Effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes

Abstract

The study was undertaken to estimate the effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and embryo development of in-vitro matured and fertilized prepubertal and pubertal goat oocytes. The oocytes were matured in maturation media (TCM–199) supplemented with either different supplements, viz. 20% estrus goat serum (EGS), 20% new calf serum (NCS), or 10% new calf serum (NCS) plus hormone (FSH + LH + E2) or 20% caprine follicular fluid (CFF). After 27 hours (h), the matured oocytes were picked up and denuded by treating with 0.1% hyaluronidase and by repeated pipetting through a fine bore pipette. The denuded oocytes were transferred in fert-TALP medium (fertilization medium). The capacitated sperm having concentration of 1×106 live sperm/ml were used for in- vitro fertilization. Oocytes were co-incubated with capacitated spermatozoa for 18 h at 38.5±1°C in an atmosphere of 5% CO2 in humidified air. The fertilized oocytes culture in EDM over their respective cell culture i.e. granulosa cells monolayer and oviductal epithelial cells were obtained for further cleavage rate and embryo development (2 celled, 4 celled, 8 celled, and >8 celled) under steriozoom microscope at every 24 h with 50% replacement of pre-equilibrated EDM media up to 7 days. Forty-eight hours post insemination the cleavage rates with granulosa cells monolayer and oviductal epithelial cells co-culture were 40.78 and 45.93% and 47.36 and 43.49% for prepubertal and pubertal goat oocytes respectively. The embryo development up to the 2 cell, 4 cell, 8 cell, and >8 cell with granulosa cells monolayer and oviductal epithelial cells co- culture were 65.32, 22.58, 6.45, 5.64 and 63.92, 27.26, 6.96, 1.89% and 50.92, 30.55, 12.03, 6.48 and 40.78, 22.36, 7.23, 6.57%, respectively, for prepubertal and pubertal goat oocytes.
In conclusion, the present study indicate that both granulosa cell monolayer and oviductal epithelial cells co-culture behaved similarly for cleavage rate and embryo development of in-vitro matured and fertilized prepubertal and pubertal goat oocytes.