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Preovulatory progestagen treatment in mares fails to delay ovulation
The Veterinary Journal 197 (2013) 324–328
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Preovulatory progestagen treatment in mares fails to delay ovulation Igor F. Canisso 1, Kirsten Gallacher, Mary-Anne Gilbert, Alexandra Korn, Christine M. Schweizer, Sylvia J. Bedford-Guaus, Robert O. Gilbert ⇑ Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA
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Article history: Accepted 21 December 2012
Keywords: Progestagen Mare Fertility Ovulation Behavior
a b s t r a c t The major objective of this study was to determine whether short-term preovulatory progestagen treatment of mares could effectively delay ovulation. Secondary objectives were to determine the effect such supplementation had on signs of estrus, follicular growth, postovulatory luteal function and pregnancy rate. Thirteen cyclic mares of different breeds were used in this study during the natural breeding season. Once mares were confirmed in estrus with a follicle of 35 mm in diameter, they were assigned in random order to receive no treatment (control), placement of a progesterone-impregnated controlled intravaginal drug releasing device (CIDR) for 2 days, or oral altrenogest treatment (0.044 mg/kg/d) for 2 days. Transrectal ultrasonography and teasing with a vigorous stallion were performed daily. Mares were inseminated every 48 h after the end of experimental treatment (progestagen groups) or beginning when the follicular diameter was 35 mm (control group) with fresh extended semen of a single fertile stallion. Each mare was followed for 3–5 cycles, allowing each treatment to be applied one or two times. Neither CIDR nor altrenogest treatment delayed ovulation. Treatment had no effect on follicular growth rate or the size of the ovulatory follicle immediately preceding ovulation. Both forms of progestagen treatment effectively abolished estrous behavior within 24 h. Estrous response to the stallion returned to the control level after cessation of treatment. Similarly, a reduction in endometrial edema was detected during progestagen treatment, which returned to normal after cessation of treatment. Altrenogest treatment tended to reduce the chance of pregnancy (P = 0.09) compared to the control group. The use of progestagens to delay ovulation in mares lacks efficacy and may threaten successful establishment of pregnancy. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Optimal fertility in mares requires breeding close to ovulation (Newcombe and Cuervo-Arango, 2011), providing an incentive to monitor and control of the estrous cycle (Allen et al., 2007). Precise control of the time of ovulation offers major advantages in broodmare management. In contrast to many other species, the duration of estrus in mares is long and variable, with time of ovulation more closely related to the end of behavioral estrus than its beginning (Carnevale, 2008). In addition, many stallions in high demand have full ‘books’ and access to these stallions can be difficult to arrange, while available breedings may not coincide with the optimal timing for mare fertility. Similarly, for those breeds that allow use of cooled, shipped semen, availability of the semen may not coincide with the optimal time for insemination. Some strategies success⇑ Corresponding author. Tel.: +1 607 253 3100. E-mail address: [email protected] (R.O. Gilbert). Current address: Reproduction Laboratory, Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40546-0099, USA. 1
1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2012.12.028
fully hasten ovulation, such as use of human chorionic gonadotrophin or gonadoliberin agonists (Loy and Hughes, 1966; Sieme et al., 2003; Hemberg et al., 2006; Davies Morel and Newcombe, 2008), but to date there is no practical way to delay ovulation in mares. Ovulation of mares has been delayed by daily administration of equine follicular fluid (Bergfelt and Ginther, 1985). This experiment demonstrated that, apart from steroid hormones, follicular fluid contained a proteinaceous component that was also capable of inhibiting follicle stimulating hormone (FSH) secretion and delaying ovulation. However, this treatment had to be applied for several days during the phase of early follicular growth, and the postponement of ovulation was not predictable. Although interesting, and potentially promising at some future time, this approach is not currently practical. Similarly, daily administration of dexamethasone (30 mg/day starting at Day 10 of the cycle) prevented estrus and ovulation in 7/8 treated mares (Asa and Ginther, 1982), but this approach may have potentially adverse side effects and does not allow predictable control of ovulation. Altrenogest is a potent progestagen commonly used in broodmare management. Previous attempts to postpone ovulation using altrenogest used small numbers of mares or yielded inconsistent
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results (James et al., 1998; Bruemmer et al., 2000). Controlled internal drug release (CIDR) has been used extensively in the cattle industry for control of estrus and ovulation (Martinez et al., 2002a,b). Intravaginal devices have been used in horses to advance ovulation in transitional mares (Newcombe, 2002; Hanlon and Firth, 2012). We therefore aimed to test the effects of administration of these exogenous progestagens on follicular development, time of ovulation, development of the corpus luteum and establishment of pregnancy in mares. Brief postponement of ovulation in superovulated cows had little effect on development competence of resulting embryos (van de Leemput et al., 2001), and growth of the equine follicle has been shown to be sensitive to exogenous progesterone (Gastal et al., 1999). Furthermore, functional removal of progesterone by administration of a luteolytic dose of prostaglandin F2a is associated with a reduced interovulatory interval in mares (Ginther et al., 2009), suggesting that the opposite effect might be produced by progesterone supplementation. Development of the equine follicle is quite consistent, with linear growth over most of the pre-ovulatory period (Ginther, 1990), allowing detection of potential changes brought about by progestagen administration. The aims of the current study were to determine whether exogenous progestagen (intravaginal device or oral altrenogest) administration in mares: (1) delayed ovulation; (2) affected incidence of spontaneous ovulation upon cessation of treatment; (3) affected luteal function (as determined by progesterone profiles) following ovulation; (4) depressed fertility; (5) affected ultrasonographically-evident endometrial edema, and (6) affected estrous behavior as elicited by teasing to an experienced stallion.
still, raised tail, winked, passed fluids and lowered pelvis [postured]) accompanied by some non-receptive behaviors (tail switching, squealing, holding ears back, attempts to kick); (8) mares showed full estrus (stood still, raised tail, winked, passed fluids and lowered pelvis [posture]) with no non-receptive behaviors.
Material and methods
Fertility trial
The present study was carried out from May to September in the Northern hemisphere physiological reproductive season of 2010. All procedures were approved by the Cornell University Institutional Animal Care and Use Committee (protocol number 2010-0020).
Pregnancy diagnosis was carried out 12 days post ovulation by transrectal palpation and ultrasonography examination using a linear 5 MHz Aloka transducer, with a mare designated as not pregnant if an embryonic vesicle was not seen by 12–14 days post ovulation, despite multiple reproductive examinations on the two previous days. If confirmed pregnant the embryonic vesicle was manually reduced using standard technique described elsewhere (Macpherson and Reimer, 2000) for pre-fixation twin reduction, and then the mare was given a 10 mg dose of prostaglandin F2a (dinoprost thromethamine, Lutalyse, Pfizer, IM). Mares were used in consecutive cycles.
