Identification of the optimal manipulation medium temperature for in vitro handling of oocytes and embryos during in vitro maturation, parthenogenesis, and in vitro culture in pigs.

Abstract

Exposure of cumulus-oocyte complexes (COCs), mature oocytes, and preimplantation embryos to subphysiological temperatures during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) has been shown to impair oocyte maturation, embryogenesis, and developmental competence. However, most previous studies have focused primarily on defining optimal embryo culture temperatures. Therefore, the present study aimed to establish optimal temperature conditions for in vitro manipulation, to minimize impairment of oocyte maturation, embryogenesis, and preimplantation development. All in vitro manipulation steps during the IVM, PG, and IVC processes were conducted using manipulation media maintained at defined temperatures (39°C, 38°C, 37°C, 35°C, 34°C, and 33°C) through the combined use of microscope warming plates and infrared irradiation. Oocyte maturation and the preimplantation developmental competence of embryos following parthenogenetic activation were subsequently evaluated. The use of infrared irradiation to control manipulation medium temperature precisely did not adversely affect oocyte maturation or embryonic developmental competence. Among the subphysiological temperature conditions examined (33°C-38°C), maintaining the in vitro manipulation media at 35°C throughout the IVM, PG, and IVC processes resulted in significantly reduced perivitelline space size, increased intra-ooplasmic glutathione levels, improved mitochondrial distribution normality, and enhanced preimplantation development compared with manipulation at 39°C, the physiological body temperature of pigs. Based on these results, we suggest that in vitro manipulation of immature COCs, oocytes, and embryos in pigs should be performed using manipulation media maintained at 35°C.