In vitro growth (IVG) culture of bovine oocyte-cumulus-granulosa complexes (OCGCs) is generally carried out for 12 or 14 days using conventional gas impermeable culture devices. The culture duration may be longer compared to follicular development in vivo. During follicular development, follicles receive oxygen from micro vessels; however, oxygen supply is limited under the culture using conventional gas impermeable devices. The purpose of this study was to investigate the effect of increasing dissolved oxygen availability using a gas permeable (GP) culture device with or without antioxidant (astaxanthin, Ax) supplementation on 8-day IVG culture systems for bovine OCGCs derived from early antral follicles. We cultured OCGCs in GP, GP supplemented with Ax (GP + Ax), and a conventional gas impermeable device (control) for 8 or 12 days. OCGC viability were significantly higher when cultured for 8 days than 12 days (p < 0.001) in all culture condition, but significant difference was not observed between groups (p > 0.05). Antrum formation rates of OCGCs were higher after 12 days than 8 days of culture in all culture condition (p < 0.001) and were significantly higher in the control than GP groups regardless of Ax supplementation (p < 0.05). Oocyte diameters were similar among day-8 GP + Ax, day-8 control and day-12 control groups (p > 0.05). Nuclear maturation rates of oocytes grown in vitro for 8 days were significantly higher in the GP + Ax group than in the control and the GP groups (p < 0.05) and similar to oocytes grown for 12 days regardless of the culture conditions (p > 0.05). The generation of reactive oxygen species in OCGCs on day 8 of IVG culture was significantly lower in the GP + Ax group than those of the GP and control groups (p < 0.05). IVG oocytes after eight days of culture developed into blastocysts, and the cleavage and blastocyst rates were similar in all treatment groups. However, in vivo-grown oocytes had significantly higher (p < 0.05) cleavage and blastocyst rates than the IVG oocytes in all groups. The present study demonstrates that increased oxygen availability using a GP culture device with Ax supplementation promotes oocyte growth and maturation competence but inhibits proliferation of granulosa cells and antrum formation compared with a conventional gas impermeable culture device, and that OCGCs can attain developmental competence after 8 days of IVG culture.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

The reproductive ability of male animal is dependent to a great extent on the effective functions of the genital glands. The present study was carried on the pelvic urethra of 32 sexually mature male donkeys. 5µm sections were prepared from the samples and stained with different stains to show the different structures of the pelvic urethra. Scanning electron microscopic studies were performed on the lumen of the pelvic urethra to show the different shape of the urethral gland opening on the surface layer of the lamina epithelialis of the pelvic urethra. The pelvic urethra of donkey is formed of prostatic and membranous parts. The lamina epithelialis of the pelvic urethra varied at its different regions. The urethral glands were observed along the entire length of the pelvic urethra within the lamina propria-submucosa. They were mostly of the branched tubulo-alveolar glands lined by high cuboidal or pyramidal-shaped epithelial cells. The activity of the urethral glands in donkey varied throughout the year. It was more pronounced during spring, which was manifested by increased epithelial height, decreased nuclear/cell ratio, decreasing interstitial connective tissue/glandular tissue ratio and increased cellular secretory activity. This activity decreased gradually during the summer and autumn to reach its minimal level during winter.