Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

INTRODUCTION
 MATERIALS and METHODS
  1. Culture media
  2. Oocyte collection and IVM
  3. Somatic cell nuclear transfer and parthenogenesis (PA)
  4. Post-activation and embryo culture
  5. Measurement of oocyte diameter
  6. Measurement of GSH and ROS contents
  7. Determination of mitochondrial oxidative activity
  8. Differential count of inner cell mass and trophectoderm cells
  9. Experimental design
  10. Statistical analyses
 RESULTS
  1. Dose-dependent effects of salubrinal during IVM on embryonic development after PA
  2. Effects of salubrinal during various stages of IVM on PA embryonic development
  3. Effect of salubrinal treatment during IVM on the quality of PA blastocysts
  4. Effects of salubrinal on intra-oocyte GSH and ROS contents
  5. Effects of salubrinal on mitochondrial oxidative activity and oocytes diameter
  6. Effects of salubrinal during IVM on SCNT embryonic development
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES

• Fazle Elahi(College of Veterinary Medicine, Kangwon National University)
• Hyeji Shin(College of Veterinary Medicine, Kangwon National University)
• Joohyeong Lee(Institute of Veterinary Science, Kangwon National University)
• Eunsong Lee(College of Veterinary Medicine, Kangwon National University, Institute of Veterinary Science, Kangwon National University) Correspondence