45 EFFECT OF OSMOLARITY IN CULTURE MEDIUM ON THE PRE-IMPLANTATION DEVELOPMENT OF PORCINE NT AND IVF EMBRYOS

Abstract

In vitro production of the pig embryo is very important as an initial step to improve its application in biotechnology. The in vitro production system for pig embryos, however, has been plagued by the high incidence of polyspermy and poor embryo quality. The present study was conducted to examine the relationship between apoptosis and osmolarity of culture medium in pre-implantation development of porcine NT and IVF embryos. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 40–44 h. Fresh semen was diluted and equilibrated at 16°C. The final concentration of motile spermatozoa was adjusted to 5 × 10⁵ cells/mL in fertilization medium. Fetal fibroblasts were prepared from a 35-day-old porcine fetus for use as donor cells. The NT and IVF embryos were cultured in PZM-3 supplemented with 0.05 M sucrose or a final concentration of 138 mM NaCl (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for the remaining days. For the control, NT and IVF embryos were cultured in PZM-3 for whole culture period. After 6 days of culture, the developmental ability of embryos, total cell numbers, ratio of ICM/TE, and apoptosis of cells in blastocysts were examined. The developmental rate to the blastocyst stage of NT embryos was significantly higher (P < 0.05) in the sucrose and NaCl groups than in the control [14.7% (21/153) and 21.7% (34/154) vs. 11.5% (18/152), respectively]. Also, the developmental rate to the blastocyst stage after IVF was slightly higher in embryos cultured in the medium supplemented with NaCl than in the control group [21.8% (49/235) and 26.4% (61/237) vs. 18.9% (44/247)]. For apoptosis, both NT and IVF blastocysts produced in the sucrose and NaCl groups showed slightly lower frequency of apoptosis compared to that of the control (2.2% and 2.8% vs. 3.1% for NT; 0.9% and 0.7% vs. 1.1% for IVF). These studies suggest that the high osmolarity in the early embryo culture stage could enhance the in vitro development of both porcine NT and IVF embryos to the blastocyst stage and could reduce the apoptosis of cells.