119 LEPTIN ENHANCED THE DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYO CULTURED IN VITRO

Abstract

Buffalo is an important livestock resource in many Asian and Mediterranean countries. In vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical factors in IVEP procedures. Recent research revealed that supplementation of leptin in the in vitro culture (IVC) medium could significantly increase embryo development (2005 Mol. Cell Endocrinol. 229, 141–147; 2006 Reproduction 132, 247–256). In this study, the effect of leptin on buffalo embryo development in vitro was assessed by supplementation of the leptin into the IVC medium. Methods: Buffalo oocytes were aspirated form 2 to 6 mm follicles from slaughterhouse ovaries and washed in TCM199 and once more with in vitro maturation (IVM) medium (TCM199, 5% ECS, 15 μg mL^–1 FSH). Oocytes with compact cumulus cells were matured in IVM medium at 38.5°C, 5% CO₂ for 22–22 h. The frozen–thawed buffalo sperm underwent a centrifugation in Percoll gradient to remove the dead sperm. Ten to 15 matured oocytes were added to a drop of 40 μL modified Tyrode’s medium supplemented with 0.6% BSA, 2.0 mm caffeine and 20 μg mL^–1 Heparin. Concentration of sperm added into the fertilization medium was 1 to 2 million per mL. Eight to 10 h after insemination, the presumptive zygotes were transferred to IVC medium (TCM199, 10% newborn cow serum) supplemented with 0 ng mL^–1 (control), 10 ng mL^–1, 100 ng mL^–1 or 500 ng mL^–1 of leptin. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8 after insemination. The experiment was repeated 10 times, and a total of 831 oocytes were used with the IVF procedures. The results revealed that the cleavage rates in the group of 0 ng mL^–1, 10 ng mL^–1, 100 ng mL^–1 and 500 ng mL^–1 of leptin were 50.1 ± 3.5%, 55.0 ± 1.3%, 50.0 ± 1.8% and 52.9 ± 2.2%, respectively. No statistical difference was observed regarding cleavage rates between treatments (P > 0.05). The percentage of oocytes developing to blastocysts in the group of 10 ng mL^–1 and 100 ng mL^–1 leptin were 26.1 ± 1.5% and 23.5 ± 1.2%, respectively, significantly higher than that of 17.5 ± 2.1% in the control (P < 0.05). The blastocyst development rate in the group of 500 ng mL^–1 leptin was 20.9 ± 1.4%, less than that of 10 ng mL^–1 (P < 0.05). In conclusion, the results of this study indicated that supplementation of leptin in the IVC medium could enhance the blastocyst development in buffalo species and the optimal concentration of leptin in the present procedures was 10 ng mL^–1.

This work was jointly supported by National Science and Technology Supporting Program (No. 2006BAD04A18), Guangxi Science Foundation (0832012) and Guangxi University Key Research Program (No. 2005ZD05).