Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle

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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 2012, 24, Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle Milo C. Wiltbank A,B, Alexandre H. Souza A, Paulo D. Carvalho A, Robb W. Bender A and Anibal B. Nascimento A A Department of Dairy Science, University of Wisconsin Madison, 1675 Observatory Drive, Madison, WI 53706, USA. B Corresponding author. wiltbank@wisc.edu Abstract. This manuscript reviews the effect of progesterone (P4) during timed AI protocols in lactating dairy cows. Circulating P4 is determined by a balance between P4 production, primarily by the corpus luteum (CL), and P4 metabolism, primarily by the liver. In dairy cattle, the volume of luteal tissue is a primary determinant of P4 production; however, inadequate circulating P4 is generally due to high P4 metabolism resulting from extremely elevated liver blood flow. Three sections in this manuscript summarise the role of P4 concentrations before breeding, near the time of breeding and after breeding. During timed AI protocols, elevations in P4 are generally achieved by ovulation, resulting in an accessory CL, or by supplementation with exogenous P4. Elevating P4 before timed AI has been found to decrease double ovulation and increase fertility to the timed AI. Slight elevations in circulating P4 can dramatically reduce fertility, with inadequate luteolysis to the prostaglandin F2a treatment before timed AI being the underlying cause of this problem. After AI, circulating P4 is critical for embryo growth, and for establishment and maintenance of pregnancy. Many studies have attempted to improve fertility by elevating P4 after timed AI with marginal elevations in fertility. Thus, previous research has provided substantial insights into mechanisms regulating circulating P4 concentrations and actions. Understanding this prior research can focus future research on P4 manipulation to improve timed AI protocols. Additional keywords: corpus luteum, dairy cows, Ovsynch, ovulation, P4. Introduction Efficient reproduction is important for optimal profitability on dairy operations. Unfortunately, most dairy farms do not attain optimal reproduction due to many factors related to the management, health and physiology of high-producing dairy cows. The physiology issues involved in the reproduction of lactating dairy cows are complex, but increasingly, the interactions between nutrition, the hormonal systems and altered reproduction in dairy cattle have been elucidated and reviewed (Lucy 2001; Wiltbank et al. 2006; Chagas et al. 2007; Sartori et al. 2010). This review will focus on the role of progesterone (P4) in reproduction in the lactating dairy cow. Factors regulating the circulating concentrations of P4 will first be reviewed. Although the effects of P4 on reproduction are numerous, this review will discuss these effects during three periods in relationship to the time of breeding. In this manuscript, we will specifically discuss this relationship during timed AI programs focussing on the role of P4 concentrations before, during or after timed AI (Fig. 1). Regulation of circulating P4 concentrations The P4 concentrations that reach the receptors within each particular cell are the key determinants of the physiological Journal compilation Ó IETS 2012 actions of P4 in an animal. Almost all tissues have sufficient blood supply that circulating P4 concentrations are the primary determinant of cellular P4 concentrations. Therefore, factors regulating circulating P4 primarily determine the magnitude of P4 responses throughout the body. Circulating P4 concentrations represent a balance between the production of P4, primarily by the corpus luteum (CL), and the metabolism of P4, primarily by the liver. If P4 production is increased by an increase in luteal tissue without a change in liver blood flow, then circulating P4 will increase. Conversely, if there is an increase in liver blood flow (e.g. from 1000 to 2000 L h 1 ) then there will be a corresponding decrease in circulating P4 even though P4 production has not been altered. Thus, there are two key pathways that primarily regulate circulating P4 in lactating dairy cows. First, high constitutive P4 production by large luteal cells is primarily a function of the mass of luteal tissue or, in other words, the mass of large luteal cells (Niswender et al. 1994). Second, elevated P4 metabolism in lactating dairy cows is primarily a function of a liver with substantial enzymatic capacity for P4 metabolism and elevated rates of liver blood flow (Sangsritavong et al. 2002).

