Ultrasonographic imaging of the reproductive tract and surgical recovery of oocytes in Cebus apella (capuchin monkeys)

Two-dimensional real-time and Doppler ultrasonography are valuable non-invasive methods to assess reproductive anatomy and physiology. In adult, postpubertal female Cebus apella (capuchin monkeys), the objectives were to determine (1) uterine and ovarian dimensions, ovarian follicular dynamics, day of ovulation, and arterial blood flow of uterus and utero-ovarian ligament during the follicular phase of the menstrual cycle and (2) the number of oocytes aspirated from antral follicles at laparotomy. Based on two-dimensional, transabdominal B-mode ultrasonography, mean (± S.E.M.) length, height, width, and volume of the uterus were 17.9 ± 0.4, 12.4 ± 0.3, 13.6 ± 0.3 mm, and 1.55 ± 0.08 mL, respectively, and of the ovary were 13.4 ± 0.2, 8.2 ± 0.1, 7.7 ± 0.1 mm, and 4.5 ± 0.2 mL. Ovarian follicles were monitored for 6 days before ovulation, which occurred on day 9.3 ± 0.5 (range, days 7–11; day 1 = start of menses), with 10 of 12 ovulations in the right ovary. Diameter and volume of the preovulatory follicle were 10.1 ± 0.2 mm and 0.55 ± 0.03 mL (on the estimated day of ovulation) and of the CL were 8.1 ± 0.4 mm and 0.3 ± 0.05 mL. Resistivity and pulsatility indices were 0.86 ± 0.02 and 2.15 ± 0.11 for uterine arteries, and were 0.69 ± 0.04 and 1.63 ± 0.15 for the utero-ovarian ligament (UOL) artery; just prior to ovulation, both indices peaked (P < 0.05) in the uterine artery ipsilateral to the side of ovulation, but both reached a nadir (P < 0.05) in the UOL artery. In the absence of ovarian stimulation, 31 oocytes (diameter, 137 ± 10 μm) were aspirated (average of 2 oocytes/(female attempt)) on days 5, 7, and 9. In conclusion, transabdominal ultrasonography facilitated assessment of reproductive anatomy and physiology in C. apella adult females. Resistance and pulsatility indices of uterine and UOL arteries changed near the time of ovulation. Dominant follicles were easiest to aspirate at 8–9 mm in diameter (∼day 9), with intact cumulus-oocyte complexes recovered from ovarian follicles 2–9 mm in diameter.     

Pattern of follicular development in high fecundity Olkuska ewes during the estrous cycle

Folliculogenesis was studied daily in the 18 oestrous cycles in six prolific Olkuska ewes from October to December using transrectal ultrasonography to record the number and size of all ovarian follicles > or =2 mm in diameter. Blood samples were taken once a day and were analyzed for concentrations of FSH, LH, estradiol and progesterone. Follicular and hormonal data were analyzed for associations between different stages of development of the follicular waves and concentrations of FSH and estradiol. The first wave during which at least one follicle reached maximum diameter of > or =4 mm after ovulation, was defined as a wave 1, and the following waves were numbered sequentially. Waves 1, 2, 3, 4 and the ovulatory one emerged on days: -2 to 4, 4 to 8, 6 to 11, 10 to 12 and 11 to 15, respectively. The mean number of follicles per wave that reached diameter of > or =4 mm was 4.15 +/- 1.1 and 16.62 +/- 8.6 follicles per estrous cycle of a total 299 follicles were observed. Significantly more follicles (p> or =0.05) emerged on days 2, 8 and 13 than in other days. Serum FSH concentrations fluctuated from 0.11 ngml(-1) on day 2 to preovulatory maximum 1.81 ngml(-1) on day 17 of the estrous cycle. The emergence of follicular waves was associated with elevations of FSH concentrations in blood serum. The mean increase in FSH concentration was followed by the recruitment of follicles of the next wave. The mean daily FSH concentration and the mean number of follicles emerging each day were negatively correlated. The length of the interwave interval (4.4 +/- 1.6 days) did not differ significantly from the interval between pulses of FSH (4.8 +/- 0.3 days). The mean serum estradiol concentrations showed fluctuations until day 14 and then gradually increased from 5.47 +/- 0.3 pgml(-1) to reach a peak 13.14 +/- 0.2 pgml(-1) on the day before ovulation. To summarize, the growth of ovarian follicles during the estrous cycle in high fecundity Olkuska sheep exhibited a distinct wave-like pattern. Ovarian follicles emerged from the pool of 2 mm follicles. The preovulatory follicles originated from the large follicle population were present in the ovary at the time of luteal regression. The initial stages of the growth of the largest follicles appears to be controlled primarily by increases in FSH secretion.     

