Ultrasonographic Observation of Follicular and Luteal Dynamics during the Estrous Cycle in Shiba Goats

Journal of Reproduction and Development, Vol. 46, No. 1, 2000 Ultrasonographic Observation of Follicular and Luteal Dynamics during the Estrous Cycle in Shiba Goats Jun ORITA, Tomomi TANAKA, Hideo KAMOMAE and Yoshihiro KANEDA Laboratory of Veterinary Reproduction, Tokyo University of Agriculture and Technology, 3 5 8 Saiwai-cho, Fuchu, Tokyo 183 8509, Japan Abstract. Transrectal ovarian ultrasonography was performed to examine follicular and luteal dynamics during the estrous cycle in four adult female Shiba goats. Detectable follicles and the corpora lutea in both ovaries were monitored daily for 44 days using a B-mode scanner with a 7.5 MHz transrectal transducer. Blood samples were collected by jugular venipuncture at the time of ultrasonography and analyzed for estradiol-17β and progesterone by radioimmunoassay to monitor ovarian activity. Three to six follicles, whose diameters ranged from 1 to 7 mm, were observed throughout the estrous cycle; a few follicles grew to more than 5 mm in diameter and most of them atrophied during the luteal phase. Ovulatory follicles appeared from 5 days before ovulation coinciding with the start of the regression of corpora lutea, and then grew rapidly into 5.5 ± l.0 mm (mean ± SD) in diameter in parallel with the increase in the plasma concentration of estradiol-17β. The corpora lutea, 6.1 ± 1.3 mm in major axis, were detected from 3.0 ± 1.6 days after ovulation and stopped growing on 6 to 8 days after ovulation, with a major axis of 8.1 ± 2.2 mm. The mean length of the major axis of fully developed corpora lutea remained constant until 14 days after ovulation and then decreased rapidly to 5.2 ± 1.7 mm on the day of the next ovulation. In the early and functional luteal phase, there was a positive correlation between mean length of the total major axis of corpora lutea and mean plasma concentration of progesterone, whereas in the late luteal phase, the mean length of the total major axis of corpora lutea decreased slower than the progesterone levels. The present study demonstrated the ovarian follicular and luteal dynamics throughout the estrous cycle, indicating that ultrasonography is a reliable means for monitoring ovarian activity in Shiba goats. Key words: Ultrasonography, Ovary, Estrous cycle, Shiba goat. (J. Reprod. Dev. 46: 31 37, 2000) he transrectal ultrasonography has been used to monitor the structural changes in the reproductive organs of domestic animals, for example, ovarian follicular growth and luteal events during the estrous cycle in cattle [1,2] and pigs [3], and resumption of ovarian activity [4 6] and morphological uterine involution throughout the early postpartum period [5] in dairy cows. This Accepted for publication: December 8, 1999 Correspondence: H. Kamomae technique has also been used in the clinical research field to examine the mechanisms responsible for ovarian disorders such as ovarian cyst [7]. Ultrasonic imaging analysis had not been used for reproductive organs in small ruminant species, however, until the recent study by Schrick et al. [8] in ewes. They applied the high-resolution transrectal transducer, which is used for human prostate examination, to investigate the changes in the ovarian structures during the estrous cycle. The Shiba goat, Japanese indigenous goat, has been proven to be a useful experimental model for

32 ORITA et al. scientific study of domestic ruminants. Some aspects of their endocrine and neuroendocrine characteristics governing the reproductive system have been studied [9 14]. However, a method for monitoring changes in the ovarian structures of this species without surgery or slaughter has not been established in this species, and, therefore, ovarian dynamics in various reproductive states remains unknown. The establishment of an ultrasonographic technique for monitoring ovarian follicular and luteal dynamics in the Shiba goat would provide an opportunity to study the mechanisms controlling ovarian function in ruminant species. In the present study, we tried to use the transrectal ultrasonography to monitor ovarian structural dynamics in Shiba goats and demonstrated changes in the follicles and corpora lutea in