Broodmares Thirteen cyclic, reproductively sound mares of various breeds (Warmbloods, Thoroughbreds, Paint Horses, and Quarter Horses) were used for this study. Every mare used on this study had had multiple regular cycles before the beginning of the trial. The mares were examined by palpation and ultrasonography per rectum every 1–3 days, according to their follicular and uterine features, and daily once in estrus. Once mares were confirmed in estrus, and had a large preovulatory follicle (P35 mm diameter), they were allocated to one of three treatments in random sequence. Each mare was followed for 4–6 cycles, allowing each treatment to be repeated one or two times. The treatments were: (1) no treatment (Control); (2) a progesterone-impregnated controlled internal drug release device intended for use in cattle (EaziBreed CIDR, Pfizer Animal Health) applied intravaginally for 48 h; and (3) altrenogest (Regu-Mate, Merck, 0.044 mg/kg/day orally for 2 days). At each examination data recording included size of the dominant follicle and significant subordinate follicles, detection of ovulation, endometrial edema (subjective scale, 0–3, with 3 maximal) and assessment of estrous behavior (Gorecka et al., 2005). Once mares were in estrus, monitoring was continued daily until ovulation was detected. Therefore time of ovulation was recorded with ±24 h of accuracy. Estrous behavior was assessed by a single evaluator. The evaluator stood laterally-caudally to the mare; every behavior displayed by the mare was recorded and the mare was given a final score according to the Gorecka et al. (2005) scoring system. During each teasing episode a mature stallion was placed in a teasing stall (with an open top, which allowed physical interaction between the mare being teased and the teaser). The mares were presented to the teaser and allowed to interact with him for at least approximately 3 min. A score was assigned as follows: (1) non-receptive behavior (tail switching, moving around, squealing, holding ears back, attempts to kick); mare attacked or kicked at the teasing stallion; (2) nonreceptive behavior (tail switching, moving around, squealing, holding ears back, attempts to kick) and no severe attack toward the stallion; (3) non-receptive behavior (tail switching, squealing, holding ears back, attempts to kick) and the mare stood still; (4) mare stood still indifferently; (5) mare showed estrus (stood still, raised tail or winked, accompanied by some non-receptive behavior tail switching, squealing, holding ears back, attempts to kick); (6) mare showed estrus (stood still, raised tail or winked) and no non-receptive behaviors; (7) mare showed full estrus (stood
Semen processing and artificial insemination Semen from a single fertile Dutch Warmblood stallion was used. Semen was collected using a dummy mount with an estrus mare present in the breeding shed. The collections were performed every 24–48 h, with the use of a Missouri artificial vagina. Immediately after each collection the semen was filtered, a sample from semen fraction gel free was subjectively evaluated for total and progressive motility. Sperm concentration was assessed by the use of a spectrophotometer (591B Equine Densimeter, Animal Reproduction Systems). The semen was then extended using a commercial skimmed milk extender (E-Z-Mixin CST containing amikacin sulfate, Animal Reproduction Systems) to 25–50 million sperm/mL and placed on a commercial semen shipment container (Equitainer II, Hamilton Research) with a minimal final volume of 120 mL per container. The semen was cooled and used within 24 h of collection. Semen was deposited in the uterine body, using standard technique for AI with cooled semen. Each artificial insemination (AI) was performed using a standard dose of 500 106 progressively motile sperm (Brinsko, 2006). Approximately 6 h post AI each mare was given a dose of 20 U of oxytocin to prevent intrauterine fluid accumulation and all mares were re-checked for intrauterine fluid accumulation and if fluid depth greater than 20 mm was present uterine lavage with 2 L of Lactate Ringer’s Solution was performed and three injections of oxytocin (10 U) were given IM, one immediately after the intrauterine flushing and the other doses were given approximately every 6 h. Every bred mare was re-checked every 24 h until no detectable intra-uterine fluid accumulation was seen. Lavage and oxytocin treatments were re-applied according to the criteria above, if necessary. During the control cycle, each mare was inseminated every 48 h beginning on the day that the dominant follicle reached a size of 35 mm until ovulation was confirmed. In the treatment cycles, inseminations were commenced after the end of treatment (i.e. immediately after CIDR removal or 24 h after the second dose of oral altrenogest) and continued every 48 h until ovulation.
Statistical analysis Maximum preovulatory follicle size, average daily growth rate of dominant follicle, time interval from follicular diameter of 35 mm to ovulation and progesterone concentrations were treated as continuous outcomes and analyzed using mixed linear regression. Fixed variables were treatment, day of cycle and treatment-by-day interactions (or cycle-day interactions when investigating relationship with pregnancy); individual mares were treated as random variables. Pregnancy was treated as a binary outcome and analyzed using mixed logistic regression with the same fixed and random variables. Uterine fluid accumulation was dichotomized (=<10 mm or >10 mm fluid depth) and analyzed similarly using mixed logistic regression. Scores for estrus behavior and endometrial edema were treated as ordinal and evaluated by ordinal logistic regression (without random variable of mare). All calculations were performed using StataIC 11.2 for Windows (Stata Corp). Graphs were produced with StataIC or with GraphPad Prism 5.04 for Windows. The sample size was chosen to be able to detect a change in follicle growth rate from 3.3 mm/d to 2.0 mm/day, and a change in time interval to ovulation of 2 days or more with a = 0.05 and b = 0.20. It was recognized, however, that this sample size would only be able to identify severe depression of fertility and that subtle alteration of pregnancy rate would be undetectable with the power of this experiment.
Results Data from 58 cycles of 13 mares were available for analysis, with 17 cycles for the altrenogest and CIDR groups and 24 control cycles. Each mare was evaluated for at least three cycles and received each treatment at least once. Nine mares were followed for five cycles each.
