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PubMed1988

Superovulation of immature rats by continuous infusion of follicle-stimulating hormone.

Immature female rats were infused s.c. continuously over a 60-h period with partially purified porcine pituitary follicle-stimulating hormone (FSH) preparations differing in degree of purity and having widely divergent luteinizing hormone (LH):FSH potency ratios as defined by radioreceptor assays. Rats infused with the more purified FSH preparation (FSH-A) ovulated a mean of 60-85 oocytes per rat on the morning of the third day (Day 1) after FSH infusion was begun (on Day -2). The same total dose of FSH administered as a single s.c. injection or as twice daily injections over the same 60-h period resulted in ovulation in only a minority of treated rats (3/16), with none achieving ovulation rates approaching those of rats infused continuously. High fertilization rates (80% of ovulated oocytes) were observed in superovulated rats joined with fertile males on the evening of the second day of infusion (Day 0). Of the 67 +/- 7 fertilized ova per rat retrieved from oviducts flushed on Day 1, 52 +/- 8, or 80%, were accounted for as morulae or blastocysts recovered when oviducts and uteri were flushed on the morning of Day 5, demonstrating essentially normal developmental rates and high survival rates in reproductive tracts of superovulated females during the preimplantation period. Infusion of rats with the same dose of a less well-purified FSH preparation (FSH-E) containing 20 times as much LH activity, or injection of rats with a superovulatory dose of pregnant mare's serum gonadotropin (PMSG) (40 IU), were much less effective in causing superovulation, with ovulation rates of 17 +/- 6 and 34 +/- 8 oocytes/rat, respectively, compared to 79 +/- 9 oocytes/rat infused with the FSH preparation (FSH-A) containing lower LH activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

Attenuation of gonadotrophin release and reserve in superovulated women by gonadotrophin surge attenuating factor (GnSAF).

In-vivo and in-vitro studies have provided evidence that a non-steroidal ovarian factor, called gonadotrophin surge attenuating factor (GnSAF), attenuates the endogenous LH surge in superovulated women. To study the mechanism of action of GnSAF, the LH response to two i.v. pulses of GnRH (10 micrograms each, 2 h apart) was investigated in eight normally ovulating women during the late follicular phase of a spontaneous and an FSH superovulated cycle. The maximal LH increase in response to the first pulse (initial release) was considered as representing the acutely releasable pool and the delta LH area under the whole curve (integrated response) the reserve pool of LH. Both the initial release and the integrated response to GnRH were markedly attenuated in the FSH as compared to the spontaneous cycles. The response to the second pulse was significantly greater than the response to the first pulse (self-priming effect of GnRH) in both the spontaneous and the FSH cycles. However, in the FSH cycles the self-priming effect of GnRH was markedly reduced as compared to the spontaneous cycles. We conclude that during superovulation induction in women the two pools of pituitary LH are markedly attenuated. It is suggested that GnSAF attenuates both the GnRH-induced initial release of LH and the self-priming effect of GnRH on the pituitary.

Adult↗

PubMed1994

Prorenin and active renin concentrations in ovarian follicular fluid increase after the LH peak in superovulated heifers.

1. The aim was to analyse the in vivo variations with time of prorenin and active renin and their relationship to steroid hormones in ovarian follicular fluid during follicular growth in heifers. 2. Thirty one beef heifers were assigned to two groups after oestrous synchronization: an unstimulated and a follicle-stimulating hormone (FSH)-treated (superovulated) group. Within each group, animals were slaughtered at different times of the follicular phase of the oestrous cycle. Ovarian follicular fluids were aspirated and analysed for the concentrations of active renin, prorenin, oestradiol-17 beta (E2) and progesterone (P4). 3. Prorenin and active renin concentrations in follicular fluid remained constant until the luteinizing hormone (LH) peak, after which time they increased four- and two-fold, respectively, in superovulated heifers. 4. In follicular fluid, prorenin and active renin correlated negatively with oestradiol and E2/P4 ratio but positively with progesterone during follicular growth in superovulated heifers. Prorenin also correlated negatively with oestradiol and E2/P4 ratio in unstimulated heifers. 5. The increase of renin concentrations in ovarian follicles after the LH peak and the correlations to steroid hormones suggest an important role of the ovarian renin-angiotensin system in bovine follicular growth and maturation.