2 Circulating progesterone and fertility Reproduction, Fertility and Development 239 High P4 Before AI Pre-AI Low P4 Near AI E 2 AI High P4 After AI Post-AI Fig. 1. Model showing the role of P4 before AI, near AI and after AI. Several studies have evaluated the effect of elevating P4 before AI with substantial improvements in fertility (.10%). It is also critical that P4 be extremely low near the time of AI (,0.5 ng ml 1 ) or fertility is greatly reduced. Finally, an elevation in P4 after AI is essential for pregnancy and increasing P4 post AI may improve fertility in some circumstances. Importance of high P4 during follicular growth before AI The key role of higher circulating P4 concentrations before AI on the subsequent fertility of lactating dairy cows was first described by Fonseca et al. (1983). More recent manipulative studies have shown some improvements (,5 7% increase in percentage pregnant) by using a progesterone-releasing vaginal device (P4 device) during the Ovsynch programme before AI (Stevenson et al. 2006, 2008; Chebel et al. 2010). Nevertheless, the results from these studies may be somewhat confounded due to improvements in synchronisation rates that may accompany the use of a P4 device in the Ovsynch protocol. We have tested the effects of elevated P4 on fertility to a timed AI during the Double Ovsynch programme (Cunha et al. 2008). Cows (n ¼ 564) were randomly assigned to have either high or low P4 during the Ovsynch protocol. The cows with low P4 had an increased double ovulation rate (20.6% vs 7.0%; P, 0.01), which would be expected to potentially increase fertility in these cows (ovulation of more follicles should result in a greater chance for pregnancy). However, cows with low P4 before AI had much lower fertility (37.1% pregnant at Day 29 pregnancy diagnosis) compared with cows with high P4 (51.0%; P, 0.001). This clearly indicates that increasing P4 before timed AI can result in a substantial improvement in fertility, suggesting that the reason for lower fertility in lactating dairy cows may be at least partly due to reduced P4 concentrations during the period before AI, as discussed extensively by Inskeep (2004). Obviously, we produced an artificial reduction in P4 during this study; however, under normal conditions, P4 concentrations may be reduced due to the high feed intake and increased P4 metabolism of lactating dairy cows (Sangsritavong et al. 2002). In the future, practical programmes may be developed that target an elevation in circulating P4 in order to increase fertility. One further observation of this study was related to pregnancy loss. Pregnancy loss averages,20% in high-producing dairy cows when pregnancy loss is evaluated between Day 28 after AI (by ultrasound) and Day 60 after AI. This period is critical for development of the embryo. We observed a decrease (P ¼ 0.05) in pregnancy loss between Day 29 and Day 57 when cows had high P4 before breeding (6.8% loss) as compared with low P4 (14.3% loss). Thus, the high P4 group not only had an increased number of pregnancies at the Day 29 pregnancy diagnosis; these cows also had less susceptibility to pregnancy loss after this time. This was not due to P4 after AI because circulating P4 concentrations after AI were actually slightly higher in the cows that had low P4 before AI (2.9 ng ml 1 ) compared with the cows with high P4 before AI (2.5 ng ml 1 ). Thus, there was a positive effect of elevated P4 before AI on subsequent maintenance of pregnancy, even after 29 days following AI. Studies by Bisinotto et al. (2010) also demonstrate the importance of elevated P4 during the growth of the final (ovulatory) follicular wave. Two studies were performed in which the effect of P4 during the Ovsynch protocol was evaluated. In the first study, cows were evaluated for P4 concentration at the time of the first gonadotrophin-releasing hormone (GnRH) of Ovsynch and 7 days before this first GnRH. Cows were classified as anovular, or as beginning Ovsynch with high P4 or low P4. Cycling cows that began Ovsynch with high P4 had higher numbers of pregnancies per AI (P per AI ¼ 43.0%) than cows that had low P4 (31.3%) or that were anovular (29.7%) at the time of initiation of Ovsynch. In the second study, cows were presynchronised with two prostaglandin (PGF) treatments, and Ovsynch was initiated either 3 or 10 days after the second PGF. This design would produce cows that ovulate a dominant follicle from either the first or second follicular wave near the timed AI. Similar to the first study, cows that ovulated from the second follicular wave (high P4 concentrations) had higher P per AI than cows ovulating from the first follicular wave (low P4; 41.7% vs 30.4%). Thus, it is clear that increasing P4 during growth of the ovulatory follicle increases fertility to the subsequent timed AI by more than 10%. The mechanisms that produce this increase in fertility are still being investigated. An elegant study in superstimulated cows (Rivera et al. 2011) showed that high P4 during superstimulation increased the subsequent quality of embryos collected on Day 7 after the superovulation oestrus. Cows began superstimulation during the second follicular wave (high P4), during