Concentrations of Circulating Gonadotropins During. Various Reproductive States in Mares

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1 BIOLOGY OF REPRODUCTION, (19) Concentrations of Circulating Gonadotropins During Various Reproductive States in Mares KURT F. MILLER, S. L. BERG, D. C. SHARP and. J. GINTHER Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 537 ABSTRACT Concentrations of circulating FSH and LH were measured in pony and horse mares during the estrous cycle. The gonadotropin profiles did not differ significantly between the two types of mares. During the estrous cycle FSH and LH were secreted in a reciprocal pattern with FSH high during diestrus and LH high during estrus. Although the patterns of FSH and progesterone secretion were similar, FSH rose before progesterone at the end of estrus, indicating that the initial FSH increase was not due to a positive feedback effect of progesterone. The patterns of LH, but not of FSH secretion, diverged between nonpregnant and pregnant mares 19 days after ovulation. In both hysterectomized and pregnant mares, FSH concentrations were elevated after the end of estrus, comparable to what occurred during diestrus. Treatment of diestrous mares with estradiol or of seasonally anovulatory mares with clomiphene citrate had no significant effect on concentrations of circulating FSH or LH. INTRODUCTION The concentrations of gonadotropins in the circulation during various reproductive states in mares are not adequately documented. When compared with horse mares, pony mares have lower incidences of multiple ovulations, diestrous ovulations, and pseudopregnancy, a longer interovulatory interval, and a shorter ovulatory season (Ginther, 1979). With these differences in mind, examinations were made of circulating concentrations of FSH and LH during the estrous cycles of both pony (Experiment 1) and horse mares (Experiment ). The patterns of progesterone secretion have been shown to diverge between nonpregnant and pregnant mares about 15 days after ovulation (Allen and Hadley, 1974; Squires et al., 1974c; Sato et a!., 1977). The patterns of gonadotropin secretion during this time are less clear (Evans and Irvine, 1975). Therefore, a contemporary comparison of gonadotropin profiles was made between nonpregnant and pregnant mares during the 3 days subsequent Accepted February 1, 19. Received July 17, Presented in part at the 9th Annual Meeting of the American Society of Animal Science, Madison, WI (1977). Animal Science Department, University of Florida, Gainesville, FL 311. to ovulation (Experiment 3). During the first two months of pregnancy, mares have marked follicular growth leading to the formation of secondary corpora lutea (CL; Squires et a!., 1974a). Hysterectomy, which results in CL maintenance (Ginther and First, 1971), also results in follicular growth similar to that found in pregnant mares (Squires et al., 1974b). This suggests that the growth of follicles in early pregnancy is due to a substance other than PMSG. To examine the role of pituitary gonadotropins in this process, comparisons were made of concentrations of circulating gonadotropins from Days 1 to 7 after the end of estrus in pregnant and hysterectomized mares (Experiment 4). Comparisons between the patterns of gonadotropin and estradiol secretion during the estrous cycle (Noden et al., 1975; Pattison et al., 1974) suggest a possible negative feedback effect of estrogens on FSH secretion in the mare. This hypothesis was tested by administration of exogenous estradiol to diestrous mares and examination of changes in gonadotropin levels (Experiment 5). From limited clinical observations, clomiphene citrate, a synthetic antiestrogen, has been reported to stimulate follicular growth in the anovulatory mare (Robinson, 1977). To test this hypothesis, seasonally anovulatory mares were injected with one of several doses of clomiphene citrate, and the changes in ovarian 744

