Characteristics of peaks in serum concentrations of follicle-stimulating hormone and estradiol, and follicular wave dynamics during the interovulatory interval in cyclic ewes.

There are three or four ovarian follicular waves in the interovulatory interval of cyclic ewes. Each follicular wave is preceded by a transient peak in serum follicle-stimulating hormone (FSH) concentrations. Serum concentrations of estradiol also increase concurrent with the growth of follicle(s) in each wave. In the current study, we investigated the patterns of follicular wave development and characteristics of FSH and estradiol peaks in all follicular waves of the interovulatory interval and after induction of a supraphysiologic FSH peak in cyclic ewes (Ovis aris). In Experiment 1, 19 ewes underwent daily ovarian ultrasonography and blood sampling for a complete interovulatory interval. In Experiment 2, seven ewes received two administrations of ovine FSH (oFSH), 8h apart (1 microg/kg; sc), at the expected time of the endogenous FSH peak preceding the second follicular wave of the interovulatory interval. In Experiment 1, the amplitude of the FSH peaks decreased (up to 50%), whereas basal serum FSH concentrations increased across the interovulatory interval (P<0.05). Maximum follicular diameter was greater (P<0.05) for Wave 1 and the Ovulatory wave (6.0+/-0.3 and 6.1+/-0.2 mm, respectively) than for Waves 2 and 3 (5.3+/-0.1 and 5.4+/-0.3 mm, respectively). Life span was greater for follicles in Wave 1 compared with other waves (P<0.05). Treatment with oFSH increased the amplitude of an FSH peak by 5- to 6-fold. This treatment increased estradiol production (P<0.05) but had little effect on other characteristics of the subsequent follicular wave. We concluded that changes in the amplitude and duration of the peaks in serum concentrations of FSH that precede follicular waves across the interovulatory interval do not influence the characteristics of the follicular waves that follow.

Ovarian antral follicular dynamics in sheep revisited: comparison among estrous cycles with three or four follicular waves.

In this study, the characteristics of ovarian follicular waves and patterns of serum concentrations of follicle-stimulating hormone (FSH), estradiol, and progesterone were compared between cycles with three (n=9) or four (n=10) follicular waves in Western White Face (WWF) ewes (Ovis aries). Transrectal ultrasonography and blood sampling were performed daily during one cycle. Estrous cycles were 17.11+/-0.3 and 17.20+/-0.2 d long in cycles with three and four waves, respectively (P>0.05). The first interwave interval and the interval from the emergence of the final wave to the day of ovulation were longer in cycles with three waves compared with those in cycles with four waves (P<0.05). The growth phase (5.1+/-0.5 vs. 3.1+/-0.4 d) and life span (5.67+/-0.3 vs. 4.3+/-0.3 d) of the largest follicle growing in the last or ovulatory wave was longer in cycles with three waves compared with that in cycles with four waves (P<0.05). The maximum diameter of the largest follicle was greater in the first wave and the ovulatory wave compared with that in other waves of the cycle (P<0.05). The regression phase of the largest follicle growing in the first wave was longer in cycles with three waves compared with that in cycles with four waves (4.44+/-0.4 vs. 3.4+/-0.4 d; P<0.05). The length of the life span, regression phase, and, although not significant in every case, FSH peak concentration and amplitude decreased across the cycle (P<0.05). We concluded that estrous cycles with three or four follicular waves were confined within the same length of cycle in WWF ewes. In this study, there were no apparent endocrine or follicular characteristics that could explain the regulation of the different number of follicular waves (three vs. four) during cycles of similar length.

Synchronization of follicular wave emergence in the seasonally anestrous ewe: the effects of estradiol with or without medroxyprogesterone acetate.

