Embryo

Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro

Abstract

This study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electro-stimulation (El-St) and cytochalasin B (CB) treatment of in vitro matured oocytes. The culture medium for development, based on Whitten's medium, contained 0.5 mg/ml hyaluronic acid (mWM), and a two-step culture system, in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In Experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01 to 5 mg/ml polyvinyl alcohol (PVA). In Experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml PVA (PVA-mWMs) for 0, 48, 72 h, and subsequently cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle's basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05 to 5 mg/ml PVA (49 to 53% vs. 63%; P>0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11 to 20% vs. 39%; P<0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 h or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In Experiments 3 to 5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In Experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of non-polar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage-rates of the diploids after compaction and the total number of cells in the blastocysts (53.7±2.7) and ICM (12±0.5). These findings indicate that the presence of non-polar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.