Recently, the activity of cathepsins B was found to be correlated inversely with the developmental competence of bovine oocytes. In this study, we investigated (1) the role of intracellular cathepsin B expression and developmental competence as well as the quality of bovine preimplantation embryos, and (2) the effect of cathepsin B inhibitor (E-64) during in vitro culture (IVC) on the development and quality of bovine embryos. After in vitro fertilization (IVF) followed by IVC for 7 days, good and poor quality embryos classified by morphology and developmental rate on days 2, 4, and 7 were assessed for cathepsin B expression and activity. To investigate the effect of cathepsin B inhibition on embryonic development, putative zygotes were cultured with or without E-64, followed by evaluation of cleavage and blastocyst rates on days 2 and 7, respectively. Embryonic quality was evaluated by both TUNEL staining and total cell number in day-7 blastocysts. In each developmental stage, cathepsin B expression and activity were significantly higher in poor quality embryos than good quality ones. Moreover, addition of E-64 during IVC significantly increased both the blastocyst rate and the total cell number. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in day-7 blastocysts. These results indicate that cathepsin B activity can be useful as a marker for inferior quality embryos. Moreover, inhibition of cathepsin B greatly improves the developmental competence of preimplantation embryos and increases the number of good quality embryos.

Recently, the quantity of cathepsin transcripts in cumulus cells was found to be associated with low-developmental competence of bovine oocytes. In the present study, we investigated (1) the relation between cathepsin B activity and the quality of in vitro-matured cumulus-oocyte complexes (IVM COCs) and denuded oocytes and (2) the effect of a cathepsin B inhibitor (E-64) on embryo development and quality. The activity of cathepsin B was evaluated in IVM COCs and denuded oocytes. After maturation of COCs with or without E-64, followed by in vitro fertilization, zygotes were cultured for 8 days. Cleavage and blastocyst rates were evaluated on days 2 and 8, respectively. Quality of embryos was evaluated by differential staining of day 8 blastocysts. TUNEL staining was conducted on IVM COCs and blastocysts. Cathepsin B activity was clearly detected in the low-quality oocytes, and in the cumulus cells of both high- and low-quality oocytes. This latter activity was diminished by addition of E-64. The presence of E-64 during IVM also significantly increased both the blastocyst rate and the total cell number, and improved blastocyst quality associated with a significant increase of trophoectoderm cells. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in both the cumulus cell layer of matured oocytes and blastocysts. These results indicate that cathepsin B activity can be a useful marker of oocyte quality. Furthermore, inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high-quality embryos.

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Key words: D-Penecillamine, Hypotaurine, Epinephrine, In vitro fertilization, BuffaloIn vitro fertilization (IVF) is a necessary procedure in the in vitro production of embryo. Spermatozoa need to undergo capacitation to be able to perform the acrosome reaction which is required for zona pellucida penetration. Different additives are used to aid in that process, such as caffeine and theophylline. In this study, the effects of IVF media supplementation with D-penicillamine, Hypotaurine and epinephrine (PHE) on fertilization and cleavage rates were assessed. Cumulus oocyte complexes (COCs) containing a compact cumulus cells and normal homogenous cytoplasm were selected. In vitro maturation (IVM) was performed in TCM199 supplied with hormones overnight in a culture conditions of 38.5°C and 5% CO 2 in air. After 20-22 hr. of maturation, oocytes were randomly assigned to five groups of sperm-TALP media (control and four different PHE concentrations) then inseminated. The fertilization rate of the expected embryos were assessed after 22 hr. after staining with aceto-orcin 1% or cultured for 48 hr. to estimate the cleavage rate. The current data revealed that, 25 µl/ml PHE significantly (P< 0.05) improved fertilization (55.93 vs 36.96%) and cleavage (46.15 vs 22.22%) rates when compared to control.

This review focused on the various methods for controlling estrous cycles in well-managed dairy cows. Because up to 70% of dairy cows may stay non-pregnant after an AI procedure, an effective approach for identifying and reinseminating open cows is essential for dairy herds to achieve optimal reproductive performance. Overall, well-managed dairy farms with effective estrus detection programs inseminate 50% or more of non-pregnant cows after behavioral estrus is detected. Cows not detected in estrus are admitted in a resynchronization of ovulation procedure to receive a timed AI (TAI) service to avoid a long interbreeding interval. In Egypt, a widely used program involves starting the Ovsynch protocol (GnRH-7 d-PGF2-56 h-GnRH-16 to 20 h-TAI) 32 days after an initial AI, regardless of pregnancy status. Previous studies have proven that there was no difference in pregnancy/artificial insemination (P/AI) between Ovsynch+P4 and Presynch-Ovsynch, both protocols were equally effective in improving the fertility of cows with a CL 15 mm. The review also addressed different methods for synchronization of ovulation and different factors affecting the selection of the management program.