92 EFFECTS OF ZONA PELLUCIDA CUTTING BY LASER ON THE CRYOSURVIVAL AND HATCHING RATES OF VITRIFIED BLASTOCYSTS IN BUFFALO (BUBALUS BULALIS)

Abstract

The objective of this study was to determine if zona pellucida (ZP) opening before cryopreservation or after thawing would effect cryosurvivability and hatching rate of vitrified blastocysts produced by IVF. The IVF blastocysts were derived from oocytes obtained from abattoir ovaries; 15–20 μm ZP opening by a XYClone^® system (Hamilton Thorne Biosciences, Beverly, MA, USA), using a pulse strength of 90% and pulse length of 800 μs, was adopted for assisted hatching. The hatching rates of fresh blastocysts of various ages with or without ZP opening were observed within 24 h. Furthermore, 20% ethylene glycol (EG) + 20% dimethyl sulfoxide (DMSO) + 0.5 M sucrose was adopted as vitrificaiton medium in the following experiments, and the expanded blastocysts harvested on Days 6, 7, 8, and 9 were respectively divided into three groups: Group 1: the blastocysts were vitrified after 15 to 20 μm ZP opening opposite to the inner cell mass, and the blastocoels were also blotted in order to outflow the blastocoelic fluid before vitrification; Group 2: the 15 to 20 μm ZP cutting by laser was performed immediately after thawing; Group 3: as control, the blastocysts were vitrified and thawed without any other treatment. The thawed blastocysts in different groups were cultured for 72 h to observe the survival and hatching rates. The results showed that 1) the hatching rate of fresh Day 9 blastocysts that underwent ZP cutting was higher than that of fresh counterpart (77.8% v. 50.0%, 28/36 v. 20/40, P < 0.05); 2) after vitrification, the ZP of blastocysts was averagely thickened for 2.8 μm; and 3) the cryosurvival and hatching rates of Days 6 to 8 blastocysts in three groups were not different from each other, whereas as for Day 9 blastocysts, the cryosurvival rate of vitrified blastocysts in Group 1 was significantly higher than Group 3 (75.0% v. 53.8%, 30/40 v. 28/52, P < 0.05), and the hatching rate of vitrified blastocysts in Group 2 was significantly higher than Group 3 (80.0% v. 42.9%, 16/20 v. 12/28, P < 0.05). In conclusion, laser-assisted hatching can promote the hatching rate of fresh and vitrified blastocysts derived from Day 9. Furthermore, ZP opening and blastocoel breaking before vitrification can also improve the cryosurvival rate of Day 9 blastocysts.

This research was supported by grants from the National Natural Science Foundation of China (31160456) and the Natural Science Foundations of China under Grant No. 0991011 and No. 2011GXSFB018045)