Developmental programming: exogenous gonadotropin treatment rescues ovulatory function but does not completely normalize ovarian function in sheep treated prenatally with testosterone

Prenatal testosterone treatment leads to LH excess as well as ovarian follicular and ovulatory defects in the adult. These disruptions may stem from LH excess, abnormal FSH input, compromised ovarian sensitivity to gonadotropins, or intrinsic ovarian defects. To determine if exogenous gonadotropins rescue ovarian and ovulatory function of testosterone-treated sheep, the release of endogenous LH and biopotent FSH in control and prenatal testosterone-treated sheep was blocked with a GnRH antagonist during the first two breeding seasons and with LH/FSH coadministered in a manner approximating natural follicular phase. An acidic mix of FSH was administered the first 36 h at 2-h intervals and a less acidic mix for the next 12 h at 1-h intervals (different FSH preparations were used each year), and ovulation was induced with hCG. Circulating FSH and estradiol responses to gonadotropins measured in 2-h samples differed between treatment groups in Year 1 but not in Year 2. Ovarian follicular distribution and number of corpora lutea (in ewes that ovulated) tracked by ultrasonography and luteal progesterone responses were similar between control and prenatal testosterone-treated females but differed between years. Furthermore, hCG administration induced large cystic and luteinized follicles in both groups of females in Year 2, although the growth rate differed between control and prenatal testosterone-treated females. Our findings provide evidence that 1) ovulatory response in prenatal testosterone-treated females can be rescued with exogenous gonadotropins, 2) resultant follicular response is dependent on the nature of gonadotropic input, and 3) an abnormal follicular milieu may underlie differences in developmental trajectory of cystic follicles in prenatal testosterone-treated females.     

Post partum patterns of circulating FSH, LH, prolactin, estradiol, and progesterone in nonsuckling cynomolgus monkeys.

Circulating levels of FSH, LH, prolactin (Prl), estradiol (E), and progesterone (P) were determined by RIA in four intact and four monkeys luteectomized (CLX) at parturition in order to a) characterize the patterns of these hormones during the puerperium, and b) examine a possible inhibitory role of the "rejuvenated" corpus luteum (CL) on the resumption of follicle growth post partum. In both groups during the first four weeks, FSH and LH were at tonic levels typical of ovulatory cycles. Recurrent puerperal "surges" of FSH, but not LH, unaccompanied by increments in serum E, were observed in both intact and CLX monkeys. No consistent pattern of serum Prl was apparent. CLX was followed by a prompt fall in serum P levels, which were elevated above typical follicular phase levels into the second week post partum in intact monkeys. Menstrual cycles resumed 2-4 months after delivery. Hormonal patterns during the first menstrual cycle post partum were indistinguishable from those observed in pregravidic ovulatory cycles. The findings indicate that in nonsuckling cynomolgus monkeys a) although it secretes progesterone, the puerperal CL does not inhibit the resumption of the ovarian cycle post partum, b) the puerperal ovary is not absolutely refractory to gonadotropins, since initial trials with Pergonal + hCG stimulated ovarian function, and c) ovarian activity during the puerperium may be limited by factors other than the tonic supply of gonadotropins.     

Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.     

Changes in plasma progesterone, estradiol, follicle-stimulating hormone and luteinizing hormone during diestrus and ovulation in rats with 5-day estrous cycles: effect of antibody against progesterone

Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5-day cycle advances ovulation and completion of the cycle by 1 day in the majority of animals (75 and 80%, respectively). Progesterone (250 micrograms) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release.     

Ovarian follicular and luteal dynamics in wapiti during seasonal transitions

Transitions from the anovulatory to the ovulatory season (n=20) and ovulatory to anovulatory season (n=11), were monitored daily by transrectal ultrasonography in wapiti. In 17 of 20 observations, the first interovulatory interval (IOI) was short (9.1+/-0.3 days; mean+/-S.E.M.) compared with later in the ovulatory season (21.3+/-0.1) and the last IOI (21.2+/-0.6 days). With one exception, the short IOI were composed of only one wave of follicular development. Subsequent IOI were composed of two or three waves. Maximum diameters of the first two ovulatory follicles were similar (11.3+/-0.4 mm versus 11.3+/-0.2 mm), but both were larger (P<0.05) than the last two ovulatory follicles of the ovulatory season (10.3+/-0.3 and 10.1+/-0.4 mm). Multiple ovulations occurred in three hinds at the first ovulation of the season and in one hind at the second ovulation, but were not at any other time. Day-to-day profiles of CL diameter and plasma progesterone concentration were smaller (P<0.05) for short versus long IOI. Maximum diameter (12.8+/-0.6 mm versus 12.5+/-0.6 mm) and the diameter profile of the last CL of the season were not different from that of the previous CL. In summary, transition to regular ovulation occurred over a 3-week interval and was preceded by one short IOI (9 days). Multiple ovulations were detected only at the onset of the ovulatory season. The characteristics of the last IOI of the ovulatory season were similar to those reported during the rut. The wave pattern of follicle development was maintained throughout both fall and winter transition periods and follicular wave emergence was preceded by a surge in serum FSH concentrations. Transition to anovulation occurred over a 3-month interval and was marked by a failure of the dominant follicle to ovulate after a typical luteal phase.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and number of embryos collected from superovulated Holstein heifers

Mature Holstein heifers were induced to superovulate with twice-daily injections of porcine follicle-stimulating hormone (FSH), and were given either 20 mg i.m. of recombinant bovine somatotrophin (rBST) or saline with each FSH injection. The animals were artificially inseminated and the embryos were collected nonsurgically at Day 7. There was no significant difference in the mean (+/-S.D) total number of embryos collected from rBST-treated animals (8.3+/-5.3) when compared with that of the controls (7.2+/-6.6), or in the mean number of transferable embryos (5.3+/-4.0 vs 5.2+/-4.5). However, co-treatment with rBST tended to increase the ovulatory response, and it significantly increased plasma progesterone concentrations at Day 6 (P = 0.04). Based on these latter observations, we conclude that treatment with rBST enhanced the superovulatory response in heifers.     

Opioidergic control of gonadotropin secretion in the female rabbit: divergent effects of morphine on secretion of follicle-stimulating hormone and luteinizing hormone

The nature of secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was followed in female rabbits on a daily basis from age 36 to 60 days by sequential 5-min blood sampling over 1- to 2-h periods each day. Both LH and FSH were found to be secreted in a pulsatile manner. The mean LH pulse amplitude over the 25 days was 0.95 +/- 0.32 ng/mL and for FSH it was 10.15 +/- 1.11 ng/mL. Mean plasma LH levels were significantly increased from 1.46 +/- 0.08 ng/mL in 36 to 42-day-old rabbits to 1.89 +/- 0.12 ng/mL in 43 to 50-day-old rabbits and remained elevated from 50 to 60 days. FSH levels during the same periods also rose significantly from 14.93 +/- 0.79 to 19.57 +/- 2.05 ng/mL. To examine the influence of endogenous opioid peptides on the release of LH and FSH in 36 to 60-day-old female rabbits, morphine sulfate at 0.2, 0.5, 2.0, and 5.0 mg/kg was administered subcutaneously after 30 min baseline sampling, and blood was taken for another 60-120 min. Morphine at all doses and at all ages inhibited the amplitude and frequency of LH pulses but had no effect on FSH secretion. To determine whether the effects of morphine on LH secretion could be reversed with naloxone, females aged 82-114 days were used. Naloxone administered 1 h after morphine reversed the inhibitory effects of morphine, whereas the simultaneous administration of naloxone with morphine had variable effects but seemed to delay the LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Associations between emergence of follicular waves and fluctuations in FSH concentrations during the estrous cycle in ewes