Animals↗

PubMed2005

Exposure of C57BL/6J male mice to an electric field improves copulation rates with superovulated females.

It is well-known that there are considerable strain differences in the relative copulation rates between male and superovulated female mice. In particular, the C57BL/6J strain of mice has a lower rate of successful copulation. We examined the effect of exposure to an electric field on sexual behavior in C57BL/6J male mice. When C57BL/6J males were exposed to a 50 Hz, 45 kV/m electric field for 30 min per day for 11 days and placed in a cage with a superovulated female of the same strain, the successful copulation rates of males was significantly improved compared with unexposed males (P<0.05). These results suggest that the exposure of C57BL/6J male mice to an electric field improves their sub-fertility activity in mating with superovulated females.

Animals↗

PubMed2005

Induction of superovulation by immunoneutralization of endogenous inhibin in immature rats.

The effects of passive immunoneutralization of endogenous inhibin on ovulation rate in immature rats were investigated. Efficiency of superovulation on production of fertilized oocytes was compared between the inhibin antiserum (inhibin-AS) and equine chorionic gonadotropin (eCG) protocols. Immature female Wistar strain rats were superovulated with a single injection of 100-200 microl inhibin-AS, with and without an injection of human chorionic gonadotropin (hCG). A total of 77.8% of the 26-30-day-old rats treated with a single injection of 100-200 microl inhibin-AS ovulated 72 h after treatment, while rats given normal goat serum (NGS; 200 microl) did not ovulate. At 28 days of age, all of the inhibin-AS treated rats ovulated when additional hCG treatment was given, whereas the number of ovulated oocytes was not affected. The number of ovulated oocytes in the inhibin-AS-hCG treated groups was significantly higher than that of the NGS-hCG treated group. In addition, plasma concentrations of FSH in the inhibin-AS-hCG treated group significantly increased compared with the NGS treated group. While the percentage of mated rats in the 200 microl inhibin-AS-hCG treated group was significantly lower than that of the 15 IU eCG-hCG treated group, the fertilization rate was comparable between the two groups. The number of fertilized oocytes in the 200 microl inhibin-AS-hCG treated group was significantly higher in comparison with the 15 IU eCG-hCG treated group. These results suggest that immunoneutralization of endogenous inhibin could be a reliable method for induction of superovulation to collect a large number of normally fertilized oocytes in immature rats.

Animals↗

PubMed1975

Embryonic mortality during the pre- and post-implantation periods of pregnancy in mature mice after superovulation.

Sexually mature mice were stimulated to superovulate by giving exogenous gonadotrophins at known stages of the oestrous cycle. Untreated animals which ovulated spontaneously served as controls. The number of oocytes ovulated by each female was estimated from counts of the number of CL of pregnancy, and the incidence of embryonic mortality during the pre- and post-implantation stages of pregnancy was assessed from the number of zygotes recovered from the reproductive tract at 2-0 and 4-0 days post coitum and of conceptuses examined at 7-5 and 11-5 days post coitum. The mean number of oocytes ovulated by treated animals was 39-54, compared with 12-80 in controls: in mice which had superovulated, 44% of the ova were lost before implantation compared with about 10% in the controls. Further losses occurred about the time of implantation and at mid-pregnancy and thus the number of embryos classified as normal rarely exceeded the maximum found in controls. Death at mid-pregnancy seemed to be preceded by developmental retardation. The possibility that genetic and environmental factors play a role in embryonic loss after superovulation is discussed.

Animals↗

PubMed1983

Oocyte normality after superovulation in immature rats.

