46 HYPOTHERMIC STORAGE FOR 10 DAYS OF BOVINE EMBRYOS USING TYPE III ANTIFREEZE PROTEIN

Abstract

Recently, we developed a medium that enabled bovine embryos to be held for up to 7 days at 4°C (Tsuchiya et al. 2014 IETS meeting). To be of practical value, mammalian embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. Antifreeze proteins (AFP) were discovered in various organisms (such as fish, insects, plants, and bacteria) living in cold regions. They show a unique ability to protect cold-sensitive cells from hypothermic damage. Here, we found that a biomolecule known as type III AFP solubilised into an optimized solvent can keep alive bovine embryos for a maximum of 10 days at hypothermic temperatures. First, human hepatoma (HepG2) and rat insulinoma (RIN-5F) cells were stored at 4°C in Euro-Collins solution (Kobayashi Seiyaku, Osaka, Japan) supplemented with or without 10 mg mL^–1 of type III AFP for 24 h. The viability rate of the cells was assessed by trypan blue (Dojindo, Kumamoto, Japan) dissolved in PBS. Second, high-quality blastocysts produced in vivo were stored at 4°C in a plastic ministraw in 25 mM HEPES medium 199 plus 20% fetal bovine serum (FBS) supplemented with or without 10 mg mL^–1 of type III AFP for 10 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO₂ in air with high humidity. The viability and hatching rate of the embryos were assessed at the end of the culture period. Finally, 4 embryos stored for 10 days with type III AFP were reloaded into plastic straws with the washing medium and transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda Electronics Co. Ltd., Toyohashi, Japan) on Day 60 of gestation. Data were analysed using chi-squared and Student's t-tests. In the absence of type III AFP, the cell viability values of HepG2 and RIN-5F after hypothermic storage for 24 h were only 5 and 22%, respectively. However, in the presence of type III AFP, the cell viability values were dramatically increased (HepG2: 71%; RIN-5F: 59%) than those measured without type III AFP. To examine the effect of type III AFP for hypothermic preservation of bovine embryos, we used 80 high-quality embryos produced in vivo and assigned them randomly to 2 experimental groups. The viability and hatching rates of the chilled embryos stored with type III AFP for 10 days were significantly higher (58 and 30%, respectively) than those of without type III AFP (28 and 0%, respectively). The pregnancy rate of the chilled embryos stored with type III AFP was 50%: 2 pregnancies continue until now. In conclusion, prolongation of short-term preservation period with AFP-containing fluid will realise LN₂-free storage of bovine embryos for a 10-day period.

This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.