Follicular activity and hormonal secretory profile in vicuna (Vicugna vicugna)

The objective of the present study was to characterize ovarian activity in non-mated vicunas, relating ovarian structures (evaluated by transrectal ultrasonography, daily for 30 days) to changes in plasma concentrations of estradiol-17beta and progesterone. Ovarian follicular activity occurred in waves, characterized by the follicle emergence, growth and regression. The mean duration of follicular waves was 7.2+/-0.5 days (mean+/-S.E.M.), with a range of 4-11 days. The follicular growth phase averaged 3.0+/-0.2 days, the static phase 1.4+/-0.1, the regression phase 2.9+/-0.3 days, and the inter-wave interval was 4.2+/-0.3 days. The mean growth rate during the growing phase was 1.8+/-0.1mm/day, while the duration of the interval from 6mm to maximum diameter was 1.4+/-0.1 days. The mean maximum diameter of the dominant follicle was 8.4+/-0.3mm (range: 6.2-11.2) and mean diameter of the largest subordinate follicle was 5.4+/-0.1mm. There was an inverse relationship between the size of the largest follicle and the total number of follicles (r=-0.21, P=0.002). Follicle activity alternated between ovaries in 77% of the waves, with 40% of dominant follicles present in the left ovary and 60% in the right ovary. Plasma estradiol-17beta concentrations also had a wave-like pattern, varying between 12.0 and 62.8 pmol/l. Plasma progesterone concentrations remained below 5.0 nmol/l and there was no ultrasonographic evidence of ovulation during the study.     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Follicular dynamics during the ovulatory season in goats

Growth and regression of ovarian follicles>or=3 mm were studied by transrectal ultrasonography for 4 interovulatory intervals in each of 5 Saanen goats. The observed number of growing identified 4-mm follicles per day differed (P<0.05) from randomness, indicating that follicles, on the average, emerged in groups (waves). Averaged over all interovulatory intervals, the number of 3-mm follicles on each day that later reached >or=6 mm followed a pattern of significant peaks on Days 0 (ovulation), 4,8 and 14. A follicular wave was defined by consecutive days of entry of follicles>or=6 mm into the wave, and the day of emergence was defined as the first day that the >or=6 mm follicles were 3 mm. In 15 of 20 (75%) interovulatory intervals, 1 wave emerged during each of Day -2 to Day 1 (Wave 1); Days 2 to 5 (Wave 2); Days 6 to 9 (Wave 3); and Days 10 to 15 (Wave 4). Ovulation occurred during Wave 4. The mean days of emergence of Waves 1 to 4 were Days -1, 4, 8 and 13, respectively. However, in 5 of these 15 interovulatory intervals, 50% of the apparent waves merged or were continuous so that a distinction could not be made between 2 waves. The largest follicle grew to a larger (P<0.05) maximum diameter for Waves 1 (8.7+/-0.3 mm) and 4 (9.7+/-0.3 mm) than for Waves 2 (7.2+/-0.2 mm) and 3 (7.3+/-0.2 mm). The following observations suggested that the phenomenon of follicular dominance was more common during Waves 1 and 4 than during Waves 2 and 3: 1) the interwave intervals (days) were longer (P<0.05) for Waves 1 (3.4+/-0.2) and 4 (4.3+/-0.6) than for Waves 2 and 3 (2.5+/-0.2 for each wave) and 2) the correlation between maximum diameter of largest follicle and the subsequent interwave interval was significant for Waves 1 and 4 but not for Waves 2 and 3. The 5 remaining interovulatory intervals were irregular and involved more than 4 waves, including 2 interovulatory intervals with prolonged follicular phases (14 and 21) and failures of ovulation. In conclusion, the predominant follicular-wave pattern was 4 waves with ovulation from Wave 4, and apparent follicular dominance was expressed during some follicular waves, especially during Waves 1 and 4.     