The developmental ability of oocytes recovered from superovulated immature rats (40 i.u. PMSG) was compared with that of oocytes from control rats (4 i.u. PMSG). Oocytes were collected from the oviducts immediately after ovulation or from large follicles, and were transferred to one ovarian bursa of normal adult recipients. Fertilization and subsequent fetal development occurred in the recipients which were killed on Day 20. The proportions of oocytes surviving after transfer from the oviducts of donors which received 4 or 40 i.u. PMSG or from the follicles of those which received 40 i.u. PMSG were not significantly different. However, the proportion of oocytes surviving after recovery from the follicles of donors receiving 4 i.u. PMSG was significantly higher (P less than 0.05). These studies suggest that oocytes recovered from superovulated or control rats are equally able to develop, and the failure to maintain pregnancy that has been reported for superovulated rats in previous studies is not attributable to defects in the oocytes.

Animals↗

PubMed1998

Improved method of superovulation in monovulatory brushtail possums (Trichosurus vulpecula) using pregnant mares' serum gonadotrophin-luteinizing hormone.

A new superovulation regimen for the monovulatory brushtail possum (Trichosurus vulpecula) has been devised. It reduces the number of hormone treatments required and elicits a better rate of ovulation than the established pregnant mares' serum gonadotrophin (PMSG)-GnRH method. Ovarian stimulation was achieved by a single intramuscular injection of 15 iu PMSG. This treatment resulted in the recruitment and development of a large number (9-12) of Graafian follicles on the ovaries. Induction of ovulation was achieved by a single intramuscular injection of 2-10 mg pig LH or multiple intramuscular injections of GnRH, (4 x 50 micrograms, 90 min apart) given 72 h after PMSG treatment. Superovulation occurred in all animals (n = 48) examined 48 h after treatment irrespective of dose of LH or type of ovulatory stimulus used. The highest ovulatory success (83%), the maximum number of ovulation sites (9.5 +/- 2.8) and the highest number of oocytes recovered (9.0 +/- 2.5) were achieved after treatment with PMSG and 4 mg LH. These results were significantly greater than the ovulatory success (43%), numbers of ovulation sites (3.9 +/- 1.1) and number of oocytes recovered (2.1 +/- 0.9) after PMSG-GnRH treatment (P < 0.05). This simpler and more effective superovulation protocol should assist with more effective manipulation of possum reproduction in captivity.

Animals↗

PubMed2006

Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers.

The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus-oocyte complexes of 16 Prim' Holstein heifers (14 +/- 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 microg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers' growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 +/- 80 g/day (n = 16). In period 2 it was 730 +/- 70 (n = 8) and 1300 +/- 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1-3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and beta-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1-3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.

3-Hydroxybutyric Acid↗

PubMed1995

Efficient induction of superovulation in adult rats by PMSG and hCG.

Adult Wistar-Imamichi rats were induced to superovulate by an i.p. injection of PMSG followed by an i.p. injection of hCG 48 hr later. The doses of PMSG and hCG administered were 0, 75, 100, 150, 300 iu/kg body weight. In this study the doses were based on iu/kg body weight rather than iu/rat to facilitate accurate induction. The following results were obtained. In the adult rats (10 to 15 weeks old) PMSG induced an increase in the number of morphologically normal and fertilized oocytes, as well as in the total number of superovulated oocytes, dose-dependently upto 150 iu/kg. The increase was significant compared with control rats, but PMSG treatment had only half this effect in young rats (6 to 9 weeks old), compared with the adult rats. These results suggest that sensitivity to PMSG changes with age. The proportion of fertilized oocytes among the morphologically normal oocytes was not significantly different in young and adult rats, and both groups had high percentages. In view of the results of this study, the authors recommend that, to collect a large number of fertilized oocytes required for generation of transgenic rats, the induction of superovulation be performed in adult rats aged 10-15 weeks rather than young rats aged 6-9 weeks and giving PMSG at a dose of 150 iu/kg and hCG at a dose of 75 iu/kg.

Aging↗

PubMed2001

Studies on factors affecting superovulation and embryo transfer in Hungarian merino ewes.

