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.     

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.     

Development of rabbit embryos during a 96-h period of in vitro culture after superovulatory treatment under conditions of elevated ambient temperature.

The effects of elevated ambient temperature on the response to exogenous gonadotropins were evaluated in female New Zealand White rabbits exposed to 33+/-1 degrees C (mean +/- SE) and 10-30% relative humidity (8 h/day) during a 5-day period. Does were treated with pFSH (0.3 mg/0.3 ml Standard Armour) twice daily during three consecutive days with a minimum interval of 8 h between injections. Six hours after the last FSH injection all does were removed from the experimental chamber, given hCG (25 IU/kg) and paired overnight. Nineteen hours after pairing, embryos were flushed from the reproductive tracts, evaluated, and subjected to in vitro culture during a 96-h period. The ovulatory responses to exogenous gonadotropins and fertilization rates did not differ significantly under conditions of elevated ambient temperature, whereas fewer blastocysts and increased number of degenerate embryos were observed after culture. We conclude that although hyperthermia was induced during exposure to elevated ambient temperature, it did not alter the ovulatory responses to gonadotropin treatment and plasma concentrations of FSH and LH compared with does in a thermoneutral environment. Exposure of donor rabbits to elevated ambient temperature before mating, however, increased embryonic degeneration.     

Superovulation in nonseasonal Japanese native goats, with special reference to the developmental progression of embryos

Treatment for superovulation with pregnant mare serum gonadotropin (PMSG) and follicle stimulating hormone (FSH) was carried out in nonseasonal breeder Japanese goats which are widely used as a substitute model for cattle in various studies in Japan. The proportion of females that came into estrus (93 and 99%) and the interval between PGF(2) administration and estrus (1.5 to 2.0 days) did not differ between females treated with PMSG and those treated with FSH. The number of normal embryos recovered was significantly higher (P<0.01) in FSH-treated (9.4 +/- 5.6) femals than in PMSG-treated females (5.7 +/- 4.4). The developmental stage of embryos recovered from 1.0 to 8.5 at 0.5-d intervals after mating is also described. The development to the two-cell, four-cell, eight-cell, morula, blastocyst and zona-free blastocyst stage was first observed 1.5, 2.5, 5.0 to 5.5, 6.0 and 6.5 d, respectively, after human chorionic gonadotropin (hCG) injection.     

Gonadotropin effects on chromosomal normality of hamster preimplantation embryos

Induction of ovulation with pregnant mare's serum (PMS) and the timing of human chorionic gonadotropin (hCG) injection on chromosomal normality were examined in preimplantation hamster embryos. Two separate experimental trials were done. The first compared superovulation (SO, PMS on Day 1 of the cycle followed by hCG on Day 4) to natural ovulation. Natural mating was used. In the second series of trials, precocious superovulation (PSO, PMS on Day 1 followed by hCG on Day 3) was used. Since there is poor sperm transport in PSO females, direct uterine artificial insemination (AI) was used to achieve fertilization. The control animals in the second series of trials were naturally ovulating females subjected to the artificial insemination procedure. Of 785 embryos analyzed in the SO group, 9 (1.1%) were aneuploid (5 hyperploidy and 4 hypoploidy) and 8 (1.0%) showed triploidy. In the PSO group, artificial insemination resulted in a normal development rate of 85.5% up to the 2-cell stage. A total of 2.6% karyotypically abnormal embryos, consisting of 5 (1.1%) aneuploid and 7 (1.5%) polyploid, were found among 460 embryos examined in PSO females. No significant difference in the incidence of chromosomal abnormalities was observed between the stages of development. The overall incidence of chromosomal imbalance in hormonally treated females was not significantly different from that in controls (2.2% in SO cycles vs. 1.2% in natural cycles, 2.6% in PSO with AI vs. 2.4% in natural cycles with AI). These results indicate that PMS-hCG treatment has no adverse effect on the chromosomal integrity of hamster preimplantation embryos.     

Effect of eCG dose and ovulation induction treatments on embryo recovery and in vitro development post-vitrification in two selected lines of rabbit does

The aim of this work was to evaluate the effect of different doses of eCG administered subcutaneously (0, 50 and 200 IU) and the hormonal induction of ovulation (GnRH or hCG) on embryo recovery and in vitro development of embryos post-vitrification in two selected lines of rabbit does. The two selected lines were line V (selected for the litter size at weaning) and line R (selected for growth rate). Administration of 200 IU of eCG significantly increased ovulation rate (19.2 +/- 1.2 versus 15.5 +/- 1.1 and 12.2 +/- 1.3, and the number of haemorrhagic follicles (13.8+/-1.6 versus 3.8+ /- 1.4 and 3.8 +/- 1.7), but significantly decreased recovery rate (28.8 +/- 6.3 versus 47.7 +/- 5.7 and 48.7 +/- 6.7, 200 IU versus 50 IU and 0 IU eCG, respectively), the number of normal embryos recovered per doe with at least one embryo (5.8 +/- 0.9 versus 8.2 +/- 0.9, 200 IU versus 50 IU eCG doses) and the in vitro development of embryos post-vitrification (51.9% versus 66.1%, 200 IU versus 50 IU eCG doses, respectively). Inducing ovulation with hCG significantly increased ovulation rate when compared with GnRH (17.3 +/- 0.8 versus 13.8+/-1.4), but no significant differences in embryo recovery and embryo development post-vitrification were observed between the two treatments. No significant differences were observed between the two selected lines in ovulation and recovery rates, the number of haemorrhagic follicles and the number of recovered embryos per doe. However, the post-vitrification in vitro rate of development was 59.7% for line R and 51.9% for line V (p < 0.05). It was concluded that the use of 50 IU of eCG subcutaneous with hCG or GnRH prior to embryo cryopreservation programmes in rabbits achieves the best results for embryo recovery, with the best development of recovered embryos post-vitrification.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Cocaine inhibits mating-induced, but not human chorionic gonadotropin-stimulated, ovulation in the rabbit.

The effects of cocaine on ovulation and corpus luteum function were investigated in New Zealand White rabbits. Forty females were randomly assigned to control and cocaine-treated groups. Controls were given vehicle s.c. daily for 5 days and cocaine-treated rabbits received 40 mg/kg cocaine hydrochloride s.c. daily for 5 days. One hour after the last cocaine dose, half the control and half of the cocaine-treated groups were mated with fertile males and the other half of each group received hCG i.v. Serial blood samples were obtained over 4 h on the day of mating or hCG treatment (Day 0), and then at intervals from Days 1-18. No mated, cocaine-treated rabbits ovulated, vs. 6 of 10 controls (chi-square: p = 0.01). In contrast, all animals given hCG had comparable numbers of corpora lutea (control: 7.1 +/- 0.8; cocaine: 5.7 +/- 0.8). Peak levels of benzoylecgonine (the major cocaine metabolite) occurred between 180 and 240 min after cocaine administration. In cocaine-treated animals that were mated, Day 0 serum LH (repeated measures MANOVA, p less than 0.01) and FSH (p less than 0.03) concentrations were lower than those in pregnant controls. Serum LH and FSH levels for all hCG recipients (cocaine-treated and control) did not differ. Serum prolactin concentrations in mated, pregnant rabbits were higher than in all other groups; cocaine treatment did not affect this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The repeatability of superovulatory response and embryo recovery in sheep

