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Summary. Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P < 0·05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P < 0·05) after intrauterine insemination (50% of ova from 8/11 ewes).
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Summary. Immature female rats (60–65 g) were injected with 4 i.u. PMSG on Day –2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65–67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16–18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20.
After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients.
These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.
Keywords: in-vitro fertilization; embryo development; implantation; asynchronous transfer; rat
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Summary. In Exp. 1, PMSG was injected to 26-day-old prepubertal rats to induce ovulations. On Day 2 (2 days later, the equivalent of the day of pro-oestrus) they received at 08:00 h 5 mg hydroxyflutamide or vehicle and at 12:00 h 2 mg progesterone or testosterone or vehicle. Animals were killed at 18:00 h on Day 2 or at 09:00 h on Day 3. Progesterone but not testosterone restored the preovulatory LH surge and ovulation in hydroxyflutamide-treated rats. In Exp. 2, 2 mg progesterone or testosterone were injected between 10:30 and 11:00 h on Day 2, to advance the pro-oestrous LH surge and ovulation in PMSG-primed prepubertal rats. Injection of hydroxyflutamide abolished the ability of progesterone to advance the LH surge or ovulation. Testosterone did not induce the advancement of LH surge or ovulation. In Exp. 3, ovariectomized prepubertal rats implanted with oestradiol-17β showed significantly (P< 0·01) elevated serum LH concentrations at 18:00 h over those observed at 10:00 h. Progesterone injection to these animals further elevated the serum LH concentrations at 18:00 h, in a dose-dependent manner, with maximal values resulting from 1 mg progesterone. Hydroxyflutamide treatment significantly (P< 0·003) reduced the serum LH values in rats receiving 0–1 mg progesterone but 2 mg progesterone were able to overcome this inhibition. It is concluded that progesterone but not testosterone can reverse the effects of hydroxyflutamide on the preovulatory LH surge and ovulation. It appears that hydroxyflutamide may interfere with progesterone action in induction of the LH surge, suggesting a hitherto undescribed anti-progestagenic action of hydroxyflutamide.
Keywords: anti-androgen; progesterone; ovulation; anti-progestagen; LH; rat
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Summary. 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 < 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.
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Summary. Oestrus was synchronized in ewes by (a) withdrawal of an intravaginal progestagen sponge after 12 days or (b) injection of PGF-2α on Day 11 of the oestrous cycle. In addition, ewes were treated with (a) no hormone, (b) 1500 i.u. PMSG 48 h before sponge removal or PG injection, or (c) 24 mg porcine pituitary FSH in multiple injections commencing 48 h before sponge removal or PG injection, in a 2 × 3 factorial design. Ewes were inseminated with 0·2 ml fresh undiluted semen into the neck of the cervix 48 h after sponge removal or PG injection. Normally cyclic ewes were similarly inseminated within 12 h of observed standing oestrus.
At 24 h after insemination one uterine horn and one oviduct were flushed for recovery of spermatozoa and ova. When compared with naturally cyclic ewes, PG synchronization resulted in a marked reduction in the numbers of spermatozoa recovered (P < 0·05), and sponge synchronization led to a small, non-significant, reduction. Within the synchronized ewes, PMSG and FSH resulted in an equivalent superovulatory response, but there was a marked reduction in sperm recovery when compared with unstimulated animals (P < 0·01), with the greatest reduction attributable to PMSG treatment. Spermatozoa were recovered from fewer ewes treated with PMSG than with FSH (P < 0·05). Despite the observed impairment of sperm transport, a high fertilization rate was observed within each group and there were no differences between treatments.
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Summary. Immature female rats (65–70 g) were injected with 4 i.u. PMSG (control) or superovulated with 8,16,24 or 40 i.u. PMSG and were killed 68–70 h later, shortly after the normally expected time of ovulation. Oocytes were recovered from the oviducts and inseminated in vitro. After 18 h oocytes were counted and classed as degenerate or 1-cell. Mean numbers of oocytes recovered were 8·2, 26·8, 50·7, 38·7 and 38·5 for each dose of PMSG respectively. The 1-cell oocytes were assessed for sperm penetration of the vitellus and pronuclear development and later for development to the 2-cell stage. Fertilization rates at the 1-cell stage were 76·8, 62·9, 53·6, 52·2 and 44·5% for the rats treated with 4, 8, 16, 24 and 40 i.u. respectively (P < 0·001). On average, 91% of fertilized 1-cell oocytes developed to the 2-cell stage and there was no difference between treatments in this respect. Significantly more of the unfertilized oocytes were degenerate in the rats treated with 24 or 40 i.u. PMSG (34·6 and 50·4%) than in those treated with 4, 8 or 16 i.u. (7·0, 13·9, and 7·5%) (P < 0·001).
