Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium.

The aim of this study was to investigate whether inhibin A and/or activin A play a role in the acquisition of oocyte competence during the final stages of oogenesis. The particular goal was to establish whether inhibin A and activin A exert development-enhancing effects during in vitro maturation in serum-free media and whether such effects are related to changes in the kinetics of meiotic resumption and/or fertilization rates. Cumulus-oocyte-complexes (COCs) were matured in two control media (Medium 199 [M199] with hormones and serum, hormone-serum control; M199 + 0.6% BSA, BSA-control) and nine treatment media (M199 with 0.6% BSA containing 100, 10, and 1 ng/ml of recombinant human inhibin A, recombinant human activin A, and the combination of the two). Oocytes were fertilized and cultured using standard procedures. Cleavage was assessed at 54 h and blastocyst development at 8 days after in vitro fertilization. Kinetics of oocyte maturation and the fertilization rates were evaluated after fixing and staining (Hoechst 33342) of oocytes at 8, 16, and 22 h after onset of in vitro maturation or of presumptive zygotes at 12 h after in vitro fertilization, respectively. Although there was no effect on cleavage rates, inhibin A and activin A significantly enhanced postcleavage development at concentrations of 10 ng/ml (57.7 +/- 7.5% and 56.6 +/- 11.7%, respectively) and 100 ng/ml (50.6 +/- 18.6% and 56.4 +/- 4.0%, respectively) compared to that in the BSA-control group (24.6 +/- 3.2%). Whereas inhibin A- and activin A-treated oocytes showed development-enhancing effects similar to those in the hormone-serum controls, these groups differed with regard to the kinetics of meiotic resumption. Likewise, the enhanced development of the hormone-serum control and the inhibin A/activin A-treated oocytes was not related to increased fertilization rates relative to the BSA-control. These results suggest that inhibin A and activin A may play important roles during the final stages of oogenesis and that recombinant inhibins and activins are useful compounds for the development of a serum-free culture system for in vitro maturation of oocytes from cattle and possibly other mammalian species.

A dominant follicle does not affect follicular recruitment by superovulatory doses of FSH in cattle but can inhibit ovulation.

It has been suggested that superovulation in cattle is impaired if FSH injections are initiated in the presence of a dominant follicle, but the results of experiments to test this hypothesis have been contradictory. However, previous experiments were conducted during mid-cycle, when the absence or presence of a dominant follicle is difficult to assess. We took a different approach by comparing the effects of initiating superovulatory injections of FSH (11 equal doses of FSH-P, every 12 h) on Day 1 of the bovine estrous cycle, when a dominant follicle clearly is not present, vs initiation on Day 6, when a dominant follicle clearly is present and actively growing (n = 17 heifers in a "crossover" design). In 8 17 heifers initiation of FSH injections in the presence of a dominant follicle (Day 6 group) caused ovulation of the dominant follicle within 1 to 2 days and formation of a smaller than normal CL. These animals had higher than normal concentrations of plasma progesterone around the time of expected estrus (P < 0.05) and failed to exhibit estrus. Although the mean number and diameter of the follicles recruited in response to FSH injections in heifers that ovulated the dominant follicle prematurely were not different from the other heifers in the Day 6 group, no ovulations were observed, and no embryos or ova were recovered 6 d after insemination. Conversely, when FSH injections were initiated on Day 1 in these 8 heifers, they exhibited estrus, and their plasma progesterone around the time of estrus, mean ovulation rate, and number of total and transferable embryos recovered did not differ from the responses observed in the remaining 9 heifers treated either on Day 1 or on Day 6. Taken together, these results indicate that a dominant follicle does not affect the ability of smaller follicles to be recruited in response to exogenous FSH, but may impair their ovulation. These findings provide an explanation for previous reports of decreased superovulatory responses during times of the cycle when a dominant follicle would be expected to be present.

Oxytocin in mares: lack of evidence for oxytocin production by or action on preovulatory follicles.

It is not known whether the equine preovulatory follicle produces oxytocin or is a target tissue for oxytocin, as has been reported for other species, especially ruminants. Bovine granulosa cells secrete oxytocin, and oxytocin modulates the production of progesterone by granulosa cells in vitro. We examined whether oxytocin plays a comparable role in the equine preovulatory follicle. To test the hypothesis that the equine preovulatory follicle produces oxytocin during estrus and that its production increases in late estrus, preovulatory follicles were isolated during early (Days 1 to 2; n = 4) and late (Days 4 to 5; n = 4) estrus. Granulosa cells, pieces of theca interna and pieces of follicle wall (theca with attached granulosa cells) were cultured for 3 d with or without equine gonadotropins. Culture media were collected, replaced at 3, 6, 12, 24, 48, and 72 hr of culture, and assayed for oxytocin. Granulosa cells from preovulatory follicles secreted negligible amounts of oxytocin during 3 d of culture, irrespective of gonadotropin treatment or stage of estrus. Likewise, negligible amounts of oxytocin were measured in theca and follicle wall cultures at both developmental stages, in the presence or absence of gonadotropins. Furthermore, follicular fluid from early or late estrous follicles contained only negligible amounts of oxytocin. To determine if oxytocin affects steroidogenesis by equine granulosa cells, granulosa cells from follicles obtained on Day 2 of estrus were cultured with graded doses of oxytocin (0, 1, 10, 100, and 1,000 ng/ml) in defined medium supplemented with testosterone (0.5 microM) and culture media were assayed for estradiol-17 beta and progesterone. Estradiol was secreted throughout the culture period, and its production was not significantly affected by oxytocin treatment (P > 0.05). Progesterone secretion was relatively low during the first 24 hr of culture, increased dramatically on the second day of culture, and remained high through the third day. No dose of oxytocin had a significant effect on progesterone secretion (P > 0.05). In conclusion, the results indicate that equine preovulatory follicles, isolated during early or late estrus, are neither a source of oxytocin nor a target for oxytocin action on steroidogenesis. Although ovarian oxytocin appears to play a role in regulating follicular function in some other mammalian species, our data provide no support for such a role for oxytocin in mares.

