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Relationships between FSH surges and follicular waves during the estrous cycle in mares
Theriogenology39:781-796,1993
RELATIONSHIPS BETWEEN FSH SURGES AND FOLLICULAR WAVES DURING THE ESTROUS CYCLE IN MARES D.R. Bergfelt and O.J. Ginther Animal Health and Biomedical Sciences Veterinary Science Building University of Wisconsin-Madison Madison, WI 53706 USA Received for publication: Accepted:
June 16, 1992 September 1, 1992
ABSTRACT Individual follicles 215 mm were monitored daily by ultrasonography in 12 mares during the estrous cycle. Follicular waves were designated as major waves (primary and secondary) and minor waves based on maximum diameter of the largest follicle of a wave (major waves, 34 to 47 mm; minor waves, 18 to 25 mm). Dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the second largest follicle. Dominant follicles of primary waves (n=12) emerged (attained 15 mm) at a mean of Day 12 and resulted in the ovulations associated with estrus (ovulation=Day 0). The dominant follicle of a secondary wave (n=l) emerged on Day 2 and subsequently ovulated in synchrony with the dominant follicle of the primary wave, which emerged on Day 9. The largest follicles of minor waves (n=4) emerged at a mean of Day 5, reached a mean maximum diameter 3 days later, and subsequently regressed. There was a significant increase in mean daily FSH concentrations either 6 days (primary wave) or 4 days (minor waves) before the emergence of a wave. Mean concentrations of FSH decreased significantly 2 days after emergence of the primary wave. Divergence between diameter of the dominant and largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than of the largest subordinate follicle 3 days after wave emergence and by the cessation of growth of the largest subordinate follicle beginning 4 days after the emergence of a wave. Surges of FSH were identified in individual mares by a cycle-detection program; surges occurred every 3 to 7 days. Elevated mean FSH concentrations over the 6 days prior to emergence of the primary wave was attributable to a significantly greater frequency of individual FSH surges before wave emergence than after emergence and to an increase in magnitude of peak concentrations of FSH associated with individual surges. Key words: estrous cycle, follicular waves, FSH surges, mare Acknowledgments Supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and by Equiculture, Inc., Cross Plains, Wisconsin. The authors thank L.J. Kulick for graphics and M. Radtke for manuscript preparation.
Copyright 0 1993 Butterworth-Heinemann
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INTRODUCTION Transrectal ultrasonic imaging has been used in mares to monitor day-to-day diameter changes of individual follicles during transition from the anovulatory to ovulatory seasons (1) and during the estrous cycle (1,2). Follicular waves were indicated by the simultaneous emergence and development of several follicles. Two follicular waves were detected in 11 of 25 interovulatory intervals, whereas during the remaining intervals only the wave that gave origin to the ovulatory follicle was detected (3). The largest follicle of each type of wave reached at least 2.5 mm and became dominant, whereas the remaining follicles regressed (subordinates). Waves that give origin to a dominant follicle have been called major waves (3). A major wave that emerges during late estrus or early diestrus has been called a secondary wave; the dominant follicle ovulates during diestrus, regresses, or becomes hemorrhagic. A major wave that emerges during mid-cycle has been called a primary wave; the dominant follicle results in the primary (estrus-associated) ovulation. Detectable waves that do not develop an apparent dominant follicle are called minor waves (4). Recently, associations between elevations in mean daily FSH concentrations and emergence of follicular waves during the interovulatory interval in heifers (5) and during pregnancy in mares (4,6) have been reported. In early pregnant mares, the emergence of major follicular waves (4,6) and minor follicular waves (4) were each preceded by a significant increase in mean FSH concentrations; subsequent to emergence of major waves, a significant decrease in mean FSH concentrations occurred. The importance of FSH to follicular development during the estrous cycle in mares has been demonstrated by diminished FSH and follicular development following treatment with a proteinaceous fraction of equine follicular fluid (7). However, the relationships between circulating concentrations of FSH and follicular dynamics during the cycle have not been adequately established. It has been stated (8), however, but not statistically documented, that maximal FSH concentrations in blood samples collected every other day occurred 2 to 4 days before a follicle reached 20 mm and subsequently declined to minimal concentrations 4 to 6 days later. Furthermore, it has been reported (9) that changes in the number of large follicles occurred most commonly 6 days after corresponding changes in FSH concentrations. The objective of the present study was to characterize the relationships between daily circulating concentrations of FSH and follicular development during the estrous cycles of mares. Consideration was given to individual FSH surges within mares as well as to FSH and follicular changes averaged over all mares. MATERIALS
AND METHODS
Twelve riding-type horse mares, 4 to 16 years of age and weighing 400 to 550 kg, were used from May to August in the study. Although the breeds were not known, they appeared to be primarily Quarter Horse and Appaloosa, based on the size, conformation and color patterns of the mares. The mares were obtained from livestock sale barns; all were nonlactating, and their reproductive histories were not known. The mares were kept
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under natural light in outdoor paddocks and had free-choice access to hay, water and trace mineralized salt, and were fed 2 to 4 kg cob corn daily per mare. Ultrasonic examination of the ovaries and uterus was performed as described elsewhere (10) with a real-time, linear-array scanner equipped with a 7.5 MHz intrarectal transducer. Mares were not used if they had ultrasonic indications of apparent ovarian or uterine abnormalities (10). The ovaries were monitored by ultrasound and blood samples were collected once daily (every 24 hours), beginning when diameter of the largest follicle reached 225 mm and continuing until 3 days after the end of an inter-ovulatory interval. An interovulatory interval was defined as the interval between the flit and second ovulation of successive ovulatory periods. If double asychronous ovulations occurred during a given ovulatory period, the first ovulation was used to assign a beginning and end to the interovulatory interval (ovulation=Day 0). Blood samples were drawn by jugular venipuncture into heparinized tubes and immediately stored on ice for transport to the laboratory. After centrifugation, plasma Circulating was collected, and aliquots were made and stored frozen (-20°C). concentrations of FSH (11) were measured in plasma samples using a radioimmunoassay previously validated in this laboratory. The intra- and inter-assay coefficients of variation for the FSH assay were 15 and 17%, respectively. The FSH data set used herein was also used for a different purpose in a previous study to examine the nature of synchronous LH and FSH surges during the estrous cycle (12). The ovaries were monitored daily by ultrasound, and the examinations were recorded with a 3/4-inch (16 mm) videotape recorder attached to the ultrasound scanner. Videotapes were subsequently viewed on a 16-inch (41mm) video monitor. Sequential identification of individual follicles >lS mm was done similarly to that described for early pregnant mares (6). A diagram de&ting relative location and size of all follicles 215 mm was made for each ovary on each day. The diameter of follicles (average of height and width of antrum) was determined using a transparent grid placed over a still image on the video monitor. The relative location of follicles and the corpus luteum were used as reference points to identify individual follicles. Each apparent follicular wave was ranked according to maximum diameter of the largest follicle; the ranking extended from the largest to the smallest diameter. The maximum diameter of the second largest follicle and the difference in maximum diameters between the 2 largest follicles were also listed for each wave. The waves were then divided into 2 categories (major and minor waves) using the point of greatest discontinuity in the rankings from the largest to the smallest follicle diameters. The follicle of a major wave reaching the largest diameter was defined as the dominant follicle, and the second largest follicle of a major wave was defined as the largest subordinate follicle if it emerged within 2 days of the dominant follicle. The day of detected wave emergence was defined as the day the retrospectively identified dominant follicle of a major wave or the largest follicle of a minor wave was 15 to
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16 mm in diameter. If the follicle was fust detected when it reached 117 mm, a reported growth rate of 3 mm per day (10) was used to project back to the day of emergence.
