336 DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING SPERM CYTOSOLIC FACTOR (SCF) AT FUSION-ACTIVATION

Abstract

At fertilization, the sperm activates the developmental program of the oocyte by inducing an elevation in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). One possible explanation is that at sperm–oocyte fusion the fertilizing spermatozoon introduces a factor into the cytoplasm of the oocyte which opens the Ca²⁺ release channels from the intracellular stores through a yet unidentified mechanism. This study investigated the development of porcine nuclear transfer embryos fused and activated in the presence of sperm cytosolic factor (SCF) isolated from porcine sperm. Ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h at 35–39°C, and rinsed in 0.9% NaCl. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus for use as donor cells. For parthenogenesis, matured oocytes were activated using 2 DC pulses of 1.2 kV cm^-1 for 30 µs in fusion medium (0.1 mM CaCl₂) supplemented with 100, 200, or 300 µg mL^-1 SCF. For NT, matured oocytes were enucleated, reconstructed, and fused. Reconstructed embryos were divided into 2 groups. The embryos in one group were fused with fusion medium (1.0 mM CaCl₂), and the embryos in the other group were fused with fusion medium (0.1 mM CaCl₂) supplemented with 100 µg mL^-1 SCF. After fusion, the embryos were cultured in PZM-3 under 5% CO₂ in air at 38.5°C. A TUNEL assay was used to assess the presence of apoptotic cells (In Situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Data were subjected to a generalized linear model procedure (PROC-GLM) of the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). Oocytes activated parthenogenetically in the presence of SCF showed significantly higher developmental rate to the blastocyst stage compared to that of controls (21.3–27.6% vs. 12.5%; P < 0.05). For NT, there was no difference between treatments in developmental rate to the blastocyst stage (18.2% vs. 17.1%). However, the apoptosis rate was slightly lower in blastocysts produced in the presence of SCF than that in controls. These results indicate that the presence of SCF in fusion medium can support a higher quality of porcine nuclear transfer embryos.