2 FSH AND LH IN VARIOUS REPRODUCTIVE STATES OF MARES 745 follicles, estrous behavior and gonadotropin concentrations were examined (Experiment ). MATERIALS AND METHODS Unless indicated otherwise, all animals were pony mares (15-3 kg) which were obtained from upper Midwest sources and maintained at the University of Wisconsin Experimental Farms. Estrus was determined as described previously (Ginther et al., 197) and ovulation was detected by rectal palpation. Blood samples were collected by venipuncture, and plasma, obtained following centrifugation, was stored frozen (-#{17}C) until assayed for hormone concentration by radioimmunoassays previously validated in this laboratory (FSH, Freedman et a!., 1979; LH, Whitinore et al., 1973; progesterone, Squires et al., 1974c). Samples were assayed in duplicate, and all samples from a mare were run in the same assay. When comparisons were made between groups, the groups were balanced across assays. Hormone data were analyzed using a linear least squares analysis of variance (ANOVA) program. The hierarchal nature of sequential sampling was taken into account with treatment effects tested by animal within Experiment treatment. I Gonadotropin patterns during the estrous cycle of the pony mare were examined. Blood samples were collected daily from three groups of mares for one estrous cycle: 1) pony mares in Wisconsin during June (n = 4); ) pony mares in Wisconsin during August- September (n = 5); and 3) pony mares in Florida during August-September (n = 3). Estrous cycle length was 5. ±. days (mean ± SEM). Cycles were normalized to 5 days for all mares by either randomly excluding days or randomly assigning days with missing data for mares with cycles longer or shorter than 5 days, respectively. Differences between groups were not found, and therefore data for the three groups were pooled. Experiment Gonadotropin patterns over the estrous cycle of the horse mare were examined. Mares were either Thoroughbreds or Quarter Horses maintained at the University of Florida. Blood samples were collected daily over one cycle during either June-July (n = 3) or August-September (n = 3). Mean cycle length was.7 ±.7 days. Cycles were normalized to 3 days by either randomly excluding days or randomly assigning days with missing data for mares with cycles longer or shorter than 3 days, respectively. When cycles were normalized to a common length and data analyzed in reference to ovulation, relationships between gonadotropins within individual animals were sometimes obscured. To examine these relationships and the relationship of FSH and progesterone, data from this experiment and Experiment 1 were combined and normalized to the occurrence of specific changes in hormone concentration. Mares were used that had no more than one missing sample for the interval from 4 days before to 4 days after the event of interest. Events of interest were the occurrence of the highest concentration of LH, the lowest concentration of FSH and the first and last days that the progesterone concentration exceeded 1 ng/ml. Experiment 3 Gonadotropin patterns of mares were compared between early pregnancy (n = 3) and the corresponding days of the estrous cycle (n = 4). The comparison involved daily samples from the day of ovulation (Day ) to Day 3, at which time the first ovulation occurred in the nonpregnant group. As part of another project, pregnancy was confirmed by removal of embryos at Day 35. Experiment 4 Gonadotropin patterns were compared between pregnant (n = ) and hysterectomized (n = 5) mares for Days 1-7 postesrrus. Mares were balanced across seasons and were randomly selected from a previously reported study (Squires et al., 1974b). Mares were either hysterectomized or sham operated on Day 3 postestrus. Beginning on Day 1, blood samples were collected at 3 day intervals until Day 19 and at 4 day intervals from Day 4 to Day 7. FSH was assayed in both groups for Days 1-7. LU was assayed for Days 1-7 in the hysterectomized group, but only for Days 1-3 in the pregnant group because of PMSG cross reactivity in the LU assay. Experiment 5 The effect of treatment with esrradiol on concentrations of circulating gonadotropins during diestrus (Days -15 or Days 5-15) was examined. On the day of ovulation (Day ), mares were randomly assigned to one of three groups: 1) control (n = 4); ) estradiol, Days -15 (n = 3); and 3) estradiol, Days 5-15 (n = 3). Mares in the control group were untreated, while mares in the estradiol groups were injected with 5 Mg/day of estradiol-17a, i.m., in cottonseed oil. Blood samples were drawn daily and assayed for gonadotropin concentrations. Experiment The effect of a single injection of clomiphene citrate on gonadotropin concentrations, estrous behavior and ovarian follicles was examined in seasonally anovulatory mares. In January, pony mares were assigned to one of six groups (n = 3) which received, 1, 5, 1, 3 or 5 mg clomiphene citrate. The clomiphene citrate was suspended in oil and given as a single i.m. injection. Blood samples were drawn at, 1, 3, and 1 h and daily for 1 days after treatment. Mares were examined for estrus and palpated on Days, 5, 1 and 3. Experiment I RESULTS The ANOVA for pony estrous cycles normalized to 5 days (Fig. 1) indicated an effect of day (P<.1) for FSH and LH. Mean FSH

