38 HIGH OSMOLARITY AT EARLY CULTURE STAGE IMPROVES IN VITRO DEVELOPMENT OF PRE-IMPLANTATION PORCINE NUCLEAR TRANSFER EMBRYOS

Abstract

Fragmentation occurs during early developmental stage of electrically activated oocytes and nuclear transfer (NT) embryos, and it might be the cause of the low developmental rate of pre-implantation porcine nuclear transfer embryos. The present study was conducted to investigate whether addition of sugars such as sorbitol and sucrose suppresses fragmentation and supports the development of NT embryos. Oocytes derived from a local abattoir were matured for 42–44 h and enucleated. Fetal fibroblast cells were obtained from a Day 35 porcine fetus. Parthenogenetically activated oocytes or NT embryos were cultured in PZM-3 for 6 days, or in PZM-3 supplemented with sorbitol or sucrose for 2 days and then cultured in PZM-3 for the remaining 4 days. The osmolarity of PZM-3, PZM-3 supplemented with 0.05 M or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose was 269 ± 6.31, 316 ± 3.13, 362 ± 4.37 and 315 ± 5.03 mOsm, respectively. When the parthogenotes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate was observed. Interestingly, sugar addition into PZM-3 at early culture stage for 2 days reduced the fragmentation rate compared to the rate in PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 significantly (P < 0.05) increased the cleavage rate (67.6 ± 5.80 vs. 77.3 ± 3.03) and developmental rate to the blastocyst stage (10.2 ± 0.79 vs. 19.4 ± 1.77). There was no significant difference between treatments for the number of nuclei in the blastocysts. In addition the fragmentation rate in sugar-supplemented PZM-3 was reduced compared to that in PZM-3 without sugar (26.1 ± 4.30 vs. 14.5 ± 1.74). In conclusion, the increased osmolarity of PZM-3 with sugar supplementation at an early developmental stage for 48 h could increase the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate.