163 SUCROSE ADDITION AT EARLY CULTURE STAGE IMPROVES DEVELOPMENT OF PRE-IMPLANTATION PORCINE NT AND IVF EMBRYOS

Abstract

Apoptosis is a form of cell death leading to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes, and cell death without lysis or damage to neighboring cells. It might contribute to the low developmental rate of in vitro-produced (IVP) embryos, but apoptosis in porcine embryos is still unclear. This study investigated the effect of sucrose in the culture medium on the development of porcine NT and IVP embryos. Oocytes were aspirated from the follicles in 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. A final concentration of motile spermatozoa was adjusted to 5 × 10⁵ cells/mL in fertilization medium. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus and used as donor cells. Embryos were cultured in PZM-3 supplemented with 0.05 M sucrose for 2 days, and then cultured in the PZM-3 without sucrose for 4 days at 38.5°C under 5% CO₂ in air. Apoptotic cell death was analyzed by using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. All data were subjected to a generalized linear model procedure (PROC-GLM) of the statistical analysis system (SAS; SAS Institute, Inc., Cary, NC, USA). NT and IVF embryos cultured in the medium supplemented with sucrose showed a significantly higher blastocyst formation rate than those cultured with no addition (28.8 vs. 20% and 32.3 vs. 17.9%; P < 0.05, respectively). For apoptosis, both NT and IVF embryos cultured in the medium with sucrose showed significantly lower frequency of apoptosis compared to embryos cultured in the medium without sucrose (3.4 vs. 6.3% and 0.6 vs. 1.8%; P < 0.05, respectively). Finally, the number of nuclei in NT blastocysts cultured in the medium with sucrose was higher than that of NT blastocysts cultured without sucrose (30.8 vs. 25.5; P < 0.05, respectively). However, the number of nuclei in the IVF blastocysts was not significantly different between groups. These results indicate that sucrose addition may increase the development of porcine NT and IVF embryos to the blastocyst stage and decrease the rate of apoptotic cells.