Over an 8-year period, a total of 328 Scottish Blackface donor ewes were involved in a MOET program. They were synchronized with fluorogestone acetate sponges and superovulated with ovine FSH. After the onset of estrus, ewes were hand-mated and laparoscopic artificial insemination was performed with fresh semen 44-46 h after sponge removal. Embryos were recovered semi-laparoscopically on either Day 5 or Day 6 after insemination. Of the 328 donor ewes used, 222 ewes were supervoulated only once, while the remaining ewes were superovulated either twice (73 ewes), 3 times (26 ewes) or 4 times (7 ewes) at yearly intervals to generate a maximum of 474 records for subsequent analysis. There was no significant change in either mean ovulation rate or the mean number of embryos recovered per donor ewe at successive treatments. However, significant (P < 0.05 at least) effects of both year and donor ewe age existed for superovulatory response and number of embryos recovered, though only the effect of year was significant (P < 0.001) for percentage embryo recovery. Repeatability was significant (P < 0.05 at least) for both superovulatory response (r = 0.55, s.e. 0.055) and number of embryos recovered (r = 0.38, SE 0.074), but not for percentage embryo recovery (r = 0.04, SE 0.102).     

Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport

Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 +/- 0.4 d with estrus lasting 6.3 +/- 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r = -0.91 and r = -0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.     

Effects of castration and estradiol treatment on the postovulatory secretion of follicle-stimulating hormone in the mated rabbit

The effects of castration on the postovulatory secretion of follicle-stimulating hormone (FSH) was measured in mated rabbits. When ovaries were removed at 12 or 18 h postcoitum, FSH increased within 24 h of surgery but without evidence of the previously observed pattern of FSH secretion in the postovulatory period. To prevent the postcastration rise in FSH, various doses of estradiol were injected into does castrated 12 h after mating. Two micrograms estradiol/kg, given daily, was found to prevent the postcastration rise of FSH but was not sufficient to suppress the postovulatory secretion of FSH in intact animals. The postovulatory pattern of FSH release was disrupted in does castrated at either 12 or 18 h postcoitum despite adequate estradiol replacement therapy. Furthermore, in chronically castrated does treated with estradiol (2 micrograms/kg per day), neither mating nor human chorionic gonadotropin (hCG) injection elicited any change in blood FSH levels even though both treatments have been previously found to cause a postovulatory FSH surge. The results of these studies indicate that the ovary, by way of some ovarian secretion, is required for the postovulatory secretion of FSH in the rabbit. The necessary ovarian factor does not appear to be estradiol.     

Effect of an asynchrony between ovulation and insemination on the results obtained after insemination with fresh or frozen sperm in rabbits

The effects of the introduction of an 8-h asynchrony between ovulation and insemination on litter size components from rabbits were assessed. A total of 202 females belonging to a maternal line were used. Fresh and frozen sperm were used to perform the inseminations. Sperm was frozen with an extender composed of 1.75 M DMSO and 0.05 M sucrose. Four experimental groups were obtained depending on the type of sperm used (fresh or frozen) and on the moment that ovulation had been induced relative to the insemination (at the same time as insemination (t(0)) or 8 h before insemination (t(8))). Laparoscopy was performed on 12th day of pregnancy in pregnant females, and the ovulation rate, normal and total implanted embryos were noted. At kindling, total and live-born rabbits were noted. Results showed that better results were obtained after insemination with fresh semen than with frozen sperm (for females in the group t(0): 79% versus 61% fertility rate, 10.2 versus 6.4 normal implanted embryos and 8.1 versus 5.2 total number born, for fresh and frozen sperm, respectively). On the other hand, after the introduction of an 8-h asynchrony between ovulation and insemination, results were lower for both fresh (50% fertility rate, 7.5 normal implanted embryos and 5.7 total number born for the group of the asynchrony) and frozen sperm (31% fertility rate, 4.6 normal implanted embryos and 3.4 total number born for the group of the asynchrony). Although an approach between the moment of insemination and ovulation is justified when sperm survival could be compromised, results observed after the induction of an 8-h asynchrony were not those expected, perhaps due to the ageing of the oocytes before being fertilised, leading to both lack of fertilisation or early embryonic mortality.     