Fertility is often lower in anestrous compared to cyclic ewes, after conventional estrus synchronization. We hypothesized that synchronization of ovarian follicular waves and ovulation could improve fertility at controlled breeding in anestrous ewes. Estradiol-17beta synchronizes follicular waves in cattle. The objectives of the present experiments were to study the effect of an estradiol injection, with or without a 12-d medroxyprogesterone acetate (MAP) sponge treatment, on synchronization of follicular waves and ovulation in anestrous ewes. Twenty ewes received sesame oil (n=8) or estradiol-17beta (350 microg; n=12). Eleven ewes received MAP sponges for 12d and were treated with oil (n=5) or estradiol-17beta (n=6) 6d before sponge removal. Saline (n=6) or eCG (n=6) was subsequently given to separate groups of ewes at sponge removal in the MAP/estradiol-17beta protocol. Estradiol treatment alone produced a peak in serum FSH concentrations (4.73+/-0.53 vs. 2.36+/-0.39 ng/mL for treatment vs. control; mean+/-S.E.M.) after a short-lived (6 h) suppression. Six of twelve ewes given estradiol missed a follicular wave around the time of estradiol injection. Medroxyprogesterone acetate-treated ewes given estradiol had more prolonged suppression of serum FSH concentrations (6-18 h) and a delay in the induced FSH peak (32.3+/-3.3 vs. 17.5+/-0.5 h). Wave emergence was delayed (5.7+/-0.3 vs. 1.4+/-0.7d from the time of estradiol injection), synchronized, and occurred at a predictable time (5-7 vs. 0-4d) compared to ewes given MAP alone. All ewes given eCG ovulated 3-4d after injection; this predictable time of ovulation may be efficacious for AI and embryo transfer.

Effects of treatment with GnRH from 4 to 8 weeks of age on the attainment of sexual maturity in bull calves.

In bull calves an early transient increase in circulating concentrations of LH occurs between 6 and 20 weeks of age. This has been shown to influence reproductive development and performance later in life. In an attempt to hasten the onset of sexual maturity, bull calves (Hereford x Charolais) were treated (im) with 120 ng/kg of GnRH (n=6) twice every day from 4 to 8 weeks of age; control calves received saline (n=6). Injection of GnRH resulted in an LH pulse in all animals. GnRH treated bulls displayed more rapid testicular growth rates between 22 and 44 weeks of age. Sexual maturity (SC>or=28 cm) was achieved earlier in GnRH treated bulls compared to saline treated bulls (41.7+/-2.22 and 47.0+/-0.45 weeks of age, respectively) and this was confirmed by age of sexual maturity based on ejaculate characteristics (>50 million spermatozoa, >10% motility; 45.0+/-0.86 and 49.0+/-1.13 weeks of age for GnRH and control treated bull calves, respectively; P<0.05). We concluded that treatment with GnRH, twice daily, from 4 to 8 weeks of age, prior to the endogenous early increase in plasma LH concentrations, could increase in plasma LH concentrations, advance testicular development and reduce age at puberty in beef bull calves. This may provide the basis for a simple regimen to hasten sexual development in the bull calf.

Short- and long-term effects of unilateral ovariectomy in sheep: causative mechanisms.

The mechanisms of ovulatory compensation following unilateral ovariectomy (ULO) are still not understood. In the present study, we investigated the short- and long-term effects of ULO in sheep using transrectal ovarian ultrasonography and hormone estimations made during the estrous cycle in which surgery was done, the estrous cycle 2 mo after surgery, and the 17-day period during the subsequent anestrus. The ULOs were done when a follicle in the first follicular wave of the cycle reached a diameter > or =5 mm, leaving at least one corpus luteum and one ovulatory-sized follicle in the remaining ovary. Ovulation rate per ewe was 50% higher in the ULO ewes compared with the control ewes at the end of the cycle during which surgery was performed, but it did not differ between groups at the end of the cycle, 2 mo later. This compensation of ovulation rate in ULO ewes was due to ovulation of follicles from the penultimate follicular wave in addition to those from the final wave of the cycle. Ovulation from multiple follicular waves appeared to be due to a prolongation of the static phase of the largest follicle of the penultimate wave of the cycle. Interestingly, the length of the static phase of waves was prolonged in ULO ewes compared with control ewes in every instance where the length of the static phase could be determined. Changes in follicular dynamics due to ULO were not associated with alterations in FSH and LH secretion. In conclusion, ovulatory compensation in ULO sheep involves ovulation from multiple follicular waves due to the lengthened static phase of ovulatory-sized follicles. These altered antral follicular dynamics do not appear to be FSH or LH dependent. Further studies are required to examine the potential role of the nervous system in the enhancement of the life span of the ovulatory-sized follicles leading to ovulatory compensation by the unpaired ovary in ULO sheep.