A technique that was developed to study episodic fluctuations in blood hormones (13) was used to detect peak concentrations of FSH in individual mares. This procedure was used recently to detect FSH and LH surges in blood samples collected daily during the menstrual cycle in monkeys (14). In brief, the program determined threshold concentrations of FSH based on the variability of the assay results between duplicate samples for each mare within each day. Concentrations that were greater than the threshold values were detected and identified as peak FSH concentrations. An FSH surge in an individual mare was defined by a progressive increase and decrease in concentrations that encompassed a peak concentration (nadir-to-peak-to-nadir). Frequency of occurrence of individual FSH surges, the number of FSH surges per mare, and the magnitude of peak concentrations of FSH associated with individual surges were determined for 8 days before and 8 days after emergence of the primary follicular wave. Data for all primary waves were grouped to characterize the dominant and the largest subordinate follicles of the wave and to examine the temporal relationships between mean daily FSH concentrations and mean days of wave emergence, and between mean FSH concentrations and mean day of divergence in diameter of the dominant and the largest subordinate follicles. Mean day of emergence of the dominant follicle of the primary wave was used to normalize the dominant follicle, largest subordinate follicle, and FSH concentrations to the day scale. The day-to-day associations among the dominant follicle, largest subordinate follicle, and FSH concentrations were maintained; that is, when a value for the dominant follicle for a given day was normalized to conform with the mean, the corresponding value for the largest subordinate follicle and the corresponding FSH value were also normalized. Mean concentrations of FSH were profiled 8 days before and 8 days after wave emergence to encompass the days before wave emergence and’ the day of divergence in diameter of the dominant and largest subordinate follicles. To examine the associations between mean FSH concentrations and mean day of emergence of a minor wave, concentrations were normalized to the first ovulation of the interovulatory interval, since in retrospect, all of the detected minor waves began soon after ovulation. Mean concentrations of FSH were studied from Day -2 to Day 4 so that the days did not extend beyond the mean day of emergence of a minor wave. The mean day of emergence of the largest follicle of a minor wave was used to place the wave on the interovulatory-interval day scale. The mean FSH values were compared between mares with a minor wave versus those with no minor wave. Analyses of variance for sequential data were used to study the day effects for diameter of the dominant and the largest subordinate follicles and FSH concentrations associated with primary waves and diameter of the largest follicle of minor waves. A split-plot analysis of variance for sequential data was used to study the main effect of group, day, and the interaction of group-by-day for FSH concentrations in mares with a minor wave versus those with no minor wave. When a significant (P
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group, day, or group-by-day interaction was indicated, Duncan’s multiple range tests were used within each end point to examine differences between days, and unpaired t-tests were used to examine differences between groups within days. Characteristics of major and minor waves were compared using unpaired t-tests for the following end points: 1) the number of follicles, 2) the maximum diameter and day attained for the largest and second largest follicles, and 3) the difference in maximum diameter between the largest and second largest follicles. Paired t-tests were used to examine mean differences in diameter between the dominant and largest subordinate follicles of the primary wave within days. Frequency of occurrence of individual FSH surges, number of FSH surges/mare, and magnitude of peak concentrations of FSH associated with individual surges were studied before and after emergence of the primary wave using a (X-square goodness-of-fit analysis, paired t-test, and unpaired t-test, respectively. RESULTS Follicular waves appeared to fall into 2 populations based on inspection of ranked data. The maximum diameter of the largest follicle was 34 to 47 mm (meanfSEM, 42.2k1.2) for 1 of the wave categories (designated as major waves; n=12) and 18 to 25 mm (21.2f1.4) for the other wave category (designated as minor waves; n=4). The difference in maximum diameter between the 2 largest follicles was 11 to 24 mm (18.0fl.l) for major waves and 1 to 5 mm (3.0f1.3) for minor waves. Characteristics of major and minor waves during the interovulatory interval with results of statistical analyses are shown below (Table 1). Mean (fSEM) length of the interovulatory interval was not different @0.05) between mares with only a primary wave (n=7; 22.7fl.O days) and mares with either primary and secondary waves or primary and minor waves (n=5; 21.4f0.7 days). The day-to-day diameters of all individually identified follicles >15 mm and of circulating concentrations of FSH beginning 3 days before the first ovulation and ending on the day of ovulation at the end of the interovulatory interval are shown for each of 12 mares in Figure 1. Based on inspection of individual follicular profiles, all 12 mares had a primary wave leading to ovulation at the end of the interovulatory interval. In addition, Mare L had a secondary wave and the dominant follicle ovulated in synchrony with the dominant follicle of the subsequent primary wave. Mares J and K also had double ovulations, with both originating from the primary wave. All double ovulations were unilateral and synchronous. Four of the 12 mares (Mares H, I, J and K) had a minor wave preceding the primary wave. Characteristics of individual FSH surges 8 days before and 8 days after emergence of the primary wave in 12 mares with results of statistical analyses are shown in Table 2. The frequency of occurrence of individual FSH surges, the number of surges per mare, and the magnitude of peak concentrations of FSH associated with individual surges were all significantly greater before wave emergence than after emergence. The number of days encompassing peak FSH concentrations were not different (PN.05) before and after wave emergence. The interval between peak concentrations of FSH associated with
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surges could not be determined after wave emergence because the 4 surges that were detected occurred in 4 separate mares.
Table 1. Characteristics (meat&EM) of major and minor waves occurring during the interovulatory interval in 12 mares
Wavesa Majorb
Minor
12
4
__
11.8 kO.7
4.8 kO.8
P<0.0001
5.2 f0.8
2.5 M-9
Largest follicle Maximum diameter (mm) Dayd attaining maximum diameter
42.2 f1.2 9.2 f0.4
21.2 f1.4 3.5 50.6
PcO.0001 P
Second largest follicle Maximum diameter (mm) Dayd attaining maximum diameter
24.2 i0.7 4.2 k0.3
18.3 f1.7 2.3 f1.9
P
Difference in maximum diameter between largest and second largest follicle (mm)
18.0 fl.1
3.0 il.3
End points
Number of detected waves Day of emergenceC Number of detected follicles (L15 mm)
Probability
NS
PcO.OOO1
Waves in which the largest attained follicle diameter was 34 to 47 mm or 18 to 25 mm were designated major and minor waves, respectively. Means included data from all primary waves (1 per mare) but did not include data from the secondary wave in Mare L. If more than 1 follicle 34 to 47 mm was detected and subsequently associated with the same wave, it was handled by averaging follicle diameters and days of emergence. Day on which the retrospectively identified largest follicle of a wave was 15 to 16 mm (ovulation=Day 0). Number of days in reference to day of wave emergence. NS=not significant. The mean diameter of the dominant and largest subordinate follicles as well as the associated mean concentrations of FSH grouped and normalized for all primary waves (upper panel), and the number of detected individual FSH surges in relation to wave emergence (lower panel) are shown in Figure 2. Mean follicle diameters did not include data for the day of wave emergence because the number of mares that had corresponding
Theriogenology
Table 2. Characteristics (meat&EM) of individual FSH surges in 12 mares during 8 days before and 8 days after emergence of the primary wavea
Individual
FSH surges
Frequency Number/mare
Before wave emergenceb
After wave emergence
Probability
29
4
P
2.4 +0.2 (range, 1 to 4)
0.3 kO.1 (range, 0 to 1)
P
Magnitude of peak FSH concentrations (rig/ml)
12.8 k2.3 (range, 3 to 36)
4.5 f0.7 (range, 3 to 6)
P
Number of days encompassing peak concentrationsc
4.5 zko.3 (range, 3 to 9)
4.2 ti.6 (range, 3 to 6)
NS
Interval (days) between peak FSH concentrationsd
4.0 f0.3 (range, 3 to 7)
_-
__
a The day the retrospectively identified dominant follicle was 15 to 16 mm. Included individual FSH surges that were detected on the day of wave emergence. Nadir-to-peak-to-nadir constituting a progressive increase and decrease in concentrations. Number of days between successive peak concentrations of FSH associated with individual surges (n=17). NS=not significant.
