265 EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO

Abstract

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta & Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5 μg mL⁻¹ FSH in 5% CO₂ incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42 h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF) + 0.8 %BSA (control), 2) mSOF + 0.8 % BSA + gelatin (1 mg mL⁻¹) 3) mSOF + 0.8% BSA + 1 mg mL⁻¹ gelatin + 10 ng mL⁻¹ epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658 ± 0.009 to 1.0331 ± 0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P < 0.05) in mSOF + 0.8% BSA + 1 mg mL⁻¹ gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF + 0.8% BSA + 10 ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.