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The results for follicular diameter and growth, estrous behavior score and endometrial edema score are graphically summarized in Figs. 1–4. Days from detection of a 35 mm follicle to ovulation were 3.71 ± 0.26 days, 4.0 ± 0.54 days and 3.82 ± 0.35 days (mean ± SEM) for control, altrenogest and CIDR groups, respectively. Neither CIDR nor altrenogest treatment delayed ovulation (P = 0.84). The maximal preovulatory size of the ovulatory follicle (40.96 ± 0.68 mm, 40.71 ± 0.85 mm and 40.82 ± 0.88 mm for control, altrenogest and CIDR groups, respectively) was not affected by the treatment (P = 0.86). Treatment had no effect on rate of follicular growth. Fig. 1 shows daily mean follicular size from the day of detection of a 35 mm follicle. On Fig. 2 the same data are shown corrected for day of ovulation. There was no significant differences in slope (P = 0.51) or intercept (P = 0.85) of the lines for follicle size (mean ± SEM) relative to day of ovulation. Ovulation occurred during progestagen therapy in five cycles of three mares (three times for altrenogest and twice for the CIDR). These mares were not inseminated. Both forms of progestagen treatment had prompt and dramatic effects on estrous behavior (P < 0.0001), effectively abolishing receptivity within 24 h. Response to the stallion returned to control levels after cessation of treatment (Fig. 3). Similarly, both forms of treatment mediated reduction in endometrial edema (assessed ultrasonographically) beginning at 24 h for the CIDR and 48 h for altrenogest. The endometrial edema score returned to control level after cessation of treatment (Fig. 4). Pregnancy was detected in 5/14 (36%) mated mares in the altrenogest group, 7/15 (47%) in the CIDR group and 14/23 (61%) in the control group. This study lacked power for comparison of pregnancy rate, but it is noteworthy that the pregnancy rate for altrenogest treated cycles was lower when analyzed using mare as a random variable (P = 0.09). The number of services per cycle was 1.82 ± 0.14 for the control cycles, 1.58 ± 0.56 for altrenogest and 1.31 ± 0.13 for CIDR-treated cycles (P = 0.17). Uterine fluid accumulation to a depth of greater than 10 mm occurred in 12/17 (71%) altrenogest-treated cycles, 7/17 (41%) CIDRtreated cycles and 7/24 (29%) of the control cycles. Multiple logistic regression (with mare as random variable) indicated that occurrence of uterine fluid was affected by altrenogest (P = 0.011), but not by CIDR treatment (P = 0.31). However, presence of a periovulatory fluid depth of >10 mm was not statistically related to pregnancy (P = 0.855). There was no effect of treatment (P > 0.7), or treatment by cycle day interaction (P > 0.5), on progesterone concentration after ovulation; not surprisingly, day of cycle (with day of ovulation designated 0) was highly significant (P < 0.0001). Progesterone concentrations are shown in Fig. 5. Examination of the association between pregnancy status and progesterone concentration in the mixed linear model revealed that the interaction term (cycle day – pregnancy status) had P < 0.15. Therefore relationship between pregnancy sta-
Fig. 1. Median follicular size beginning on first day of treatment (35 mm).
Fig. 2. Follicular size (mean ± SEM) relative to day of ovulation (D0, ovulation). There is no difference in slope (P = 0.51) or intercept (P = 0.85) of the lines.
Fig. 3. Daily teasing score (mean ± SEM) after Gorecka et al. (2005). Both forms of progestin treatment reduced teasing score (P < 0.0001).
Fig. 4. Median daily edema score. CIDR reduced edema after 24 and 48 h of treatment (P < 0.001) and altrenogest only after 48 h (P = 0.008).
tus and progesterone concentration was evaluated separately for Days 0, 5 and 12. The model included mare as random variable. There was no effect of progesterone concentration on day of ovulation on pregnancy in that cycle (P = 0.5), but progesterone concentration on Day 5 was related to pregnancy outcome. Mares found to be pregnant on Day 12 of the cycle had Day 5 progesterone concentration of 38.12 ± 3.21 nmol/L (11.99 ± 1.01 ng/mL) and those not pregnant had a progesterone concentration of 28.56 ± 2.00 nmol/L (8.98 ± 0.63 ng/mL) on Day 5 (P = 0.03). However, this difference was not related to treatment. On Day 12 of the cycle progesterone concentrations were 37.08 ± 4.96 nmol/L (11.66 ± 1.56 ng/mL) and 27.16 ± 2.70 nmol/L (8.54 ± 0.85 ng/mL; P = 0.688) for pregnant and non-pregnant mares, respectively. Again, these concentrations were unrelated to treatment. Fig. 6 depicts progesterone concentrations for pregnant and non-pregnant cycles.
I.F. Canisso et al. / The Veterinary Journal 197 (2013) 324–328
Fig. 5. Serum progesterone concentration by day of cycle for each treatment group (Day 0, day of ovulation). For treatment, P > 0.7; for treatment-by-day interaction P > 0.5; for day, P = 0.0001.
Fig. 6. Progesterone concentration by pregnancy status. Pregnant mares had higher progesterone concentration on Day 5 after ovulation (P = 0.03).
Mares tolerated the intravaginal progesterone releasing devices well. No devices were lost in this study. Some mares developed mild vaginitis with a small amount of visible exudate that resolved promptly upon removal. Application of a CIDR device consistently increased serum progesterone concentrations for the period that the device was left in the vagina. Progesterone concentrations were basal within 1 day after withdrawal of the device (Fig. 7).
Discussion The time from identification of a 35 mm follicle to its ovulation was not different between treatment and control groups, indicating that exogenous progestagen treatment, as administered in this experiment, did not delay ovulation. Size of the preovulatory follicle (approximately 41 mm) and growth rate of the largest domi-
Fig. 7. Serum progesterone concentrations for mares with intravaginal CIDR (Day 0, day of insertion). Devices were removed on Day 2.
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nant follicle were similarly unaffected. The preovulatory size of the dominant follicle is reported to be relatively consistent within a given broodmare (Cuervo-Arango and Newcombe, 2008), which was accounted for in the experimental design (each mare participated at least once in each treatment) and in the statistical analysis (mare was treated as a random variable in mixed model analyses). Although Gastal et al. (1999) demonstrated an effect of exogenous progesterone on follicular growth in mares, in that study the progesterone was administered for a prolonged period starting early in follicular growth. Thus, it may be that earlier initiation of progestagen treatment might have affected follicular growth and ovulation. Nonetheless, progestagen treatment was not without effect. Estrous behavior was effectively suppressed within 24 h in both progestagen treatment groups and endometrial edema was markedly reduced in both treatment groups (albeit more promptly for the CIDR group than the altrenogest-treated mares). Both parameters returned to pretreatment levels after cessation of treatment. Abolition of receptive behavior would be a serious disadvantage in naturally bred horses, with risks of injury to animals and personnel. The role of progestagens in suppression of estrous behavior is well documented (Loy and Swan, 1966) and it is also known that progesterone potentiates non-estrous behaviors (Ginther, 1979). Endometrial edema is characteristic of estrus in mares, but its abolition by progestagen treatment has apparently not previously been reported. In addition, progestagen treatment resulted in numerically reduced pregnancy rate for both progestagen groups relative to the control, which is cause for concern, and may indicate a real depression of fertility. Previous studies have found inconsistent effects of altrenogest on interval to ovulation (James et al., 1998; Bruemmer et al., 2000). In the small study of James et al. (1998), altrenogest-treated mares had numerically lower pregnancy rate than controls, but Bruemmer et al. (2000) reported no effect. In the later trial, however, mares were treated with altrenogest at 0.088 mg/kg and several mares