values for the dominant and largest subordinate follicles was small (n=4). There was a main effect of day (P
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40 Mare A
Mare B
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Number of days from ovulation Figure 1. Diameter profiles of all follicles 215 mm and individually identified (solid lines) and circulating concentrations of FSH (dashed lines) for each of Mares A to L beginning 3 days before the first ovulation and ending on the day of ovulation terminating the end of the interovulatory interval. The dotted line
Theriogenolog
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Number of days from ovulation at 15 mm indicates that follicles 45 mm were not studied. Stars indicate peak concentrations of FSH associated with individual FSH surges. Thick arrows indicate days of emergence of major waves (primary and secondary) and thin arrows indicate days of emergence of minor waves. Ovulation=OV.
790
Theriogenology 12
1
Primary waves
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(n=lZ mares)
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Number of days from wave emergence Mean (LSEM) diameter of the dominant and largest subordinate follicles and associated concentrations of FSH grouped for all primary waves (upper panel) and number of detected individual FSH surges in relation to wave emergence (lower panel). Data for the 3 end points in the upper panel were normalized to the mean day of emergence of the dominant follicle (Day 12). There was a main effect of day (P
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The mean diameter of the largest follicle of minor waves and mean concentrations of FSH for mares with a minor wave and with no minor wave as well as the results of statistical analyses are shown in Figure 3. The mean day of emergence of the minor wave occurred 4.8 days after ovulation (TabIe 1); Day 5 was used to place the mean follicle profile of the wave on the day scale. The diameter of the largest follicle increased (PcO.05) between Days 5 and 7 and reached a maximum diameter (mean, 21.2 mm; Table 1) on Day 8. Concentrations of FSH in mares with a minor wave increased (PcO.05) between 2 days before and 1 day after ovulation; concentrations were higher (PcO.05) on Days 1 to 3 in mares with a minor wave than in mares with no minor wave. Subsequent to Day 3, concentrations of FSH in mares with no minor wave increased (PcO.05) and were not different from concentrations in mares with a minor wave.
- 25
Largest follicle of 7-
G: Pco.02 D: P
D: PcO.0008
.
5-
L
......................................_...__.........................._.___.__.......___ _.15
-2
-1
0
1
2
3
4
5
6
7
6
9
10
11
Number of days from ovulation Figure 3. Mean (&SEM) concentrations of FSH normalized to the first ovulation of the interovulatory interval in mares with and without minor waves and diameter of the largest follicle of minor waves. Mean day of emergence of the minor wave was used to place the mean follicle profile of the wave on the day scale. Results of statistical analyses are indicated: 1) main effect of group (G); 2) main effect of day (D); 3) interaction of group-by-day (GD). The first significant increase in mean FSH concentrations and in diameter of the largest follicle in mares with a minor wave and days in which concentrations were signifkantly different between mares with and without a minor wave are indicated by a star. The dotted line at 15 mm indicates that follicles cl5 mm were not studied.
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DISCUSSION Major and minor follicular waves occurred during the estrous cycle in mares as determined by monitoring diameter of individual follicles 115 mm. It was reported previously that 1 or 2 follicular waves occurred during the first estrous cycle following transition from the anovulatory to ovulatory seasons (1) and during the estrous cycle of mares that had already entered the ovulatory season (2); however, no attempts were made to group the waves into major and minor categories. In the present study, the day of emergence of the dominant follicle of the primary wave was not altered in regard to whether or not a secondary wave, or a minor wave, preceded it. The dominant follicle of the secondary wave in Mare L emerged on Day 2 and the dominant follicle of the primary wave emerged on Day 9; subsequent to Day 9, the 2 follicles developed concurrently, and a synchronous, double ovulation occurred involving the dominant follicle of each wave. That is, a set of double ovulations involved the primary wave only (Mares J and K) while the other set involved both a secondary wave and a primary wave (Mare L). That is, a double ovulation may not always involve 2 dominant follicles of the primary wave, but may include the dominant follicle of a secondary wave. In previous studies (1,2), the dominant follicles of secondary waves either ovulated during die&us, regressed, or became hemorrhagic. Differences in characteristics between major and minor waves in the present study were as follows: 1) the largest follicle of major waves reached a maximum diameter later after wave emergence than did the largest follicle of minor waves (means, 9 days and 42 mm versus 4 days and 21 mm) and 2) dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the 2nd largest follicle, whereas the 2 largest follicles of minor waves differed by a mean of only 3 mm. These differences appear similar to reported (4) differences between major and minor waves during early pregnancy in mares. Apart from the follicles associated with major and minor waves, it appeared from the graphs of individual follicles that other minor waves emerged, especially in Mare K. By design, follicles of a major wave were defined as those originating within f2 days of emergence of the dominant follicle. However, other follicles emerged 3 to 4 days before (mean, 3 days) and 3 to 7 days after (mean, 4 days) emergence of the dominant follicle of the primary waves. Characterizing follicles as part of a minor wave or as subordinates of a major wave seemed dependent upon an arbitrary definition. That is, in some interovulatory intervals, the dominant follicle emerged from a broad base of detected follicular activity and some of the follicles appeared to regress (minor wave) before emergence of follicles that seemed more closely associated with the primary wave. The statement that follicles of a wave emerge in synchrony therefore must be tempered with the concept that many days may be involved in follicle emergence. Furthermore, since only follicles 115 mm were studied, it is not known whether or not there were other patterns or types of waves for follicles ~15 mm. The duration of individual surges of FSH that were detected before and after emergence of the primary wave encompassed a mean of 4.5 days. That is, in relation to peak concentrations of FSH associated with an individual surge, the number of days from nadir-to-peak-to-nadir constituted, on the average, more than 3 days. Pulses of FSH have
