Alanine Supplementation during Pig Oocyte Maturation Improved Embryonic Development by Increasing Glutathione Level and Gene Expression Associated with Embryo Development

Alanine known as non-essential amino acid was detected at high concentration in reproductive tracts and follicular fluid in developing porcine antral follicle. The purpose of this study was to determine the effect of alanine supplementation during in vitro maturation (IVM) of porcine oocyte. We investigated nuclear maturation, intraoocyte glutathione (GSH) contents in metaphase II (MII) oocytes, and subsequent embryonic development. And also, We detected the gene expression pattern in MII oocytes, early embryo and blastocyst derived parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). The base medium for IVM was North Carolina State University-23 (NCSU-23) medium, modified by supplementing 10 ng/mL EGF, 0.5 ug/mL FSH, and 0.5 ug/ LH, replacing BSA with 0.1% (w/v) PVA, and deleting glutamine. Alanine of various concentrations (0, 0.363, 1, 5, and 10 mM) were added to base IVM medium. The proportion of mature oocyte after IVM did not increase by alanine treatment at various concentrations. However, The intraoocyte GSH content was higher (p<0.05) in oocytes treated with 0.363 mM alanine (1.17±0.01 pixels per oocyte) than non-treated oocytes (1.00±0.02 pixels per oocyte). Blastocyst formation of PA (31.2±2.5% vs. 19.8±2.5%) and SCNT (20.5±1.9% vs. 10.1±2.0%) embryos was significantly (p<0.05) improved by treatment with 0.363 mM alanine during IVM compared with embryos derived from the non-treated oocytes. Supplementation of oocytes IVM medium with 0.363 mM alanine significantly (p<0.05) increased the gene expression of POU5F1 and FGFR2 levels in early embryo and blastocyst derived PA and SCNT embryos. In MII oocytes, transcript levels of POU5F1 and FGFR2 as well as CDK1 gene were significantly (p<0.05) increased in 0.363 mM alanine-treated oocytes. Our results demonstrate that treatment with 0.363 mM alanine during pig oocyte maturation improves developmental competence after PA and SCNT by influencing cytoplasmic maturation, such as improved GSH content in IVM oocyte and increasing gene expression associated with embryonic development in oocyte and embryo.