270 IN VITRO FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) OOCYTES: EFFECTS OF MEDIA AND SPERM MOTILITY INDUCING AGENTS

Abstract

The identification of an optimal in vitro fertilization system is critical in order to improve the in vitro embryo production efficiency in buffalo species. The aim of this work was to evaluate the effects of fertilization media and sperm motility inducing factors (SMIF) on cleavage and blastocyst rates in buffalo species. Cumulus-oocytes complexes (n = 516), recovered from slaughtered animals, were matured in vitro in TCM 199 + 10 % FCS, 0.5 μg mL⁻¹ FSH, 5 μg mL⁻¹ LH, 1 μg mL⁻¹ 17β-estradiol and 50 μM cysteamine, at 38.5°C under 5% CO₂ in humidified air for 24 hours. The mature oocytes were randomly assigned to four groups for fertilization. In particular, IVF was carried out at 38.5°C under 5% CO₂ in humidified air in either Tyrode’s modified medium or Brackett Oliphant medium, in the presence of 0.01 mM heparin;; each medium was supplemented with either a mixture of 0.2 mM penicillamine and 0.1 mM hypotaurine or 5 mM caffeine. Frozen-thawed sperm from a tested bull was treated by the swim-up procedure and used at a final concentration of 20⁶ mL⁻¹. After 20–22 h presumptive zygotes were cultured in SOF medium, supplemented with essential and non-essential amino acids and BSA, in a gas atmosphere of 5% CO₂, 7% O₂ , and 88% N₂, up to the blastocyst stage. Cleavage rates and blastocyst yields were analyzed by a full factorial model 2 × 2 with medium and SMIF effects (SPSS 11.0). The analysis used permits the identification of statistical differences between treatments irrespective of an interaction (Searle SR. 1971. Linear model. Ed. John Wiley & Sons;; XXI:533). The comparison of the two media, irrespective of the SMIF used, did not show any difference in cleavage rate (43.7% v. 39.3%, respectively, in TALP and BO). On the contrary higher cleavage rates were recorded with hypotaurine-penicillamine v. caffeine (47.7% v. 35.3%; P < 0.05), regardless of the medium employed. However, a significant interaction between media and SMIF was found;; in fact the addition of hypotaurine and penicillamine significantly improved cleavage rate compared with caffeine in TALP medium (59.6% v. 27.9%; P < 0.05) whereas no differences were observed in BO (35.9% v. 42.7%, respectively). With regard to blastocyst yield a significant effect of medium was also found, with the highest embryo production in TALP v. BO (13.9% v. 6.8%; P < 0.05). Blastocyst rate was improved in the presence of hypotaurine-penicillamine v. caffeine (13.6% v. 7.2%; P < 0.05). Furthermore there was a significant interaction between medium and SMIF, with the highest embryo yields in the presence of hypotaurine-penicillamine v. caffeine in TALP (20.7% v. 7.1%, respectively;; P < 0.05) but not in BO (6.4% v. 7.2%, respectively). The differences we found disappeared when the embryo yield was calculated in relation to the cleaved eggs, with the exception of a lower efficiency of BO v. TALP (15.9% v. 31.1%; P = 0.057), suggesting an influence of BO also on post-fertilization development.