Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39degrees C under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean +/- SD) per prepubertal and pubertal goats were 5.2 +/- 0.6 and 6.8 +/- 0.6, and 77.3 +/- 0.1% and 80.5 +/- 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 +/- 0.8 vs. 2.6 +/- 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode's albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 +/- 0.9% vs. 32.7 +/- 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 +/- 1.7 vs. 32.2 +/- 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.
Animals ; Culture Techniques ; Embryonic Development/*physiology ; Female ; Fertilization in Vitro/*veterinary ; Goats/*embryology ; Male ; Oocyte Retrieval/veterinary ; Oocytes/*physiology ; Ovary/cytology ; Sexual Maturation ; Sperm Capacitation ; Tissue and Organ Harvesting/veterinary

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.
Animals ; Cloning, Organism/*veterinary ; Culture Media ; Embryo, Mammalian ; Karyotyping ; Nuclear Transfer Techniques/*veterinary ; *Oocytes ; Swine/*embryology ; Whales/*embryology