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been detected in blood samples collected frequently within 24 hours on discrete days of the estrous cycle in mares (15-17). In one study (17), the interval between pulses was significantly shorter during the mid- to late-lute& phase (5 pulses/hour) than during estrus (3 pulses/hour). Individual surges (as defined here) occurred over several days, whereas pulses (as previously described; 15-17) occurred frequently within a day. Therefore, it is likely that once-a-day samples would involve a portion of a pulse in some mares on some days. Presumably, pulses of FSH, or a portion of them, were superimposed on the individual surges of FSH described here, but this has not been studied. The frequency of occurrence of individual FSH surges was significantly greater during the 8 days preceding emergence of the primary wave (mean day of emergence, Day 12) than during the 8 days following wave emergence. During the 8 days preceding wave emergence, the mean number of days between successive peak concentrations of FSH associated with individual surges was 4.0 days (range, 3 to 7 days). In a recent study (14), using the same technique to detect individual surges as reported in our study, similar short intervals between FSH surges appeared to occur during the menstrual cycle in monkeys (mean, 4 days; range, 2 to 7 days). Furthermore, based on a sinusoidal regression analysis, periodic surges of FSH occurred at short intervals during the estrous cycle in ewes (4 to 6 days; 18). The interval between peak concentrations of sequential FSH surges appear to be similar among mares, monkeys, and ewes; however, critical studies are needed involving many species to further investigate this phenomenon. It should also be emphasized, that surges reported in our study were detected in plasma samples collected once every 24 hours and therefore may not have detected individual surges that occurred between sampling times. Wave emergence was characterized by a significant mean increase in the diameters of the dominant and largest subordinate follicles of the primary wave as well as the largest follicle of the minor wave when the waves were normalized and grouped for the 12 mares. The temporal relationships between mean FSH concentrations and emergence of primary and minor follicular waves were characterized by a significant increase in FSH concentrations either 6 days (primary wave) or 4 days (minor wave) before wave emergence. The elevated mean FSH concentrations prior to emergence of the primary wave were attributable to both a significant increase in frequency of individual FSH surges and in the magnitude of peak concentrations of FSH associated with individual surges. The extensive variability encompassing the mean daily FSH concentrations is attributable to individual surges occurting at different times within and among mares. It has been reported recently that an elevation in mean FSH concentrations was temporally associated with emergence of follicular waves during the estrous cycle in heifers (5) and with the emergence of both major (4,6) and minor (4) follicular waves during early pregnancy in mares. Results following cautery of the dominant follicle or follicular fluid treatment in heifers indicated that an increase in mean FSH concentrations necessarily preceded the emergence of a wave (5). Mean concentrations of FSH significantly decreased 2 days after emergence of the dominant follicle of the primary wave. In a previous study (12), utilizing the FSH data reported here, mean concentrations of immunoreactive inhibin during the interovulatory
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interval significantly increased between Days 7 and 12, reaching high levels on Day 0, whereas mean FSH concentrations significantly decreased between Days 11 and 14, reaching low levels on Days 18 to 20. Perhaps, the reduced FSH concentrations following emergence of the primary wave was attributable, at least in part, to the suppressive effect of increasing circulating concentrations of inhibin in association with the development of follicles. It is not known to what extent follicles of various size categories in major and minor waves contribute to systemic levels of inhibin. In this regard, administration of a proteinaceous FSH-inhibiting component of equine follicular fluid collected from preovulatory-sized follicles had a greater suppressing effect on systemic FSH concentrations than did follicular fluid collected from smaller follicles (19); however, the smaller follicles presumably were more likely to be undergoing attesia. Divergence between the diameter of the dominant follicle and the largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than the largest subordinate follicle 3 days after wave emergence as well as by cessation of growth of the largest subordinate follicle, as indicated by a plateau beginning 4 days after wave emergence. Selection of the dominant follicle of the primary wave (time of divergence in growth profiles of dominant versus subordinate follicles) occurred in association with decreasing mean FSH concentrations. Similar results have been reported in association with follicular waves in cyclic heifers (5), and major waves in pregnant mares (4,6). It is not known whether the drop in FSH is functionally related to the follicle selection process leading to divergence and whether the mechanism is similar in mares and heifers. It has been reported, however, that exogenous FSH delayed the time of divergence in heifers (5), and an FSH-rich pituitary extract can override divergence and lead to superovulation in mares (3). Further examination of the hormonal and follicular events associated with the time of divergence is needed to determine the role, if any, of decreasing FSH concentrations in the selection mechanism. The present results indicate the occurrence of the following in the estrous cycle of mares: 1) either major waves or major and minor waves in individual esttous cycles, 2) individual FSH surges with a greater degree of frequency and magnitude before emergence of the primary wave than after its emergence, 3) elevated mean daily concentrations of FSH preceding emergence of major and minor waves and an FSH decrease subsequent to emergence of major waves, 4) individual FSH surges at different times accounting for the prolonged overall mean elevation in FSH levels in association with wave emergence, and 5) divergence in diameter of the dominant and largest subordinate follicles of primary waves in association with a decrease in mean daily FSH concentrations. REFERENCES 1.
Ginther, O.J. Folliculogenesis during the transitional period and early ovulatory season in mares. J. Reprod. Fertil. %311-320 (1990).
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2.
Sirois, J., Ball, B.A. and Fortune, J.E. Patterns of growth and regression of ovarian follicles during’ the oestrous cycle and after hemiovariectomy in mares. Equine Vet. J. &(Suppl.):43-48 (1989).
3.
Ginther,, O.J. Reproductive Biology of the Mare: Basic and Applied Aspects Equiservices, Inc., Cross Plains, WI 1992, pp. 173-290.
4.
Ginther, O.J. and Bergfelt, D.R. Associations between FSH surges and major and minor follicular waves in pregnant mares. Theriogenology. In press. (1992).
5.
Adams, G.P., Matteri, R.L., Kastelic, J.P., Ko, J.C.H. and Gin&r, O.J. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J. Reprod. Fertil. 9&177-188 (1992).
6.
Bergfelt, D.R. and Ginther, O.J. Relationships between circulating concentrations of FSH and follicular waves during early pregnancy in mares. J. Equine Vet. Sci. (1992). (In press).
7.
Bergfelt, D.R. and Ginther, O.J. Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare. Theriogenology &99-108 (1985).
8.
Palmer E. New results on follicular growth and ovulation in the mare. &: Follicuhu Growth and Ovulation Rate in Farm Animals. Roche, J.F., and O’Callaghan, D. (eds), Martinus Nijhoff Publishers, Dordrecht_Boston/Lancaster, 1987, pp. 237-255.
9.
Ginther, O.J. Reproductive Biology of the Mare: Basic and Applied Aspects. Equiservices, Inc., Cross Plains, WI, 1979, pp. 169-216.
10.
Ginther, O.J. Ultrasonic Imaging and Reproductive Equiservices Inc., Cross Plains, WI, 1986, pp. 378.
11.
Freedman, L.J., Garcia, M.C. and Ginther, O.J. Influence of photoperiod and ovaries on seasonal reproductive activity in mares. Biol. Reprod. 20567-574 (1979).
12.
Bergfelt, D.R., Mann, B.G., Schwartz, N.B. and Ginther, O.J. Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle in mares. J. Equine Vet. Sci. 11:319-322 (1991).
13.
Clifton, D.K. and Steiner, R.A. Cycle detection: A technique for estimating the frequency and amplitude of episodic fluctuations in blood hormone and substrate concentrations. Endocrinology &z1057-1064 (1983).
Events
in the Mare.
796
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14.
Matter-i, R.L., Bridson, W.E., Dierschke, D.J., Wegner, F.H. and Durning, M. The secretion of bioactive and immunoreactive follicle-stimulating hormone(FSH) and luteinizing hormone (LH) throughout the menstrual cycle of the rhesus monkey. Am. J. Primatol. 26243-257 (1992).
15.
Alexander, S.L., Irvine, C.H.G. and Turner, J.E. Comparison by three different radioimmunoassay systems of the polymorphism of plasma FSH in mares in various reproductive status. J. Reprod. Fertil. 35(Suppl.):9-18 (1987).
16.
Thompson, D.L., McNeill, D.R., Wiest, J.J., St. George, R.L., Jones, L.S. and Garza, F. Secretion of luteinizing hormone and follicle stimulating hormone in intact and ovariectomized mares in summer and winter. J. Anim. Sci. j&247-253 (1987).
17.
Evans, J.W. FSH secretion patterns in cycling mares: Equine Vet. Sci. B:203-207 (1990).
18.
Miller, K.F., Nordheim, E.V. and Ginther, O.J. Periodic flucuations in FSH concentrations during the ovine estrous cycle. Theriogenology j&669-679 (1981).
19.
Miller, K.F., Wesson, J.A. and Ginther, O.J. Changes in concentrations of circulating gonadotropins following administration of equine follicular fluid to ovariectomized mares. Bio. Reprod. 23:867-872 (1979).
Ultradian rhythms.
J.
RELATIONSHIPS BETWEEN FSH SURGES AND FOLLICULAR WAVES DURING THE ESTROUS CYCLE IN MARES D.R. Bergfelt and O.J. Ginther Animal Health and Biomedical Sciences Veterinary Science Building University of Wisconsin-Madison Madison, WI 53706 USA Received for publication: Accepted:
June 16, 1992 September 1, 1992
ABSTRACT Individual follicles 215 mm were monitored daily by ultrasonography in 12 mares during the estrous cycle. Follicular waves were designated as major waves (primary and secondary) and minor waves based on maximum diameter of the largest follicle of a wave (major waves, 34 to 47 mm; minor waves, 18 to 25 mm). Dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the second largest follicle. Dominant follicles of primary waves (n=12) emerged (attained 15 mm) at a mean of Day 12 and resulted in the ovulations associated with estrus (ovulation=Day 0). The dominant follicle of a secondary wave (n=l) emerged on Day 2 and subsequently ovulated in synchrony with the dominant follicle of the primary wave, which emerged on Day 9. The largest follicles of minor waves (n=4) emerged at a mean of Day 5, reached a mean maximum diameter 3 days later, and subsequently regressed. There was a significant increase in mean daily FSH concentrations either 6 days (primary wave) or 4 days (minor waves) before the emergence of a wave. Mean concentrations of FSH decreased significantly 2 days after emergence of the primary wave. Divergence between diameter of the dominant and largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than of the largest subordinate follicle 3 days after wave emergence and by the cessation of growth of the largest subordinate follicle beginning 4 days after the emergence of a wave. Surges of FSH were identified in individual mares by a cycle-detection program; surges occurred every 3 to 7 days. Elevated mean FSH concentrations over the 6 days prior to emergence of the primary wave was attributable to a significantly greater frequency of individual FSH surges before wave emergence than after emergence and to an increase in magnitude of peak concentrations of FSH associated with individual surges. Key words: estrous cycle, follicular waves, FSH surges, mare Acknowledgments Supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and by Equiculture, Inc., Cross Plains, Wisconsin. The authors thank L.J. Kulick for graphics and M. Radtke for manuscript preparation.
Copyright 0 1993 Butterworth-Heinemann
782
Theriogenology
INTRODUCTION Transrectal ultrasonic imaging has been used in mares to monitor day-to-day diameter changes of individual follicles during transition from the anovulatory to ovulatory seasons (1) and during the estrous cycle (1,2). Follicular waves were indicated by the simultaneous emergence and development of several follicles. Two follicular waves were detected in 11 of 25 interovulatory intervals, whereas during the remaining intervals only the wave that gave origin to the ovulatory follicle was detected (3). The largest follicle of each type of wave reached at least 2.5 mm and became dominant, whereas the remaining follicles regressed (subordinates). Waves that give origin to a dominant follicle have been called major waves (3). A major wave that emerges during late estrus or early diestrus has been called a secondary wave; the dominant follicle ovulates during diestrus, regresses, or becomes hemorrhagic. A major wave that emerges during mid-cycle has been called a primary wave; the dominant follicle results in the primary (estrus-associated) ovulation. Detectable waves that do not develop an apparent dominant follicle are called minor waves (4). Recently, associations between elevations in mean daily FSH concentrations and emergence of follicular waves during the interovulatory interval in heifers (5) and during pregnancy in mares (4,6) have been reported. In early pregnant mares, the emergence of major follicular waves (4,6) and minor follicular waves (4) were each preceded by a significant increase in mean FSH concentrations; subsequent to emergence of major waves, a significant decrease in mean FSH concentrations occurred. The importance of FSH to follicular development during the estrous cycle in mares has been demonstrated by diminished FSH and follicular development following treatment with a proteinaceous fraction of equine follicular fluid (7). However, the relationships between circulating concentrations of FSH and follicular dynamics during the cycle have not been adequately established. It has been stated (8), however, but not statistically documented, that maximal FSH concentrations in blood samples collected every other day occurred 2 to 4 days before a follicle reached 20 mm and subsequently declined to minimal concentrations 4 to 6 days later. Furthermore, it has been reported (9) that changes in the number of large follicles occurred most commonly 6 days after corresponding changes in FSH concentrations. The objective of the present study was to characterize the relationships between daily circulating concentrations of FSH and follicular development during the estrous cycles of mares. Consideration was given to individual FSH surges within mares as well as to FSH and follicular changes averaged over all mares. MATERIALS
AND METHODS
Twelve riding-type horse mares, 4 to 16 years of age and weighing 400 to 550 kg, were used from May to August in the study. Although the breeds were not known, they appeared to be primarily Quarter Horse and Appaloosa, based on the size, conformation and color patterns of the mares. The mares were obtained from livestock sale barns; all were nonlactating, and their reproductive histories were not known. The mares were kept
Theriogenology
783
under natural light in outdoor paddocks and had free-choice access to hay, water and trace mineralized salt, and were fed 2 to 4 kg cob corn daily per mare. Ultrasonic examination of the ovaries and uterus was performed as described elsewhere (10) with a real-time, linear-array scanner equipped with a 7.5 MHz intrarectal transducer. Mares were not used if they had ultrasonic indications of apparent ovarian or uterine abnormalities (10). The ovaries were monitored by ultrasound and blood samples were collected once daily (every 24 hours), beginning when diameter of the largest follicle reached 225 mm and continuing until 3 days after the end of an inter-ovulatory interval. An interovulatory interval was defined as the interval between the flit and second ovulation of successive ovulatory periods. If double asychronous ovulations occurred during a given ovulatory period, the first ovulation was used to assign a beginning and end to the interovulatory interval (ovulation=Day 0). Blood samples were drawn by jugular venipuncture into heparinized tubes and immediately stored on ice for transport to the laboratory. After centrifugation, plasma Circulating was collected, and aliquots were made and stored frozen (-20°C). concentrations of FSH (11) were measured in plasma samples using a radioimmunoassay previously validated in this laboratory. The intra- and inter-assay coefficients of variation for the FSH assay were 15 and 17%, respectively. The FSH data set used herein was also used for a different purpose in a previous study to examine the nature of synchronous LH and FSH surges during the estrous cycle (12). The ovaries were monitored daily by ultrasound, and the examinations were recorded with a 3/4-inch (16 mm) videotape recorder attached to the ultrasound scanner. Videotapes were subsequently viewed on a 16-inch (41mm) video monitor. Sequential identification of individual follicles >lS mm was done similarly to that described for early pregnant mares (6). A diagram de&ting relative location and size of all follicles 215 mm was made for each ovary on each day. The diameter of follicles (average of height and width of antrum) was determined using a transparent grid placed over a still image on the video monitor. The relative location of follicles and the corpus luteum were used as reference points to identify individual follicles. Each apparent follicular wave was ranked according to maximum diameter of the largest follicle; the ranking extended from the largest to the smallest diameter. The maximum diameter of the second largest follicle and the difference in maximum diameters between the 2 largest follicles were also listed for each wave. The waves were then divided into 2 categories (major and minor waves) using the point of greatest discontinuity in the rankings from the largest to the smallest follicle diameters. The follicle of a major wave reaching the largest diameter was defined as the dominant follicle, and the second largest follicle of a major wave was defined as the largest subordinate follicle if it emerged within 2 days of the dominant follicle. The day of detected wave emergence was defined as the day the retrospectively identified dominant follicle of a major wave or the largest follicle of a minor wave was 15 to
784
Theriogenology
16 mm in diameter. If the follicle was fust detected when it reached 117 mm, a reported growth rate of 3 mm per day (10) was used to project back to the day of emergence.
A technique that was developed to study episodic fluctuations in blood hormones (13) was used to detect peak concentrations of FSH in individual mares. This procedure was used recently to detect FSH and LH surges in blood samples collected daily during the menstrual cycle in monkeys (14). In brief, the program determined threshold concentrations of FSH based on the variability of the assay results between duplicate samples for each mare within each day. Concentrations that were greater than the threshold values were detected and identified as peak FSH concentrations. An FSH surge in an individual mare was defined by a progressive increase and decrease in concentrations that encompassed a peak concentration (nadir-to-peak-to-nadir). Frequency of occurrence of individual FSH surges, the number of FSH surges per mare, and the magnitude of peak concentrations of FSH associated with individual surges were determined for 8 days before and 8 days after emergence of the primary follicular wave. Data for all primary waves were grouped to characterize the dominant and the largest subordinate follicles of the wave and to examine the temporal relationships between mean daily FSH concentrations and mean days of wave emergence, and between mean FSH concentrations and mean day of divergence in diameter of the dominant and the largest subordinate follicles. Mean day of emergence of the dominant follicle of the primary wave was used to normalize the dominant follicle, largest subordinate follicle, and FSH concentrations to the day scale. The day-to-day associations among the dominant follicle, largest subordinate follicle, and FSH concentrations were maintained; that is, when a value for the dominant follicle for a given day was normalized to conform with the mean, the corresponding value for the largest subordinate follicle and the corresponding FSH value were also normalized. Mean concentrations of FSH were profiled 8 days before and 8 days after wave emergence to encompass the days before wave emergence and’ the day of divergence in diameter of the dominant and largest subordinate follicles. To examine the associations between mean FSH concentrations and mean day of emergence of a minor wave, concentrations were normalized to the first ovulation of the interovulatory interval, since in retrospect, all of the detected minor waves began soon after ovulation. Mean concentrations of FSH were studied from Day -2 to Day 4 so that the days did not extend beyond the mean day of emergence of a minor wave. The mean day of emergence of the largest follicle of a minor wave was used to place the wave on the interovulatory-interval day scale. The mean FSH values were compared between mares with a minor wave versus those with no minor wave. Analyses of variance for sequential data were used to study the day effects for diameter of the dominant and the largest subordinate follicles and FSH concentrations associated with primary waves and diameter of the largest follicle of minor waves. A split-plot analysis of variance for sequential data was used to study the main effect of group, day, and the interaction of group-by-day for FSH concentrations in mares with a minor wave versus those with no minor wave. When a significant (P
785
Theriogenology
group, day, or group-by-day interaction was indicated, Duncan’s multiple range tests were used within each end point to examine differences between days, and unpaired t-tests were used to examine differences between groups within days. Characteristics of major and minor waves were compared using unpaired t-tests for the following end points: 1) the number of follicles, 2) the maximum diameter and day attained for the largest and second largest follicles, and 3) the difference in maximum diameter between the largest and second largest follicles. Paired t-tests were used to examine mean differences in diameter between the dominant and largest subordinate follicles of the primary wave within days. Frequency of occurrence of individual FSH surges, number of FSH surges/mare, and magnitude of peak concentrations of FSH associated with individual surges were studied before and after emergence of the primary wave using a (X-square goodness-of-fit analysis, paired t-test, and unpaired t-test, respectively. RESULTS Follicular waves appeared to fall into 2 populations based on inspection of ranked data. The maximum diameter of the largest follicle was 34 to 47 mm (meanfSEM, 42.2k1.2) for 1 of the wave categories (designated as major waves; n=12) and 18 to 25 mm (21.2f1.4) for the other wave category (designated as minor waves; n=4). The difference in maximum diameter between the 2 largest follicles was 11 to 24 mm (18.0fl.l) for major waves and 1 to 5 mm (3.0f1.3) for minor waves. Characteristics of major and minor waves during the interovulatory interval with results of statistical analyses are shown below (Table 1). Mean (fSEM) length of the interovulatory interval was not different @0.05) between mares with only a primary wave (n=7; 22.7fl.O days) and mares with either primary and secondary waves or primary and minor waves (n=5; 21.4f0.7 days). The day-to-day diameters of all individually identified follicles >15 mm and of circulating concentrations of FSH beginning 3 days before the first ovulation and ending on the day of ovulation at the end of the interovulatory interval are shown for each of 12 mares in Figure 1. Based on inspection of individual follicular profiles, all 12 mares had a primary wave leading to ovulation at the end of the interovulatory interval. In addition, Mare L had a secondary wave and the dominant follicle ovulated in synchrony with the dominant follicle of the subsequent primary wave. Mares J and K also had double ovulations, with both originating from the primary wave. All double ovulations were unilateral and synchronous. Four of the 12 mares (Mares H, I, J and K) had a minor wave preceding the primary wave. Characteristics of individual FSH surges 8 days before and 8 days after emergence of the primary wave in 12 mares with results of statistical analyses are shown in Table 2. The frequency of occurrence of individual FSH surges, the number of surges per mare, and the magnitude of peak concentrations of FSH associated with individual surges were all significantly greater before wave emergence than after emergence. The number of days encompassing peak FSH concentrations were not different (PN.05) before and after wave emergence. The interval between peak concentrations of FSH associated with
Theriogenology
786
surges could not be determined after wave emergence because the 4 surges that were detected occurred in 4 separate mares.
