Duration and pattern of follicular evacuation during ovulation in the mare

Animal Reproduction Science, 15 (1987) 131 - 138

131

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Duration and Pattern of Follicular Evacuation during Ovulation in the Mare D.H. TOWNSON and O.J. G I N T H E R

Department of Veterinary Science, University of Wisconsin-Madison, 1655 Linden Drive, Madison, WI53706 (U.S.A.) (Accepted 29 June 1987)

ABSTRACT Townson, D.H. and Ginther, O.J., 1987. Duration and pattern of follicular evacuation during ovulation in the mare. Anita. Reprod. Sci., 15: 131-138. The extent and pattern of follicular-fluid release at the time of ovulation was studied using transrectal ultrasonography in 12 mares. Mares with large ( >/35 mm) preovulatory follicles were administered human chorionic gonadotrophin (hCG) to induce ovulation. Each mare was examined beginning at 30 ( n = 2 ) , 34 ( n = 3 ) , 35 ( n = 3 ) , or 36 ( n = 4 ) hours following hCG treatment. An examination schedule of every hour, half-hour, 9 to 16 rain, or continuously was used to detect the onset of follicular collapse (duration of schedule, 6 to 12 h). In 10 of 12 mares, initial fluid loss occurred between examinations (range, 9 to 27 min), but residual fluid loss was followed continuously until: (1) no detectable fluid remained, (2) residual fluid had reached a minimal level ( 5% or less of original fluid area), or (3) no decrease was detected during 5 rain of continuous examination. If it is assumed that the initial fluid loss occurred, on the average, midway between two examinations, there was a decrease of 81% of the original antral area in 7.3 rain. During the subsequent continuous examinations, there was an average additional 16% area decrease in an average of 5.2 rain. For the remaining two mares, follicular-fluid loss was observed continuously from preovulation to completion of fluid release. Two extremes of fluid loss patterns were observed in these two mares. One pattern was an abrupt loss of follicular fluid in which the majority (83% of the antral area) disappeared in less than 1 min. The other pattern was a slow, gradual loss of fluid which took 4 min for 83% of the area to be lost. However, the time required for evacuation of approximately 96% of the antral area in both mares was 6 rain.

INTRODUCTION Investigations describing the nature of follicular evacuation during the ovulation process in mares have utilized cinematography and laparoscopy ( Withe r s p o o n , 1970; M e r k t a n d H e i n z e , 1 9 7 6 ) a n d u l t r a s o n o g r a p h y ( G i n t h e r a n d Pierson, 1984a). However, these studies provided limited information. The m a j o r i t y o f k n o w l e d g e o f t h e o v u l a t o r y p r o c e s s is b a s e d o n s t u d i e s i n c a t t l e ( B e r n a r d e t al., 1 9 8 4 ) , r a t s ( B l a n d a u , 1955 ), r a b b i t s ( B j e r s i n g a n d C a j a n d e r ,

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132 1974 ), non-human primates ( Nigi, 1977 ), and women (Crespigny et al., 1981 ). The study in women (Crespigny et al., 1981) and the chance observations in mares ( Ginther and Pierson, 1984a) appear to be the only investigations where ultrasonography was used to observe follicular-fluid loss without manipulation of anatomical structures of surgical intervention. The mare provides several advantages for studying ovulatory events by ultrasound. First, mares that are accustomed to transrectal examinations will tolerate repeated or continuous examinations with minimal restraint. Second, intrarectal placement of the ultrasound transducer reduces the distance between the transducer and the ovary, permitting the use of a high resolution transducer. Furthermore, the preovulatory follicle is large (e.g., 40 m m in diameter) and easily detected and monitored. The objective of this investigation was to characterize follicular evacuation associated with the ovulatory process in mares by examining the rate and extent of follicular-fluid release. MATERIALSAND METHODS Twenty pony mares weighing 200-400 kg were used during the ovulatory season (1 June to 4 September). The ultrasound scanner was a real time, Bmode instrument equipped with a linear-array, 5 MHz transducer {Equisonics 310, Equisonics Corporation, Bensenville, IL). Mares were selected for later detailed study by daily ultrasound monitoring of ovarian follicle growth and uterine endometrial changes. Mares were assigned to the experiment when they developed a single follicle that was >i 35 m m and had an endometrial score characteristic of estrus (heterogeneous ultrasound image with obvious endometrial hypertrophy and folding; Ginther and Pierson, 1984b; Hayes et al., 1985). When the largest follicle reached 35 mm, 2000 international units of human chorionic gonadotrophin (hCG) were given intramuscularly to induce ovulation. Each mare was examined for the presence of the preovulatory follicle 12 h following hCG treatment. Beginning at 24 ( n = 1 ), 30 ( n-- 3 ), 34 ( n-- 6), 35 ( n = 3 ), or 36 ( n = 7 ) hours after hCG administration, examinations were conducted with increasing frequency at intervals of one hour, half-hour, 9 to 16 min, or continuously according to time restraints and the operator's subjective, nondefined opinion on the imminence of the onset of antral collapse (duration of examination schedule, 6 to 12 h). Mares that did not ovulate during the examination schedule were not used. Follicular evacuation was defined, retrospectively, as being underway when an area decrease of 20% or more was first observed. Although initial fluid loss occurred between examinations in some mares, all mares were examined on a continual basis following the detection of initial loss until no detectable fluid remained, residual fluid appeared