The objectives of this study were (a) to assess the ovulatory response and embryo production of Hungarian Merino ewes after superovulation, (b) to investigate the factors influencing the efficiency of embryo transfer (ET) in Hungarian Merino ewes, (c) to compare the results of two ovarian stimulation protocols (PMSG and PMSG + FSH treatment) in Hungarian Merino ewes, and (d) to study how superovulation, laparoscopic insemination and surgical embryo retrieval (ER) affect the subsequent reproduction of Hungarian Merino donor females after an ET programme. There was no significant difference between the ovarian stimulation protocols in the ratio of donor ewes responding to superovulation nor in the average number of corpora lutea. However, the number of transferable embryos recovered per donor ewe was higher in the PMSG + FSH group. The proportion of transferable embryos, unfertilized oocytes and degenerated embryos did not differ between the treatment protocols. The total pregnancy rate was 53.4% (179/335). Neither the developmental stage of the embryo nor the number of transferred embryos affect the implantation of embryos. However, the increased number of transferred embryos positively influenced the pregnancy rate. No difference was found in the pregnancy rate between synchronised and non-synchronised groups of recipients. Thirty-six out of 45 donor ewes (80%) became pregnant within one year after the ET programme, indicating that ovarian stimulation and surgical ER did not affect adversely their reproduction.

Animals↗

PubMed1994

Some morphological and hormonal aspects of ovulation and superovulation in the rat.

In the first experiment, mature female Wistar rats, displaying a regular 4-day oestrous cycle, were killed in succession every 2 or 3 h on the day of pro-oestrus and oestrus until the time of ovulation. In the second experiment, immature female Wistar rats (aged 24 days) were injected s.c. with 30 IU pregnant mare serum gonadotrophin (PMSG) and 56 h later with 20 IU human chorionic gonadotrophin (hCG). They were killed in groups at 0, 24, 48, 56 and 57 h, and then every 2 h until 72 h. Excised ovaries were homogenized and analysed for steroid content or they were submitted to a routine histological procedure. The cyclic and PMSG/hCG-treated rats exhibited some similarities and differences in the general pattern of steroid content. Either a presumptive endogenous LH surge or administration of hCG resulted in an increase in the ovarian androgen concentration which preceded a rise in progesterone; the progesterone peak, in turn, was accompanied by a fall in the amount of androgens and oestradiol. However, in comparison with cyclic rats, superovulated animals displayed a significantly higher ovarian androgen level for a prolonged period; ovarian oestradiol concentration was also raised while the progesterone content was much lower. Histological analysis revealed large differences between the ovaries of superovulated and cyclic rats, especially with regard to the maturing follicles. The majority of PMSG/hCG-derived follicles showed hypertrophied theca interna and degenerated or luteinized granulosa. A large number of preovulatory follicles did not ovulate. These results clearly indicate that PMSG/hCG-induced follicles are not equal to the follicles developing during a normal oestrous cycle. This should be taken into consideration when using superovulated animals in experiments.

Androgens↗

PubMed1996

Induction of superovulation by immunoneutralization of endogenous inhibin through the increase in the secretion of follicle-stimulating hormone in the cyclic golden hamster.

The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17 beta and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1 = day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17 beta markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 microliters inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 microliters inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17 beta in the animals treated with 200 microliters inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 microliters inhibin-AS induced superovulation on day 1 of the third cycle. These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.

Animals↗

PubMedDate not supplied

Intrauterine insemination for cervical and male factor without superovulation.

Intrauterine insemination (IUI) has been used for the treatment of various causes of infertility, including unexplained infertility, male factor, and cervical factor. Some centers frequently use superovulation combined with IUI. The study presented herein attempted to evaluate the efficacy of IUI without superovulation in cases where all causes of infertility other than cervical or male factors have been eliminated. However, in the case of poor or absent cervical mucus, the use of controlled ovarian hyperstimulation (COH) may obscure the actual importance of the IUI, since it is possible that the poor cervical mucus is related to poor timing, inadequate follicular maturation, or low estradiol levels, which if corrected will obviate the need for IUI. In this study IUI was targeted for 36-40 h following the sera luteinizing hormone surge. A total of 108 patients were enrolled in this study: 47 with male factor, 61 with cervical factor. Patients were followed for a maximum of three cycles unless a pregnancy occurred within 3 months of treatment. Comparison of pregnancy rates (PRs) were based on diagnosis. The cumulative PRs per cycle for each of the three cycles studied were as follows: cervical factor--19.7, 36.8, and 36.8%; male factor--12.8, 29.3, and 38.3%. Thus, PRs were comparable for both groups after three treatment cycles. These data demonstrate that IUI is an effective therapy for cervical and/or male factor, even without superovulation.