3 74 MILLER ET AL. E ) C - 4., OIv 4 FSH oy 3 FIG. 1. Concentrations of circulating FSH and LH during the estrous cycle of pony mares (mean ± SEM; n = 1). Vertical bars (Isd) indicate the magnitude of the least significant difference (P<.5). OV = ovulation. concentrations were low for several days prior to ovulation (<3 ng/ml). Following ovulation mean FSI-I concentrations began to increase and by Day 3 the mean concentration (4.4 ng/mi) was significantly greater than on the day of ovulation (.3 ng/ml). From Days 3 to 15 mean FSH concentrations, although fluctuating, remained elevated (4.4 to 7.7 ng/ml). After Day 15 mean FSH concentrations decreased consistently, reaching low preovulatory levels (<3 ng/ml) on Day 1 (4 days prior to ovulation). Mean LI-I concentrations increased prior to ovulation, reached a high on Day 1 (. ng/ml) and subsequently declined. From Days to 15 mean LI-I concentrations remained low (< ng/ml). After Day 14 mean LH concentrations increased slowly to Day (3 days prior to ovulation; 3.5 ng/ml). After Day the rate of change appeared to increase, with mean LH concentrations reaching a high point again at Day 1 (. ng/ml). Experiment The ANOVA for horse estrous cycles normalized to 3 days (Fig. ) indicated an effect of Day (P<.1) for FSH and LH. Mean FSH concentrations were low (<3 ng/ml) for several days prior to ovulation. Mean FSH concentrations appeared to begin rising before ovulation; the concentration at the time of the first ovulation (Day, 3.4 ng/ml) tended (P<O.1) to be greater than on Day -, and the concentration on Day 1 was greater (P<.5) than on Day -. From Days 3 to 15 mean concentrations of FSH, although fluctuating, remained elevated ( ng/ml). After Day 15, mean FSH concentrations decreased, reaching low preovulatory levels (<3 ng/ml) on Day 1 (5 days prior to ovulation). Mean concentrations of FSH appeared to rise before the second ovulation. Mean LH concentrations increased prior to ovulation, reached a high on Day 1 (11.1 ng/ml) and subsequently declined. From Days to 19 mean LH concentrations remained low (< ng/ml). Mean LU concentrations increased from 3 days prior to ovulation (Day ) to a high point again at Day 1(13.7 ng/ml) o.j 14 E 1 ) C #{149} 4. - ov FSH. t FIG.. Concentrations of circulating FSH and LH during the estrous cycle of horse mares (mean ± SEM; n = ). Vertical bars (Isd) indicate the magnitude of the least significant difference (P<.5). OV = ovulation. ov

4 FSH AND LU IN VARIOUS REPRODUCTIVE STATES OF MARES 747 ) C When data were combined for Experiments 1 and, the highest FSH value occurred on the average 11.3 ± 1.1 days after the first ovulation and 13. ± 1. days before the second ovulation (n = 1). The lowest FSH value occurred. ±. days before the second ovulation (n = 9). Normalization of gonadotropin data to the lowest FSH value resulted in significant effects of day for both FSH and LH (Fig. 3). It appeared that the slope of the LH curve increased dramatically between Days and 1 (Day = day of lowest FSH value). The highest LH value occurred on the average 1. ±. days (n = 14) and the lowest value 1.3 ± 1. days (n = 13) after the first ovulation. Normalization of gonadotropin data to the highest LU value resulted in significant effects of day for both LH and FSH (Fig. 3). Concentrations of FSH increased before LU reached its highest value. When FSH and progesterone data were normalized to the first occurrence of a sample with progesterone concentration greater than 1 ng/ml (n = 5), a significant effect of day was found for both FSH and progesterone concentrations (Fig. 4). The FSH concentrations were significantly higher on Day --1 than on Day -4 (Day = day of first occurrence of a sample with greater than 1 ng/ml of progesterone). Concentrations of progesterone were not significantly elevated over baseline until Day 1. When data were normalized to the first occurrence of a sample with progesterone concen- F SH 11 MINIMUM j sd FIG. 3. Concentrations of circulating gonadotropins normalized (Day ) to the minimum FSH (n = 9) or maximum LU (n = 14) concentration during