Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro1

Abstract

The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of 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 0.01-5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle 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 0.05-5.0 mg/ml of PVA (49%-53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%-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 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3-5, 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 nonpolar 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 inner cell mass (12 +/- 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.

Adding Essential Amino Acids at a Low Concentration Improves the Development of In Vitro Fertilized Porcine Embryos

“…Our results suggest that the presence of EAA at a low concentration during the first 72 h of culture is not overtly inhibitory although neither were the EAA beneficial and there is evidence that they inhibit the development of cleavage stage mouse [20] and, under certain conditions, pig embryos [10]. As such, we decided to include the EAA at a 1:100 dilution in our culture system from day 3 onwards only.…”

“…Ammonium in the culture medium has been shown to be detrimental to the in vitro development of mouse embryos [21,22] and it has been suggested that a decrease in the rate of ammonium accumulation may be partially responsible for the improved development seen by reducing the concentrations of EAA in the culture mouse embryos [11]. However, further research is required to determine how much these factors contribute to the effect seen in our experiment.Our results suggest that the presence of EAA at a low concentration during the first 72 h of culture is not overtly inhibitory although neither were the EAA beneficial and there is evidence that they inhibit the development of cleavage stage mouse [20] and, under certain conditions, pig embryos [10]. As such, we decided to include the EAA at a 1:100 dilution in our culture system from day 3 onwards only.…”

“…Early cleavage development was found to be stimulated by the addition of NEAA to the culture medium but inhibited by EAA in both the mouse [7,8] and cow [9]. The presence of EAA during the first 48 h of culture in a protein free medium inhibited the development of porcine embryos, but stimulated development when added after 48 h [10]. Lane et al…”

Fetal bovine serum influences apoptosis and apoptosis-related gene expression in porcine parthenotes developing in vitro

“…Therefore, diploid parthenotes have frequently been used to study early development in the pig (Van Thuan et al 2002). In the present study, we determined the effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and BSA on the developmental ability and apoptosis of porcine 2-cell parthenotes at 24 h after activation (before embryonic genome activation) and late 4-cell diploid parthenotes at 72 h after activation (after embryonic genome activation) developing in vitro.…”

Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs

“…In the present study, addition of EAAs and NEAAs to NCSU-23 had no effect on the in vitro development of IVF and SCNT embryos. However, previous studies have suggested that addition of EAAs and NEAAs to the culture medium promotes the development of embryos in mice [42], cows [43,44] and pigs [22,45]. These previous studies reported that EAAs and NEAAs are components that promote embryo development when cultured in media containing lactate and pyruvate as energy substrates.…”