underwent follicular regression, preventing their insemination during that cycle. Given the lack of efficacy of this intervention for postponing ovulation, the possibility of depressed fertility makes its use for this purpose even less justifiable. In the current study, altrenogest treatment was associated with higher risk of periovulatory intrauterine fluid accumulation, although this was not directly linked to fertility in a statistically significant way. Possible mechanisms for such an effect might include an effect on myometrial activity and physical clearance, or impairment of innate immune function, but would require further investigation. Conversely, the preovulatory administration of progestagens did not alter the postovulatory profile of endogenous progesterone secretion. All treated mares ovulated and had circulating progesterone profiles similar to those in control mares. Interestingly, however, pregnant mares had significantly higher progesterone concentration on Day 5 after ovulation than non-pregnant mares, regardless of treatment group. Although a similar observation has been made in dairy cows (Larson et al., 1997) this has not been previously reported in mares. Data from a larger group of mares may provide evidence of whether a range of progesterone values may potentially be used as an early indicator of pregnancy. Notably, mares tolerated the intravaginal progesterone releasing devices well. Mild vaginitis with a small amount of purulent exudate that occurred in some mares resolved promptly upon removal of the device. The nylon thread attached to the device to facilitate removal was removed before insertion because we found that it tended to irritate mares. We also found it more convenient and hygienic to insert the devices using a sterile-gloved hand after cleansing of the perineum, rather than using the insertion instrument provided with the product. All CIDR-treated mares displayed physiologically relevant concentrations of circulating progesterone
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during treatment. CIDR treatment (with a device manufactured specifically for use in mares) has been used in managing spring transition in mares (Hanlon and Firth, 2012), and CIDR may represent a reasonable choice for temporary (and prompt) repression of signs of estrus, which may be useful in management of show horses or management/diagnosis of horses with purported estrus cycle-related behavioral problems. Conclusion Treatment with altrenogest or CIDR did not alter follicular growth rate or time to ovulation in mares. Estrous behavior was significantly suppressed 24 h post CIDR vaginal insertion or oral altrenogest. Fertility was not significantly depressed by the treatment with CIDR compared to the control group, but reduction approached significance for the altrenogest group. Use of progestagens to delay ovulation in mares cannot be recommended for lack of efficacy and possible impairment of fertility. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors are thankful to Ms. Sophie Trowbridge and Drs. Valeria Tanco and Katie Beltaire for their help during the project, and to an anonymous reviewer for helpful suggestions. This research was supported by the Harry Zweig Memorial Fund for Equine Research. These results have been presented in part as an abstract at the Annual Meeting of the Society for Theriogenology, Baltimore MD, USA, August 2012. References Allen, W.R., Brown, L., Wright, M., Wilsher, S., 2007. Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and stallions in England. Equine Veterinary Journal 39, 438–445. Asa, C.S., Ginther, O.J., 1982. Glucocorticoid suppression of oestrus, follicles, LH and ovulation in the mare. Journal of Reproduction and Fertility Suppl. 32, 247–251. Bergfelt, D.R., Ginther, O.J., 1985. Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare. Theriogenology 24, 99–108. Brinsko, S.P., 2006. Insemination doses: How low can we go? Theriogenology 66, 543–550. Bruemmer, J.E., Coy, R.C., Olson, A., Squires, E.L., 2000. Efficacy of altrenogest administration to postpone ovulation and subsequent fertility in mares. Journal of Equine Veteterinary Science 20, 450–453.
Carnevale, E.M., 2008. The mare model for follicular maturation and reproductive aging in the woman. Theriogenology 69, 23–30. Cuervo-Arango, J., Newcombe, J.R., 2008. Repeatability of preovulatory follicular diameter and uterine edema pattern in two consecutive cycles in the mare and how they are influenced by ovulation inductors. Theriogenology 69, 681–687. Davies Morel, M.C., Newcombe, J.R., 2008. The efficacy of different hCG dose rates and the effect of hCG treatment on ovarian activity: Ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare. Animal Reproduction Science 109, 189–199. Gastal, E.L., Bergfelt, D.R., Nogueira, G.P., Gastal, M.O., Ginther, O.J., 1999. Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares. Biology and Reproduction 61, 1492–1498. Ginther, O.J., 1979. Reproductive Biology of the Mare: Basic and Applied Aspects, First Ed. Equiservices, Cross Plains, WI. Ginther, O.J., 1990. Folliculogenesis during the transitional period and early ovulatory season in mares. Journal of Reproduction and Fertility 90, 311–320. Ginther, O.J., Siddiqui, M.A., Beg, M.A., 2009. Physiologic and nonphysiologic effects of exogenous prostaglandin F2alpha on reproductive hormones in mares. Theriogenology 72, 417–424. Gorecka, A., Jezierski, T.A., Sloniewski, K., 2005. Relationships between sexual behaviour, dominant follicle area, uterus ultrasonic image and pregnancy rate in mares of two breeds differing in reproductive efficiency. Animal Reproduction Science 87, 283–293. Hanlon, D.W., Firth, E.C., 2012. The reproductive performance of Thoroughbred mares treated with intravaginal progesterone at the start of the breeding season. Theriogenology 77, 952–958. Hemberg, E., Lundeheim, N., Einarsson, S., 2006. Successful timing of ovulation using deslorelin (Ovuplant) is labour-saving in mares aimed for single AI with frozen semen. Reproduction in Domestic Animals 41, 535–537. James, A.N., Vogelsang, M.M., Forrest, D.W., Stott, G.G., 1998. Efficacy of short-term administration of altrenogest to postpone ovulation in mares. Journal of Equine Veterinary Science 18, 329–331. Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone postbreeding and increased milk urea nitrogen in lactating cows. Journal of Dairy Science 80, 1288–1295. Loy, R.G., Hughes, J.P., 1966. The effects of human chorionic gonadotrophin on ovulation, length of estrus, and fertility in the mare. Cornell Veterinarian 56, 41–50. Loy, R.G., Swan, S.M., 1966. Effects of exogenous progestogens on reproductive phenomena in mares. Journal of Animal Science 25, 821–826. Macpherson, M.L., Reimer, J.M., 2000. Twin reduction in the mare: Current options. Animal Reproduction Science 60–61, 233–244. Martinez, M.F., Kastelic, J.P., Adams, G.P., Cook, B., Olson, W.O., Mapletoft, R.J., 2002a. The use of progestins in regimens for fixed-time artificial insemination in beef cattle. Theriogenology 57, 1049–1059. Martinez, M.F., Kastelic, J.P., Adams, G.P., Mapletoft, R.J., 2002b. The use of a progesterone-releasing device (CIDR-B) or melengestrol acetate with GnRH, LH, or estradiol benzoate for fixed-time AI in beef heifers. Journal of Animal Science 80, 1746–1751. Newcombe, J.R., 2002. Field observations on the use of a progesterone-releasing intravaginal device to induce estrus and ovulation in seasonally anestrous mares. Journal of Equine Veterinary Science 22, 378–382. Newcombe, J.R., Cuervo-Arango, J., 2011. The effect of time of insemination with fresh cooled transported semen and natural mating relative to ovulation on pregnancy and embryo loss rates in the mare. Reproduction in Domestic Animals 46, 678–681. Sieme, H., Schafer, T., Stout, T.A., Klug, E., Waberski, D., 2003. The effects of different insemination regimes on fertility in mares. Theriogenology 60, 1153–1164. van de Leemput, E.E., Vos, P.L., Hyttel, P., van den Hurk, R., Bevers, M.M., van der Weijden, G.C., Dieleman, S.J., 2001. Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/ prostaglandin-treated heifers. Theriogenology 55, 573–592.
