL, Beltranena E, Ambrose DJ, Foxcroft GR, Dyck MK. Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (plh) and its effects on reproductive function. Theriogenol. 2008;70(7): De Rensis F, Kirkwood RN. Review: Control of estrus and ovulation: Fertility to timed insemination of gilts and sows. Theriogenol. 2016;86(6): Estienne MJ, Harper AF, Horsley BR, Estienne CE, Knight JW. Effects of P.G. 600 on the onset of estrus and ovulation rate in gilts treated with Regu-mate. J Anim Sci. 2010: Francisco C, Johnston M, Webel S, Swanson M & Kraeling R. Evaluation of reproductive performance using OvuGel with a single fixed-time artificial insemination protocol in six commercial swine farms. Am Assoc Swine Veterinarians, Proceedings. 2015: Hühn U, Jöchle W, Brüssow KP. Techniques developed for the control of estrus, ovulation and parturition in the East German pig industry: A review. Theriogenol. 1996;46(6):

28 Kauffold J, Beckjunker J, Kanora A, Zaremba W. Synchronization of estrus and ovulation in sows not conceiving in a scheduled fixed-time insemination program. Anim Reprod Sci. 2007;97: Knox RV, Esparza-Harris KC, Johnston ME, Webel SK. Effect of numbers of sperm and timing of a single, post-cervical insemination on the fertility of weaned sows treated with OvuGel. Theriogenol. 2017;92: Knox RV. Review: Artificial insemination in pigs today. Theriogenol. 2016;85: Knox RV. Review: Recent advancements in the hormonal stimulation of ovulation in swine. Vet Med: Res Rep. 2015;6: Knox RV, Taibl JN, Breen SM, Swanson ME, Webel SK. Effects of altering the dose and timing of triptorelin when given as an intravaginal gel for advancing and synchronizing ovulation in weaned sows. Theriogenol. 2014;82(3): Kraeling RR, Webel SK. Review: Current strategies for reproductive management of gilts and sows in North America. J Anim Sci Biotechnol. 2015;6:3. Manjarín R, Cassar G, Friendship RM, Garcia JC, Dominguez JC, Kirkwood RN. Effect of additional human chorionic gonadotrophin (hcg) on follicular growth and ovulation in gonadotrophin-treated gilts. Can J Vet Res. 2015: Martinat-Botté F, Venturi E, Guillouet P, Driancourt MA, Terqui M. Induction and synchronization of ovulations of nulliparous and multiparous sows with an injection of gonadotropin-releasing hormone agonist (Receptal). Theriogenol. 2010;73(3): Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa. Theriogenol. 2003;60: Rosales F, Quintero V, Gonzalez M, Aguilera A, Fernadez M, Martens M. Fixed time insemination; improving fertility and saving labour. Proceedings of the 20th International Pig Veterinary Congress. 2008;2:427. Rozeboom KJ, Troedsson MHT, Shurson GC, Hawton JD, Crabo BG. Late estrus or metestrus insemination after estrual inseminations decreases farrowing rate and litter size in swine. J Anim Sci. 1997;75(9): Schlegel W, Wähner M, Stenzel S. Different combinations of PMS and hcg use for cycle stimulation in synchronized ovulation of gilts and effects on results of pregnancy. Monatsh Veterinärmed. 1978;33:

29 Senger PL. Pathways to pregnancy and parturition. 3rd edition. Current Conceptions Inc., Redmon, OR Shinde KP, Gupta SK. Review: Scientific artificial insemination in swine. Asian J Anim Sci. 2016;11: Soede NM, Langendijk P, Kemp B. Reproductive cycles in pigs. Anim Reprod Sci. 2011;124: Stewart KR, Flowers WL, Rampacek GB, Greger DL, Swanson ME, Hafis HD. Endocrine, ovulatory and reproductive characteristics of sows treated with an intravaginal GnRH agonist. Anim Reprod Sci. 2010;120: Turner AI, Hemsworth PH, Tilbrook AJ. Susceptibility of reproduction in female pigs to impairment by stress or elevation of cortisol. Domest Anim Endocrinol. 2005;29(2): Ulguim RR, Fontana DL, Bernardi ML, Wentz I, Bortolozzo FP. Single fixed-time artificial insemination in gilts and weaned sows using plh at estrus onset administered through vulvar submucosal route. Theriogenol. 2016;86(4): Wongkaweewit K, Prommachart P, Raksasub R, Buranaamnuay K, Techakumphu M, De Rensis F, Tummaruk P. Effect of the administration of GnRH or hcg on time of ovulation and the onset of estrus-to-ovulation interval in sows in Thailand. Trop Anim Health Prod. 2012;44(3): Youngquist RS. Current therapy in large animal theriogenology. 1st edition. W.B. Saunders Company, Philadelphia, PA

30 CHAPTER 2: COMPARISON OF SINGLE, FIXED-TIME ARTIFICIAL INSEMINATION IN WEANED SOWS USING ecg-plh OR TRIPTORELIN ACETATE TO SYNCHRONIZE OVULATION ON TWO FARMS 2.1 Introduction An important aspect of swine production is reproductive efficiency. Maximizing sow productivity and fully utilizing available farrowing crates requires that the sows return to estrus promptly after weaning (Kauffold et al., 2007). It is especially true for producers operating batch farrowing systems, that the variation in time from weaning to rebreeding be consistent and predictable. The wean-to-estrus interval, the duration of estrus, and the estrus-to-ovulation interval affect the sow-to-sow variation with regard to the timing of conception (Degenstein et al., 2008; Knox et al., 2014). In addition, the onset of estrus is not a reliable predictor for time of ovulation (Knox et al., 2014). Since optimal fertility is achieved by insemination during the 24- hour period prior to ovulation, it is general practice to inseminate sows every 24 hours while they remain in standing heat, which usually involves at least two breedings per estrus period (Cassar et al., 2005; Ulguim et al., 2016). If insemination is performed too early relative to ovulation it will result in decreased fertility and inseminating too late has the same effect (Castagna et al., 2003; Rozeboom et al., 1997). Conventionally, sows will not be bred if they do not display estrus or if signs of standing heat are not observed (Knox et al., 2017). With the help of hormonal treatment protocols, ovulation can be manipulated so that it is predictable, allowing for fixedtime artificial insemination (FTAI) to be performed (Knox et al., 2017). As the name suggests, single FTAI allows for a single insemination to be performed at a specific predetermined time resulting in reproductive performance that is comparable to the traditional multiple inseminations (De Rensis & Kirkwood, 2016). When an FTAI protocol is followed, all weaned sows are bred at a specific time without determining the time of onset of signs of standing heat or judging the 22