Effects of unilateral ovariectomy on follicular development and ovulation in cattle

In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers.     

In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.     

Ultrasound and endocrine evaluation of the ovarian response to PGF2alpha given at different stages of the luteal phase in ewes

The purpose of this study was to evaluate the ovarian response of ewes to two treatments with PGF2alpha using transrectal ovarian ultrasonography and hormone measurements. Fifteen milligrams of PGF2alpha was given to six cyclic Western White Face (WWF) ewes early in the estrous cycle (Days 4 to 7) and to six late in the cycle (Days 10 to 12 after ovulation), and a second treatment was given 9 days after the first. Ultrasound scanning and blood sampling started 7 days prior to the first PGF2alpha treatment and ended 10 days (scanning) or 19 days (blood sampling) after the second PGF2alpha treatment, for both groups of ewes. Mean ovulation rate (2.6 +/- 0.7) did not differ significantly between the ewes first treated early or late in the cycle, or after the first or second treatments with PGF2alpha. The time from treatment to ovulation was longer in ewes first treated early (4.0 +/- 0.3 days) compared to late (2.8 +/- 0.4 days) in the cycle (P < 0.05). Both the number of ovulations (range: 0-7) and time from treatment to ovulation (range: 1-9 days) were highly variable. This variability appeared to be due to the extension of the life span of ovulating follicles that emerged prior to PGF2alpha administration and also ovulation of some follicles that emerged after treatment. When results for first and second treatments were pooled, the total number of follicles > 5 mm in diameter on the day of treatment that failed to ovulate in response to PGF2alpha was higher in ewes first treated early (0.8 +/- 0.2/ewe) compared to late (0.3 +/- 0.2/ewe) in the cycle (P < 0.05). The proportion of detected luteal structures relative to the number of ovulations was lower in ewes first treated early compared to late in the cycle (60 and 86%, respectively; P < 0.05). Disruption of ovulatory follicle dynamics and normal luteogenesis, and variability in the timing of ovulation after PGF2alpha treatments could all contribute to poor or variable fertility when prostaglandins are used for estrus synchronization.     

Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high‐resolution ultrasonography

To date, ultrasonography of monkey ovaries is rare and typically of low resolution. The objectives of this study were to use state‐of‐the‐art, high‐resolution, transabdominal ultrasonography with real‐time Doppler capabilities to: (1) determine whether one can reliably detect in real time the large dominant follicle, the corpus luteum (CL), and small (<2 mm) antral follicles on the ovaries of rhesus monkeys during the natural menstrual cycle; and (2) predict the follicular response of rhesus ovaries to controlled ovarian stimulation (COS) protocols. Rhesus monkeys were selected for transabdominal ultrasonography using a GE Voluson 730 Expert Doppler System at discrete stages of the menstrual cycle. Subsequently, serial ultrasound scanning was employed to observe growth of antral follicles and the CL. Finally, females were scanned to assess follicular growth during COS. The dominant structure and small antral follicles (<2 mm) were reliably visualized in real time. The follicle destined to ovulate could be identified by size differential by day 3 of the follicular phase. The number of small antral follicles present before onset of COS protocol correlated positively with the number of metaphase II‐stage oocytes collected after treatment. The results of this study demonstrate that the population dynamics of antral follicle pools can be noninvasively evaluated in monkeys during natural and pharmacologic ovarian cycles. Am. J. Primatol. 71:384–392, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of luteinizing hormone on superovulatory and steroidogenic responses of rat ovaries to infusion with follicle-stimulating hormone