Folliculogenesis was studied daily in 16 interovulatory intervals in 5 Polypay ewes from mid February through April using transrectal ultrasonic imaging. The 3-mm follicles attaining > or = 5 mm on Days--1 (ovulation=Day 0) to 11 showed significant peak numbers on Days 0, 5 and 10. The number of 3- and 4-mm follicles that did not reach > 4 mm was not significant, indicating that these follicles did not manifest a wave pattern. A follicular wave was defined as one or more follicles growing to > or = 5 mm; the day the follicles were 3 mm was the day of wave emergence, and the first wave after ovulation was Wave 1. Waves 1, 2 and 3 emerged on Days -1 to 2,4 to 7 and 8 to 10, respectively. Four interovulatory intervals in April were short (9 to 14 d), indicating the end of the ovulatory season. In the remaining 12 intervals, the ovulatory wave was Wave 3 in one interval, Wave 4 in 8 intervals, and Wave 5 or 6 in 3 intervals. The ovulatory wave followed the rhythmic pattern of Waves 1 to 3 by emerging on Days 11 to 14 in 50% of the intervals. In the remaining intervals, either the ovulatory wave was Wave 4 but did not emerge until Day 16 or other waves intervened between Wave 3 and the ovulatory wave. The longest intervals (22, 24 and 24 d) had >4 waves. Based on a cycle-detection program, peak values of FSH fluctuations were temporally associated with the emergence of waves as indicated by the following: 1) tendency (P < 0.08) for an increase in FSH concentrations between 3 and 2 days before emergence of a wave; 2) close agreement between mean number of waves per interval (mean +/- SEM, 4.1 +/- 0.3) and mean number of identified FSH fluctuations (4.5 +/- 0.3); 3) close agreement in length of interwave intervals (4.0 +/- 0.3) and interpeak (FSH) intervals (3.6 +/- 0.2); 4) positive correlation (r(2)=0.8) for number of the 2 events (follicular waves and FSH fluctuations) within intervals; and 5) a closer (P < 0.01) temporal relationship between the 2 events than would have been expected if the events were independent. The results support a relationship between transient increases in FSH concentrations and emergence of follicular waves throughout the interovulatory interval in Polypay ewes, with the 2 events occurring approximately every 4 d.     

Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary

In isolated, perfused ovaries of rats treated with pregnant mare's serum gonadotropin (PMSG), purified preparations of ovine follicle-stimulating hormone (FSH) (oFSH-211B) and rat FSH (rFSH-I-6), 100 ng/ml, were found to induce ovulations (4.8 +/- 0.9, n = 4, and 6.4 +/- 2.0, n = 5, ovulations per ovary, respectively). Indomethacin (5 micrograms/ml) added to the perfusate inhibited this ovulatory effect and exogenous prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), or prostaglandin E2 (PGE2) (0.5 microgram/ml), reversed the blockade. Ovine FSH and rFSH had only a weak stimulatory effect on estradiol release, and only rFSH caused a significant increase in progesterone accumulation. Indomethacin reduced the stimulatory effect of rFSH on progesterone release, and this effect was reversed by PGE2 but not by PGF2 alpha. In a 6-h incubation experiment with preovulatory rat follicles, we tested the biological activity of gonadotropins used to induce oocyte maturation. The concentration of FSH used in the perfusion experiments induced oocyte maturation in more than 88% of the oocytes studied. The data confirm earlier findings that FSH can induce ovulations and show that prostaglandins are involved in this process. The data also indicate that prostaglandins might be involved in the FSH-induced increase of progesterone levels.     

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.     

Follicular dynamics and concentrations of steroids and gonadotropins in lactating cows and nulliparous heifers

Differences in follicular development and circulating hormone concentrations, between lactating cows and nulliparous heifers, that may relate to differences in fertility between the groups, were examined. Multiparous, cyclic, lactating Holstein cows (n=19) and cyclic heifers (n=20) were examined in the winter, during one estrous cycle. The examinations included ultrasound monitoring and daily blood sampling. Distributions of two-wave and three-wave cycles were similar in the two groups: 79 and 21% in cows, 70 and 30% in heifers, respectively. Cycle lengths were shorter by 2.6 days in heifers than in cows, and in two-wave than in three-wave cycles. The ovulatory follicle was smaller in heifers than in cows (13.0+/-0.3 mm versus 16.5+/-0.05 mm). The greater numbers of large follicles in cows than in heifers corresponded well to the higher concentrations of FSH in cows. The duration of dominance of the ovulatory follicle tended to be longer in cows than in heifers. Estradiol concentrations around estrus and the preovulatory LH surge were higher in heifers than in cows (20 versus 9 ng/ml). Progesterone concentrations were higher in heifers than in cows from Day 3 to Day 16 of the cycle. Circulating progesterone did not differ between two-wave and three-wave cycles. The results revealed differences in ovarian follicular dynamics, and in plasma concentrations of steroids and gonadotropins; these may account for the differences in fertility between nulliparous heifers and multiparous lactating cows.     

