145 Comparison of various buffalo sera collected during different phases of estrous cycle for in vitro maturation and culturing of Nili-Ravi buffalo oocytes

The objective of current study was to compare different types of buffalo sera collected during different phases of the oestrous cycle for IVM and cleavage rate of Nili-Ravi buffalo oocytes. Buffaloes (n = 5) were synchronised using the Ovsynch protocol and serum was harvested after centrifugation (400 × g) of blood collected via jugular venipuncture at luteal phase (LPS), onset of oestrus (ES0), and 30 h after oestrus onset (ES30). All sera were analysed for oestradiol-17β to progesterone ratio (E2:P4) using radioimmunoassay. Later, sera were heat-inactivated at 56°C for 30 min and added to maturation media (Medium 199). Fetal bovine serum (FBS) was also added as a control in the study. Oocytes were aspirated from 380 pairs of Nili-Ravi ovaries, and 1037 oocytes of grade A and B were incubated (38.6°C with 5% CO₂, and 98% relative humidity) in maturation medium containing 20% of LPS (n = 245), ES0 (n = 280), ES30 (n = 250), and FBS (n = 262) for 24h. Oocyte maturation was assessed based on the cumulus-oocyte complex (COC) expansion and graded as fully expanded, partially expanded, and not expanded. Fully expanded oocytes were further processed for IVF for 12 h in BO medium. Nuclear maturation of oocytes was assessed in each serum at 8, 22, and 24 h using Hoechst 33342 stain in a different set of oocytes. After IVF, oocytes from each serum were in vitro cultured for 48 h and assessed for cleavage (2 or more cells). Data were analysed using PROCMIX (SAS version 9.0; SAS Institute Inc.), considering days of follicular aspiration as a random factor while different sera were fixed effects in the model. Comparison of oocyte maturation and cleavage rates among different sera was made using Tukey’s multiple comparison test with P < 0.05 was considered as significant. The results revealed a greater ratio (P < 0.01) of fully expanded oocytes in ES0 (79.5%) and FBS (74.5%) as compared LPS (44%) and ES30 (63.3%). The ratio of MI stage oocytes after 8 h of maturation in ES0 and FBS was comparable (P > 0.05) but differed (P < 0.05) with LPS and ES30. The ratio of MII stage oocyte at 22 h of maturation was greater in ES0 than LPS and ES30, whereas FBS had a comparable ratio of MII stage oocytes to that of ES0 and ES30 (P > 0.05). The ratio of MII stage oocytes at 24 h of maturation was similar between ES0 and FBS whereas the least ratio of MII stage was observed in LPS. Maximum cleavage rate was observed in ES0 (55.7%) compared with LPS (18.4%), ES30 (35.7%), and FBS (43.1%). The least (P < 0.05) ratio of E2:P4 was observed in LPS (0.34) whereas ES30 (12.5) and ES0 (8.0) had comparable E2:P4 ratio (P > 0.05), suggesting that a delicate balance of E2:P4 ratio may explain the better oocyte maturation and cleavage rate in ES0. We concluded that oocyte maturation and cleavage rates were better in ES0 than in LPS and ES30. Moreover, ES0 had a better cleavage rate than FBS.