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The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Preovulatory progestagen treatment in mares fails to delay ovulation Igor F. Canisso 1, Kirsten Gallacher, Mary-Anne Gilbert, Alexandra Korn, Christine M. Schweizer, Sylvia J. Bedford-Guaus, Robert O. Gilbert ⇑ Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA
a r t i c l e
i n f o
Article history: Accepted 21 December 2012
Keywords: Progestagen Mare Fertility Ovulation Behavior
a b s t r a c t The major objective of this study was to determine whether short-term preovulatory progestagen treatment of mares could effectively delay ovulation. Secondary objectives were to determine the effect such supplementation had on signs of estrus, follicular growth, postovulatory luteal function and pregnancy rate. Thirteen cyclic mares of different breeds were used in this study during the natural breeding season. Once mares were confirmed in estrus with a follicle of 35 mm in diameter, they were assigned in random order to receive no treatment (control), placement of a progesterone-impregnated controlled intravaginal drug releasing device (CIDR) for 2 days, or oral altrenogest treatment (0.044 mg/kg/d) for 2 days. Transrectal ultrasonography and teasing with a vigorous stallion were performed daily. Mares were inseminated every 48 h after the end of experimental treatment (progestagen groups) or beginning when the follicular diameter was 35 mm (control group) with fresh extended semen of a single fertile stallion. Each mare was followed for 3–5 cycles, allowing each treatment to be applied one or two times. Neither CIDR nor altrenogest treatment delayed ovulation. Treatment had no effect on follicular growth rate or the size of the ovulatory follicle immediately preceding ovulation. Both forms of progestagen treatment effectively abolished estrous behavior within 24 h. Estrous response to the stallion returned to the control level after cessation of treatment. Similarly, a reduction in endometrial edema was detected during progestagen treatment, which returned to normal after cessation of treatment. Altrenogest treatment tended to reduce the chance of pregnancy (P = 0.09) compared to the control group. The use of progestagens to delay ovulation in mares lacks efficacy and may threaten successful establishment of pregnancy. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Optimal fertility in mares requires breeding close to ovulation (Newcombe and Cuervo-Arango, 2011), providing an incentive to monitor and control of the estrous cycle (Allen et al., 2007). Precise control of the time of ovulation offers major advantages in broodmare management. In contrast to many other species, the duration of estrus in mares is long and variable, with time of ovulation more closely related to the end of behavioral estrus than its beginning (Carnevale, 2008). In addition, many stallions in high demand have full ‘books’ and access to these stallions can be difficult to arrange, while available breedings may not coincide with the optimal timing for mare fertility. Similarly, for those breeds that allow use of cooled, shipped semen, availability of the semen may not coincide with the optimal time for insemination. Some strategies success⇑ Corresponding author. Tel.: +1 607 253 3100. E-mail address: [email protected] (R.O. Gilbert). Current address: Reproduction Laboratory, Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40546-0099, USA. 1
1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2012.12.028
fully hasten ovulation, such as use of human chorionic gonadotrophin or gonadoliberin agonists (Loy and Hughes, 1966; Sieme et al., 2003; Hemberg et al., 2006; Davies Morel and Newcombe, 2008), but to date there is no practical way to delay ovulation in mares. Ovulation of mares has been delayed by daily administration of equine follicular fluid (Bergfelt and Ginther, 1985). This experiment demonstrated that, apart from steroid hormones, follicular fluid contained a proteinaceous component that was also capable of inhibiting follicle stimulating hormone (FSH) secretion and delaying ovulation. However, this treatment had to be applied for several days during the phase of early follicular growth, and the postponement of ovulation was not predictable. Although interesting, and potentially promising at some future time, this approach is not currently practical. Similarly, daily administration of dexamethasone (30 mg/day starting at Day 10 of the cycle) prevented estrus and ovulation in 7/8 treated mares (Asa and Ginther, 1982), but this approach may have potentially adverse side effects and does not allow predictable control of ovulation. Altrenogest is a potent progestagen commonly used in broodmare management. Previous attempts to postpone ovulation using altrenogest used small numbers of mares or yielded inconsistent
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results (James et al., 1998; Bruemmer et al., 2000). Controlled internal drug release (CIDR) has been used extensively in the cattle industry for control of estrus and ovulation (Martinez et al., 2002a,b). Intravaginal devices have been used in horses to advance ovulation in transitional mares (Newcombe, 2002; Hanlon and Firth, 2012). We therefore aimed to test the effects of administration of these exogenous progestagens on follicular development, time of ovulation, development of the corpus luteum and establishment of pregnancy in mares. Brief postponement of ovulation in superovulated cows had little effect on development competence of resulting embryos (van de Leemput et al., 2001), and growth of the equine follicle has been shown to be sensitive to exogenous progesterone (Gastal et al., 1999). Furthermore, functional removal of progesterone by administration of a luteolytic dose of prostaglandin F2a is associated with a reduced interovulatory interval in mares (Ginther et al., 2009), suggesting that the opposite effect might be produced by progesterone supplementation. Development of the equine follicle is quite consistent, with linear growth over most of the pre-ovulatory period (Ginther, 1990), allowing detection of potential changes brought about by progestagen administration. The aims of the current study were to determine whether exogenous progestagen (intravaginal device or oral altrenogest) administration in mares: (1) delayed ovulation; (2) affected incidence of spontaneous ovulation upon cessation of treatment; (3) affected luteal function (as determined by progesterone profiles) following ovulation; (4) depressed fertility; (5) affected ultrasonographically-evident endometrial edema, and (6) affected estrous behavior as elicited by teasing to an experienced stallion.
still, raised tail, winked, passed fluids and lowered pelvis [postured]) accompanied by some non-receptive behaviors (tail switching, squealing, holding ears back, attempts to kick); (8) mares showed full estrus (stood still, raised tail, winked, passed fluids and lowered pelvis [posture]) with no non-receptive behaviors.
Material and methods
Fertility trial
The present study was carried out from May to September in the Northern hemisphere physiological reproductive season of 2010. All procedures were approved by the Cornell University Institutional Animal Care and Use Committee (protocol number 2010-0020).
Pregnancy diagnosis was carried out 12 days post ovulation by transrectal palpation and ultrasonography examination using a linear 5 MHz Aloka transducer, with a mare designated as not pregnant if an embryonic vesicle was not seen by 12–14 days post ovulation, despite multiple reproductive examinations on the two previous days. If confirmed pregnant the embryonic vesicle was manually reduced using standard technique described elsewhere (Macpherson and Reimer, 2000) for pre-fixation twin reduction, and then the mare was given a 10 mg dose of prostaglandin F2a (dinoprost thromethamine, Lutalyse, Pfizer, IM). Mares were used in consecutive cycles.
