Miniature ponies: 1. Follicular, luteal and endometrial dynamics during the oestrous cycle

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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 8,, Miniature ponies: 1. Follicular, luteal and endometrial dynamics during the oestrous cycle E. L. Gastal A,D, A. P. Neves B, R. C. Mattos B, B. P. L. Petrucci B, M. O. Gastal C and O. J. Ginther A,C A Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 376, USA. B Department of Animal Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. C Eutheria Foundation, Cross Plains, WI 328, USA. D Corresponding author. egastal@svm.vetmed.wisc.edu Abstract. Follicular dynamics were studied during 12 interovulatory intervals (IOIs) and 36 preovulatory periods in Miniature mares. The percentage of IOIs with the following follicle events was: ovulatory wave with only one follicle 1 mm (%), diameter deviation similar to previous reports in larger mares (2%) and minor waves emerging before or after the ovulatory wave (%). Follicle data were compared among Miniature ponies, large ponies and Breton horses (n = 12 IOIs per breed). The IOI was longer (P <.1) in Miniature ponies (23.3 ±.9 days) and in large ponies (23.9 ±. days) than in Breton horses (.3 ±.7 days). The Miniature ponies had fewer (P <.1) growing follicles 1 mm per ovulatory wave (1. ±.3) and more (P <.4) ovulatory waves (6/11) with only one follicle 1 mm than large ponies (9.8 ±.8 and /12) and horses (.8 ±.9 and /12). Maximum diameter of the preovulatory follicle was smaller (P <.3) in the Miniature ponies (38.3 ±.7 mm) than in the horses (44. ± 1.4 mm), but the difference between breeds was slight (6%) compared with the difference in bodyweight (6%). Considering the small number of follicles per ovulatory wave, Miniature mares are a potential model for comparative studies in folliculogenesis within and among species. Additional keywords: corpus luteum, endometrial echotexture, follicles, ultrasound. Introduction In spite of numerical, economical and social importance (Frankeny 3), knowledge of the reproductive patterns of Miniature equids remains scarce and fragmentary. The few scientific reproductive studies for these breeds have been done in stallions (Metcalf et al. 1997; Paccamonti et al. 1999; Neves et al. ). Therefore, information regarding reproduction in Miniature mares has been extrapolated from studies of the larger conventional breeds. Ultrasonographic images of uterine, ovarian, follicular and luteal structures in Miniature mares (Campbell 1992) and Miniature jennies (Purdy ) seem comparable to those of larger mares (Ginther 199b). Although there are a few clinical reports (Judd 1994; Tibary 4), no critical study on follicular dynamics and the associated luteal and endometrial changes in Miniature mares was found in the literature. Studies in this area would have fundamental and practical importance for formulating and testing hypotheses and for diagnosing and prognosing reproductive events in Miniature mares. Follicular wave refers to several follicles that emerge and initially grow in synchrony. Various numbers and types of follicular waves develop during an equine interovulatory interval (IOI; Ginther 199b). In a major wave, the largest follicle attains the diameter of a dominant follicle ( 28 mm). In minor waves, the largest follicle does not become dominant. A major anovulatory wave sometimes precedes the wave that originates the ovulation associated with oestrus or the follicular phase. The ovulatory wave in mares emerges midway during an IOI of days. On average, the future dominant follicle emerges earlier than the future largest-subordinate follicle in ponies (Gastal et al. 1997, 4a), horses (Ginther et al. 4b), cattle (Ginther et al. 1997) and women (Ginther et al. 4b). After emergence, the follicles of a wave develop in a common-growth phase for several days (Gastal et al. 4a).At the end of the common-growth phase, a distinctive change in growth rates begins, wherein the developing dominant follicle maintains a constant growth rate, and the remaining follicles (subordinate follicles) grow at a reduced rate and regress. This process is called deviation and in mares begins when the diameter of the two largest follicles on average are 22. mm and 19. mm (Gastal et al. 1997, 1999; Ginther et al. 4a). Preovulatory follicles in horses and large ponies generally reach 4 4 mm the day before ovulation (Ginther 199b; Ginther et al. 4a). Limited data suggest that preovulatory follicles are mm smaller in Miniature mares and 1 mm larger in Clydesdales than in Quarter Horses and large ponies (Ginther 199b). These and other findings (reviewed in Ginther 199b) indicate that differences in follicle diameter among breeds and types of mares are relatively small, despite great differences in CSIRO /RD /8/376

2 Ovarian and uterine dynamics in Miniature ponies Reproduction, Fertility and Development 377 body size. Knowledge on the diameter of the preovulatory follicle in Miniature mares would be useful in comparison of breeds and types, considering the extremely small body size. In Miniature stallions, the size of the accessory glands, excluding the vesicular glands (Pozor and McDonnell 2), and testes and sperm output have been reported to be smaller than in large breeds (Metcalf et al. 1997; Paccamonti et al. 1999; Neves et al. ); however, measurements of the male reproductive organs relative to differences in body size among breeds have not been reported. In Miniature mares, studies are needed on number and size of follicles, corpus luteum size and endometrial echotexture during the oestrous cycle. The purposes of the present study in Miniature ponies using ultrasonography were: (1) to study follicle dynamics during an interovulatory interval with emphasis on the ovulatory follicle during the preovulatory period; (2) to characterise the changes in endometrial echotexture score and corpus luteum size; and (3)to compare number of follicles per ovulatory wave and diameter and growth rate of the ovulatory follicle among Miniature ponies, large ponies and Breton horses. Materials and methods Experiment 1. Follicles, corpus luteum and endometrial echotexture Animals All procedures were approved by the Research Ethics Committee of the Federal University of Rio Grande do Sul, Brazil (approval number 27). Twelve interovulatory intervals (IOIs) in nine non-lactating Miniature pony mares, aged 4 to 12 years (mean ± s.e.m., 7.3 ±.9 years), weighing 11 to 136 kg (124.3 ± 3. kg), and measuring 8 to 1 cm at the withers were studied. The study was done in Brazil where the animals are classified as the Brazilian pony breed, which originates from breedings of Shetland or Miniature ponies with Fallabella Miniature Horses and more recently with American Miniature Horses (Bergmann et al. 1997). Age was based on dental characteristics (American Association of Equine Practitioners 2). Data were collected from January to March during two ovulatory seasons in the southern hemisphere (latitude S). Daylength in January is equivalent to daylength in July in the northern hemisphere. Mares were kept under natural daylight in pastures with free access to water and trace-mineralised salt. Feed consisted of pasture and supplementation with commercial ration and alfalfa or grass hay. Mares were selected with docile temperament and with no apparent abnormalities of the reproductive tract as determined by transrectal ultrasound examination (Ginther 199b). Ultrasonography An experienced operator with small hands and forearms performed the transrectal examinations with the mare on an elevated platform.the majority of the mares had been used in the previous season for artificial insemination and embryo transfer programs (Neves et al. 4) and were accustomed to handling and transrectal procedures. During the daily ultrasound examinations, which lasted 1 min per mare, no sedative, rectal relaxant, or caudal epidural anaesthesia was used. Rectal lubricant was used liberally and rectal irritation was not observed in any mare during the experiments. The mares maintained good body condition throughout the study (scores of 6 to 8 on a scale of 1 = thin to 9 = obese; Henneke et al. 1983). An ultrasound B-mode instrument (Aloka SSD-V; Aloka, Wallingford, CT, USA) equipped with a MHz linear-array transrectal transducer was used for evaluation of the ovaries and uterus (Ginther 199a, 199b; Gastal et al. 1998). The mares were scanned by ultrasound daily to determine the day of ovulation by disappearance of the preovulatory follicle and confirmed by subsequent detection of a corpus luteum (CL). The experimental period began 1 days after a pre-experimental ovulation and continued throughout the IOI until days after the second ovulation (ovulation = Day ). The beginning of the experiment was truncated, retrospectively, so that daily observations extended from 6 days before an ovulation until days after the next ovulation. Sequential data were analysed during one IOI and during both preovulatory periods (Days 6 to 1). End points Diameters of follicles from both ovaries were obtained from the average of height and width of the antrum using the apparent maximal area from each of two frozen images. The total number of follicles 2 mm was counted and the follicles were grouped into five classes: 2 1 mm, mm, 1.1 mm,.1 2 mm, and >2 mm. In addition, the follicle classes and the total number of follicles 2 mm were considered for the left and right ovaries. On each day, the four largest follicles for each mare were ranked from F1 (largest) to F4, without consideration of day-to-day identity. Follicles F1 to F4 and the five diameter classes were used to represent follicle activity throughout the experimental period. The mean length of the IOI (23.3 ±.9 days) rounded to 23 days was utilised to normalise the data to a common day scale. For this purpose, daily continuity was maintained from Day 6 to Day 4 relative to the first ovulation, followed by a break, and then daily from Day 18 to Day relative to the second ovulation. The day of the break in continuity was chosen to minimise disrupting expected events (e.g. day of deviation) and to allow comparisons with a previous study in larger (> kg) ponies (Ginther et al. 6). Follicle identity was also used to determine the day of emergence and history of each follicle throughout each IOI and during deviation of follicles in the ovulatory wave. The identity method involved day-to-day tracking of each follicle retrospectively from sketches of ovaries showing diameter and location of identified follicles, as described (Ginther 199b; Gastal et al. 1997). Follicles with maintenance of individual identity from day-to-day were displayed for each of the 12 IOIs from Day at the beginning of the interval to days after the end of the interval. The day of emergence of the future ovulatory follicle was defined as the day it reached 1 mm. The diameter of 1 mm was used to minimise the likelihood of error in follicle identification. If not found until >1 mm, the day of emergence was estimated by retroprojection of the mean of the first three consecutive growth rates; this was necessary in two ovulatory waves. Follicles that emerged with no more than 1 day elapsed between them were considered to be part of the same wave. When 2days elapsed and a follicle reached >1 mm, the follicles were considered to be part of different waves (Gastal et al. 1999, ).

3 378 Reproduction, Fertility and Development E. L. Gastal et al. The term follicular wave was used even when only one follicle was detected. The day of deviation in individual IOIs was detected by retrospective study of the diameter changes. The beginning of deviation was designated as the day before the day with an apparent change in differences in diameter between the two largest follicles, as described (Gastal et al. 1997). Deviation was assigned to the follicular wave only when the second-largest follicle was apparently from the same wave and reached 18 mm. Deviations that began when the largest follicle was 19 to 26 mm (mean, 22. mm) were defined as being similar to those reported for larger breeds (Ginther et al. 4a). The number of major and minor anovulatory waves per IOI was evaluated in reference to the day of emergence of the ovulatory wave. A major anovulatory wave was identified by the presence of a dominant follicle that reached 28 mm and regressed. A minor wave did not contain a dominant follicle. These definitions of waves and dominance have been used previously for mares (Ginther 1993; Ginther et al. 4b). The diameter of the CL was obtained from the average of height and width of the apparent maximal area from each of two frozen images. The area (cm 2 ) of a cross section of the CL for each examination was determined from the maximal area averaged from two still images, using the scanner s tracing function. Echotexture of the endometrium (Ginther and Pierson 1984) was evaluated daily and scored from 1 to 4 (minimal to maximal oedema of the endometrial folds); scores of 3 and 4 were considered representative of oestrus (Gastal et al. 1998). Experiment 2. Ovulatory follicle One year after the end of Experiment 1 the same mares plus four additional mares (n = 13) were monitored daily by ultrasound during 1 IOIs from January to March for follicle development and ovulation. Experiment 2 was carried out to obtain more data regarding the ovulatory follicle and to confirm findings from Experiment 1 performed in a different ovulatory season. Diameter of the ovulatory follicle was obtained daily from Days 6 to in 1 preovulatory periods. Follicle diameter was measured similarly as in Experiment 1. End points evaluated were: interval from maximum diameter of the ovulatory follicle to ovulation, diameter of the ovulatory follicle at maximum and on Day 1, and growth rates of the ovulatory follicle from Days 6 to 3 and Days 3to 1. Preliminary comparisons among breeds After the experiments were performed, a preliminary comparison was made among 12 IOIs in the Miniature ponies (Experiment 1), 12 large ponies and 12 Breton horses. The end points evaluated were number of follicles 1 mm associated with the ovulatory wave, diameter and growth rate of the ovulatory follicle, and length of the IOI. The large ponies (Gastal et al. 4a) and Breton horses (Ginther et al. 4b) were randomly selected from the control mares used in reported projects in which follicles were identified from day-to-day; data were obtained from the records and have not been reported previously. The number of follicles refers to growing follicles that reached 1 mm during the days that the future ovulatory follicle was between 1 and mm (common-growth phase). Diameter of the preovulatory follicle was obtained at maximum and on Day 1. Growth rates of the ovulatory follicle were obtained for Days 6 to 3 and Days 3 to 1. To consider the relative differences between breeds in mean maximum diameter of the ovulatory follicle v. median bodyweight, the percentage differences between breeds were compared between the two characteristics as follows: breed A (largest value) minus breed B divided by breed A 1. Statistical analyses Sequential data were tested for normality according to Kolmogorov-Smirnov tests. When the normality test was significant (P <.), data were transformed by either natural logarithms or a ranking procedure. One-way ANA was used to study the effect of day. Sequential data for F1 to F4, follicle classes and ovulatory follicle were analysed by the SAS MIXED procedure to determine the main effects (group and day) and the interaction, using a repeated statement to account for the autocorrelation between measurements (version 8.2; SAS Institute Inc., Cary, NC, USA). Duncan s multiple range tests were used for comparisons among days when a significant day effect was obtained. Comparisons among breeds were made by one-way ANA and Duncan s multiple range tests. Paired or unpaired t-tests were utilised to locate differences for single end points within or between Experiments 1 and 2. Chi-square analysis was used to examine differences in frequency data. A probability of P. indicated that a difference was significant. A probability between P>. and P.1 indicated that significance was approached and is defined as a tendency. Data are presented as the mean ± s.e.m., unless otherwise indicated. Results Experiment 1. Follicles, corpus luteum and endometrial echotexture The mean diameters of the four largest follicles without day-today identity are shown from Day 6 before the first ovulation to Day after the second ovulation for the 12 interovulatory intervals (IOIs) in Fig. 1. Main effects of follicle, day and an interaction were found during the IOI. Significant effects of day were also found for each of F1, F2, F3 and F4. The mean numbers of follicles in the various follicle classes are depicted in Fig. 2. There were significant effects of follicle class, day and an interaction. Significant main effects of day were observed for each class (P <.1 to P<.1). The beginnings of the first increase and first decrease are shown for every end point during the normalised IOI (23 days). More.1 2 mm and >2 mm follicles per day were in the right than the left ovary (day effects, P<.4 and P<.7, respectively). There were no other effects of the side of the body or day-by-side interactions for any of the other follicle classes. A day effect was found for the score of endometrial echotexture and for diameter and area of the CL (Fig. 2). The first significant decreases (P <.3) in CL diameter and area occurred between Day 2 and Days 6 and, respectively. The first significant increase (P <.1) for the mean endometrial echotexture score occurred between Days 13 and 9 (corresponds to 1 to 14 days after ovulation). Maximal mean

4 Ovarian and uterine dynamics in Miniature ponies Reproduction, Fertility and Development Diameter (mm) Days from an ovulation Equivalent days after first ovulation Fig. 1. Mean (± s.e.m.) diameter of the four largest follicles for 12 IOIs normalised to a common day scale, using the mean length of the interovulatory interval (IOI, 23 days). Daily continuity of the data was maintained from Days 6 to 4 relative to the first ovulation, followed by a break and then daily from Days 18 to relative to the second ovulation. Diameter rankings (F1 to F4) are for each day without regard to day-to-day identity. Main effects (P <.1) of follicle, day, and an interaction and significant effects (P <.2) of day for each follicle were found., ovulation. endometrial score occurred on Day 4, followed by a decrease (P <.1) by Day 2. Based upon inspection of the individual follicle profiles with day-to-day identity (Fig. 3), the following situations were detected for the 12 IOIs: ovulatory wave with only one identified growing follicle 1 mm (n = 6), deviation similar to previous reports in mares with the second largest follicle 18 mm (n = 3), apparent deviation involving a small (<16 mm) secondlargest follicle and with only one growing follicle thereafter (n = 1), deviation not identified owing to unknown origin of the second-largest follicle (n = 1), minor wave preceding the major or ovulatory wave (n = 1), minor wave emerging after the ovulatory wave (n = 4), and minor waves emerging before and after the ovulatory wave (n = 1).The individual mares with these variations are identified in the legend for Fig. 3. In most IOIs (1/12, 83%), two to four apparently-growing follicles did not grow beyond 1 mm. Mean length of intervals, diameters and growth rates of the future ovulatory follicle, follicle populations and number of minor follicular waves during the IOI are shown in Table 1. The maximum diameter of the ovulatory follicle tended to be larger (P <.1) than the diameter on Day 1 (day before ovulation). The growth rates from emergence of the future ovulatory follicle to maximum diameter were greater (P <.1) than the rates from maximum diameter to ovulation. No differences in the ovulatory follicle between the first and second preovulatory periods were observed for the interval from maximum diameter to ovulation, for diameters at maximum and on Day 1, and for growth rates from Days 6to 3 and Days 3to 1. The number of follicles per day within follicle classes, the total number of follicles and the frequency of minor waves during the IOI are shown in Table 1. The number of follicles per day within IOIs and the mean total number of follicles per IOI ranged from 4 to 14 and 4.7 to 8. follicles, respectively. All follicles