Immature female rats were infused s.c. continuously over a 60-h period with a partially purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH activity 4.2 x NIH-FSH-S1 and luteinizing hormone (LH) activity 0.022 x NIH-LH-S1. High rates of superovulation were observed in rats receiving 1 U FSH/day, with 69 +/- 11 oocytes/rat recovered as cumulus-enclosed oocytes from oviducts on Day 1 (equivalent to the day of estrus). Addition of LH to the FSH, at dosages equivalent to 2.5-100 micrograms/day NIH-LH-S1 equivalents (2.5-100 mU) resulted in a dose-related inhibition of superovulation, reaching a nadir of 15 +/- 7 oocytes recovered from rats receiving 50 mU LH/day together with 1 U FSH/day. At the two highest LH doses, 50 and 100 mU/day, ovulation was advanced so that 12 +/- 3 and 15 +/- 4 oocytes, respectively, were recovered from oviducts of these rats flushed on the morning of Day 0, compared to none in rats infused with FSH alone. Ovarian steroid concentrations (ng/mg) observed on the morning of Day 0 in rats infused with FSH alone were progesterone, 0.50 +/- 0.13; testosterone, 0.16 +/- 0.08; androstenedione, 0.06; and estradiol, 0.23 +/- 0.05. On the morning of Day 1, ovarian progesterone concentrations in rats infused with FSH alone had risen to 3.30 +/- 0.33 ng/mg, whereas concentrations of testosterone, androstenedione, and estradiol, had fallen to essentially undetectable levels. Addition of LH to the FSH infusion resulted in dose-related increases, on Day 0, of all four steroids up to a dosage of 25 mU LH/day. At higher LH dosages, Day 0 ovarian concentrations of androgens and estradiol fell markedly, while progesterone concentrations continued to increase. Histological examination of ovaries revealed increases in the extent of luteinization of granulosa cells in follicles with retained oocytes on both Days 0 and 1 in rats infused with 25 and 50 mU LH/day together with 1 U FSH/day, compared to those observed in rats receiving FSH alone. These findings indicate that the elevated progesterone levels on Day 0 and inhibition of ovulation observed at these LH doses were due to premature luteinization of follicles, thus preventing ovulation. At lower LH doses, no sign (based on histologic or steroidogenic criteria) of premature luteinization was evident, suggesting that the decreased superovulation in these rats was due to decreased follicular maturation and/or increased atresia rather than to luteinization of follicles without ovulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicular growth monitoring in the female cat during estrus

Follicular growth in the feline ovary is usually detected indirectly, through behavior observation, vaginal smears, or more invasively, by estradiol assay in blood. This study was designed to describe follicular dynamics by transabdominal ultrasonography. Secondly, the stage of follicular growth was associated to behavioral and vaginal changes. Ovarian ultrasonography was performed during nine anovulatory and 12 ovulatory cycles. Forty-eight follicles were followed during anovulatory cycles: on the first day of estrus behavior, 4.8 ± 0.2 follicles (2 to 7 per female) of 2.3 ± 0.01 mm mean diameter were present. Follicular growth continued at a rate of 0.2 ± 0.04 mm per day. At least one follicle in the cohort reached a diameter greater than 3.0 mm. Maximal follicular growth (when one follicle of the cohort reached the maximal diameter observed for the whole estrus) was reached 3.8 ± 0.3 days after the onset of estrus with the largest follicle reaching a diameter of 3.5 ± 0.04 mm. Growth of the various follicles within a cohort was not exactly synchronous. When no ovulation took place, the follicular diameter decreased by 0.1 ± 0.01 mm per day until the end of estrus. The first day after the end of behavioral estrus, the diameter of the largest follicle in each cohort was 2.7 ± 0.05 mm. No correlation was found between follicular development and either vaginal smear characteristics, or time elapsed since the onset of estrus. When ovulations were mechanically induced after one follicle had reached 3.0 mm in diameter, artificial insemination produced normal pregnancy rate and litter size: four pregnant females out of nine, and 2 to 4 kittens per litter. Ultrasonography proved thus to allow the monitoring of follicular growth in the female cat, with low correlation with behavior and vaginal smear modifications. Further studies are needed to evaluate the interest of an ultrasonographic ovarian follow-up to determine the optimal moment for ovulation induction prior to artificial insemination.     