the first follicular wave (low P4) or during the first follicular wave with P4 supplementation using two progesterone-releasing devices to increase P4 concentrations. Although the total structures that were collected (embryos and oocytes) did not different between groups, the percentage of structures that were transferable embryos were less for cows superstimulated during the first follicular wave (55.9%), compared to during the second follicular wave (88.5%) or during the first follicular wave with P4 supplementation (78.6%). Similarly, Nasser et al. (2011) reported a dramatic increase in transferable embryos in beef cattle when P4 was supplemented during superstimulation of the first follicular wave. These results are consistent with an effect of elevated P4 during follicle growth on subsequent embryo development after ovulation. However, in a recent study in which embryos were recovered from single-ovulating cows that had follicles grow during low or high P4, there was no difference in embryo quality on Day 7 (58.3% vs 53.3%; Cerri et al. 2011b). A companion study (Cerri et al. 2011a) indicated that although

3 240 Reproduction, Fertility and Development M. C. Wiltbank et al. cows with low P4 had increased basal luteinising hormone concentrations, altered follicular dynamics and follicular fluid composition that could alter oocyte quality, a particularly distinct difference in cows with low P4 was the premature development of pathways leading to uterine PGF2a secretion. Thus, altered uterine function could also have an important role in reducing fertility in cows that have low P4 concentrations before AI. Our laboratory has recently completed a unpublished study evaluating Day 7 embryo quality in single-ovulating cows with follicle development occurring in low versus high P4 environments. We found a greater percentage of Grade 1 and 2 embryos in cows with high P4 than low P4 before AI (86.5% vs 61.5%; P ¼ 0.02). Thus, our recent results are consistent with the effects of low P4 on fertility being evident by Day 7 after AI, even in single-ovulating lactating cows, consistent with the results of Rivera et al. (2011) in superovulated cows. At this time, it is not possible to provide a definitive explanation for the reason why high P4 before AI produces higher fertility. Nevertheless, the fertility-enhancing effects are dramatic and relatively consistent in lactating dairy cows (Inskeep 2004). Future studies are needed to define the mechanism for this effect and to design reproductive management programmes that optimise these fertility-enhancing effects. Importance of low progesterone near the time of AI Inadequate luteolysis can result in an elevation in circulating P4 near AI and a reduction in fertility. This is clearly a problem with some cows during timed AI programmes (Souza et al. 2007; Brusveen et al. 2008), but also may be a problem in AI programmes based on detection of oestrus. Studies on cows that are inseminated based on oestrus detection have generally reported that minor elevations in P4 near AI are detrimental to fertility (De Silva et al. 1981; Waldmann et al. 2001; Ghanem et al. 2006), although some studies did not show any effect (Erb et al. 1976; Plym Forshell et al. 1991). For instance, De Silva et al.(1981) studied the effects of circulating P4 in dairy heifers (n ¼ 76) and cows (n ¼ 123) that were observed twice daily for visual signs of oestrus. They described a linear relationship between plasma P4 near AI and fertility in cows with low P4 (0.2 ng ml 1 on average) having over 50% P per AI; whereas, P per AI was only 20% in cows with elevated P4 (0.8 ng ml 1 on average) near AI. Similarly, Waldmann et al. (2001), using milk P4 measurements at the time of visually observed oestrus (n $ 2000 recorded first post partum inseminations done in 458 dairy herds), also described a strong negative relationship between circulating P4 near AI and fertility measured as nonreturn rates. A third study (Ghanem et al. 2006), also using milk samples at the time of AI in repeat-breeder lactating cows, found a drastic decrease in fertility as P4 increased, as well as greater embryonic losses in cows with higher P4 near AI. At this time, it is not clear whether elevated P4 near oestrus is the result of inaccurate detection of oestrus (false positive oestrus for cows in dioestrus) or incomplete regression of the CL with behavioural oestrus demonstrated in the presence of slightly elevated P4. In addition, the percentage of dairy cows in current commercial dairy herds that are inseminated in the presence of elevated P4 due to either of these two potential problems is not clear. The problems with elevations in circulating P4 near AI during timed AI programmes have been more extensively studied during the last few years. The percentage of cows that do not have complete luteal regression following the PGF of Ovsynch has been reported to range from 5% to 30% (Moreira et al. 2000; Gümen et al. 2003; Souza et al. 2007; Brusveen et al. 2009; Martins et al. 2011). A recent extensive study of incomplete luteolysis evaluated multiple blood samples in cows at first AI (n ¼ 652) and second or greater AI (n ¼ 394; Martins et al. 2011). They