Adult↗

PubMed1990

Effect of variability in response to superovulation on donor cow selection differentials in nucleus breeding schemes.

The effectiveness of nucleus breeding schemes based on multiple ovulation and embryo transfer in practice is highly dependent upon the achieved reproductive rate. This study quantifies the effects of donor cow variability in response to superovulation (random and phenotypically correlated with milk yield) and failure rates (proportion of selected cows producing no transferable embryos) on the mean and variance of realized female selection differentials as affected by herd size, average embryo yield, and the coefficient of variation for embryo yield. Results show that differences in failure rates (0 to 40%) reduced realized female selection differentials up to 64%. The existence of a negative phenotypic correlation (-.3) between donor cow embryo yield and milk yield caused smaller reductions (up to 19%). Variability in realized female selection differentials is largest for small nucleus units and should not be neglected when planning the establishment of a nucleus breeding unit. The use of multiple flushes rather than a single flush per cow to produce the same average number of transferable embryos can reduce the variability in realized selection differentials but increases generation interval. Variability in response to superovulation will also affect inbreeding rates and the average accuracy of selection. Simulation studies are needed to quantify the total effect of variability in response to superovulation on the genetic responses possible in nucleus breeding schemes relying on multiple ovulation and embryo transfer.

Analysis of Variance↗

PubMed1998

Superovulation in vesper mouse Calomys musculinus (Rodentia-Sigmodontinae).

Optimal conditions for pharmacological induction of ovulation of vesper mouse, Calomys musculinus, were analyzed. The best superovulation (a mean of about 21 eggs per female, range 12-45) was induced by the administration of 12 IU of PMSG followed 48 hr later by injection of 15 IU of hCG. Ovulation started about 10 hr after administration of hCG and was completed during the next 4-5 hr. The induction of ovulation was achieved irrespective of the stage of the oestrus cycle at the moment of PMSG administration. The majority of females (105, 82.7%) responded to the treatment with either an ovulatory (53.4%) or superovulatory (49.7%) response. Oocyte recovery and egg quality were clearly influenced by the age of females, 30 days to more than 120 days old. The majority (90.3%) of superovulated eggs was morphologically normal, and only a small proportion of eggs showed morphological abnormalities (7.4%) or were spontaneously activated (2.3%). Superovulated oocytes under these conditions, were able to undergo normal fertilization in vitro. After 6 hr of sperm-egg interaction in vitro, 87.5% of the oocytes had extruded the second polar body and/or developed pronuclei.

Age Factors↗

PubMed2001

Biochemical status of ovaries after induction of superovulation on different days of estrus cycle in mice.

To investigate the role of ovarian status and to find out a suitable hormonal dose for induction of superovulation and its effect on biochemical status of the ovaries, the mice were injected with PMSG in doses of 5, 7.5, and 10 IU on different days of the estrous cycle i.e. proestrus, estrus, metestrus and diestrus followed by hCG injection 48 hr later. All these treatments increased the mean ovarian weight and ovulation rate when compared with that of control animals. Maximum response was observed by treatment with 7.5 IU PMSG on the day of estrus. This treatment resulted in a non-significant decrease in total proteins but a significant increase in the lipid concentrations while no change in cholesterol content of the ovaries of superovulated mice. The activity of acid phosphatase and lactate dehydrogenase significantly increased and alanine aminotranseferase significantly decreased in the ovaries of mice after superovulatory treatment when compared with that of control animals. This reveals that treatment with PMSG and hCG results in metabolic alterations in the ovaries which may perhaps be inducing biosynthetic deficiencies in oocytes as indicated by increased prenatal mortality in superovulated pregnant mice when compared with that of controls in the present studies.

Animals↗