IN VITRO DEVELOPMENTAL POTENTIAL OF BLASTOMERES SEPARATED FROM 4- AND 8-CELL RABBIT EMBRYOS AND CULTURED IN MEDIUM SUPPLEMENTED WITH AMINO ACIDS.

Abstract

This study aimed to compare the potential competence of blastomeres separated from intact 4-cell and 8-cell APRI rabbit embryos, and the effect of addition of essential (EAA) and non-essential (NEAA) amino acids on their in vitro developmental. Embryos were recovered from 16 APRI doe rabbits line superovulated by Equine ChorionicGonadotropin (eCG), followed by 0.2 ml GnRH analogue immediately after natural mating with bucks of the same line. Embryos were collected 32–40 h post-coitum and only embryos at 4-cell and 8-cell stages were used in this study. Intact embryo or separated blastomeres were culture in vitro in TCM-199 unsupplemented (M1) or supplemented with 25 µl/ml (M2) or 50 µl/ml (M3) from each of EAA and NEAA. Results showed that separation rat of blastomeres was higher (P<0.05) from intact 4-cell than 8-cell embryos (95.3 vs. 81.7%). Cleavage (80.56 vs. 72.73%) andmorula/blastocyst production (65.74 vs. 58.18%) rates were higher (P≥0.05) of intact embryos at 4-cell stage than those at 8-cell stage. Percentage of degenerated embryos showed an opposite trend. Cleavage (68.89 vs. 63.85%) and morula/blastocyst production (47.11 vs. 44.06%) rates were higher of blastomeres of 4-cell than 8-cell embryos. Percentage of degenerated embryos showed an opposite trend. M3 improved the cleavage of blastomeres of 4-cell embryos (76.92 vs. 61.33%, P<0.05) and those of 8-cell embryos (67.46 vs. 56.69%, P≥0.5) as compared to M1, meanwhile M2 insignificantly improved cleavage rate of blastomeres of 4-cell or 8-cell embryos. Also, M3 improved (P<0.05) morula/blastocyst production rate of blastomeres of 4-cell or 8-cell embryos as compared to M1 and M2. Percentage of degenerated embryos was insignificantly improved by amino acid addition (M2 and M3) as compared to M1. In conclusion, potential competence of blastomeres to morula/blastocysts was better when blastomeres were separated from intact embryos at 4-cell than at 8-cell stage. This potential competence of blastomeres of 4-cell or 8-cell embryos to morula/blastocyst stage improved when in vitro culture medium (TCM-199) was supplemented with essential and non-essential amino acids at a level of 50 µl/ml from each.