196 IN VITRO DEVELOPMENT OF RECONSTRUCTED WATER BUFFALO (BUBALUS BUBALIS) OOCYTES AFTER FETAL SKIN FIBROBLAST CELL NUCLEAR TRANSFER

Abstract

The present study was undertaken to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to determine the developmental competence of embryos following transfer of these nuclei to in vitro-matured enucleated buffalo oocytes. Skin cells were isolated from 1–2-month-old fetuses, obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in Dulbecco's PBS) for 15–20 min. The cells were washed four times with Dulbecco's PBS and then once with RPMI-1640 medium + 10% FBS by centrifugation at 600g. 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. COCs collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 1 μg mL⁻¹ E-β + 5 μg mL⁻¹ FSH-P + 10 μg mL⁻¹ LH + 10% FBS) for 22–24 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 and 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 electrofused and incubated in the activation medium (TCM-199 + 8 μg mL⁻¹ 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. Next, the cleaved embryos were co-cultured with buffalo oviductal cells in embryo development medium. Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electrofused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to the 2-cell stage, 3 (3.8%) reached the 4-cell stage, and 1 (1.3%) reached the 8-cell stage. In the synchronized group, out of 100 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (40%) were electrofused, activated, and cultured, out of which 4 (10%) developed to the 2-cell stage, 3 (7.5%) to the 4-cell stage, 2 (5.0%) to early morula stage, and 1 (2.5%) to blastocyst 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.