126 EFFECT OF ASSISTED HATCHING ON THE SURVIVAL AND THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO AFTER CRYOPRESERVATION

Abstract

The aim of this work is to investigate the effect of assisted hatching (AH) by partial zona pellucida (ZP) dissection on the survival and the development of bovine IVP embryos after ultra-rapid vitrification and slow freezing. COC obtained from abattoir bovine ovaries were matured and fertilized in vitro, and then cultured in IVD101 (Research Institute for the Functional Peptides, Yamagata, Japan) at 38.5°C in 5% CO₂, 5% O₂, 90% N₂. The treatment of AH was done on compacted morulae by partially dissecting ZP with a micromanipulator. As a control, non-treated embryos with intact ZP were used. For vitrification, the blastocysts at days 7 and 8 were placed into a vitrification solution (Dulbecco's PBS (D-PBS) supplemented with 20% glycerol, 20% ethylene glycol (EG), 0.3 m sucrose (SUC), 0.3 m xylose, and 3% polyethylene glycol) for 30 s after two-step equilibration. Then, they were immediately placed on a custom-made vitrification tool made of nylon fishing line with a small piece of iron attached to one end (V-tool), and immersed into liquid nitrogen (LN₂). After cooling, the embryos on the V-tool were placed into frozen 0.25 mL straws filled with a diluting solution (D-PBS supplemented with 0.5 m SUC and 20% new born calf serum) using a magnet, and then they were preserved in LN₂. For warming, the straws were immersed into 25°C water. The V-tool was then introduced into the column of diluting solution using a magnet. For freezing, the blastocysts at days 7 and 8 were frozen by the conventional procedure with 10% EG. For thawing, the straws were immersed into 30°C water. In this study, 120 embryos were vitrified and 128 embryos were frozen. Warmed and thawed embryos were washed more than two times, and cultured in TCM199 supplemented with 20% fetal bovine serum and 0.1 mm β-mercaptoethanol for 72 h for assessment of survivability and developmental capacity of post-thaw embryos. Data were analyzed with the chi-square test. The survival rates of vitrified embryos were the same with or without AH (81.1 and 82.0%, P > 0.05). The survival rates of frozen embryos were also the same with or without AH (76.3 and 66.7%, P > 0.05). The survival rates of vitrified embryos without AH was significantly higher than that of frozen embryos without AH (82.0 v. 66.7%, P < 0.05). The hatched rates of frozen embryos without AH were significantly lower than that of frozen embryos with AH and those of vitrified embryos with and without AH (43.5 v. 64.4%, 67.9 and 68.9%, P < 0.05). These results indicated that AH enhanced the development of frozen bovine IVP embryos and that our vitrification method using a V-tool did not require AH for development of embryos.