Ultrasonographic characteristics of ovulatory follicles and associated endocrine changes in cyclic ewes treated with medroxyprogesterone acetate (MAP)-releasing intravaginal sponges and equine chorionic gonadotropin (eCG).

The aim of this study was to assess the ultrasonographic characteristics of ovulatory follicles in cyclic Western White Face ewes (December) that had received intravaginal sponges containing medroxyprogesterone acetate (MAP; 60 mg) for 12 days, with or without an injection of 500 IU of equine chorionic gonadotropin (eCG) at sponge removal. We hypothesized that quantitative echotextural attributes of the follicles in ewes treated only with MAP would differ from those in MAP/eCG-treated ewes, reflecting the increased antral follicular growth and secretory function under eCG influence. Digital images of ovulatory follicles obtained at 0 and 24 h after MAP sponge removal and at 24 h before ovulation in the eCG-treated (five ewes, 13 follicles) and control (six ewes, 9 follicles) animals, were subjected to computerized analyses. The mean diameter of ovulatory follicles increased (p < 0.001) 24 h after eCG treatment. The mean pixel intensity and heterogeneity of the follicular antrum (p < 0.001), as well as mean pixel intensity of the follicular wall and perifollicular ovarian stroma (p < 0.05), were greater in eCG-treated animals compared with control ewes 24 h after sponge removal and at 24 h before ovulation. Mean serum concentrations of oestradiol-17beta tended to increase (p = 0.06) 24 h after eCG treatment and the eCG-treated ewes exceeded (p < 0.05) control animals in progesterone concentrations from days 9-15 after ovulation. Our results support the hypothesis that large antral follicles in eCG-treated ewes exhibit distinctive echotextural characteristics. Follicular image attributes in eCG-treated ewes appear to be indicative of the changes in follicular morphology and secretory activity caused by the administration of the exogenous gonadotropin, which has both FSH- and LH-like activities.

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle.

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.

Does injection of prostaglandin F(2alpha) (PGF2alpha) cause ovulation in anestrous Western White Face ewes?

In a previous study in our laboratory, treatment of non-prolific Western White Face (WWF) ewes with PGF(2 alpha) and intravaginal sponges containing medroxyprogesterone acetate (MAP) on approximately Day 8 of a cycle (Day 0 = first ovulation of the interovulatory interval) resulted in ovulations during the subsequent 6 days when MAP sponges were in place. Two experiments were performed on WWF ewes during anestrus to allow us to independently examine if such ovulations were due to the direct effects of PGF(2 alpha) on the ovary or to the effects of a rapid decrease in serum concentrations of progesterone at PGF(2 alpha)-induced luteolysis. Experiment 1: ewes fitted with MAP sponges for 6 days (n = 12) were injected with PGF(2 alpha) (n = 6; 15 mg im), or saline (n = 6) on the day of sponge insertion. Experiment 2: ewes received progesterone-releasing subcutaneous implants (n = 6) or empty implants (n = 5) for 5 days. Six hours prior to implant removal, all ewes received a MAP sponge, which remained in place for 6 days. Ewes from both experiments underwent ovarian ultrasonography and blood sampling once daily for 6 days before and twice daily for 6 days after sponge insertion. Additional blood samples were collected every 4 h during sponge treatment. Experiment 1: 4-6 (67%) PGF(2 alpha)-treated ewes ovulated approximately 1.5 days after PGF(2 alpha) injection; these ovulations were not preceded by estrus or a preovulatory surge release of LH, and resulted in transient corpora hemorrhagica (CH). The growth phase was longer (P < 0.05) and the growth rate slower (P < 0.05) in ovulating versus non-ovulating follicles in PGF(2 alpha)-treated ewes. Experiment 2: in ewes given progesterone implants, serum progesterone concentrations reached a peak (1.7 2 ng/mL; P < 0.001) on the day of implant removal and decreased to basal concentrations (<0.17 ng/mL; P < 0.001) within 24 h of implant removal. No ovulations occurred in either the treated or the control ewes. We concluded that ovulations occurring after PGF(2 alpha) injection, in the presence of a MAP sponge, could be due to a direct effect of PGF(2 alpha) at the ovarian level, rather than a sudden decline in circulating progesterone concentrations.

