Variations in bovine embryo production between individual donors for OPU-IVF are closely related to glutathione concentrations in oocytes during in vitro maturation.

Variations in embryo production between individual oocyte donors represent a serious problem in cattle production, when implementing ovum pick-up (OPU) and in vitro maturation (IVM) of oocytes. However, the precise cause of this problem is unknown. Here, we aimed to investigate the relationship between the glutathione (GSH) concentration in IVM oocytes and embryo development to explore a potential cause of the variations between individual donors. First, we found a high positive correlation (r = 0.80) between the GSH concentration in IVM oocytes and the blastocyst development rate of oocytes collected in the same OPU session for each donor (N = 11). Second, we selected two donors with significantly different blastocyst development rates. In samples from these donors, we examined the dynamics of oocytes GSH concentrations, and the gene expression of the glutathione synthetase (GSS) and glutathione peroxidase (GPX) genes in cumulus-oocyte complexes (COCs) during IVM. At 0 and 24 h after IVM, oocytes from the donor with the highest blastocyst development rate (high donor) exhibited significantly higher oocyte GSH concentrations than oocytes from the donor with the lowest blastocyst development rate (low donor, P < 0.05). At 4 and 9 h after IVM, GSH concentrations gradually decreased in oocytes from both donors. The GSS expression levels at 0, 4, and 9 h after IVM were significantly higher in COCs from the high donor than in COCs from the low donor (P < 0.05). The expression levels of GPX, a marker of oxidative stress, at 4 and 9 h after IVM were significantly higher in COCs from the low donor than in COCs from the high donor. Finally, adding cysteine into the IVM medium of oocytes collected in the same OPU session from the low donor significantly increased oocytes GSH concentrations and blastocyst development rates (P < 0.05). In conclusion, we showed that oocyte GSH concentrations were related to the differences in embryo development between individual donors. Our results suggested that increasing the GSH concentration in IVM oocytes could reduce the individual differences in embryo production between donors.

Estradiol benzoate treatment before ovum pick-up increases the number of good quality oocytes retrieved and improves the production of transferable embryos in Japanese Black cattle.

The aim of this study was to evaluate the efficacy of treatment with estradiol benzoate (EB) at luteal phase prior to the ovum pick-up (OPU) during in vitro production of transferable embryos in Japanese Black cattle. A total of 15 cows were used as oocyte donors for OPU. Of those, four donors were randomly allocated (three times) into each of two treatment groups as a crossover study, and OPU session was carried out six times per one donor. Another eleven donors were used in a paired difference test by one crossover trial. Donors in the control group received no hormonal treatment; whereas, donors in the EB group received 1 mg of EB as a single injection. First, we observed dynamics of ovarian follicles and emergence of follicular wave after EB injection using transrectal ultrasonography. The number and proportion of medium-sized follicles with 4 to 6 mm in diameter increased gradually and achieved a peak at 72 and 96 hours after EB injection. The OPU was performed 88 hours after EB injection. The EB-treated donors had a higher proportion of follicles with 4 to 6 mm in diameters at the time of OPU. The stimulation with EB significantly increased the numbers of follicles aspirated, and the good quality cumulus-oocyte complexes per OPU. Furthermore, in the EB group, the percentage of transferable blastocysts was significantly greater than that in the control group (P<0.05). In conclusion, a single EB injection before OPU increases the number of medium-sized follicles and can produce more transferable embryos.

Effective embryo production from Holstein cows treated with gonadotropin-releasing hormone during early lactation.

The low efficiency of embryo production in Holstein cows during early lactation presents many challenges for animal production. To improve its efficiency, the outcomes of single GnRH injections 48 hours before each of three cycles of ovum pick up (OPU; weeks 2, 4, and 6) were compared with three cycles of unstimulated OPU (controls; weeks 1, 3, and 5) in 35 Holstein cows during 6 weeks of early lactation (40-80 days postpartum). More total follicle numbers (19.5 vs. 16.0; P < 0.05) but fewer dominant follicles (0.5 vs. 1.4; P < 0.01) were observed by ultrasound, and more cumulus-oocyte complexes were collected in a single OPU in the treatment cycles compared with controls (15.3 vs. 11.5; P < 0.05). The numbers of morphologically "good" cumulus-oocyte complexes graded A and B in the stimulated OPUs were significantly greater than in controls (2.8 vs. 1.7 and 5.8 vs. 4.2, respectively; P < 0.05). Significantly, more oocytes stained positively with brilliant cresyl blue after GnRH treatment compared with the control cycles (13.7 vs. 9.6; P < 0.05). After in vitro fertilization, embryos in the treatment cycles had improved development (P < 0.01) during each developmental stage compared with the controls (9.0 vs. 6.2 two-cell embryos; 4.7 vs. 3.0 four-cell embryos; 3.3 vs. 2.0 morulae; and 3.0 vs. 1.7 blastocysts, respectively). Moreover, there was no significant difference in pregnancy rate of the recipient cows after embryo transfer (57.1% vs. 42.1%; P > 0.05) no matter if the embryos came from the GnRH-treated cycles or not. Thus, GnRH-stimulated OPUs improved the efficiency of embryo production in Holstein cows during early lactation. This novel method for in vitro embryo production should benefit the dairy industry.

Estrone sulfate and progesterone profiles during late gestation in recipient cows transferred embryos produced by nuclear transfer and in vitro fertilization.

The aim of present experiment was to evaluate the plasma concentrations of estrone sulfate (E(1)S) and progesterone (P(4)) during late gestation in recipient cows transferred embryos produced by nuclear transfer (NT) and in vitro fertilization (IVF). Blood samples were collected from recipients transferred embryos produced by NT (n=9) and IVF (n=13) at 160, 220, 240, 260 and 270 d of gestation and then at 5 d intervals until parturition. Plasma samples were analyzed for E(1)S and P(4) by ELISA. One NT and three IVF cows aborted between days 220 and 260 of gestation. Two NT and one IVF cow had prolonged gestation (over 290 d). One IVF cow had an overweight fetus (50 kg) after abortion (257 d). The patterns of changes in the concentrations of E(1)S during late gestation in the NT and IVF cows were almost identical. The NT and IVF cows that aborted had prolonged gestation and much higher E(1)S concentrations than the average. One NT cow aborted after 220 d of gestation and had a sudden high increase in its E(1)S concentration from 160 d to 220 d of gestation. The NT and IVF cows that had prolonged gestation also had significantly higher (P<0.05) P(4) concentrations than the average. These results raise the possibility that the E(1)S and P(4) profiles can be used to monitor some late gestational problems, such as higher birth weight, abortion and prolonged gestation.