133 to reach a minimal level (approximately 5% or less of original antral area), or no decrease was detected over the course of 5 rain. A three-quarter-inch video tape recorder (Sony U-matic V0-5600, Sony, Itasca, IL) was connected to the ultrasound scanner during the examination schedule. Reference structures within the ovary (e.g., smaller follicles, corpora albicantia) were noted so that a given orientation of the ovary could be recognized in subsequent examinations. An image showing the reference structures was frozen on the ultrasound screen adjacent to the real-time images. Estimates of the duration of follicular-fluid discharge (antral-collapse time) were based on intrafollicular cross-sectional areas traced from the ultrasound images recorded on video tape during rectal examinations. The tapes were examined through a high-resolution 19-inch video monitor (Panasonic WV-5490, Panasonic, Secaucus, NJ) using the underscan mode. The real-time images were frozen so that cross-sectional images could be traced from the monitor screen. The two-dimensional image that encompassed the largest area was used. Tracings of the antrum were drawn by following the margin between the nonechogenic antral cavity and echogenic ovarian stroma. Calculations for area were made utilizing a computerized (IBM-PC AT, International Business Machines, Inc., Danbury, CT) morphometry program ( The Morphometer, Woods Hole Educational Associates, Woods Hole, MA). i

RESULTS Seven mares in which follicular evacuation occurred before ( n = 2) or after (n = 5) the examination schedule were not used. In another mare, complete follicle collapse occurred between examinations ( 30-min interval ) so that no portion of the process was observed. Evacuation of the follicle was observed continuously in two mares from preovulation to completion of fluid release. Antral area measurements were calculated from tracings for each minute (Figs. 1 and 2). For Mare A, the antral area decreased abruptly from 10.6 cm 2 to 1.9 cm 2 (8.7 cm 2 decrease or 82% reduction in fluid area) in the first minute. During the next 2 min, the fluid area decreased at a rate of 2% per min and at 4 min after the beginning of fluid loss, the antrum was reduced to 6% of the preovulatory area. The remaining fluid was lost over a period of several minutes so that only 1% of the original antral area was detected at 9 min. In contrast, the size of the antrum in Mare B reduced slowly, decreasing from 9.7 cm 2 to 7.2 cm 2 ( 2.5 cm 2 reduction or 26% loss in fluid area) in the first minute. The initial 26% decrease was followed by losses of 24%, 10%, 23 %, 4%, 9% and 1% during the second through seventh minutes, respectively. Despite the difference in antral area decrease patterns, the decrease in Mare A and Mare B was approximately the same in 6 min (97% and 96% area reduction in 6 min, respectively), and the antrum subsequently

134 E 2

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Mere A o--o

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10-

g::i:

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I 0 Minutes

I 1

I 2 After

I 3

i 4

Beginning

I 5

i 6 of

I 7 Fluid

I 8

I 9

Loss

Fig. I. Cross-sectional areas of the antrum from two mares examined continuously for follicular evacuation during the ovulatory process. Broken lines represent 7.5 rain and 1 rain prior to the onset of fluid loss for Mares A and B, respectively.