When rats were killed 63–65 h after PMSG, just before the normally expected time of ovulation, some of the rats treated with 24 and 40 i.u. PMSG had partly ovulated: of the oocytes recovered from the oviducts only 12·3% (24 i.u.) and 26·6% (40 i.u.) were fertilized.
These results demonstrate that proportionately fewer oocytes recovered from superovulated rats are competent to undergo in-vitro fertilization than are oocytes recovered from control rats.
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Summary. Follicular fluid from small- (1–2 mm) or medium- (3–8 mm) sized pig follicles was collected under conditions designed to minimize possible alteration or degradation of native fluid components. The effects of follicular fluid with or without benzamidine, an inhibitor of proteolytic activity, on oocytes collected 20 or 44 h after PMSG treatment of rats were examined. A follicular fluid fraction of Mr < 10 000 (PM-10 membrane filter) was also tested. Follicular fluid from small- and medium-sized follicles and control medium alone supported maturation of oocytes collected 20 or 44 h after PMSG, but follicular fluid (50%) from medium-sized follicles containing 5·0 mm-benzamidine significantly inhibited oocyte maturation. Comparable inhibition was also observed with medium containing 5·0 mm-benzamidine. The PM-10 filtrate failed to inhibit oocyte maturation as assessed by germinal vesicle breakdown but did significantly inhibit first polar body formation and therefore restricted the extent of maturation. The results indicate that native pig follicular fluid is unable to inhibit the initiation of maturation of rat oocytes in vitro.
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Summary. The ovarian bursa was peeled from around one ovary of each rat and the rats were killed 1, 2, 3, 4 and 5 weeks later. The proportion of rats that maintained a bursa-free ovary did not change over the 5-week period (80–89%). Ovulation from the peeled ovary occurred in all rats but oocytes (1–4) were found in the ipsilateral oviduct in only 18% of the rats. The presence of oocytes in the oviduct was normally associated with some degree of re-encapsulation of the ovary. In another experiment rats were mated within 1 week of removal of the bursa from around the ovary. Unilateral pregnancy resulted in 92% of the rats. In a third experiment fertilized oocytes from mated donor rats were transferred into the oviduct next to the peeled ovary in 15 mated recipients. Of 85 zygotes transferred, 51 survived to be viable fetuses on Day 20. A single fetus developing from an endogenous oocyte was found in the transfer uterine horn in only one rat.
This preparation may be useful in studies which attempt to determine the viability of oocytes that have undergone various manipulations in vivo or in vitro.
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Summary. Immature female rats (75 g body wt, aged 29 days) were injected with 4 or 40 i.u. PMSG on Day –2 and were killed at intervals between 18:00 h on Day –2 and 09:00 h on Day 1. Control animals (4 i.u.) ovulated between 00:30 and 05:30 h on Day 1 whereas the number of ova recovered from superovulated rats (40 i.u. PMSG) increased slowly between 06:00 h on Day –1 and 24:00 h on Day 0 and markedly between 24:00 on Day 0 and 06:00 on Day 1.
Similarly treated rats were caged overnight on Day 0 with males of proven fertility and killed between 14:00 and 16:00 h on Day 1. A significantly lower percentage of normal 1-cell ova was recovered from the superovulated rats compared to control animals (71·6 and 98·5% and of these 1-cell ova a lower percentage was fertilized (69·7 and 99·1%). In the control group all mated animals had a high proportion of ova fertilized whereas 26% of superovulated rats had none or a very low proportion fertilized. In the control animals there was evidence of sperm penetration and pronucleus fromation; in superovulated rats significantly fewer ova had pronuclei than were penetrated.
These results suggest that reduced fertility of superovulated immature rats is due to complete or partial failure of fertilization in some animals. The extended period during which ovulation occurs may be a contributory factor.
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Summary. Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (< 0·1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1–2 mm), medium (3–5 mm) and large >5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6–12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4–12 ng/mg protein). Addition of PGE-2 (1 μg/ml) caused a further significant (P < 0·05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2α, FSH and LH were ineffective when added to culture media.
Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0·4 pg/ml to 2·4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0·1 ng/mg protein) but rose to 1·4 and 3·5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. Oestradiol secretion by granulosa cells cultured with androstenedione as an aromatizable substrate was essentially unchanged when obtained from 0 to 12 h after GnRH injection (31–37 ng/mg protein), but fell significantly to 7·4 ng/mg in cells obtained 18 h after GnRH injection, indicative of decreasing aromatase activity as luteinization progresses during the preovulatory period. It is concluded that granulosa cells, in response to the preovulatory LH surge, acquire the ability to synthesize and secrete oxytocin in parallel with the increased progesterone and decreased oestradiol secretion which occurs during luteinization.
Keywords: oxytocin; granulosa cells; goat; LH; luteinization
