The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design.
Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

Abstract
 INTRODUCTION
 MATERIALS AND METHODS
  1. Culture media and reagents
  2. Oocytes maturation
  3. Preparation of nuclei donor cells
  4. Somatic cell nuclear transfer and parthenogenetic activation
  5. Post-activation treatment and embryo culture
  6. Differential staining of the inner cell mass (ICM) andtrophectoderm (TE)
  7. Experimental design
  8. Statistical analysis
 RESULTS
  1. Effect of various osmolarities of medium during IVC onembryonic development after PA
  2. Effect of alanine or/and glycine in IVC medium withvarious osmolarities on embryonic development after PA
  3. Effect of glycine in IVC medium with various osmolaritieson embryonic development and cell number of ICM andTE after SCNT
 DISCUSSION
 REFERENCES

• Joohyeong Lee(Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Institute of Veterinary Science, Kangwon National University)
• Yongjin Lee(Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University)
• Hae Hong Jung(Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University)
• Seung Tae Lee(Division of Applied Animal Science, College of Animal Life Science, Kangwon National University)
• Geun-Shik Lee(Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Institute of Veterinary Science, Kangwon National University)
• Eunsong Lee(Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Institute of Veterinary Science, Kangwon National University) Correspondence