were identified and tracked; that is, the number of follicles was the same for both the identity and non-identity methods. Experiment 2. Ovulatory follicle The maximum diameter of the ovulatory follicle tended to be larger (P <.1) than the diameter on Day 1 (Table 1). The growth rate of the ovulatory follicle between Days 6 and 3 was greater (P <.4) than between Days 3 and 1. Comparisons between Experiments 1 and 2 No differences were found between experiments regarding the ovulatory follicle for the following five end points: interval from maximum diameter to ovulation (overall, 1.7 ±.1 day); diameter at maximum (37.3 ±. mm) and on Day 1 (3.9 ±.6 mm); and growth rates from maximum diameter to ovulation ( 1. ±.4 mm day 1 ), between Days 6 and 3 (2.7 ±.2 mm day 1 ), and Days 3 and 1 (1.2 ±.3 mm day 1 ) (Table 1). The mean diameter of the ovulatory follicle at maximum was greater (P <.4) than on Day 1 when data from both experiments were combined. In 17/36 (47%) preovulatory periods the ovulatory follicle did not increase in diameter during Days 2 to 1. The mean growth rate from Days 6to 3 was also greater (P <.1) than from Days 3 to 1 for both experiments combined. The growth profile of the ovulatory follicle from Days 6 to 1 did not differ (P >.) between Experiments 1 and 2 and between left and

5 38 Reproduction, Fertility and Development E. L. Gastal et al. (a) Total number of follicles (b) 6 Number of follicles within each follicle class mm mm 1.1 mm.1 2 mm 2.1 mm (c) 4 28 Endometrial echotexture score (1 4) CL area (cm 2 ) CL diameter (mm) Days from ovulations Equivalent days after ovulation Fig. 2. (a) Mean (± s.e.m.) total number of follicles; (b) number of follicles in various diameter classes; and (c) score for endometrial echotexture ( ), corpus luteum (CL) diameter ( ) and area ( ), normalised to a common day scale, using the mean length of the interovulatory intervals (IOI, 23 days; n = 12), except for the CL end points. Main effects (P <.1) of follicle class and day, and an interaction for the five follicle classes and a significant main effect (P <.1) of day within each class were found. A day effect (P <.1) was found for the score of endometrial echotexture and for diameter and area of the CL. A circle encompassing a mean indicates the beginning of an increase or decrease (P <.) in each end point. right ovaries for both experiments combined (data not shown). The frequency of an ovulatory follicle and future CL in the right ovary (61%, 22/36) tended (P <.6) to be greater than in the left ovary (39%, 14/36) when data from all preovulatory periods were combined for both experiments. Preliminary comparisons among breeds The comparisons among the Miniature ponies, large ponies and Breton horses are shown in Table 2. The Miniature ponies and the large ponies had a longer IOI than did the Breton horses. The Miniature ponies had fewer growing follicles 1 mm per

6 Ovarian and uterine dynamics in Miniature ponies Reproduction, Fertility and Development 381 Diameter (mm) Mare 1 Mare 2A Mare 2B 1F D i mw E mw E Mare 3 Mare 4 Mare 1F D 1F h E mw E mw E Mare 6A Mare 6B Mare 7 D h d i E E mw E Mare 8 Mare 9A Mare 9B 1F 1F 1F E E mw mw E Days after ovulation Fig. 3. Experiment 1. Profiles of individual identified follicles for 12 IOIs from nine Miniature mares starting at the day of the first ovulation and ending days after the second ovulation. An A and a B refers to consecutive intervals in the same mare. Follicles that were decreasing in diameter at the first ovulation were omitted to reduce the complexity. A circle around a value indicates a follicle that was identified as decreasing in diameter and then increasing; diameters were considered only from the day of the beginning of the increase to minimise identity errors (Mares 1, 2B, 6A, 9A). d, apparent deviation, but at small follicle diameters (Mare 6B); D, deviation that seems similar to previous reports for larger breeds (Mares 2A, 3, 6A); E, emergence of the ovulatory follicle at 1 mm, excluding Mare 2B because of possible follicle identity errors between the ovulatory follicle and a possible minor wave preceding the ovulatory wave; mw, minor wave preceding the major or ovulatory wave by 2 days (Mares 1, 9B) or occurring after the ovulatory wave (Mares 1, 3, 4, 6B, 9A); i, follicle with uncertain identity preceding apparent emergence of the ovulatory follicle (Mares 2A, 6A); h, follicles of unknown origin and uncertain history (Mares, 7); 1F, only one identified follicle in ovulatory wave (Mares 1, 4,, 8, 9A, 9B);, ovulation.