Progesterone treatment, FSH plus eCG, GnRH administration, and Day 0 Protocol for MOET programs in sheep

The effect of various superstimulatory treatments on the number of corpora lutea, fertilization rate, and embryo yield was studied in sheep. Overall, data from 708 Merino donors and 4262 embryos were analyzed in four experiments. In Experiment 1, varying intervals of progesterone treatment (5 to 14 d) before follicle-stimulating hormone (FSH) administration did not significantly affect the proportion of responding donors, the mean number of corpora lutea, or the mean number of recovered and transferable embryos per donor. In Experiment 2, a single dose of equine chorionic gonadotropin (eCG, 200 or 300 IU) combined with the FSH treatment (i.e., given at CIDR removal) reduced the number and the quality of embryos compared with that for not giving eCG (P < 0.05). In Experiment 3, one dose of gonadotropin-releasing hormone (GnRH) given 24 h after CIDR removal improved the number of transferable embryos compared with that for not giving GnRH (P < 0.05). In Experiment 4, the new superstimulatory Day 0 Protocol, which includes starting FSH treatment at the emergence of Wave 1 (i.e., soon after ovulation, in the absence of a large follicle), improved ovarian response, with a tendency to produce more embryos compared with that for the Traditional Protocol. In summary, this study, analyzing data from various pharmacologic treatments, allows an improvement from four to eight transferable embryos per treated donor in multiple ovulation and embryo transfer programs in sheep.     

GnRH agonist Lupron (leuprolide acetate) pre-treatments prevent ovulation in response to gonadotropin stimulation in the clouded leopard (Neofelis nebulosa)

In many species, controlling the ovary prior to induction of ovulation improves the success of ovarian response and artificial insemination (AI). We assessed the impact of suppression of estrus with the GnRH agonist, Lupron, on ovarian sensitivity to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the clouded leopard. Seven female clouded leopards were given two injections of Lupron (3.75 mg IM) 23 d apart, followed 44 d later by eCG and hCG. Daily fecal samples were collected from 60 d before Lupron to 60 d after hCG. Fecal metabolites of estrogen (E) and progesterone (P) were measured by radioimmunoassay. Lupron decreased (P < 0.05) the number of E peaks during Lupron treatment compared to pre-Lupron. All females had baseline E and six of seven (86%) had nadir P on day of eCG. Exogenous gonadotropins induced E elevations in all females. However, mean E in the gonadotropin-provoked estrus was decreased (P < 0.05) compared to pre-Lupron estrous periods. Only one of seven (14%) females ovulated after eCG/hCG. In conclusion, estrous cycle control with Lupron resulted in predictable ovarian suppression prior to gonadotropin stimulation but altered ovarian sensitivity by an as yet unknown mechanism so that ovulation was inhibited, even when using a proven exogenous gonadotropin protocol.     

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine stimulates reproduction in rabbit females

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate and cyclic guanosine monophosphate phosphodiesterases, on the reproductive efficiency of gonadotropin-stimulated rabbits (Oryctolagus cuniculus, Leporidae, Lagomorpha). The ovarian cycle and ovulation of control rabbits was induced by pregnant mare serum gonadotropin (PMSG) followed by administration of human chorionic gonadotropin (hCG; first series of experiments) or gonadotropin-releasing hormone (GnRH; second series of experiments). Experimental animals received PMSG and hCG together with IBMX (at 5 or 25 μg/animal) or GnRH together with IBMX (at 50 or 500 μg/animal). After ovulation and mating, in the first series of experiments, animals were killed; the pronuclear-stage eggs were flushed from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian corpora lutea, ovulated oocytes, and oocyte-derived embryos reaching blastocyst stage were determined. In the second series of experiments, all the animals were kept until parturition, when the pregnancy and birth rate, litter size, and number, viability, and body weight of pups were recorded. IBMX injections at doses of 5 or 25 μg/animal significantly increased the number of ovulations/corpora lutea, harvested zygotes, and embryos derived from these zygotes. Administration of IBMX at doses of 500 μg/animal or 50 μg/animal to nulliparous young animals (4.5 mo of age) significantly increased their pregnancy rate and birth rate, litter size, and litter weight. In multiparous old animals (2 yr of age), IBMX at a dose of 50 μg/animal, but not 500 μg/animal, significantly increased their pregnancy rate and litter size, but not the birth rate, number of pups per female, or litter weight. These data demonstrate that (1) IBMX can enhance the stimulatory effect of GnRH/gonadotropins on rabbit ovulation, oocyte maturation, embryo yield and development, pregnancy and birth rate, and number, viability, and body weight of pups; (2) nulliparous young females (4.5 mo of age) are more sensitive to IBMX treatments than the multiparous old animals (2 yr of age); and (3) cyclic nucleotides-dependent intracellular mechanisms are involved in control of rabbit reproductive functions and IBMX, an activator of these mechanisms, can be a stimulator of reproduction and fertility.     