reduced in size at a rate of 2% between the seventh and the eighth minutes and 1% during the ninth minute, respectively. In the ten remaining mares, the onset of fluid loss was not observed, but sequential examinations ranging from 9 to 27 min between observations permitted an estimation of the duration of the emptying process. The initial portion of the fluid-loss time was based on discontinuous examinations whereas the remaining fluid-loss time was determined by continuous observation. The portion based on discontinuous examinations ranged from 26% of the preovulatory area for a 27-min interval to 100% for a 9-min interval between examinations (Table 1). The average initial area decrease was 81% for an average 14.6-min time span between observations. The residual area decrease during the subsequent continuous examination ranged from 74% in 3 min to 32% in 11 min with an average of 16% in 5.2 min. DISCUSSION The duration of the follicle emptying process in two mares in which evacuation was observed continuously was calculated on a per minute basis. In these two mares, the antral cavity was nearly emptied (96 to 97% of the original antral area) 6rain after the onset of follicular evacuation. However, the patterns of follicular-fluid loss differed. One pattern was an abrupt collapse of the antrum to 83% of its original size in 1 min or less, followed by a much reduced rate of loss (Mare A). During the first minute of follicular evacuation, approximately 25 s of the process were not recorded on tape because additional fecal material had to be removed from the rectum. Therefore, the initial reduction could have occurred in less than 1 min. In contrast, the second pattern of fluid loss was a slow decrease in the size of the antral cavity with fluid loss

135 MARE A

0 Min : 100%

5 Min : 6%

1 Min : 18%

6 Min : 3%

2 Min : 15%

7 Min : 3%

3 Min : 13%

8 M i n : 1%

4 Min : 6%

9 M i n : 1%

MARE B

0 Min : 100%

4 M i n : 17%

I Min : 74%

5 Min : 13%

2 Min : 50%

6 Min : 4%

7 Min : 3%

3 Min : 40%

8 Min : 3%

Fig. 2. Antral area tracings taken from ultrasound images for Mares A and B at various times during follicular evacuation. Two patterns of fluid loss were observed: a rapid loss ir. the first minute followed by gradual loss (Mare A), and a gradual loss evenly distributed over several minutes (Mare B). more gradually distributed over time ( M a r e B ) . In another mare (Mare C; Table 1) the initial fluid loss (26% decrease in the fluid area) was not observed, but the remaining 74% of the antral area decreased during the next 3 min of the continuous examination; during these 3 min, there were decreases of 25% and 46% during the first two 30-s intervals and additional decreases of 2% and 1% over the last 2 min. Similar variations for follicular-fluid loss have been described in women (Crespigny et al., 1981), macacques (Nigi, 1977), and rats (Blandau, 1955 ). The report utilizing rats properly defined ovulation time as the time required for discharge of the oocyte. Other investigations, including the present study, characterize follicular-fluid loss or follicle collapse without knowledge of the time of oocyte discharge or ovulation. In the Blandau

136 TABLE 1

Cross-sectional areas (cm 2) of the antrum during follicular evacuation based on initial discontinuous examinations followed by a continuous examination after the detection of an initial fluid loss

Mare

C D E F G H I J K L

Mean SD N

Initialdiscontinuous examinations Preovulatory antral area

Area at first detection of fluid loss

(cm2)

(cm2)

Decrease in area (%)

Subsequent continuous examinations Interval between examinations (rain)

8.0 10.1 6.7 12.7 10.5 9.9 11.0 7.0 7.7 8.1

5.9 3.7 2.0 1.5 1.3 0.9 0.8 0.3 0.2 0.0

26 63 70 88 88 91 93 96 97 100

27 14 14 12 15 16 13 14 12 9

9.2 _+2.0 10

1.7 ±1.8 10

81 ±23 10

14.6 ±4.8 10

Area at start (cm 2)

Area at end (cm 2)

Decrease in area (%)

5.9 3.7 2.0 1.5 1.3 0.9 0.8 0.3 0.2 0.0

0.0 0.5 2.2 0.9 0.2 0.0 0.3 0.1 0.0 0.0

74 32 0 5 10 9 5 3 3 -

1.7 _+1.8 10

0.4 ±0.7 10

16 ±24 9

Duration of examination (rain) 3 11 6 6 3 4 7 5 2 5.2 2.7 9

study (1955), part of the variation observed in ovulation times was attributed to the location of the cumulus oophorus within the follicle. That is, if the oocyte and cumulus were in close proximity to the rupture point when ovulation occurred, the cumulus mass acted as a plug to block the rapid release of follicular fluid and the oocyte. This caused ovulation times to be lengthened. In contrast, if the cumulus mass and oocyte were deep within the follicle, initial fluid release occurred rapidly until the cumulus mass was able to inhibit oocyte release and follicular fluid flow, but shorter ovulation times resulted. It appears that similar variation in the nature of follicular evacuation associated with the ovulatory process occurs in the mare. The factor (s) which cause the variable evacuation patterns in the mare have not been determined. In ten mares, the initial portion of the loss of fluid occurred between two discontinuous observations. However, if it is assumed that loss began, on the average, midway between the two examinations, an estimation of the length of the process can be calculated. On this basis, the group average was an 81% decrease in the initial antral area in an average of 7.3 rain (14.6 min divided by 2). This estimate of the mean is close to that obtained by continuous observations in Mares A and B. The rate of loss was slower during the subsequent continuous examinations (mean, an additional 16% reduction in 5.2 min, Table 1 ). These results indicated that at some point during evacuation, the rate of

137 follicular-fluid loss tapers off and the remaining fluid is lost more slowly. In all but two of the 12 mares, some degree of gradual loss of residual fluid was detected toward the end of the process. The exceptions were Mare L ( Table 1 ), in which the antrum had collapsed entirely between examinations (9-min interval) so that no residual fluid was detected and Mare E, where 70% of the antrum disappeared by 14 min after the previous examination; the antrum enlarged by 3% in 6 min during the continuous examination. Presumably, the antral cavity became larger as a result of fluid infiltration associated with follicular rupture or the apparent increase was due to experimental error. Previous authors (Witherspoon, 1970; Merkt and Heinze, 1976) have utilized cinematography to study follicular evacuation in a limited number of mares (n = 2 and n = 1, respectively). Briefly, the process was described as an initial rush of follicular fluid to the outside of the follicle which took approximately 20 to 25 s. Subsequently, oozing continued for another 1-3 min as the follicle collapsed. Cinematography studies involve direct manipulation of reproductive structures to obtain photographic records within the peritoneal cavity. In addition, because the ovaries were examined grossly, an accurate determination of the time of complete emptying could not be made by the cinematographic approach. Ultrasound technolagy enabled follicular evacuation associated with the ovulatory process to be visualized, as it occurred, without direct manipulation of the ovary or its associated structures or the use of surgical procedures which may influence the rate and extent of follicular-fluid loss. ACKNOWLEDGEMENTS Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison. The authors thank D.H. Norback and D.K. Peters, Department of Pathology and Laboratory Medicine, for the use of the morphometer. Appreciation is also expressed to G.J. Breur and R.A. Pierson for technical assistance.

REFERENCES Bernard, C., Lambert, R.D., Beland, R. and Belanger,A., 1984. Laparoscopic investigationof the bovine ovary in the periovulatoryphase of the cycle.Theriogenology, 22; 143-149. Bjersing,L. and Cajander, S., 1974. Ovulation and the mechanism of follicularrupture.I. Light microscope changes in rabbitovarian folliclespriorto induced ovulation.CellTissue Res., 149:

287-300. Blandau, R.J., 1955. Ovulationin the livingalbino rat. Fertil. Steril., 6: 391-404. Crespigny,L.C., O'Herlihy,C.O. and Robinson, H.P., 1981. Ultrasonicobservationof the mechanism of human ovulation.Am. J. Obstet. Gynecol.,139:636-639. Ginther,O.J. and Pierson,R.A., 1984a.Ultrasonicevaluationofthe reproductivetract ofthe mare: ovaries. J. EquineVet. Sci.,4: 11-16.

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Ginther,O.J.and Pierson,R.A., 1984b. Ultrasonicanatomy of the equine ovaries.Theriogenology, 21: 471-483. Hayes, K.E.N., Pierson, R.A., Scraba, S.T. and Ginther, O.J., 1985. Effectsof estrous cycle and season on ultrasonicuterineanatomy in mares. Theriogenology, 24: 465-477. Merkt, H. and Heinze, H., 1976. Die Ovulation bei der Stute (mit Ku/zfilln).In: Proc. 20th World Veterinary Congress, 6-12 July 1975, Thessaloniki,Greece, Vol. 2, pp. 960-962. Nigi,H., 1977. Laparoscopic observationsof follicularrupture in the Japanese macacque (Macaca

fuscata). J. Reprod. Fertil., 50: 387-388. Witherspoon, D.M., 1970. Ovulation site in the mare. J. Am. Vet. Med. Assoc., 157: 1452-1459.