association with circulating concentrations of ovarian steroid hormones during the estrous cycle. been identified, the probe was advanced slowly until the small antral part of the urinary bladder came into the image of the scanner and then rotated clockwise or counter-clockwise across the reproductive tract until an ovary was scanned. The follicles were detected as echo-free black spheres and the corpora lutea were detected as spheres or ellipsoids which were imaged a little more echo-free than echo-genic ovarian stroma (Fig. 1). During the experiment, the position and size of follicles and corpora lutea in both ovaries were sketched in relation to each other, and the sketch was compared to those of the preceding days to identify individual follicle and corpus luteum imaged previously. The diameter of each follicle and the major axis of the corpus luteum were measured with the Materials and Methods Animals Four adult female Shiba goats (4 to 5 years old and approximately 25 kg of weight) with regular estrous cycles, were used. They were kept in a paddock or in indoor cages under a 12 h light and 12 h dark cycle during the experiment. Ultrasonic evaluations Ovarian images were obtained using a B-mode scanner (Aloka SUPER EYE SSD-500; Fujihira Industry Co., Ltd, Tokyo, Japan) equipped with a 7.5 MHz transducer (UST-660-7.5; Aloka Co., Ltd, Tokyo, Japan) which was designed for prostate examination in humans. The procedure for ultrasonographic observation of ovaries was based on the method originally developed for ewes [8] with some modifications. During observation, the ultrasonographic probe was covered with a special balloon fixed with a rubber band, and the space between the balloon and the probe was filled with boiled water to take clear images. The animals were tied loosely in the stanchion stalls at the standing position, and the probe was introduced with the sound beam directed ventrally into the rectum through the anus. Then the probe was forwarded to look for the urinary bladder. Once the urinary bladder had Fig. 1. Ultrasound images of Shiba goat ovaries (#16) produced by using a transrectal, 7.5 MHz human prostate transducer. (A) An ovary with a follicle. The distance between plus marks (scanner s electric caliper) indicates the diameter of the follicle observed two days before ovulation. The diameter of the follicle was 6 mm. (B) An ovary with a corpus luteum. The distance between plus marks indicates the major axis of a corpus luteum observed five days after ovulation. The major axis of the corpus luteum was 10 mm.

ULTRASONOGRAPHY OF OVARIES IN SHIBA GOATS 33 scanner s electronic calipers (Count as one fractions of 0.5 mm and over and cut away those of less). Experimental procedure All ovarian follicles and corpora lutea were observed for 44 days and the follicles that grew to more than 5 mm in diameter were recorded for analysis of growth, ovulation and atrophy in each goat. The day of ovulation (day 0) was determined as the day when a large antral follicles, which had previously been identified and had continued to grow for several days, suddenly disappeared. Blood samples were collected every day by jugular venipuncture at the time of the observations. Plasma was separated by centrifugation immediately after the blood collection and stored frozen at 20 C until the assays for concentrations of progesterone and estradiol-17β. Radioimmunoassay Plasma concentrations of estradiol-17β and progesterone were measured by the radioimmunoassay as described by Taya et al. [15]. Intraand inter-assay coefficients of variation for estradiol-17β were 11.4% and 10.3%, respectively, and intra- and inter-assay coefficients of variation for progesterone were 7.9% and 4.6%, respectively. The sensitivity for estradiol-17β and progesterone assays were 0.19 pg/ml and 3.0 pg/ml, respectively. Statistical analyses Data were expressed as mean ± SD and Student s t test was used for evaluation of the results. p< 0.05 was considered to be statistically significant. Results Estrous cycle The estrous cycle durations, i.e., the interovulatory intervals, in 4 goats were 19, 19, 21 and 22 days, respectively, and the mean duration was 20.3 ± 1.5 days. Follicular dynamics Three to six follicles with diameters of 1 to 7 mm were detected