Reversible inhibition of fertility in mice by passive immunization with anticumulus oophorus antibodies

The mucified cumulus oophorus represents an outer enveloping layer around ovulated mammalian oocytes. This coat in its definitive expanded form appears late in the preovulatory development as a result of intensive secretion of intercellular matrix by cumulus cells. We have shown recently that antibodies to the cumulus matrix inhibit human fertilization in vitro. This study was undertaken to assess, in an animal model, the effects of anticumulus oophorus antibodies on fertility by use of different passive immunization protocols. A purified anticumulus immunoglobulin fraction was prepared from hyperimmune rabbit serum and administered at different times before and after mating to mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A dose-dependent negative effect of this anticumulus antibody preparation on the number of fertilized eggs recovered from the oviducts of treated animals was observed when the antibodies were given before mating. High antibody doses also interfered with oocyte maturation and ovulation if applied on the day of eCG treatment, but no effects on these processes were found when the antibodies were given on the day of hCG treatment. The antifertility effect of anticumulus antibodies was reversible and the antibodies did not affect postfertilization development. These findings make cumulus oophorus antigens serious candidates for the development of a contraceptive vaccine.     

Cytogenetic analysis of Day-4 embryos from PMSG/hCG-treated prepuberal gilts

Prepuberal gilts were treated with pregnant mare serum gonadotropin (PMSG) to study the effects of its dosage on ovulation rate, fertilization rate after artificial insemination, embryo viability, and rate of development and incidence of chromosome abnormalities in Day-4 embryos. Gilts received 750 IU, 1250 IU or 1500 IU of PMSG, followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Gilts were inseminated 28 to 30 h following the hCG injection, and resulting embryos were collected on Day 4 post ovulation. Ovulation rate was higher in the 1250 IU group than in the 1500 IU group or the 750 IU group. The 1500 IU dose caused excessive stimulation of the ovary, resulting in the occurrence of large (>10mm diameter) unovulated follicles, reduced fertilization rate and low embryo recovery rate. There was no difference in the incidence of chromosome abnormalities among the three groups, although the 1500 IU group had higher embryonic mortality than the two lower dose groups. A dose of 1250 IU PMSG increased ovulation rate above that achieved by 750 IU and, therefore, increased the number of oocytes or embryos available for transfer or for other studies, without sacrificing embryo viability or increasing the incidence of chromosome abnormalities.     

Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle-stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (401U), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continu ous s.c. infusion over a 72-h period with a purified FSH preparation containing an opti mum ratio of luteinizing hormone (LH): FSH (FSH-hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1–4 of preg-nancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH-hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH-hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovu lation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and andro-gens on days 1–3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH-hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considera bly higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rate (FSH-hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of emyryos and low levels of circulating andorgens and estradiol-17β during the preimplantation period.     

FSH-induced ovulation in intact and hypophysectomized mice

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.     

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.