Ovarian follicular dominance in cattle: relationship between prolonged growth of the ovulatory follicle and endocrine parameters.

In cattle, the development of ovarian follicles 5 mm or larger occurs in either two or three consecutive follicular waves per estrous cycle. When the luteal phase is artificially lengthened with an intravaginal progesterone-releasing device (CIDR) that maintains subluteal levels (i.e. levels of progesterone that are below normal luteal levels, but higher than basal follicular phase levels), prolonged development of the ovulatory follicle is observed. To study the endocrinological correlates of prolonged follicular dominance and to test the hypothesis that it is mediated by effects of plasma progesterone on LH pulse frequency, heifers (n = 6/group) were treated with blank CIDRs (no progesterone, control group), with one CIDR for 14 days from day 14 of the cycle (1 CIDR group), or with one CIDR for 14 days from day 14 plus a second CIDR during days 24-28 (2 CIDR group). Cycle length was significantly longer in the 1 and 2 CIDR groups than in the controls (30.2 +/- 0.2 and 31.8 +/- 0.5 vs. 21.6 +/- 0.4 days, respectively; P < 0.0001). Follicular dynamics were normal in the control heifers. In the 1 CIDR group, the ovulatory follicle grew larger than in control or 2 CIDR animals, was maintained as the largest follicle on the ovaries for a much longer time, and ovulated after CIDR removal. In the 2 CIDR group, a similar growth pattern was observed until day 24; after insertion of a second CIDR, however, prolonged dominance was reversed, a new wave was recruited, and the dominant follicle of this wave ovulated after CIDR removal. The size of the ovulatory follicle and the length of the dominance phase in the 2 CIDR group were similar to those in control animals. Reversal of prolonged dominance in the 2 CIDR group was associated with changes in progesterone. Progesterone remained at subluteal levels (1.5-2.3 ng/ml) in both CIDR groups until day 24, when insertion of the second CIDR in the 2 CIDR group restored progesterone concentrations to normal luteal levels (3.5-6 ng/ml). Pulsatile LH secretion was assessed by frequent blood sampling every 12 min for 6 h (0800-1400 h) on selected days of the treatment cycle. LH pulse frequency was not different among groups before treatment started (days 12 and 13). However, LH pulse frequency was significantly higher in the 1 CIDR than in the 2 CIDR group on both day 26 (P < 0.03) and day 28 (P < 0.05), i.e. during the reversal of prolonged dominance in the 2 CIDR group.(ABSTRACT TRUNCATED AT 400 WORDS)

Assay of embryo-derived platelet activating factor (EDPAF) by an equine platelet aggregation assay: Preliminary data concerning its presence in bovine embryo culture media.

This study was designed to establish a sensitive bioassay for bovine platelet-activating factor (PAF), to determine if the bovine embryo secretes PAF in vitro and if PAF release is correlated with the embryo's potential to establish a pregnancy. Using an equine platelet aggregation assay, lipid extracted culture media from 33 Day-7 embryos (individually cultured for 18 hours in 1 ml of Ham's F10 containing 0.4% BSA at 37 degrees C in an air: CO2 mixture of 95:5 prior to their transfer to recipient heifers) and from control media (n=15, Ham's F10+0.4% BSA incubated simultaneously without embryos) were investigated. In addition, culture media from Day-6 (n=6) and Day-1 (2-cell, n=12) bovine embryos that were cultured for 4 hours but not transferred were examined. The aggregation assay proved to be sensitive to 5 pg of PAF. The assay proved to be specific, since the PAF receptor antagonist SRI 63-441 inhibited platelet aggregation induced by culture media in dosages comparable to aggregation induced by synthetic PAF18. From the 15 Day-7 embryos that established a pregnancy 2 contained measurable amounts of PAF in their culture media. No PAF was detected in the culture media from 13 embryos that succeeded, in the 18 embryos that failed to establish a pregnancy, or in the control media. One of 6 Day-6 embryos and 3 of 12 Day-1 (2-cell) embryos secreted detectable amounts of PAF into the culture media. Although the results indicate that some bovine embryos release PAF or a PAF-like substance in vitro, PAF measurements in the culture medium seem not to be a suitable method for the evaluation of bovine embryos prior to transfer.

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle.

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.

Effect of cholesterol oxidation products on cholesterol metabolism in the laying hen.

Two experiments were conducted to determine the effect of purified cholesterol and oxidized cholesterol in the diet of the laying hen on egg production characteristics, in vitro - in ovo utilization of acetate for cholesterol biosynthesis, and the activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in biosynthesis of cholesterol. Previous work has demonstrated inhibition of cholesterol synthesis by cholesterol oxides in tissue culture cells but not in hepatic tissues of animals through dietary administration. Feeding .5% of either purified or oxidized cholesterol had no effect on egg production, egg weight, body weight, or diet consumption. In both experiments egg yolk cholesterol was significantly increased by both cholesterol sources, but eggs from hens fed oxidized cholesterol had lower cholesterol contents than those from hens fed purified cholesterol. Relative utilization of acetate for cholesterol biosynthesis was significantly reduced by feeding both cholesterol sources. Hepatic enzyme activity measured by production of mevalonic acid was significantly inhibited by feeding purified cholesterol. A further significant reduction in enzyme activity was observed when oxidized cholesterol was fed, indicating that dietary cholesterol oxides are much more potent than purified cholesterol in limiting the activity of the enzyme.