throughout the estrous cycle and almost all of them atrophied without ovulation. Developmental and atrophic profiles of follicles that grew to more than 5 mm in diameter throughout the estrous cycle of individual goats are shown in Fig. 2. The follicles that grew in the early- to middle-estrous cycle atrophied and disappeared without ovulating (non-ovulatory follicles). On the other hand, all of the follicles that appeared in the latter half of the estrous cycle grew to more than 5 mm in diameter and then ovulated (ovulatory follicles). The mean ovulation rate (synonym for ovulation number) was 2.8 ± 1.0 (range, 2 to 5) for 8 ovulatory events in 4 animals, and the total number of ovulatory follicles in the right and left ovaries were 13 (59%) and 9 (41%), respectively. The characteristics of the nonovulatory and ovulatory follicles are shown in Table 1. The duration of follicular growth was defined as the period from the day on which a follicle was observed for the first time to the day on which a follicle grew to its maximum diameter. The duration of follicular atrophy was defined as the period from the day on which the diameter of a follicle began to decrease to the day on which that follicle was no longer detectable. Nonovulatory and ovulatory follicles developed for 5.5 ± 2.5 and 5.7 ± 1.8 days, respectively, and grew to maximum diameters of 5.9 ± 0.8 and 5.5 ± 1.0 mm, respectively. No significant difference was detected in the duration of growth and the maximum diameter between non-ovulatory and ovulatory follicles (p>0.1). The relationship between total diameter of ovulatory follicles and mean plasma concentrations of progesterone and estradiol-17β during the follicular phase is shown in Fig. 3. Almost all of the ovulatory follicles were detected from 5 days before ovulation, coinciding with the decline in progesterone concentrations. Then they rapidly developed, reaching a peak diameter of 5.5 ± 1.0 mm on the day before ovulation, and the plasma concentration of estradiol-17β increased in parallel with the follicular growth until ovulation. Corpora lutea The corpora lutea were detected from 3.0 ± 1.6 (range, day 1 to 6) days after ovulation for 8 ovulatory events in 4 animals, and they stopped growing on day 6 to 8. The mean major axis of the corpus luteum increased gradually from 6.1 ± 1.3 mm on day 3 to 8.1 ± 2.2 mm on day 9 and remained at about 8 mm in diameter until day 14. It then decreased rapidly to 5.2 ± 1.7 mm on the

34 ORITA et al. Fig. 2. Developmental profiles of individual follicles that grew to more than 5 mm in diameter throughout a representative estrous cycle in four experimental goats. The arrows indicate the ovulation of ovulatory follicles. Table 1. Characteristics of follicles that grew to 5 mm in diameter Non-ovulatory follicle Ovulatory follicle Duration of growth a) (days) 5.5 ± 2.5 5.7 ± 1.8 Maximum diameter (mm) 5.9 ± 0.8 5.5 ± 1.0 Duration of regression b) (days) 4.8 ± 2.8 Mean ± SD. a) from first appearance until maximum diameter was reached. b) from the start of diminution of diameter to disappearance. day of the next ovulation. The relationship between the mean length of the total major axis of corpora lutea and the plasma concentration of progesterone over a period of 10 days before and after ovulation is shown in Fig. 4. In the early and functional luteal phase, mean length of the total major axis of corpora lutea changed in parallel with the mean plasma concentration of progesterone, whereas in

ULTRASONOGRAPHY OF OVARIES IN SHIBA GOATS 35 Fig.4. Correlation between plasma concentrations (mean ± SD) of progesterone ( ) and mean total major axis (mean ± SD) of corpora lutea ( ) in both ovaries from day 0 (day of ovulation) to day 10 (left panel) and from day -10 to day 0 (right panel). Fig. 3. Correlation between growth of ovulatory follicles and changes in plasma concentrations of ovarian steroids during the follicular phase (4 animals, 8 ovulatory events). (a) total diameter (mean ± SD) of ovulatory follicles ( ). (b) plasma concentrations (mean ± SD) of progesterone ( ) and estradiol-17β ( ). the late luteal phase, the plasma concentration of progesterone, which began to decrease 2 days before the start of the regression of corpora lutea, decreased rapidly. The correlation