the estrous cycle (mean ± SEM). Vertical bars (Isd) indicate the magnitude of the least significant difference (P<.5). flsd. 1 P4 l ngmi p4 Ing /ml sd C: -H 1lsd t FIG. 4. Concentrations of circulating FSH and progesterone normalized (Day ) to the progesterone increase to greater than I ng/ml (n = 5) or to the progesterone decline to less than I ng/ml (n = 5; mean ± SEM). Vertical bars (lsd) indicate the magnitude of the least significant difference (P<.5). tration less than 1 ng/ml (n = 5), significant effects of day were found for progesterone and FSH concentrations. The FSH concentrations were maximal on Day -1 (Day = day of first occurrence of a sample with less than 1 ng/ml of progesterone) and then declined. Experiment 3 The ANOVA for the comparisons between nonpregnant and pregnant mares indicated no significant effect of reproductive state, day or interaction for FSH concentrations (Fig. 5). Mean FSH concentrations from day of ovulation (Day ) to Day 3 ranged from.5 to.4 ng/mi for nonpregnant mares and. to 9.4 ng/ml for those during early pregnancy. The ANOVA for LU concentrations indicated a highly significant effect of reproductive state, day and interaction of state and day (Fig. 5). Examination of means by use of Fisher s LSD indicated that the interaction was due primarily to significantly increased LU concentrations in the nonpregnant group for Days 19 to 3 and to a slower decline in LH concentration postovulation in the nonpregnant group. Experiment 4 The ANOVA for the comparison of pregnant and hysterectomized mares indicated no effect of treatment (pregnant vs hysterectomized) on concentrations of FSH. There was a significant effect of day as averaged over the two treatments, but no treatment-by-day interaction. The data were therefore pooled and the sd

5 74 MILLER ET AL. 1 FSH 1-1 E ) LH - NONPREGNANT PREGNANT FIG. 5. Concentrations of circulating gonadotropins in nonpregnant (n = 4) and pregnant (n = 3) mares from day of ovulation (Day ) to Day 3 (mean ± SEM). The vertical bar (Isd) indicates the magnitude of the least significant difference (P<.5). day effect examined by the multiple range test (Fig. ). Mean concentration of FSH were initially high, comparable to the corresponding days of diestrus, and decreased significantly between Days 1 and 13. Mean concentrations remained low until Day 4 and tended (P<.1) to increase between Days 4 and, followed by a decrease (P<.1) between Days and 3. Thereafter fluctuations occurred in the mean concentrations, but these were not significant. The lowest mean values occurred on Days --7, and these means were significantly lower than on Days 7, 1 and. The ANOVA for LH concentrations from Days 1 to 3 indicated no effect of treatment, but a highly significant effect of day. Pooling data across treatments and examination of means by use of Fisher s LSD indicated that LH concentrations were higher on Day 1 than on all other days (Fig. ). In the pregnant group, E ) C FIG.. Concentrations of circulating gonadotropins pooled across pregnant (n = ) and hysterectomized (n = 5) mares from Days 1 to 7 after the end of estrus (mean ± SEM). After Day 3 LH data from the hysterectomized mares alone are shown, as PMSG crossreacted in the LH assay. Vertical bars (lsd) indicate the magnitude of the least significant difference (P<.5). the concentration of LH was less than ng/ml on Day 3 in all 5 mares for which a sample was available; on Day 3 one mare had less than ng/ml of LU activity and 4 mares had concentrations great enough to be off the standard curve of the LU assay, due to the appearance of PMSG. By Day 4, all mares had sufficient PMSG to completely inhibit the binding of the labeled LU to the antibody in the LH assay. Experiment 5 The ANOVA for the effect of estradiol on LH and FSU concentrations during diestrus indicated no difference between treatment groups and no interaction of treatment with day. There was a significant effect of day on FSH and LH concentrations. The changes among days were similar to those found in Experiment 1. Experiment The ANOVA for treatment of seasonally anovulatory mares with clomiphene citrate indicated no significant effects either of treatment or of day for FSH or LH concentrations. There were no significant effects of treatment on number or size of palpable follicles. Although mares were occasionally found to be in estrus (9 of 7 observations), there was no consistent pattern of occurrence in any group.