Broodmares Thirteen cyclic, reproductively sound mares of various breeds (Warmbloods, Thoroughbreds, Paint Horses, and Quarter Horses) were used for this study. Every mare used on this study had had multiple regular cycles before the beginning of the trial. The mares were examined by palpation and ultrasonography per rectum every 1–3 days, according to their follicular and uterine features, and daily once in estrus. Once mares were confirmed in estrus, and had a large preovulatory follicle (P35 mm diameter), they were allocated to one of three treatments in random sequence. Each mare was followed for 4–6 cycles, allowing each treatment to be repeated one or two times. The treatments were: (1) no treatment (Control); (2) a progesterone-impregnated controlled internal drug release device intended for use in cattle (EaziBreed CIDR, Pfizer Animal Health) applied intravaginally for 48 h; and (3) altrenogest (Regu-Mate, Merck, 0.044 mg/kg/day orally for 2 days). At each examination data recording included size of the dominant follicle and significant subordinate follicles, detection of ovulation, endometrial edema (subjective scale, 0–3, with 3 maximal) and assessment of estrous behavior (Gorecka et al., 2005). Once mares were in estrus, monitoring was continued daily until ovulation was detected. Therefore time of ovulation was recorded with ±24 h of accuracy. Estrous behavior was assessed by a single evaluator. The evaluator stood laterally-caudally to the mare; every behavior displayed by the mare was recorded and the mare was given a final score according to the Gorecka et al. (2005) scoring system. During each teasing episode a mature stallion was placed in a teasing stall (with an open top, which allowed physical interaction between the mare being teased and the teaser). The mares were presented to the teaser and allowed to interact with him for at least approximately 3 min. A score was assigned as follows: (1) non-receptive behavior (tail switching, moving around, squealing, holding ears back, attempts to kick); mare attacked or kicked at the teasing stallion; (2) nonreceptive behavior (tail switching, moving around, squealing, holding ears back, attempts to kick) and no severe attack toward the stallion; (3) non-receptive behavior (tail switching, squealing, holding ears back, attempts to kick) and the mare stood still; (4) mare stood still indifferently; (5) mare showed estrus (stood still, raised tail or winked, accompanied by some non-receptive behavior tail switching, squealing, holding ears back, attempts to kick); (6) mare showed estrus (stood still, raised tail or winked) and no non-receptive behaviors; (7) mare showed full estrus (stood
Semen processing and artificial insemination Semen from a single fertile Dutch Warmblood stallion was used. Semen was collected using a dummy mount with an estrus mare present in the breeding shed. The collections were performed every 24–48 h, with the use of a Missouri artificial vagina. Immediately after each collection the semen was filtered, a sample from semen fraction gel free was subjectively evaluated for total and progressive motility. Sperm concentration was assessed by the use of a spectrophotometer (591B Equine Densimeter, Animal Reproduction Systems). The semen was then extended using a commercial skimmed milk extender (E-Z-Mixin CST containing amikacin sulfate, Animal Reproduction Systems) to 25–50 million sperm/mL and placed on a commercial semen shipment container (Equitainer II, Hamilton Research) with a minimal final volume of 120 mL per container. The semen was cooled and used within 24 h of collection. Semen was deposited in the uterine body, using standard technique for AI with cooled semen. Each artificial insemination (AI) was performed using a standard dose of 500 106 progressively motile sperm (Brinsko, 2006). Approximately 6 h post AI each mare was given a dose of 20 U of oxytocin to prevent intrauterine fluid accumulation and all mares were re-checked for intrauterine fluid accumulation and if fluid depth greater than 20 mm was present uterine lavage with 2 L of Lactate Ringer’s Solution was performed and three injections of oxytocin (10 U) were given IM, one immediately after the intrauterine flushing and the other doses were given approximately every 6 h. Every bred mare was re-checked every 24 h until no detectable intra-uterine fluid accumulation was seen. Lavage and oxytocin treatments were re-applied according to the criteria above, if necessary. During the control cycle, each mare was inseminated every 48 h beginning on the day that the dominant follicle reached a size of 35 mm until ovulation was confirmed. In the treatment cycles, inseminations were commenced after the end of treatment (i.e. immediately after CIDR removal or 24 h after the second dose of oral altrenogest) and continued every 48 h until ovulation.
Statistical analysis Maximum preovulatory follicle size, average daily growth rate of dominant follicle, time interval from follicular diameter of 35 mm to ovulation and progesterone concentrations were treated as continuous outcomes and analyzed using mixed linear regression. Fixed variables were treatment, day of cycle and treatment-by-day interactions (or cycle-day interactions when investigating relationship with pregnancy); individual mares were treated as random variables. Pregnancy was treated as a binary outcome and analyzed using mixed logistic regression with the same fixed and random variables. Uterine fluid accumulation was dichotomized (=<10 mm or >10 mm fluid depth) and analyzed similarly using mixed logistic regression. Scores for estrus behavior and endometrial edema were treated as ordinal and evaluated by ordinal logistic regression (without random variable of mare). All calculations were performed using StataIC 11.2 for Windows (Stata Corp). Graphs were produced with StataIC or with GraphPad Prism 5.04 for Windows. The sample size was chosen to be able to detect a change in follicle growth rate from 3.3 mm/d to 2.0 mm/day, and a change in time interval to ovulation of 2 days or more with a = 0.05 and b = 0.20. It was recognized, however, that this sample size would only be able to identify severe depression of fertility and that subtle alteration of pregnancy rate would be undetectable with the power of this experiment.
Results Data from 58 cycles of 13 mares were available for analysis, with 17 cycles for the altrenogest and CIDR groups and 24 control cycles. Each mare was evaluated for at least three cycles and received each treatment at least once. Nine mares were followed for five cycles each.