7 382 Reproduction, Fertility and Development E. L. Gastal et al. Table 1. Mean (± s.e.m.) length of intervals, diameters and growth rates of the future ovulatory follicle, follicle populations and frequency of minor waves during the interovulatory interval (IOI) in Miniature ponies End points Experiment 1 Experiment 2 No. IOIs 12 1 Intervals (days) from: Ovulation to ovulation 23.3 ±.9 Ovulation to emergence at 1 mm 12.3 ± 1. Emergence to ovulation 11.2 ±.7 Ovulation to maximum diameter 21. ±.8 Maximum diameter to ovulation 1.6 ±.2 (19) A 1.8 ±.2 Emergence to maximum diameter 9. ±.7 Ovulation to last detection of corpus luteum 14.3 ±.6 Diameter of ovulatory follicle (mm): At emergence 11.6 ±.4 At maximum 37.7 ±.7 (19) 36.8 ±.8 On Day ± 1. (21) B 3.3 ±.9 B Growth rates (mm day 1 ) from: Emergence to maximum diameter 3. ±.3 a Maximum diameter to ovulation 1.2 ±.8 b 2. ±. Days 6 to 3 2. ±.4 (17) x 2.9 ±.4 x Days 3 to ±.4 (21) y 1.1 ±.4 y No. follicles per day during the IOI: 2 1 mm 4.9 ± mm.6 ± mm.3 ± mm.2 ±. >2 mm.2 ±. Total 6.3 ±.3 No. IOI with minor follicular waves C 6/11 (%) A Number in parenthesis refers to preovulatory periods; data for 9 preovulatory periods were obtained before the first ovulation of Experiment 1 and the remainder before the second ovulation. B Mean on Day 1 tends to be different (P <.1) from the mean at maximum for each experiment. C Waves that did not develop a dominant follicle. a,b,x,y Means with different superscript letters within columns are different (P <.2 to.1). Means within rows are not different (P >.). Table 2. Mean (± s.e.m.) number of follicles per ovulatory wave and diameter and growth rate of the ovulatory follicle in Miniature ponies, large ponies and horse mares n = 12 mares per group, except for 11 Miniature mares for number of waves with one follicle End points Miniature ponies Large ponies Breton horses Probability Bodyweight (kg, range) 11 to 136 to 4 4 to Age (years, range) 4 to 12 4 to 13 6 to 13 Interovulatory interval (days) 23.3 ±.9 a 23.9 ±. a.3 ±.7 b P<.1 No. growing follicles 1 mm per ovulatory wave 1. ±.3 a 9.8 ±.8 b.8 ±.9 c P<.1 No. ovulatory waves with only one follicle 1 mm 6/11 a /12 b /12 b P<.4 Diameter of ovulatory follicle (mm): At maximum 38.3 ±.7 a 4.7 ± 1.4 a 44. ± 1.4 b P<.3 On Day ± 1.2 a 4.4 ± 1.3 a,b 44. ± 1.3 b P<.3 Growth rates (mm day 1 ) from: Days 6 to ±.3 X 3.8 ±.2 X 3. ±.4 P<.9 Days 3 to 1 1. ±.6 a,y 2.6 ±.4 b,y 1.9 ±.4 A,a,b P<.4 A Mean on Days 3 to 1 tends to be different (P <.7) from the mean on Days 6 to 3. a,b,c Means with different superscript letters within rows are different (P <.). X,Y Means with different superscript letters within columns are different (P <.2).

8 Ovarian and uterine dynamics in Miniature ponies Reproduction, Fertility and Development 383 Diameter (mm) Days before ovulation Fig. 4. Mean (± s.e.m.) diameter of the ovulatory follicle from Days 6 to 1 in Miniature ponies ( ), large ponies ( ) and horses ( ). There were effects of mare type (P <.), day (P <.1) and type-byday interaction (P <.3; n = 12 mares per type). ovulatory wave and more ovulatory waves with only one growing follicle 1 mm than for large ponies and horses. The diameter of the ovulatory follicle was smaller at maximum and on Day 1 in the Miniature ponies than in the horses, but was not different from the diameter in large ponies. The percentage differences in the median bodyweight between the Miniature ponies and the large ponies and the Miniature ponies and the Breton horses were 6 and 74%, respectively, whereas the corresponding differences in maximum attained diameter of the ovulatory follicle were 6 and 14%. Growth rate of the ovulatory follicle between Days 3 and 1 was smaller in the Miniature ponies than in the large ponies, but was not different from the growth rate in the horses. The growth rate of the ovulatory follicle between Days 6 and 3 was greater (P <.2) than between Days 3 and 1 for Miniature ponies and large ponies, and tended (P <.7) to be greater for the Breton horses. The diameter of the ovulatory follicle from Days 6 to 1 showed an effect of mare type (Miniature ponies, large ponies, horses), day and type-by-day interaction (Fig. 4). Discussion This is apparently the first report on ovarian follicle dynamics in Miniature mares and possibly in miniature animals of any species. Transrectal ultrasonography was utilised to investigate the dynamics of follicle growth, time of ovulation, and subsequent formation and demise of the CL, and changes in score for endometrial echotexture. In addition, number of follicles per ovulatory wave and diameter and growth rate of the ovulatory follicle were compared among Miniature ponies, large ponies and Brazilian Breton horses. The findings revealed several follicle characteristics that were specific to the Miniature ponies compared with other types and breeds. The comparisons among Miniature ponies, large ponies and Breton horses were considered to be preliminary, owing to the use of different