110 Enhancement of developmental competence of immature oocytes supplementing with leukaemia inhibitory factor as a media supplement

The present study aims to improve the in vitro production of cattle embryos by supplementing culture media with leukaemia inhibitory factor (LIF). For the current study, fresh ovaries and oviducts were collected from indigenous cattle from a local abattoir in 0.9% saline solution (30–35°C) supplemented with antibiotics (400 IU mL⁻¹ penicillin and 50 mG⁻¹ streptomycin) and were transported to the laboratory within 2–3 h of animal slaughter. These slaughterhouse ovaries were aspirated to obtain cumulus-oocyte complexes, washed 5–6 times in washing media and cultured in maturation media (TCM-199 + 10% fetal bovine serum [FBS] + 10 mg mL⁻¹ FSH-P + 0.81 mM sodium pyruvate + 50 mg mL⁻¹ gentamicin sulfate) for 24 h in a 5% CO₂ incubator at 38.5°C with maximum humidity. After 24 h of culture, matured oocytes were co-incubated with in vitro-capacitated sperms for fertilisation in Bracket and Oliphant’s medium for 15–18 h. After co-incubation, surrounding cumulus cells were stripped off by repeated gentle pipetting and presumptive zygotes were cultured for embryo development in mCR2aa culture medium. After 40 to 42 h, cleavage was observed, and embryos were cultured for 7–9 days in the same environmental conditions. Culture media used were replaced with fresh media after every 24 h. In this study, leukaemia inhibitory factor was supplemented in culture media with three different concentrations, i.e. 15, 30, 45 ng mL⁻¹. A total of 542 cumulus-oocyte complexes were used in the study. All data of four replicates were analysed by one-way ANOVA (post hoc Tukey’s test). There was no effect of LIF supplementation on cleavage rate (in control group, 71.9 ± 0.6; 72.2 ± 0.8, 78.1 ± 0.8, and 76.7 ± 0.0 in 15 ng mL⁻¹, 30 ng mL⁻¹, and 45 ng mL⁻¹, respectively). However, the addition of 30 ng mL⁻¹ LIF during culture increased (P < 0.05) blastocyst development (in control group, 7.0 ± 1.3; 7.1 ± 1.2, 12.9 ± 0.4, and 10.2 ± 1.5 in 15 ng mL⁻¹, 30 ng mL⁻¹, and 45 ng mL⁻¹, respectively) in Indian cattle.

The authors acknowledge sincere thanks to the director, National Dairy Research Institute, Karnal, joint director (Research), National Dairy Research Institute, Karnal and Head, Eastern Regional Station, National Dairy Research Institute, Kalyani, for providing funds and necessary facilities to carry out the work.