In vitro growth culture in a medium with reduced sodium chloride improves maturation and developmental competence of pig oocytes derived from small antral follicles

摘要

The objective of this study was to evaluate the effects of reducing the sodium chloride content in in vitro growth (IVG) medium to 61.6mM on in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium with 108mM NaCl (alpha MEM-108) or porcine zygote medium (PZM) containing 61.6mM (PZM-61.6) or 108mM (PZM-108) NaCl. These media were further supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 10% (v/v) fetal bovine serum. After IVG culture, oocytes were matured for 44 h in our standard IVM medium. The IVG culture in PZM-61.6 significantly increased nuclear maturation (88.0 +/- 2.2%) of SAF oocytes compared to that in PZM-108 (77.3 +/- 3.9%) or alpha MEM-108 (75.9 +/- 3.8%). After parthenogenesis (PA), the proportions of blastocysts, based on the number of metaphase II (MII) oocytes, induced for PA were not different among IVG oocytes cultured in PZM-61.6 (50.2 +/- 3.0%), PZM-108 (46.8 +/- 2.9%), or alpha MEM-108 (45.6 +/- 2.9%). The IVM oocytes derived from IVG in PZM-61.6 showed increased perivitelline space (PVS) (12.1 +/- 0.6 mm) and intra-oocyte glutathione (GSH) content (1.19 +/- 0.04 pixels/oocyte) compared to PVS (8.0 +/- 0.5 and 7.4 +/- 0.4 mm) and GSH (1.03 +/- 0.04 and 1.00 +/- 0.04 pixels/oocyte) of oocytes derived from PZM-108 and alpha MEM-108, respectively. The IVG culture in PZM-61.6 stimulated meiotic resumption after IVG and faster nuclear progression after IVM than that in alpha MEM-108. After somatic cell nuclear transfer (SCNT), the blastocyst formation of SAF oocytes grown in PZM-61.6 (17.8 +/- 3.3%) was higher than that of oocytes grown in PZM-108 (7.5 +/- 2.7%) but not different from that of oocytes in alpha MEM-108 (11.4 +/- 3.4%). Regardless of the different osmotic pressures, nuclear maturation was significantly increased by IVG culture in PZM with reduced NaCl (86.8 +/- 2.3 and 84.9 +/- 4.2% in PZM-61.6 and PZM-61.6 with sorbitol, respectively) than in PZM-108 (70.5 +/- 3.4%). Blastocyst formation was not affected by the differences in NaCl content and osmotic pressure of the IVG medium, whereas the mean number of cells in blastocysts was significantly higher following IVG culture in PZM-61.6 than in the other groups. In conclusion, the results demonstrate that, following SCNT in pigs, IVG culture of SAF oocytes in a medium with a reduced NaCl concentration stimulates oocyte maturation and improves subsequent embryonic development. (C) 2020 Elsevier Inc. All rights reserved.