Table 1. Characteristics (meat&EM) of major and minor waves occurring during the interovulatory interval in 12 mares
Wavesa Majorb
Minor
12
4
__
11.8 kO.7
4.8 kO.8
P<0.0001
5.2 f0.8
2.5 M-9
Largest follicle Maximum diameter (mm) Dayd attaining maximum diameter
42.2 f1.2 9.2 f0.4
21.2 f1.4 3.5 50.6
PcO.0001 P
Second largest follicle Maximum diameter (mm) Dayd attaining maximum diameter
24.2 i0.7 4.2 k0.3
18.3 f1.7 2.3 f1.9
P
Difference in maximum diameter between largest and second largest follicle (mm)
18.0 fl.1
3.0 il.3
End points
Number of detected waves Day of emergenceC Number of detected follicles (L15 mm)
Probability
NS
PcO.OOO1
Waves in which the largest attained follicle diameter was 34 to 47 mm or 18 to 25 mm were designated major and minor waves, respectively. Means included data from all primary waves (1 per mare) but did not include data from the secondary wave in Mare L. If more than 1 follicle 34 to 47 mm was detected and subsequently associated with the same wave, it was handled by averaging follicle diameters and days of emergence. Day on which the retrospectively identified largest follicle of a wave was 15 to 16 mm (ovulation=Day 0). Number of days in reference to day of wave emergence. NS=not significant. The mean diameter of the dominant and largest subordinate follicles as well as the associated mean concentrations of FSH grouped and normalized for all primary waves (upper panel), and the number of detected individual FSH surges in relation to wave emergence (lower panel) are shown in Figure 2. Mean follicle diameters did not include data for the day of wave emergence because the number of mares that had corresponding
Theriogenology
Table 2. Characteristics (meat&EM) of individual FSH surges in 12 mares during 8 days before and 8 days after emergence of the primary wavea
Individual
FSH surges
Frequency Number/mare
Before wave emergenceb
After wave emergence
Probability
29
4
P
2.4 +0.2 (range, 1 to 4)
0.3 kO.1 (range, 0 to 1)
P
Magnitude of peak FSH concentrations (rig/ml)
12.8 k2.3 (range, 3 to 36)
4.5 f0.7 (range, 3 to 6)
P
Number of days encompassing peak concentrationsc
4.5 zko.3 (range, 3 to 9)
4.2 ti.6 (range, 3 to 6)
NS
Interval (days) between peak FSH concentrationsd
4.0 f0.3 (range, 3 to 7)
_-
__
a The day the retrospectively identified dominant follicle was 15 to 16 mm. Included individual FSH surges that were detected on the day of wave emergence. Nadir-to-peak-to-nadir constituting a progressive increase and decrease in concentrations. Number of days between successive peak concentrations of FSH associated with individual surges (n=17). NS=not significant.
values for the dominant and largest subordinate follicles was small (n=4). There was a main effect of day (P
Theriogenology
788
40 Mare A
Mare B
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J
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30
20
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Number of days from ovulation Figure 1. Diameter profiles of all follicles 215 mm and individually identified (solid lines) and circulating concentrations of FSH (dashed lines) for each of Mares A to L beginning 3 days before the first ovulation and ending on the day of ovulation terminating the end of the interovulatory interval. The dotted line
Theriogenolog
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789
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Number of days from ovulation at 15 mm indicates that follicles 45 mm were not studied. Stars indicate peak concentrations of FSH associated with individual FSH surges. Thick arrows indicate days of emergence of major waves (primary and secondary) and thin arrows indicate days of emergence of minor waves. Ovulation=OV.
790
Theriogenology 12
1
Primary waves
$, :s::>_
(n=lZ mares)
r”
Dominant follicle
65432lODay of wave emergence I
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2
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4
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6
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Number of days from wave emergence Mean (LSEM) diameter of the dominant and largest subordinate follicles and associated concentrations of FSH grouped for all primary waves (upper panel) and number of detected individual FSH surges in relation to wave emergence (lower panel). Data for the 3 end points in the upper panel were normalized to the mean day of emergence of the dominant follicle (Day 12). There was a main effect of day (P
Theriogenology
791
The mean diameter of the largest follicle of minor waves and mean concentrations of FSH for mares with a minor wave and with no minor wave as well as the results of statistical analyses are shown in Figure 3. The mean day of emergence of the minor wave occurred 4.8 days after ovulation (TabIe 1); Day 5 was used to place the mean follicle profile of the wave on the day scale. The diameter of the largest follicle increased (PcO.05) between Days 5 and 7 and reached a maximum diameter (mean, 21.2 mm; Table 1) on Day 8. Concentrations of FSH in mares with a minor wave increased (PcO.05) between 2 days before and 1 day after ovulation; concentrations were higher (PcO.05) on Days 1 to 3 in mares with a minor wave than in mares with no minor wave. Subsequent to Day 3, concentrations of FSH in mares with no minor wave increased (PcO.05) and were not different from concentrations in mares with a minor wave.
- 25
Largest follicle of 7-
G: Pco.02 D: P
D: PcO.0008
.
5-
L
......................................_...__.........................._.___.__.......___ _.15
-2
-1
0
1
2
3
4
5
6
7
6
9
10
11
Number of days from ovulation Figure 3. Mean (&SEM) concentrations of FSH normalized to the first ovulation of the interovulatory interval in mares with and without minor waves and diameter of the largest follicle of minor waves. Mean day of emergence of the minor wave was used to place the mean follicle profile of the wave on the day scale. Results of statistical analyses are indicated: 1) main effect of group (G); 2) main effect of day (D); 3) interaction of group-by-day (GD). The first significant increase in mean FSH concentrations and in diameter of the largest follicle in mares with a minor wave and days in which concentrations were signifkantly different between mares with and without a minor wave are indicated by a star. The dotted line at 15 mm indicates that follicles cl5 mm were not studied.
Theriogenology
792
DISCUSSION Major and minor follicular waves occurred during the estrous cycle in mares as determined by monitoring diameter of individual follicles 115 mm. It was reported previously that 1 or 2 follicular waves occurred during the first estrous cycle following transition from the anovulatory to ovulatory seasons (1) and during the estrous cycle of mares that had already entered the ovulatory season (2); however, no attempts were made to group the waves into major and minor categories. In the present study, the day of emergence of the dominant follicle of the primary wave was not altered in regard to whether or not a secondary wave, or a minor wave, preceded it. The dominant follicle of the secondary wave in Mare L emerged on Day 2 and the dominant follicle of the primary wave emerged on Day 9; subsequent to Day 9, the 2 follicles developed concurrently, and a synchronous, double ovulation occurred involving the dominant follicle of each wave. That is, a set of double ovulations involved the primary wave only (Mares J and K) while the other set involved both a secondary wave and a primary wave (Mare L). That is, a double ovulation may not always involve 2 dominant follicles of the primary wave, but may include the dominant follicle of a secondary wave. In previous studies (1,2), the dominant follicles of secondary waves either ovulated during die&us, regressed, or became hemorrhagic. Differences in characteristics between major and minor waves in the present study were as follows: 1) the largest follicle of major waves reached a maximum diameter later after wave emergence than did the largest follicle of minor waves (means, 9 days and 42 mm versus 4 days and 21 mm) and 2) dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the 2nd largest follicle, whereas the 2 largest follicles of minor waves differed by a mean of only 3 mm. These differences appear similar to reported (4) differences between major and minor waves during early pregnancy in mares. Apart from the follicles associated with major and minor waves, it appeared from the graphs of individual follicles that other minor waves emerged, especially in Mare K. By design, follicles of a major wave were defined as those originating within f2 days of emergence of the dominant follicle. However, other follicles emerged 3 to 4 days before (mean, 3 days) and 3 to 7 days after (mean, 4 days) emergence of the dominant follicle of the primary waves. Characterizing follicles as part of a minor wave or as subordinates of a major wave seemed dependent upon an arbitrary definition. That is, in some interovulatory intervals, the dominant follicle emerged from a broad base of detected follicular activity and some of the follicles appeared to regress (minor wave) before emergence of follicles that seemed more closely associated with the primary wave. The statement that follicles of a wave emerge in synchrony therefore must be tempered with the concept that many days may be involved in follicle emergence. Furthermore, since only follicles 115 mm were studied, it is not known whether or not there were other patterns or types of waves for follicles ~15 mm. The duration of individual surges of FSH that were detected before and after emergence of the primary wave encompassed a mean of 4.5 days. That is, in relation to peak concentrations of FSH associated with an individual surge, the number of days from nadir-to-peak-to-nadir constituted, on the average, more than 3 days. Pulses of FSH have