defined complete luteolysis and low P4 (,0.5 ng ml 1 ) at 56, 72 and 96 h after PGF. At first AI, 79% of cows underwent complete luteolysis, whereas at second and later AI, only 71% underwent complete luteal regression (P ¼ 0.03), suggesting that reduced fertility at later inseminations may be partially caused by incomplete luteolysis. Surprisingly, higher P4 concentrations at the time of PGF were associated with a higher probability of luteolysis after PGF treatment and higher fertility (50% vs 28%, for cows with high vs low P4 at time of PGF). There may be multiple physiological mechanisms that result in the reduced fertility when P4 is elevated near AI. First, P4 may alter spermatozoon or oocyte transport by altering uterine or oviducal contractility, and thus reduce fertilisation (Hunter 2005). Second, addition of P4 to in vitro fertilisation media reduced the blastocyst rate (Silva and Knight 2000), suggesting that there may be direct effects of P4 on subsequent embryo development. This detrimental effect was reversed with a P4 receptor antagonist (mifepristone RU486), indicating a specific role for P4 receptors in this action. Elevated P4 in vitro also increased total a-inhibin production by the cumulus oocyte complex, which may reduce embryo development after cleavage (Silva et al. 1999). Further, the reduced endometrial thickness with slight elevations in P4 (Souza et al. 2011) may indicate other major effects of P4 on the uterus that could result in reduced embryo development. In summary, circulating P4 near AI has been shown by several large field studies to be detrimental to fertility in dairy cattle, but the underlying physiological mechanisms that reduce fertility are not well understood. Further, the causes of elevated P4 near AI probably differ for cows inseminated after natural oestrus compared with those inseminated following timed AI protocols such as Ovsynch. However, an additional PGF treatment in cows receiving Ovsynch and more precise detection of oestrus may help to minimise the percentage of cows having somewhat higher circulating P4 concentrations near AI (Brusveen et al. 2009). Importance of high progesterone after AI Although there is unequivocal evidence that there is an absolute requirement for P4 in pregnancy maintenance (Inskeep 2004), the results have been somewhat more equivocal about the relationship between levels of circulating P4 after AI and fertility in lactating dairy cows. Several studies have reported lower P4 in non-pregnant than pregnant cows, whereas, other studies reported no relationship between post AI P4 concentrations and fertility (Bulman and Lamming 1978; Larson et al. 1997; Mann and Lamming 1999; Gümen et al. 2003; Stronge et al. 2005;

4 Circulating progesterone and fertility Reproduction, Fertility and Development 241 Lonergan et al. 2007; Morris and Diskin 2008). More extensive modelling of P4 concentrations with pregnancy, using logistic regression, have demonstrated a relationship between circulating P4 on Days 5, 6 and 7 after AI with P per AI in dairy cows, and a relationship between the rate of P4 increase and P per AI (Stronge et al. 2005). They reported that 60 85% of dairy cows had suboptimal circulating P4 for maintenance of pregnancy, based on absolute P4 concentrations during the early luteal phase or the rate of P4 increase. Many recent studies attempting to unravel the mechanisms involved in the complex relationship between circulating P4 and levels of fertility in lactating dairy cows are discussed below. Early embryos express different types and concentrations of P4 receptors (Clemente et al. 2009), raising the possibility that P4 may be acting directly on the embryo to improve embryo development. An elegant series of experiments found no effect of P4 supplementation in vitro on blastocyst yield in the presence or absence of bovine oviducal epithelial cells (Clemente et al. 2009). Thus, it appears that development of the early embryo is not directly altered by treatment with P4. In an intriguing follow-up study, these researchers treated recipient cows with an intravaginal P4-releasing device starting on Day 3 after oestrus, with in vitro produced blastocysts transferred on Day 7. Circulating P4 concentrations were elevated in the recipient cows from Days 3 to 6, but not after that time. Thus, the rise in P4 concentrations in treated cows occurred before the transfer of embryos. Nevertheless, embryos that were transferred into recipients that had received prior P4 exposure (Days 3 to 6 of increased P4) had longer embryos on Day 14 and an embryo area that was larger than in untreated recipients (Clemente et al. 2009). The authors conclude that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation. These results are consistent with the studies of Larson et al. (2011) that also failed to find a direct effect of P4 during either Days 1 to 3 or Days 4 to 7 of culture on the percentage of embryos that developed to the morula or blastocyst stage, although small differences in glucose metabolism were observed. Further evidence for a lack of an effect of P4 in the early embryo is found in the studies of Carter et al. (2008, 2010). In the first experiment, 210 crossbred