The effect of the manipulation of follicle-stimulating hormone (FSH)-peak characteristics on follicular wave dynamics in sheep: does an ovarian-independent endogenous rhythm in FSH secretion exist?

We designed three experiments to investigate the relationship between FSH peaks and ovarian follicular waves and to examine whether an endogenous rhythm of FSH peaks exists in sheep. In experiment 1, anestrous ewes were treated with ovine FSH (oFSH) or vehicle (6 ewes per group) at the expected time of an endogenous FSH peak, to double the FSH-peak amplitude in treated ewes. In experiment 2, anestrous ewes were treated with either oFSH or vehicle (6 ewes per group) at the expected time of two consecutive interpeak nadirs, such that the treated ewes had 5 FSH peaks in the time frame of 3 FSH peaks in control ewes. In experiment 3, to measure FSH concentrations, daily blood samples were collected from 5 cyclic ewes for a control period during the estrous cycle and then for three 17-day periods after ovariectomy. Daily blood samples were collected from another group of 8 ovariectomized ewes that were treated with estradiol-releasing implants and intravaginal progestogen sponges. Doubling the FSH-peak amplitude did not alter the characteristics of the following follicular wave. Increasing the frequency of FSH peaks stimulated the emergence of additional follicular waves, but did not alter the rhythmic occurrence of FSH peaks and follicular wave emergence. Endogenous follicular waves in oFSH-treated ewes emerged and grew in the presence of the growing largest follicle of the induced follicular waves. Finally, based on the observation of serum FSH concentrations in ovariectomized ewes, it appears that there exists an endogenous rhythm for peaks in daily serum FSH concentrations, which is, at least in part, independent of regulation by ovarian follicular growth patterns.

Effects of treatment with LH or FSH from 4 to 8 weeks of age on the attainment of puberty in bull calves.

A transient increase in gonadotropin secretion between 6 and 20 weeks of age is critical for the onset of puberty in bull calves. To try and hasten the onset of puberty, bull calves were treated (s.c.) with 3 mg of bLH (n = 6) or 4 mg of bFSH (n = 6) once every 2 days, from 4 to 8 weeks after birth; control calves received saline (n = 6). At 4 and 8 weeks of age, mean LH concentrations were higher (P < 0.05) in bLH-treated (2.3 +/- 0.04 ng/ml and 1.20 +/- 0.04 ng/ml) as compared to control calves (0.50 +/- 0.1 ng/ml and 0.70 +/- 0.10 ng/ml). Mean serum FSH concentrations at 4 and 8 weeks of age, were higher (P < 0.05) in bFSH-treated (1.60 +/- 0.20 ng/ml and 1.10 +/- 0.2 ng/ml) as compared to control calves (0.38 +/- 0.07 ng/ml and 0.35 +/- 0.07 ng/ml). The age at which scrotal circumference (SC) first reached > or = 28 cm, occurred earlier (P < 0.05) in bFSH-treated calves as compared to saline-treated calves (39.3 +/- 1.3 and 44.8 +/- 1.3 weeks of age, respectively). Based on testicular histology at 56 weeks of age, treatment with bFSH resulted in greater (P < 0.05) numbers of Sertoli cells (5 +/- 0.2, 6 +/- 0.3 and 5 +/- 0.3 in bLH-, bFSH- and saline-treated calves, respectively); elongated spermatids (42 +/- 2, 57 +/- 8 and 38 +/- 5 in bLH-, bFSH- and saline-treated calves, respectively) and spermatocytes (31 +/- 3, 38 +/- 3 and 29 +/- 2 in bLH-, bFSH- and saline-treated calves, respectively) per seminiferous tubule. We concluded that treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC); hastened onset of puberty (SC > or = 28 cm); and enhanced spermatogenesis.

Ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 IU of eCG following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes.

A standard dose of 500 IU of eCG is commonly given to progestogen pre-treated anestrous ewes for induction of estrus. Twelve seasonally anestrous and 12 cyclic Western White Face ewes were treated for 12 days with intravaginal sponges impregnated with medroxyprogesterone acetate (MAP). In trials in both the breeding and nonbreeding seasons, six randomly selected ewes were given 500 IU of eCG at sponge removal to determine the effects of low dose of eCG on ovarian antral follicular dynamics and ovulation. Ultrasound scanning and blood sampling were done daily. Treatment with eCG did not have marked effects on antral follicular growth. All ewes ovulated, except for five of six control anestrous ewes. Luteal structures and progesterone secretion were confirmed in all but the control anestrous ewes. In the breeding season, peak progesterone concentrations were greater (P<0.05) in eCG-treated compared to control ewes. Daily serum estradiol concentrations were greater in the periovulatory period in eCG-treated compared to control ewes (treatment-by-day interaction; P<0.05), particularly in anestrus. Progestogen-treated ewes ovulated follicles from several follicular waves, in contrast to ovulations of follicles from the final wave of the cycle in untreated, cyclic ewes. Anestrous ewes exhibited more frequent follicular waves and FSH peaks compared to cyclic ewes after a progestogen/eCG treatment. In conclusion, 500 IU of eCG given after 12 days of progestogen treatment had limited effects on the dynamics of ovarian follicular waves. However, eCG treatment increased serum concentrations of estradiol during the periovulatory period, particularly in anestrous ewes; this probably resulted in the synchronous estrus and ovulation in anestrous ewes.

Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes.

Transrectal ovarian ultrasonographic studies have shown that, in cattle, follicular wave emergence is associated with a large increase in the number of small antral follicles (4-6mm in diameter); an analogous association has not been found for small follicles (2-3mm in diameter) in the ewe. In previous studies in ewes, accurate assessment of the number of follicles has been limited to follicles > or =2 or 3mm in size. Newer, high-resolution equipment allowed us to identify follicles > or =0.4mm and to quantify all antral follicles > or =1mm in diameter in seven cyclic Western White Face ewes. This allowed us to expand the small follicle pool examined, from 1 to 4 follicles/day (2-3.5mm in diameter) in earlier studies, to 8-18 follicles/day (1-3mm in diameter). Total number of small follicles (> or =1 and < or =3mm in diameter) increased between Days -1 and 0 (Day 0=day of ovulation), and declined between Days 1 and 3 (P<0.05). There were no significant changes in the number of small or medium (4mm in diameter) follicles around days of follicle wave emergence (+/-2 days). The 1-3 follicles in the 2-3mm size range, which constituted a follicle wave (i.e. grew to > or =5mm in size before regression or ovulation), were the only small follicles to emerge in an orderly succession during the estrous cycle, approximately every 3-5 days. Thus, unlike in cattle, there is no apparent increase in numbers of small follicles at follicle wave emergence in cyclic sheep, and little evidence for selection of recruited follicles and follicular dominance.

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.