operators, equipment, locations, time and environmental conditions. The follicle population per animal was considerably reduced in Miniature ponies, compared with the large ponies and horses in the present study and to reported data, as shown by the following: (1) a mean of six follicles 2mmday 1 during the IOI in miniatures, compared with a reported 1 or 14 follicles in large ponies and horses (Pierson and Ginther 1987; Gastal et al. 4a); (2) 1. follicles 1 mm per ovulatory wave, compared with 6 to 1 follicles 1 mm in the larger breeds and a reported 12 follicles in large ponies (Gastal et al. 4a); (3) no double dominant follicle ( 28 mm) in any of 36 preovulatory periods in the miniatures, compared with 11 of 32 periods in large ponies (Jacob et al. 7); and (4) 6/11 ovulatory waves with only one follicle 1 mm, compared with /12 in the larger breeds and in only 3 of 48 IOIs in large ponies (Ginther et al. 7a). In regard to point (4), only one or two follicles per wave were detected in 4/11 old horse mares (> years; Ginther et al. 1993), buttheoldestminiaturemareinthepresent study was 12 years old and was considered to be middle-aged.as the ovulatory wave progressed, the follicles in the small diameter classes moved to the larger classes, similar to reported plots for horses (Pierson and Ginther 1987), reflecting the dynamics of the turnover in follicle growth and atresia. However, the Miniature ponies had fewer follicles in each diameter class than the horses. As an example, the mean maximum number of follicles mm during the ovulatory wave was.6 in the miniatures compared with 6. for the 11 1 mm class reported for horses. The low incidence of deviations (2%) in Miniature ponies that were similar to those reported for larger mares contrasts with the high incidence in large ponies and horses (>8%; Ginther et al. 4a, 4b). The low incidence in the Miniature mares was related to the small number of follicles per wave and the high incidence of only one follicle 1 mm. The clear and profound reduction in the numbers of follicles of various diameters and statuses in the Miniature ponies, compared with larger breeds, indicates that Miniature ponies are a potential model for study of follicle recruitment and development without the complexities and difficulties associated with studying large numbers of follicles. The greater number of follicles.1 2 mm and >2.1 mm day 1 on the right ovary is consistent with the tendency for a greater frequency of ovulations from the right ovary. This result in the Miniature mares seems to conflict with the conclusion (Ginther 1992) from an extensive survey of literature that ovulation was more frequent from the left ovary in the larger breeds, especially in maiden mares. These apparently contrasting results indicate that Miniature ponies are a potential comparative model for studying the factors that favour ovulation from the ovary on a given side of the body. The intervals (days) between events (ovulation, emergence, maximum follicle diameter, last detection of CL) were similar to those previously reported for large ponies (Gastal et al. 1997, ). The Miniature ponies and the large ponies had a longer IOI than the Breton horses, agreeing with previous comparisons between large ponies and horses (reviewed in Ginther 1992). No major secondary anovulatory waves, as indicated by a nonovulatory dominant follicle, were detected during the 12 IOIs in the Miniature ponies. In contrast, major anovulatory waves preceded

9 384 Reproduction, Fertility and Development E. L. Gastal et al. the wave that originated the ovulation associated with oestrus or the follicular phase in 24% of Quarter Horses (Ginther 1993) and in 2% of Bretons (Ginther et al. 4b). The % incidence of IOIs with minor follicular waves in the Miniature mares is less than has been reported for horses (97%; Ginther et al. 4b). In most IOIs (83%), 2 to 4 tracked follicles did not grow beyond 1 mm, and 86% of these did not seem to be associated with the ovulatory wave. The dynamics of the underlying follicles, including the origin of the future ovulatory follicle, often were not clear. Some appeared to grow and regress, whereas others remained in an apparent static phase or approximately constant diameter for many days (Fig. 3). This is the first use of the identity method in Miniature mares and the incidence of identity errors is not known. Therefore, we prefer to forgo further study of follicles <1 mm from these data, pending further experience and experiments. The profile of the mean diameter of the four largest follicles without day-to-day identity in the Miniature ponies was similar to what has been shown for large ponies (Gastal et al. ; Ginther et al. 7b) and horses (Gastal et al. 4b). However, the maximum diameters of F2, F3 and F4 were smaller for the Miniature ponies than has been reported for large ponies and horses. For example, the maximum mean diameter of F2 was 17 mm in the present study in Miniature ponies and 24 mm in a study with large ponies (Ginther et al. 7b). All detectable follicles were accounted for with the identity method in the Miniature ponies, owing to the small number of follicles. The difference in maximum diameter of the ovulatory follicle in Miniature ponies compared with the other breeds was slight when the relative difference in bodyweight was considered. The 6 and 74% greater difference in bodyweight in large ponies and in horses, respectively, than in Miniature ponies contrasts with the 6 and 14% greater difference in follicle diameter. Thus, the relative difference in bodyweight was 1-fold (Miniature v. large ponies) and -fold (Miniature ponies v. horses) greater than the difference in maximum follicle diameter. These findings and the reported (Ginther 199b) similarity between ponies and Quarter Horses in the diameter of the ovulatory follicle indicates that a critical diameter of the follicle is associated with development of the oocyte, regardless of body size. The small number of follicles in a wave and the small follicle diameters during the preovulatory period may reflect a physical limitation of the size of the ovary in the Miniature ponies. Further study is indicated to establish the relationships between Miniature and larger mares in size of ovary and preovulatory follicle, number of follicles and bodyweight. The reduction in growth rate of the ovulatory follicle between maximum diameter (1 or 2 days before ovulation) and ovulation in the Miniature ponies is consistent with what has been reported for large ponies (Gastal et al. 6) and horses (Palmer and Driancourt 198; Koskinen et al. 1989). Prior to the preovulatory cessation or reduction in growth, the growth rate ( 3mmday 1 ) was similar among breeds in the present study and for reported studies in ponies and horses (reviewed in Ginther 199b). Increasing oedema in the endometrial folds (increase in endometrial scores) during oestrus is indicated by the presence of anechoic areas on B-mode images and is used in the management of mares for breeding (Ginther and Pierson 1984). In the present study, the initial portion of the mean changes in scores of endometrial oedema was similar to those of previous reports in larger breeds (Ginther and Pierson 1984; Hayes et al. 198; Gastal et al. 7). The first significant increase in mean score for endometrial echotexture between Days 1 and 14 seemed to be associated with luteolysis and final regression of the CL, based on reduced diameter and area. As the number of follicles increased in the largest follicle classes (.1 2 mm and >2.1 mm), the endometrial echotexture also increased, which is consistent with the indications that endometrial echotexture and oestradiol changes are temporally related (Pycock et al. 199; Gastal et al. 6). However, the scores for endometrial echotexture in the Miniature mares seemed to begin decreasing earlier (Day 4) than in larger mares (Day 3 or 2; Ginther and Pierson 1984; Hayes et al. 198; Gastal et al. 7). The mean rate of ultrasonically-measured growth and regression of the CL was similar to what has been previously shown for large ponies and horses (Bergfelt and Ginther 1996). In conclusion, comparisons of transrectal ultrasonographic data from Miniature mares with larger mares during the oestrous cycle directly (present study) or indirectly (literature), demonstrated both similarities and differences between the breeds. Similarities included: (1) length of IOI when compared with large ponies; (2) relative changes in numbers of follicles within each follicle class as the oestrous cycle progressed; (3) corpus luteum lifespan, diameter and area; and (4) reduced growth rate of the preovulatory follicle during 1 or 2 days before ovulation. Main differences from larger mares were: (1) a greatly reduced daily ovarian follicle population within each of several follicle diameter classifications; (2) a smaller number of growing follicles 1 mm per ovulatory wave; (3) a much higher frequency of ovulatory waves with only one growing follicle 1 mm; and (4) a lower incidence of detectable diameter deviation. Miniature ponies had a smaller maximum diameter of the preovulatory follicle than in horses, but the difference was slight when compared with the extreme difference in bodyweight. Considering the differences in follicle dynamics and the small number of follicles per ovulatory wave, Miniature mares are a potential experimental model for comparative studies in folliculogenesis within and among species. Acknowledgements This research (Project MP1-APN-4) was supported by the Federal University of Rio Grande do Sul (Porto Alegre, RS, Brazil), PRONEX CNPq/FAPERGS, and the Eutheria Foundation, Inc., Cross Plains, Wisconsin. The authors thank Mr. Santo Sérgio Feoli, Cabanha Morada das Pôneis, for use of the mares; Alisul Alimentos S. A., Brazil for a gift of the commercial ration; and Dee Cooper for assistance with the figures. R. C. Mattos thanks the CNPq for a Research Fellowship. 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