60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

Abstract

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3%, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3%, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4%) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3%) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9%) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development.

This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.