6 FSH AND LH IN VARIOUS REPRODUCTIVE STATES OF MARES 749 DISCUSSION Although interovulatory intervals were different (P<.5) between ponies (5. ±. days) and horses (.7 ±.7 days) the patterns of circulating concentrations of FSH and LU during the estrous cycle appeared to be similar. Mean concentrations of FSU in both types of mares were high during mid-diestrus and low during estrus (Figs. 1, ). This pattern of FSH secretion is similar to patterns previously reported (Evans and Irvine, 1975; Nett et al., 1979). Evans and Irvine (1975) reported a bimodal pattern for FSU during the estrous cycle of the horse mare, with high concentrations of FSI-I during late estrus-early diestrus and again during late diestrus. Examination of data from individual mares in Experiments 1 and (n = 1) indicated that only 3 mares ( ponies, 1 horse) had obviously bimodal patterns of FSU secretion. The majority of the mares were bled late in the breeding season, in agreement with a report that bimodality is less common late in the breeding season (Turner et al., 1979). It appeared that FSU levels began to rise earlier in the horse mares than in the pony mares (several days prior to ovulation vs day of ovulation). Mean concentrations of LH in both types of mare were low during mid-diestrus and higher during estrus (Figs. 1, ) in agreement with many other reports. It appeared that LU concentrations began to increase sooner and rose at a slower rate in pony mares than in horse mares (initial increase 9 days vs 3 days before ovulation). In this regard, however, 3 ponies from Experiment 1 and 3 horses from Experiment were housed in adjacent paddocks during August and September in Florida; no significant differences in either FSH or LH concentrations were found between the two types of mares during Days -3 to +3 (Day = ovulation) over two successive estrous cycles. It was concluded that FSH and LU were secreted in an approximately reciprocal pattern with FSH high and LU low during diestrus, and FSU low and LU high during estrus. The only departure from this pattern was between the time of the lowest FSH value (. days before ovulation) and the highest LH value (1. days after ovulation). During this period, both FSU and LU concentrations increased significantly (Fig. 3). This was in contrast to other times during the estrous cycle, when changes in FSH and LH concentrations were in opposite directions. Although the patterns of FSH and progesterone secretion during the estrous cycle are similar, suggesting a positive feedback, a significant rise in FSU concentrations occurred days before the rise in progesterone concentrations at the beginning of diestrus (Fig. 4). This indicates that a positive feedback effect of progesterone could not be the sole mechanism controlling FSH secretion, even though progesterone and FSU began to decline simultaneously at the end of diestrus. This is in agreement with data from ovariectomized (OVX) mares in which progesterone and/or estradiol treatment was without effect on FSH concentrations (Garcia et a!., 1979). Considerable fluctuation in concentrations of FSU with large SEMs were obtained during early pregnancy. The times of appearance of apparent surges of FSH were inconsistent among mares. Despite this variation, however, significant changes were detected among days when data were pooled, for pregnant and hysterectomized mares, thereby increasing the number of animals to 11. Mares during pregnancy and after hysterectomy were found to have high concentrations of circulating FSU similar to the levels found during the corresponding days of diestrus, in agreement with an earlier report for pregnant horses (Evans and Irvine, 1975). The decrease in FSH concentrations after Day 1, occurred even though progesterone concentrations in these same mares did not decrease significantly during this time (Squires et al., 1974c). This observation supports the interpretation, noted above, that changes in progesterone concentrations do not adequately account for changes in FSU concentrations. The surge of FSU at Day, reported by Evans and Irvine (1975), was not found in the present study (Fig. ); means were low between Days En agreement with a statement by Irvine and Evans (197), mean FSH increased between Days 4-. However, the reported increase at Day 3 (Irvine and Evans, 197) did not occur in the present project. The increase in FSH concentrations which occurred, on the average, between Days 4- may contribute to the growth of follicles which occurred in these same hysterectomized mares and pregnant mares before the appearance of PMSG (Squires et al., 1974b). The minimal concentrations of LH subsequent to the surge during estrus is in agreement with previous reports (Evans and Irvine, 1975; Nett et a!., 197). The reason for delayed return of

7 75 MILLER ET AL. LH to baseline in the nonpregnant group of Experiment 3 is unknown. Estradiol or clomiphene citrate treatment did not alter the circulating concentrations of either gonadotropin. Estradiol has been demonstrated to have a positive effect on LH when administered alone, but this effect was abolished when administered in combination with progesterone in the OVX mare (Garcia and Ginther, 197). When administered to mares in diestrus, exogenous estradiol may act in combination with endogenous progesterone to inhibit LH secretion. Such inhibition of LU would not be apparent, as concentrations were already at baseline levels. The lack of effect of estradiol on FSH during diestrus, found in the present project, is in agreement with results from OVX mares. Circulating concentrations of FSH in OVX mares did not change following treatment with estradiol or progesterone either alone or in combination (Garcia et al., 1979). An additional possibility is that the 5 jig/day dose was inadequate. Clomiphene citrate treatment of seasonally anovulatory mares did not alter gonadotropin concentrations, estrous behavior, or number of palpable follicles, thus failing to confirm the results of a clinical study (Robinson, 1977). ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison. Antiserum to human FSH, used in the equine FSH assay, was donated by NIAMDD. Clomiphene citrate was a gift of Merrell-National Laboratories, Cincinnati, OH. The authors acknowledge the technical assistance of Dr. L. C. Nuti, Dr. E. L. Squires and Mr. L. J. Freedman. The authors appreciate the assistance of Mr. A. Hardie with the statistical analyses and Mr. T. E. Ladell with the preparation of figures. REFERENCES Allen, W. E. and Hadley, J. C. (1974). Blood progesterone concentrations in pregnant and nonpregnant mares. Equine Vet. J., Evans, M. J. and Irvine, C.H.G. 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