coefficient of the mean length of the total major axis of corpora lutea and plasma concentration of progesterone was 0.80 throughout the estrous cycle and 0.96 from day 0 to day 9. Some regressed corpora lutea were observed even after the subsequent ovulation. Discussion In the present study, we successfully applied transrectal ovarian ultrasonography, which had been used originally for human prostate examination and had also been used in ewes [8], to investigation of structural changes in the ovaries of Shiba goats. The follicles and corpora lutea were clearly scanned and their growth and regression accorded well with the changes in the plasma concentrations of estradiol-17β and progesterone, indicating that this technique is feasible for monitoring the ovarian dynamics in female Shiba goats. However, simultaneous occurrence of growth and regression in several follicles and corpora lutea caused technical difficulties in identification of individual follicles and corpora lutea that had been observed in the preceding days. Therefore, careful observation (including, for example, sketching of imaged ovaries) is required to monitor the dynamics of follicles and corpora lutea in the polyovular species throughout the estrous cycle. Ultrasonographic studies in cattle have revealed two or three follicular waves during the estrous cycle, in which one of several growing follicles became the dominant follicle and the other follicles atrophied [1, 2]. In the ewe, serial laparotomy and ink labeling of large follicles led to the conclusion that three or possibly more phases of follicular growth and atresia occurred during the estrous cycle [16]. On the other hand, Schrick et al. [8], who used transrectal ultrasonography in a recent study on ewes, reported that the growth and atrophy of large antral follicles were observed in the luteal phase but that the two largest follicles grew simultaneously at the same rate and did not suppress the growth of the other follicles, suggesting that the follicular wave was lacking in

36 ORITA et al. the ewe. In the present study, several large follicles appeared and grew coincidentally, and the follicular dominance associated with growth in cattle was not prominent in Shiba goats. The present results support the speculation by Schrick et al. [8] that follicular dominance is weak or lacking in the polyovular ruminants (such as ewes and goats), which is probably involved in the regulation of multiple ovulation. The present study demonstrated that corpora lutea stopped growing 6 to 8 days after ovulation and began to regress from 15 days after ovulation. It was reported that the corpora lutea completed development approximately 7 days and 5 days after ovulation in the cow [17] and ewe [8,18], respectively, and started to regress approximately 18 days and 11 days after ovulation in the cow [17] and ewe [8, 18], respectively. Considering that the lengths of the estrous cycles are about 21 days in cattle and Shiba goats [9] and about 17 days in ewes, it is clarified that the number of days required for the completion of corpora lutea development in Shiba goats is similar to that in cattle and ewes, and that the timing of the start of the regression of corpora lutea during the estrous cycle in Shiba goats is intermediate between that in cattle and ewes. The area of corpora lutea has been used as an index of the plasma concentrations of progesterone during the estrous cycle and early pregnancy in the cow [19, 20], ewe [8, 18], llama [21] and mare [22]. In the present study, the total major axis of corpora lutea in both ovaries was used as an index, and it was found to be associated with changes in the plasma concentration of progesterone. These results indicate that the total major axis of corpora lutea is a reliable index of peripheral progesterone level in Shiba goats. The rate of decrease in the length of the total major axis of corpora lutea during luteolysis was less than that of the plasma concentration of progesterone, which is similar to the results reported previously in cows [19]. These findings indicate that the corpora lutea lose their function to produce progesterone before they begin to decrease in size. In conclusion, the present study demonstrated changes in the ovarian follicles and corpora lutea throughout the estrous cycle by transrectal ultrasonography, indicating that ultrasonography is reliable for evaluating ovarian activity in Shiba goats. Since the Shiba goat has proven to be a useful experimental animal, this technique could be used in studies to elucidate the mechanisms regulating normal and abnormal ovarian activities in ruminant species. Acknowledgments We are grateful to Fujihira Industry Co., Ltd and Aloka Co., Ltd for technical advice and concerning transrectal ultrasonography. References 1. Pierson RA, Ginther OJ. Ultrasonic imaging of the ovaries and uterus in cattle. Theriogenology 1988; 29: 21 37. 2. Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Fertil 1988; 83: 663 671. 3. Kähn W. Veterinary reproductive ultrasonography. London: Mosby-Wolfe; 1994. 4. Carroll DJ, Pierson RA, Hauser ER, Grummer RR, Combs DK. Variability of ovarian structures and plasma progesterone profiles in dairy cows with ovarian cysts. Theriogenology 1990; 34: 349 370. 5. Kamimura S, Ohgi T, Takahashi M, Tsukamoto T. Postpartum resumption of ovarian activity and uterine involution monitored by ultrasonography in Holstein cows. J Vet Med Sci 1993; 55 (4): 643 647. 6. Kamimura S, Ohgi T, Takahashi M, Tsukamoto T. Turnover of dominant follicles prior to first ovulation and subsequent fertility in postpartum dairy cows. Reprod Dom Anim 1993; 28: 85 90. 7. Yoshioka K, Iwamura S, Kamomae H. Ultrasonic observations in the turnover of ovarian follicular cysts and associated changes of plasma LH,FSH,progesterone and oestradiol-17β in cows. Res Vet Sci 1996; 61: 240 244. 8. Schrick FN, Surface RA, Pritchard JY, Dailey RA, Townsend EC, Inskeep EK. Ovarian structures during the estrous cycle and early pregnancy in ewes. Biol Reprod 1993; 49: 1133 1140. 9. Kano Y, Mori Y. Shiba goat. In: Suzuki Z (eds.), Mammalian Reproductive Physiology-Aspects of Its Animal Experimentation. Tokyo: Soft Science Inc; 1982: 367 379 (In Japanese) 10. Manabe Y, Yamaguchi M, Tanaka T, Mori Y. Estradiol-induced GnRH surge in ovariectomized

ULTRASONOGRAPHY OF OVARIES IN SHIBA GOATS 37 goats. J Reprod Dev 1993; 39: 91 96. 11. Mori Y, Kano Y. Changes in plasma concentrations of LH, progesterone and oestradiol in relation to the occurrence of luteolysis, oestrus and time of ovulation in the Shiba goat (Capra hircus). J Reprod Fertil 1984; 72: 223 230. 12. Mori Y, Takedomi T, Mizomoto Y, Hoshino K. Induction of preovulatory endocrine events by programmed administration of progesterone and estradiol in ovariectomized goats. Jpn J Anim Reprod 1987; 33: 36 40. 13. Mori Y, Nishihara M, Tanaka T, Shimizu T, Yamaguchi M, Takeuchi Y, Hoshino K. Chronic recording of electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator activity in the goat. Neuroendocrinology 1991; 53: 392 395. 14. Yamaguchi M, Manabe Y, Tanaka T, Mori Y, Hoshino K. Microdialysis measurement of in vivo Gonadotropin-releasing hormone secretion in the goat. J Reprod Dev 1992; 38: 143 150. 15. Taya K, Watanabe G, Sasamoto S. Radioimmunoassay for progesterone, testosterone and estradiol-17β using 125 I-iodohistamine radioligands. Jpn J Anim Reprod 1985; 31: 186 197. 16. Smeaton TC, Robertson HA. Studies on the growth and atresia of Graafian follicles in the ovary of the sheep. J Reprod Fertil 1971; 25: 243 252. 17. Ginther OJ, Knopf L, Kastelic JP. Temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J Reprod Fertil 1989; 87: 223 230. 18. Ravindra JP, Rawlings NC, Evans ACO, Adams GP. Ultrasonographic study of ovarian follicular dynamics in ewes during the oestrous cycle. J Reprod Fertil 1994; 101: 501 509. 19. Sprecher DJ, Nebel RL, Whitman SS. The predictive value, sensitivity and specificity of palpation per rectum and transrectal ultrasonography for the determination of bovine luteal status. Theriogenology 1989; 31: 1165 1172. 20. Kastelic JP, Bergfelt DR, Ginther OJ. Relationship between ultrasonic assessment of the corpus luteum and plasma progesterone concentration in heifers. Theriogenology 1990; 33: 1269 1278. 21. Adams GP, Sumar J, Ginther OJ. Form and function of the corpus luteum in llamas. Anim Reprod Sci 1991; 24: 127 138. 22. Bergfelt DR, Pierson RA, Ginther OJ. Resurgence of the primary corpus luteum during pregnancy in the mare. Anim Reprod Sci 1989; 21: 261 270.