Theriogenology
793
been detected in blood samples collected frequently within 24 hours on discrete days of the estrous cycle in mares (15-17). In one study (17), the interval between pulses was significantly shorter during the mid- to late-lute& phase (5 pulses/hour) than during estrus (3 pulses/hour). Individual surges (as defined here) occurred over several days, whereas pulses (as previously described; 15-17) occurred frequently within a day. Therefore, it is likely that once-a-day samples would involve a portion of a pulse in some mares on some days. Presumably, pulses of FSH, or a portion of them, were superimposed on the individual surges of FSH described here, but this has not been studied. The frequency of occurrence of individual FSH surges was significantly greater during the 8 days preceding emergence of the primary wave (mean day of emergence, Day 12) than during the 8 days following wave emergence. During the 8 days preceding wave emergence, the mean number of days between successive peak concentrations of FSH associated with individual surges was 4.0 days (range, 3 to 7 days). In a recent study (14), using the same technique to detect individual surges as reported in our study, similar short intervals between FSH surges appeared to occur during the menstrual cycle in monkeys (mean, 4 days; range, 2 to 7 days). Furthermore, based on a sinusoidal regression analysis, periodic surges of FSH occurred at short intervals during the estrous cycle in ewes (4 to 6 days; 18). The interval between peak concentrations of sequential FSH surges appear to be similar among mares, monkeys, and ewes; however, critical studies are needed involving many species to further investigate this phenomenon. It should also be emphasized, that surges reported in our study were detected in plasma samples collected once every 24 hours and therefore may not have detected individual surges that occurred between sampling times. Wave emergence was characterized by a significant mean increase in the diameters of the dominant and largest subordinate follicles of the primary wave as well as the largest follicle of the minor wave when the waves were normalized and grouped for the 12 mares. The temporal relationships between mean FSH concentrations and emergence of primary and minor follicular waves were characterized by a significant increase in FSH concentrations either 6 days (primary wave) or 4 days (minor wave) before wave emergence. The elevated mean FSH concentrations prior to emergence of the primary wave were attributable to both a significant increase in frequency of individual FSH surges and in the magnitude of peak concentrations of FSH associated with individual surges. The extensive variability encompassing the mean daily FSH concentrations is attributable to individual surges occurting at different times within and among mares. It has been reported recently that an elevation in mean FSH concentrations was temporally associated with emergence of follicular waves during the estrous cycle in heifers (5) and with the emergence of both major (4,6) and minor (4) follicular waves during early pregnancy in mares. Results following cautery of the dominant follicle or follicular fluid treatment in heifers indicated that an increase in mean FSH concentrations necessarily preceded the emergence of a wave (5). Mean concentrations of FSH significantly decreased 2 days after emergence of the dominant follicle of the primary wave. In a previous study (12), utilizing the FSH data reported here, mean concentrations of immunoreactive inhibin during the interovulatory
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interval significantly increased between Days 7 and 12, reaching high levels on Day 0, whereas mean FSH concentrations significantly decreased between Days 11 and 14, reaching low levels on Days 18 to 20. Perhaps, the reduced FSH concentrations following emergence of the primary wave was attributable, at least in part, to the suppressive effect of increasing circulating concentrations of inhibin in association with the development of follicles. It is not known to what extent follicles of various size categories in major and minor waves contribute to systemic levels of inhibin. In this regard, administration of a proteinaceous FSH-inhibiting component of equine follicular fluid collected from preovulatory-sized follicles had a greater suppressing effect on systemic FSH concentrations than did follicular fluid collected from smaller follicles (19); however, the smaller follicles presumably were more likely to be undergoing attesia. Divergence between the diameter of the dominant follicle and the largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than the largest subordinate follicle 3 days after wave emergence as well as by cessation of growth of the largest subordinate follicle, as indicated by a plateau beginning 4 days after wave emergence. Selection of the dominant follicle of the primary wave (time of divergence in growth profiles of dominant versus subordinate follicles) occurred in association with decreasing mean FSH concentrations. Similar results have been reported in association with follicular waves in cyclic heifers (5), and major waves in pregnant mares (4,6). It is not known whether the drop in FSH is functionally related to the follicle selection process leading to divergence and whether the mechanism is similar in mares and heifers. It has been reported, however, that exogenous FSH delayed the time of divergence in heifers (5), and an FSH-rich pituitary extract can override divergence and lead to superovulation in mares (3). Further examination of the hormonal and follicular events associated with the time of divergence is needed to determine the role, if any, of decreasing FSH concentrations in the selection mechanism. The present results indicate the occurrence of the following in the estrous cycle of mares: 1) either major waves or major and minor waves in individual esttous cycles, 2) individual FSH surges with a greater degree of frequency and magnitude before emergence of the primary wave than after its emergence, 3) elevated mean daily concentrations of FSH preceding emergence of major and minor waves and an FSH decrease subsequent to emergence of major waves, 4) individual FSH surges at different times accounting for the prolonged overall mean elevation in FSH levels in association with wave emergence, and 5) divergence in diameter of the dominant and largest subordinate follicles of primary waves in association with a decrease in mean daily FSH concentrations. REFERENCES 1.
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Adams, G.P., Matteri, R.L., Kastelic, J.P., Ko, J.C.H. and Gin&r, O.J. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J. Reprod. Fertil. 9&177-188 (1992).
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Palmer E. New results on follicular growth and ovulation in the mare. &: Follicuhu Growth and Ovulation Rate in Farm Animals. Roche, J.F., and O’Callaghan, D. (eds), Martinus Nijhoff Publishers, Dordrecht_Boston/Lancaster, 1987, pp. 237-255.
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Clifton, D.K. and Steiner, R.A. Cycle detection: A technique for estimating the frequency and amplitude of episodic fluctuations in blood hormone and substrate concentrations. Endocrinology &z1057-1064 (1983).
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Matter-i, R.L., Bridson, W.E., Dierschke, D.J., Wegner, F.H. and Durning, M. The secretion of bioactive and immunoreactive follicle-stimulating hormone(FSH) and luteinizing hormone (LH) throughout the menstrual cycle of the rhesus monkey. Am. J. Primatol. 26243-257 (1992).
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Alexander, S.L., Irvine, C.H.G. and Turner, J.E. Comparison by three different radioimmunoassay systems of the polymorphism of plasma FSH in mares in various reproductive status. J. Reprod. Fertil. 35(Suppl.):9-18 (1987).
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Thompson, D.L., McNeill, D.R., Wiest, J.J., St. George, R.L., Jones, L.S. and Garza, F. Secretion of luteinizing hormone and follicle stimulating hormone in intact and ovariectomized mares in summer and winter. J. Anim. Sci. j&247-253 (1987).
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Evans, J.W. FSH secretion patterns in cycling mares: Equine Vet. Sci. B:203-207 (1990).
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Miller, K.F., Nordheim, E.V. and Ginther, O.J. Periodic flucuations in FSH concentrations during the ovine estrous cycle. Theriogenology j&669-679 (1981).
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Miller, K.F., Wesson, J.A. and Ginther, O.J. Changes in concentrations of circulating gonadotropins following administration of equine follicular fluid to ovariectomized mares. Bio. Reprod. 23:867-872 (1979).
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