INFLUENCE OF GAMETE CO-INCUBATION TIME, SIRE AND SPECIAL ADDITIVES ON THE IN VITRO FERTILIZATION OF CUMULUS-ENCLOSED OR DENUDED BUFFALO OOCYTES

Abstract

The present study was designed to investigate the influence of cumulus removal before the in vitro fertilization step and the impact of co-incubation time, sire, and additives to the fertilization medium on the efficiency of in vitro fertilization of buffalo oocytes. In vitro matured oocytes were fertilized either as cumulus-oocyte complex (COCs) or after removal of cumulus cells (denuded). Cumulus-enclosed or denuded oocytes were co-incubated with sperm cells for 6, 12 or 18 h (experiment 1), fertilized with sperm cells from one of three sires (experiment 2) or fertilized in medium supplemented with 20μg∕ml heparin, 5 mM theophylline or a mixture 20μM penicillamine, 10μM hypotaurine and 1 μM epinephrine (PHE) (experiment 3). In all experiments, a group of oocytes was fixed and stained to evaluate the fertilization pattern (penetration, normal and abnormal fertilization rates) and the rest were cultured up to 8 days to assess the developmental competence (cleavage and blastocyst yield). In all experiments, removal of cumulus cells before fertilization step significantly retarded the fertilization pattern and the developmental competence. Various co-incubation times did not significantly influence the fertilization pattern or the developmental competence of denuded or COCs. However, 6 h tended to decrease the abnormal fertilization (15.74±1.70 vs 28.46±6.06, P=0.069) and to improve the blastocyst/oocyte (11.70±3.41 vs 5.53±1.75) and the blastocyst/cleavage (27.14±6.19 vs 11.98±3.81, P=0.082) when compared with 18 h. Sperm cells from the three sires resulted in similar fertilization pattern and developmental competence in COCs and denuded oocytes. In COCs and denuded oocytes, PHE tended to improve the blastocyst/oocyte (7.44±2.58 vs 14.67±4.29) and blastocyst/cleavage (18.43±6.08 vs 33.13±9.27) in comparison to heparin (P˃0.05). Thus, none of the investigated factors could counteract the adverse effect of cumulus removal otherwise, the addition of PHE showed promising results but it need further investigations.