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The results for follicular diameter and growth, estrous behavior score and endometrial edema score are graphically summarized in Figs. 1–4. Days from detection of a 35 mm follicle to ovulation were 3.71 ± 0.26 days, 4.0 ± 0.54 days and 3.82 ± 0.35 days (mean ± SEM) for control, altrenogest and CIDR groups, respectively. Neither CIDR nor altrenogest treatment delayed ovulation (P = 0.84). The maximal preovulatory size of the ovulatory follicle (40.96 ± 0.68 mm, 40.71 ± 0.85 mm and 40.82 ± 0.88 mm for control, altrenogest and CIDR groups, respectively) was not affected by the treatment (P = 0.86). Treatment had no effect on rate of follicular growth. Fig. 1 shows daily mean follicular size from the day of detection of a 35 mm follicle. On Fig. 2 the same data are shown corrected for day of ovulation. There was no significant differences in slope (P = 0.51) or intercept (P = 0.85) of the lines for follicle size (mean ± SEM) relative to day of ovulation. Ovulation occurred during progestagen therapy in five cycles of three mares (three times for altrenogest and twice for the CIDR). These mares were not inseminated. Both forms of progestagen treatment had prompt and dramatic effects on estrous behavior (P < 0.0001), effectively abolishing receptivity within 24 h. Response to the stallion returned to control levels after cessation of treatment (Fig. 3). Similarly, both forms of treatment mediated reduction in endometrial edema (assessed ultrasonographically) beginning at 24 h for the CIDR and 48 h for altrenogest. The endometrial edema score returned to control level after cessation of treatment (Fig. 4). Pregnancy was detected in 5/14 (36%) mated mares in the altrenogest group, 7/15 (47%) in the CIDR group and 14/23 (61%) in the control group. This study lacked power for comparison of pregnancy rate, but it is noteworthy that the pregnancy rate for altrenogest treated cycles was lower when analyzed using mare as a random variable (P = 0.09). The number of services per cycle was 1.82 ± 0.14 for the control cycles, 1.58 ± 0.56 for altrenogest and 1.31 ± 0.13 for CIDR-treated cycles (P = 0.17). Uterine fluid accumulation to a depth of greater than 10 mm occurred in 12/17 (71%) altrenogest-treated cycles, 7/17 (41%) CIDRtreated cycles and 7/24 (29%) of the control cycles. Multiple logistic regression (with mare as random variable) indicated that occurrence of uterine fluid was affected by altrenogest (P = 0.011), but not by CIDR treatment (P = 0.31). However, presence of a periovulatory fluid depth of >10 mm was not statistically related to pregnancy (P = 0.855). There was no effect of treatment (P > 0.7), or treatment by cycle day interaction (P > 0.5), on progesterone concentration after ovulation; not surprisingly, day of cycle (with day of ovulation designated 0) was highly significant (P < 0.0001). Progesterone concentrations are shown in Fig. 5. Examination of the association between pregnancy status and progesterone concentration in the mixed linear model revealed that the interaction term (cycle day – pregnancy status) had P < 0.15. Therefore relationship between pregnancy sta-
Fig. 1. Median follicular size beginning on first day of treatment (35 mm).
Fig. 2. Follicular size (mean ± SEM) relative to day of ovulation (D0, ovulation). There is no difference in slope (P = 0.51) or intercept (P = 0.85) of the lines.
Fig. 3. Daily teasing score (mean ± SEM) after Gorecka et al. (2005). Both forms of progestin treatment reduced teasing score (P < 0.0001).
Fig. 4. Median daily edema score. CIDR reduced edema after 24 and 48 h of treatment (P < 0.001) and altrenogest only after 48 h (P = 0.008).
tus and progesterone concentration was evaluated separately for Days 0, 5 and 12. The model included mare as random variable. There was no effect of progesterone concentration on day of ovulation on pregnancy in that cycle (P = 0.5), but progesterone concentration on Day 5 was related to pregnancy outcome. Mares found to be pregnant on Day 12 of the cycle had Day 5 progesterone concentration of 38.12 ± 3.21 nmol/L (11.99 ± 1.01 ng/mL) and those not pregnant had a progesterone concentration of 28.56 ± 2.00 nmol/L (8.98 ± 0.63 ng/mL) on Day 5 (P = 0.03). However, this difference was not related to treatment. On Day 12 of the cycle progesterone concentrations were 37.08 ± 4.96 nmol/L (11.66 ± 1.56 ng/mL) and 27.16 ± 2.70 nmol/L (8.54 ± 0.85 ng/mL; P = 0.688) for pregnant and non-pregnant mares, respectively. Again, these concentrations were unrelated to treatment. Fig. 6 depicts progesterone concentrations for pregnant and non-pregnant cycles.
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Fig. 5. Serum progesterone concentration by day of cycle for each treatment group (Day 0, day of ovulation). For treatment, P > 0.7; for treatment-by-day interaction P > 0.5; for day, P = 0.0001.
Fig. 6. Progesterone concentration by pregnancy status. Pregnant mares had higher progesterone concentration on Day 5 after ovulation (P = 0.03).
Mares tolerated the intravaginal progesterone releasing devices well. No devices were lost in this study. Some mares developed mild vaginitis with a small amount of visible exudate that resolved promptly upon removal. Application of a CIDR device consistently increased serum progesterone concentrations for the period that the device was left in the vagina. Progesterone concentrations were basal within 1 day after withdrawal of the device (Fig. 7).
Discussion The time from identification of a 35 mm follicle to its ovulation was not different between treatment and control groups, indicating that exogenous progestagen treatment, as administered in this experiment, did not delay ovulation. Size of the preovulatory follicle (approximately 41 mm) and growth rate of the largest domi-
Fig. 7. Serum progesterone concentrations for mares with intravaginal CIDR (Day 0, day of insertion). Devices were removed on Day 2.
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nant follicle were similarly unaffected. The preovulatory size of the dominant follicle is reported to be relatively consistent within a given broodmare (Cuervo-Arango and Newcombe, 2008), which was accounted for in the experimental design (each mare participated at least once in each treatment) and in the statistical analysis (mare was treated as a random variable in mixed model analyses). Although Gastal et al. (1999) demonstrated an effect of exogenous progesterone on follicular growth in mares, in that study the progesterone was administered for a prolonged period starting early in follicular growth. Thus, it may be that earlier initiation of progestagen treatment might have affected follicular growth and ovulation. Nonetheless, progestagen treatment was not without effect. Estrous behavior was effectively suppressed within 24 h in both progestagen treatment groups and endometrial edema was markedly reduced in both treatment groups (albeit more promptly for the CIDR group than the altrenogest-treated mares). Both parameters returned to pretreatment levels after cessation of treatment. Abolition of receptive behavior would be a serious disadvantage in naturally bred horses, with risks of injury to animals and personnel. The role of progestagens in suppression of estrous behavior is well documented (Loy and Swan, 1966) and it is also known that progesterone potentiates non-estrous behaviors (Ginther, 1979). Endometrial edema is characteristic of estrus in mares, but its abolition by progestagen treatment has apparently not previously been reported. In addition, progestagen treatment resulted in numerically reduced pregnancy rate for both progestagen groups relative to the control, which is cause for concern, and may indicate a real depression of fertility. Previous studies have found inconsistent effects of altrenogest on interval to ovulation (James et al., 1998; Bruemmer et al., 2000). In the small study of James et al. (1998), altrenogest-treated mares had numerically lower pregnancy rate than controls, but Bruemmer et al. (2000) reported no effect. In the later trial, however, mares were treated with altrenogest at 0.088 mg/kg and several mares