The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

Response of sows to oestradiol benzoate treatment after weaning at two stages of lactation

The timing and dosage of oestradiol benzoate injected after weaning was critical with respect to the pattern of behavioural oestrus and the ovarian stimulation achieved; treatment on the day of weaning (Day 0) and Day 1 with 60 micrograms oestradiol benzoate/kg body wt produced poor ovulatory responses and abnormal oestrous behaviour. Treatment on Day 2 with 30 micrograms oestradiol benzoate/kg resulted in consistent oestrus and ovulatory responses although the ovulation rates (10 . 6 +/- 1 . 1 in 3-week and 12 . 2 +/- 1 . 7 in 5-week weaned sows) were below those expected in fertile untreated sows weaned at these times. The timing of the preovulatory LH surge (53 . 6 +/- 2 h after oestradiol benzoate) was closely synchronized in all sows and a similar synchronous rise in plasma progesterone concentrations 100 +/- 4 h after oestradiol benzoate suggests a similar synchrony of ovulation. The magnitude of the LH and FSH responses to oestradiol benzoate were similar to those that occur in untreated sows and similar differences also existed in gonadotrophin secretion related to the length of lactation.     

Effect of long-term treatment with recombinant bovine somatotropin and estradiol on hormone concentrations and ovulatory response of superovulated cattle

The objective was to assess effects of long-term treatment with recombinant bovine somatotropin (bST) and estradiol-17beta (E2) on the number of follicles that ovulated in response to FSH. Non-lactating Holstein and Jersey cows (Trial 1, n=27) and Angus cows and heifers (Trial 2, n=35) received two ear implants of E2 and biweekly injections of bST in a 2 x 2 arrangement of treatments. Estradiol implants were removed 74.6 +/- 1.1 d after insertion and 18.1 +/- 0.9 d after the last biweekly injection of bST. Cows were stimulated with FSH-P beginning 2 d after removal of E2 implants, and PGF2alpha (PGF) was given on the third day of FSH treatment. Ovaries were collected to determine the number of CL at 1 to 2 wk after treatment with PGF. In Trial 2 only, cattle were inseminated at estrus and embryos were collected 6 to 8 d later. Implants of E2 increased (P < 0.01) serum E2 8-fold initially and E2 was still elevated 5-fold at removal of implants. Injections of bST increased (P < 0.01) serum growth hormone (GH) 15-fold and insulin-like growth factor-I (IGF-I) 3-fold. In Trial 1, number of CL was increased by the combination of bST+E2 (P < 0.01). In Trial 2, E2 increased the number of CL (P < 0.05), and bST increased the number of total ova and transferable embryos (P < 0.01). We conclude that long-term treatment with bST and E2 may interact to enhance follicular development and ovulatory response to FSH.     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.     

Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep

The GnRH-antagonist suppression-ovarian autotransplant model (n = 18) was used to examine the relative roles of temporal changes in FSH and LH stimulation on follicle development and selection. Follicle development was stimulated by infusion with oFSH for 3 days and treatments applied for 60 h after progestagen sponge withdrawal and before delivery of an ovulatory stimulus. In Expt 1, there was continuous infusion of FSH with or without small amplitude high frequency LH pulses, or withdrawal of FSH with or without pulsatile LH. In Expt 2, there was acute or gradual withdrawal of FSH at sponge withdrawal with pulsatile LH. The patterns of follicle development and basal and pulsatile ovarian hormone secretion were determined. The maintenance of FSH throughout the artificial follicular phase resulted in multiple follicle development and ovulation (3.3 +/- 0.3). Pulsatile LH stimulated steroid secretion (P < 0.001) but had little effect on ovulation rates (3.8 +/- 0.8) when FSH was maintained. However, withdrawal of FSH in the absence of LH resulted in atresia of the ovulatory follicles and anovulation whereas, when FSH was withdrawn in the presence of LH, preovulatory follicle development was maintained in some animals (3/6 and 5/9 in Expts 1 and 2, respectively) and these ewes had lower (P < 0.05) ovulation rates (1-2 ovulations per ewe). When FSH was withdrawn gradually in the presence of pulsatile LH, 9/9 animals ovulated with ovulation rates in the normal range. These results indicate that ovulatory follicles can transfer their gonadotrophic dependence from FSH to LH. It is hypothesized that the ability of a follicle to respond to this switch in gonadotrophic support is central to the mechanism of follicle selection.