beef heifers were used to analyse the effects of in vivo supplementation with P4 on embryo development. They observed no difference in early embryo development by Day 5 or 7 after AI; however, dramatic effects of P4 supplementation on embryonic length could be observed on Days 13 and 16 after AI (Carter et al. 2008). In an elegant study that continued this research focus (Carter et al. 2010), in vitro produced embryos were transferred to the oviduct of beef heifers that either received or did not receive a P4-releasing device on Day 3 after oestrus. There was no detectable effect of P4 on the proportion of embryos that developed to the blastocyst stage by Day 7, when embryos were recovered, or during subsequent culture of the embryos in vitro. However, there were subtle but intriguing differences in gene expression detected by microarray in the embryos recovered from recipients that received P4 supplementation (Carter et al. 2010). Thus, it seems clear that increased P4 during Days 3 to 7 induces changes in the uterus that increase embryo elongation by Day 14. Whether a P4-induced increase in embryo development can improve fertility in lactating dairy cows continues to be an area of investigation, as discussed below. Many studies have investigated the P4-induced changes in gene expression that occur in the endometrial tissue and these will not be extensively reviewed in this manuscript. However, it seems clear that there are dramatic differences in endometrial gene expression as the luteal phase progresses, and that early supplementation with P4 can induce earlier expression of these P4-induced differences (McNeill et al. 2006; Forde et al. 2009, 2011). The P4-induced changes in uterine gene expression can have dramatic consequences for the development of the embryos (Forde et al. 2011). There have been numerous studies that have evaluated the effects of P4 supplementation on fertility in cattle with the earliest experiments conducted in the 1950s (Herrick 1953; Wiltbank et al. 1956). Throughout the last 60 years (reviewed in Mann and Lamming 1999), there have been numerous methods described to increase P4 including treatment with exogenous P4 (injectable P4; P4-releasing intravaginal devices) or treatments attempting to induce the ovulation of a follicle and produce an accessory CL (e.g. human chorionic gonadotrophin (hcg) or GnRH). These experiments have varied considerably in regard to type of animal (beef vs dairy; heifers vs cows), day of supplementation or administration relative to AI, utilisation of synchronisation before AI, and number of animals in the trial (n). Of the 30 trials that we evaluated, most (25 out of 30) showed a numeric improvement in fertility with P4 supplementation, although only six of these trials showed statistical significance (P, 0.05). Of these six trials, only two (Stevenson et al and our unpublished results with hcg) used more than 100 animals per comparison. The larger trials generally have observed less dramatic effects than the smaller trials. Conclusions This manuscript has attempted to describe the underlying physiology that produces the changes in circulating P4 in lactating dairy cows and the potential reproductive challenges associated with suboptimal P4 concentrations. Metabolism of P4 appears to be the primary cause of lowered P4 in lactating dairy cows, although changes in P4 production by the CL have not yet been experimentally excluded. This manuscript reviewed the scientific literature on P4 and fertility with clear evidence for the effects of P4 at all three periods that were analysed (see Fig. 1). Prior to AI, there were very dramatic effects observed with more than 10% differences in P per AI observed by increasing P4 concentrations. The lactating dairy cow may have insufficient P4 during this period, and insufficient P4 at this time may, at least partially, underlie the high rate of double ovulations and low fertility that are characteristic of high-producing dairy cows. Near the time of AI, it is critical that P4 concentrations reach a nadir concentration. Even small increases in P4 near the time of AI were associated with dramatic reduction in fertility, either in cows bred to natural oestrus

5 242 Reproduction, Fertility and Development M. C. Wiltbank et al. or after timed AI protocols. Following AI, there are dramatic effects of increasing P4 on embryo elongation; however, these dramatic effects have generally not been observed in field trials focussed on improving fertility by supplementing P4 after AI. Thus, although substantial research has investigated the role of P4 on fertility in lactating dairy cows for more than six decades, it seems clear that future focus in this research area is likely to continue to yield exciting research results related to the physiology and practical management of dairy cows. Acknowledgements The authors thank Wisconsin Experiment Station and Pfizer Animal Health Global Academic Alliance. References Bisinotto, R. S., Chebel, R. C., and Santos, J. E. P. (2010). Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows. J. Dairy Sci. 93, doi: /jds Brusveen, D. J., Cunha, A. P., Silva, C. 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