170 INFLUENCE OF OOCYTE QUALITY AND DIFFERENT MEDIA SUPPLEMENT ON IN VITRO MATURATION, CLEAVAGE, AND EMBRYO DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES

Abstract

A series of 4 factorial-arranged experiments were conducted to study the effect of oocyte quality and different in vitro maturation (IVM) media supplements on IVM, cleavage, and embryo development of buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected at a local abattoir in a warm (32–35°C) saline (0.9% NaCl), and oocytes were aspirated using an 18-gauge needle. In experiment 1, oocytes (n = 320) were classified according to the number of cumulus cell layers and morphology of ooplasm as excellent, good, or fair. Oocytes were cultured for IVM, fertilization, and embryo culture (IVMFC) in TCM-199 + 10% FCS. In experiment 2, excellent quality oocytes (n = 237) were subjected to IVM in TCM-199 enriched with either 10% FCS or oestrous buffalo serum (EBS; 20–40 pg mL^–1) and then fertilized using frozen–thawed buffalo semen capacitated in Bracket and Oliphant's (BO) medium containing heparin (0.02 mg mL^–1) and sodium caffeine benzoate (3.89 mg mL^–1). In experiment 3, oocytes (n = 290) were classified into 2 groups; Group 1, without gonadotropins, served as a control; Group 2, in which IVM medium was supplemented with 20 IU mL^–1 equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent added to BO medium, either heparin or caffeine or both (n = 210 fertilized oocytes). In all experiments (multiple replicates), oocytes (2–6 mm in diameter) were kept at 39°C under 5% CO₂ for IVMFC and examined several times for cleavage and embryo development (morula and blastocyst). Statistical analysis was carried out using Chi-squared test. Excellent and good quality oocytes produced a higher (P < 0.05) maturation, cleavage, and morula development rates than poor quality oocytes (70% and 65% v. 33.3%), (50% and 46.2% v. 25%), and (42.9% and 33.3% v. 10%), respectively. Blastocyst production rate was also higher (P < 0.05) for excellent compared with good quality oocytes (28.6% v. 16.7%, respectively). In experiment 2, the IVM and cleavage rates were significantly higher (P < 0.05) in IVM medium plus 10% EBS than those cultured in 10% FCS (73% v. 45% and 50% v. 33.3%, respectively). In experiment 3, the addition of eCG to maturation medium increased (P < 0.05) developmental competence of buffalo oocytes (IVMFC) compared with control medium (16% v. 4%). In experiment 4, the addition of heparin together with caffeine to BO medium produced significantly higher (P < 0.05) cleavage and embryo developmental rates compared with heparin or caffeine alone (56.3% v. 33.3% and 35.7%, respectively; 22.2% v. 10% and 8%, respectively). In conclusion, excellent quality oocytes cultured in IVM medium supplemented with either EBS or eCG and fertilized with capacitated buffalo spermatozoa in BO medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocytes.