Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbeccoʹs PBS) for 15–20 min. The cells were washed 4 times with Dulbeccoʹs PBS and then once with RPMI-1640 + 10% FBS by centrifugation at 600 × g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 °C for 2–3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 μg/ml FSH-P + 10 μg/ml LH + 10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 °C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10–15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 μg/ml cytochalasin-B + 10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199 + 10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 °C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.