Cytologic, hormonal, and ultrasonographic correlates of the menstrual cycle of the New World monkey Cebus apella

Few reports on the reproductive physiology of Cebus apella have been published. In this study we characterized menstrual cycle events by means of vaginal cytology, ultrasonography (US), and hormonal measurements in serum during three consecutive cycles in 10 females, and assessed the probability that ovulation would occur in the same ovary in consecutive cycles in 18 females. The lengths and phases of the cycles were determined according to vaginal cytology. Taking the first day of endometrial bleeding as the first day of the cycle, the mean cycle length +/- SEM was 19.5+/-0.4 days, with follicular and luteal phases lasting 8.2+/-0.2 and 11.3+/-0.4 days, respectively. The follicular phase included menstruation and the periovulatory period, which was characterized by the presence of a large number of superficial eosinophilic cells in the vaginal smear. The myometrium, endometrium, and ovaries were clearly distinguished on US examination. During each menstrual cycle a single follicle was recruited at random from either ovary. The follicle grew from 3 mm to a maximum diameter of 8-9 mm over the course of 8 days, in association with increasing estradiol (E(2)) serum levels (from 489+/-41 to 1600+/-92 pmol/L). At ovulation, the mean diameter of the dominant follicle usually decreased by >20%, 1 day after the maximum E(2) level was reached. Ovulation was associated with an abrupt fall in E(2), a decreased number of eosinophilic cells, the presence of leukocytes and intermediate cells in the vaginal smear, and a progressive increase in progesterone (P) levels that reached a maximum of 892+/-65 nmol/L on days 3-6 of the luteal phase. The menstrual cycle of Cebus apella differs in several temporal and quantitative aspects from that in humans and Old World primates, but it exhibits the same correlations between ovarian endocrine and morphologic parameters.     

Waves of follicle development during the estrous cycle in sheep

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.     

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n=37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6+/-0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5+/- 0.6 versus day 10 = 10.4 +/-0.6 primary follicles/section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.     

Ovarian dynamics and their associations with peripheral concentrations of gonadotropins,ovarian steroids,and inhibin during the estrous cycle in goats

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.     

Ovarian follicles, ovulations and progesterone concentrations in aged versus young mares

The objectives of this study were: 1) to document age-related ovulation failure in mares and 2) to contrast the number of ovarian follicles, occurrence of ovulations, and postovulatory concentrations of progesterone in aged versus young mares. In Experiment 1, 4 of 10 aged (25- to 33-years-old) mares were anovulatory between July 1 and September 1, 1989. In Experiment 2, two of 25 aged (20- to 30-years-old) and none of 21 young (3- to 12-years-old) mares were anovulatory between February 1 and June 30, 1990. The average (+/- SEM) day of the first ovulation was later (P<0.05) for aged versus young mares (May 9 +/- 7.1 vs April 25 +/- 7.4 days, respectively). There tended (P<0.10) to be fewer 11- to 20-mm ovarian follicles in aged versus young mares (2.8 +/- 0.2 vs 5.3 +/- 0.1, respectively), but there was no difference (P>0.10) in the total number of ovarian follicles in aged versus young mares (21.0 +/- 0.3 vs 26.1 +/- 0.2, respectively) during the pooled periovulatory period of the first and second (single) ovulations. The number of ovulatory cycles during the study period was less (P=0.01) for aged versus young mares (2.2 +/- 0.3 vs 3.2 +/- 0.3). Plasma progesterone concentrations on Days 10 and 15 of the first ovulatory cycle were higher (P<0.05) in aged versus young mares.     

Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats

Ovarian changes determined by daily transrectal ultrasonic scanning, and its correlation with serum progesterone (P4) and estradiol (E2) concentrations were studied in seven cyclic Saanen goats. Estrous cycles were synchronized with 2 injections of a PGF2 alpha analogue 9 d apart. All follicles > or = 2 mm in diameter and CL were measured each day. One goat showed a longer interestrous interval, associated with development of a cystic-luteinized structure. The mean interovulatory interval for the other 6 goats was 20.8 +/- 0.4 d. The incidence of goats with 4, 3, and 2 follicular waves was 3, 1 and 2 respectively; follicular waves emerged on Days 0.5 +/- 0.6, 7.2 +/- 0.7, 10.7 +/- 0.5 and 13.7 +/- 0.8 for Wave 1, 2, 3 and the Ovulatory wave, respectively. The largest follicle of Wave 2 was smaller (4.9 +/- 0.1 mm) than the largest follicles of Wave 3 (6.2 +/- 0.1 mm; P < or = 0.01) and of the Ovulatory wave (7.0 +/- 0.5 mm; P < or = 0.01), and tended to be smaller than the largest follicle of Wave 1 (6.3 +/- 0.6 mm; P < or = 0.09). Interval between emergence of Wave 1 and Wave 2 was longer than interval between emergence of Wave 2 and Wave 3 (7.3 +/- 0.9 d vs 4.0 +/- 0.4 d; P < or = 0.01), and between Wave 3 and the Ovulatory wave (3.8 +/- 1.1 d; P < or = 0.05). Two days before ovulation, the diameter of the ovulatory follicle was larger (P < or = 0.01) than the first subordinate follicle. Serum E2 concentrations increased from the day of ovulation (2.7 +/- 0.3 pg/mL) to Day 2 (7.6 +/- 0.9 pg/mL; P < or = 0.01), associated with the early-mid growing phase of the largest follicle of Wave 1, and then decreased to basal levels on Day 5 (P < or = 0.01) and peaked again (16.5 +/- 2.4 pg/mL) 2 d before ovulation. The CL were detected ultrasonically on Day 3 post ovulation and attained a mean maximum diameter of 13.5 +/- 0.8 mm between Days 8 and 14. The following characteristics were observed: 1) ovarian follicular development in goats is wave-like; 2) increased P4 concentrations may be promoting follicular wave turnover; 3) it is suggested that the presence of follicular dominance and the production of E2 are different among waves. While in Wave 1 and in the Ovulatory wave, follicular dominance is present and production of E2 is consistent, no changes in serum E2 concentrations were found in other stages of the interovulatory interval. In the intervening waves, no indicators of follicular dominance could be firmly documented.     

Two days of pulsatile GnRH infusion beginning 4 days before weaning in sows initiates a wave of follicular growth that is not sustained after weaning

Intervals to estrus and ovulation in weaned sows depend partially on the diameter of ovarian follicles at weaning. The objective was to determine if follicular diameter in sows could be increased by a 48h period of GnRH infusion before weaning and whether this pre-weaning growth would advance follicular development after weaning. The posterior vena cava was cannulated in eight sows at 10+/-1 day after farrowing. Sows were randomly assigned to receive intravenous treatment with either 2mL of GnRH (1microg/mL; n=4) or 2mL of saline (n=4) every 0.5h for 48h beginning 94h before weaning. The average follicular diameter and the number of follicles within diameter classes were determined daily by ultrasonography. Serum LH concentrations increased on the first infusion day but serum LH was equal to control on the last infusion day (P<0.077). The GnRH infusion increased the average diameter of ovarian follicles (P<0.001). Serum estradiol increased (P<0.001) and serum FSH decreased (P<0.016) coincident with GnRH-induced follicular development but these changes were reversed within 24h after the end of the infusion period. Follicles that grew in response to GnRH regressed and were replaced by a new population of follicles within 4 days after weaning. Within the experimental model for the present study, a GnRH infusion increased follicular growth in lactating sows but follicles could not be sustained beyond the end of GnRH infusion.     

Differences in ovarian activity between booroola X merino ewes which were homozygous, heterozygous and non-carriers of a major gene influencing their ovulation rate

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.