underwent follicular regression, preventing their insemination during that cycle. Given the lack of efficacy of this intervention for postponing ovulation, the possibility of depressed fertility makes its use for this purpose even less justifiable. In the current study, altrenogest treatment was associated with higher risk of periovulatory intrauterine fluid accumulation, although this was not directly linked to fertility in a statistically significant way. Possible mechanisms for such an effect might include an effect on myometrial activity and physical clearance, or impairment of innate immune function, but would require further investigation. Conversely, the preovulatory administration of progestagens did not alter the postovulatory profile of endogenous progesterone secretion. All treated mares ovulated and had circulating progesterone profiles similar to those in control mares. Interestingly, however, pregnant mares had significantly higher progesterone concentration on Day 5 after ovulation than non-pregnant mares, regardless of treatment group. Although a similar observation has been made in dairy cows (Larson et al., 1997) this has not been previously reported in mares. Data from a larger group of mares may provide evidence of whether a range of progesterone values may potentially be used as an early indicator of pregnancy. Notably, mares tolerated the intravaginal progesterone releasing devices well. Mild vaginitis with a small amount of purulent exudate that occurred in some mares resolved promptly upon removal of the device. The nylon thread attached to the device to facilitate removal was removed before insertion because we found that it tended to irritate mares. We also found it more convenient and hygienic to insert the devices using a sterile-gloved hand after cleansing of the perineum, rather than using the insertion instrument provided with the product. All CIDR-treated mares displayed physiologically relevant concentrations of circulating progesterone
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during treatment. CIDR treatment (with a device manufactured specifically for use in mares) has been used in managing spring transition in mares (Hanlon and Firth, 2012), and CIDR may represent a reasonable choice for temporary (and prompt) repression of signs of estrus, which may be useful in management of show horses or management/diagnosis of horses with purported estrus cycle-related behavioral problems. Conclusion Treatment with altrenogest or CIDR did not alter follicular growth rate or time to ovulation in mares. Estrous behavior was significantly suppressed 24 h post CIDR vaginal insertion or oral altrenogest. Fertility was not significantly depressed by the treatment with CIDR compared to the control group, but reduction approached significance for the altrenogest group. Use of progestagens to delay ovulation in mares cannot be recommended for lack of efficacy and possible impairment of fertility. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors are thankful to Ms. Sophie Trowbridge and Drs. Valeria Tanco and Katie Beltaire for their help during the project, and to an anonymous reviewer for helpful suggestions. This research was supported by the Harry Zweig Memorial Fund for Equine Research. These results have been presented in part as an abstract at the Annual Meeting of the Society for Theriogenology, Baltimore MD, USA, August 2012. References Allen, W.R., Brown, L., Wright, M., Wilsher, S., 2007. Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and stallions in England. Equine Veterinary Journal 39, 438–445. Asa, C.S., Ginther, O.J., 1982. Glucocorticoid suppression of oestrus, follicles, LH and ovulation in the mare. Journal of Reproduction and Fertility Suppl. 32, 247–251. Bergfelt, D.R., Ginther, O.J., 1985. Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare. Theriogenology 24, 99–108. Brinsko, S.P., 2006. Insemination doses: How low can we go? Theriogenology 66, 543–550. Bruemmer, J.E., Coy, R.C., Olson, A., Squires, E.L., 2000. Efficacy of altrenogest administration to postpone ovulation and subsequent fertility in mares. Journal of Equine Veteterinary Science 20, 450–453.
Carnevale, E.M., 2008. The mare model for follicular maturation and reproductive aging in the woman. Theriogenology 69, 23–30. Cuervo-Arango, J., Newcombe, J.R., 2008. Repeatability of preovulatory follicular diameter and uterine edema pattern in two consecutive cycles in the mare and how they are influenced by ovulation inductors. Theriogenology 69, 681–687. Davies Morel, M.C., Newcombe, J.R., 2008. The efficacy of different hCG dose rates and the effect of hCG treatment on ovarian activity: Ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare. Animal Reproduction Science 109, 189–199. Gastal, E.L., Bergfelt, D.R., Nogueira, G.P., Gastal, M.O., Ginther, O.J., 1999. Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares. Biology and Reproduction 61, 1492–1498. Ginther, O.J., 1979. Reproductive Biology of the Mare: Basic and Applied Aspects, First Ed. Equiservices, Cross Plains, WI. Ginther, O.J., 1990. Folliculogenesis during the transitional period and early ovulatory season in mares. Journal of Reproduction and Fertility 90, 311–320. Ginther, O.J., Siddiqui, M.A., Beg, M.A., 2009. Physiologic and nonphysiologic effects of exogenous prostaglandin F2alpha on reproductive hormones in mares. Theriogenology 72, 417–424. Gorecka, A., Jezierski, T.A., Sloniewski, K., 2005. Relationships between sexual behaviour, dominant follicle area, uterus ultrasonic image and pregnancy rate in mares of two breeds differing in reproductive efficiency. Animal Reproduction Science 87, 283–293. Hanlon, D.W., Firth, E.C., 2012. The reproductive performance of Thoroughbred mares treated with intravaginal progesterone at the start of the breeding season. Theriogenology 77, 952–958. Hemberg, E., Lundeheim, N., Einarsson, S., 2006. Successful timing of ovulation using deslorelin (Ovuplant) is labour-saving in mares aimed for single AI with frozen semen. Reproduction in Domestic Animals 41, 535–537. James, A.N., Vogelsang, M.M., Forrest, D.W., Stott, G.G., 1998. Efficacy of short-term administration of altrenogest to postpone ovulation in mares. Journal of Equine Veterinary Science 18, 329–331. Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone postbreeding and increased milk urea nitrogen in lactating cows. Journal of Dairy Science 80, 1288–1295. Loy, R.G., Hughes, J.P., 1966. The effects of human chorionic gonadotrophin on ovulation, length of estrus, and fertility in the mare. Cornell Veterinarian 56, 41–50. Loy, R.G., Swan, S.M., 1966. Effects of exogenous progestogens on reproductive phenomena in mares. Journal of Animal Science 25, 821–826. Macpherson, M.L., Reimer, J.M., 2000. Twin reduction in the mare: Current options. Animal Reproduction Science 60–61, 233–244. Martinez, M.F., Kastelic, J.P., Adams, G.P., Cook, B., Olson, W.O., Mapletoft, R.J., 2002a. The use of progestins in regimens for fixed-time artificial insemination in beef cattle. Theriogenology 57, 1049–1059. Martinez, M.F., Kastelic, J.P., Adams, G.P., Mapletoft, R.J., 2002b. The use of a progesterone-releasing device (CIDR-B) or melengestrol acetate with GnRH, LH, or estradiol benzoate for fixed-time AI in beef heifers. Journal of Animal Science 80, 1746–1751. Newcombe, J.R., 2002. Field observations on the use of a progesterone-releasing intravaginal device to induce estrus and ovulation in seasonally anestrous mares. Journal of Equine Veterinary Science 22, 378–382. Newcombe, J.R., Cuervo-Arango, J., 2011. The effect of time of insemination with fresh cooled transported semen and natural mating relative to ovulation on pregnancy and embryo loss rates in the mare. Reproduction in Domestic Animals 46, 678–681. Sieme, H., Schafer, T., Stout, T.A., Klug, E., Waberski, D., 2003. The effects of different insemination regimes on fertility in mares. Theriogenology 60, 1153–1164. van de Leemput, E.E., Vos, P.L., Hyttel, P., van den Hurk, R., Bevers, M.M., van der Weijden, G.C., Dieleman, S.J., 2001. Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/ prostaglandin-treated heifers. Theriogenology 55, 573–592.