Effects of hormone sources on developmental competence of oocytes by ovum pickup in Japanese black cattle.

Japanese Black (Wagyu) cattle donors were primed with different protocols and sources of follicle-stimulating hormone (FSH) for successive ovum pickup (OPU) and embryo development after in vitro fertilization (IVF). Following OPU, retrieved cumulus oocyte complexes (COCs) were subjected to IVF, and resulting blastocysts were transferred into recipients to evaluate implantation capability. Experiment 1: The best blastocyst development (45.3 %) and embryo yields (5.0/donor/OPU) were found with oocytes retrieved from donors treated with FSH (STIMUFOL®, Belgium) at a dosage of 150 IU per donor, compared to two others commercial FSH sources. Experiment 2: There were no differences in embryo development or yield with STIMUFOL FSH (total FSH 150 IU/donor) at a priming duration of either 60-h (Regime 1, six FSH injections) or 36-h (Regime 2, four FSH injections). Experiment 3: Compacted COCs required 22-26-h maturation in vitro (IVM) before IVF for optimal blastocyst development (36.1-41.1 %); however, short (18-h) and prolonged (30-h) IVM duration resulted in lower embryonic development. In contrast, expanded COCs resulted in inferior blastocyst development compared to compacted COCs. Immunofluorescence microscopy revealed that the ratio of 89.8 % cumulus compacted COCs were at the germinal vesicle (pachytene) phase while 98.9 % cumulus expanded COCs went through spontaneous meiosis from meiotic metaphase I, anaphase I, telophase I to metaphase II upon OPU retrieval (P<0.05). Pregnancy rates were not different among three FSH sources or different FSH treatments as long as embryos reached the blastocyst stage. Our study found that different sources of FSH used for Wagyu donor priming prior to OPU resulted in differential embryo development potentials, but those embryos that reached out to blastocysts had a competent implantation ability.

Effects of donor age and reproductive history on developmental potential of ovum pickup oocytes in Japanese Black cattle (Wagyu).

The objectives of this study were to analyze the (1) effects of donor age and multiparity on development of in vitro fertilization (IVF) embryos after ovum pickup (OPU), (2) effects of repeated and consecutive OPU-IVF procedures on embryo development, and (3) embryo production from OPU-IVF in donors with differing embryo yields after multiple ovulation and embryo transfer technology (MOET) in Japanese Black cattle (Wagyu). Donors were pre-treated with low-dosage follicle-stimulating hormone (FSH; 200 IU total), and oocytes were collected via OPU and fertilized by IVF to generate blastocysts. The number of oocytes collected per OPU session per donor was lower in heifers (2-4 years old, 5.3 oocytes) than in primiparous and pluriparous cows (2-10 years old, 13.6-19.1 oocytes; P < 0.05). Rates of blastocyst development for oocytes from heifers (33.1%) were lower than for those from cows (2-10 years old, 44.1-54.3%; P < 0.05), and average blastocyst yield/OPU/animal was lower in heifers (3.7) than in 5-6 years old cows (10.1; P < 0.05). Donors undergoing five consecutive OPU-IVF sessions after low-dosage FSH showed similar oocyte retrieval (12.2-15.1 oocytes per OPU/animal), blastocyst development rates (35.6-45.0%), and embryo yield/OPU/animal (4.8-5.8; P > 0.05) across sessions. Additionally, embryo yield from OPU-IVF was significantly improved in animals with previous low embryo yield from MOET (5.9 vs. 2.6, respectively, P < 0.05). These results indicate that Wagyu cows with previous births can be more productive as OPU-IVF donors than heifers, and oocytes from donors undergoing to five consecutive OPU-IVF cycles are competent for embryo development without loss of embryo yield/OPU/animal. Moreover, OPU-IVF can be used for embryo production and breeding from all elite Japanese Black cattle, regardless of previous low embryo yield in routine MOET.

Synergistic effect of cysteamine, leukemia inhibitory factor, and Y27632 on goat oocyte maturation and embryo development in vitro.

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0-48.0%; P > 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P < 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P < 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P < 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P < 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P < 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.