81 INDUCED BLASTOCOEL COLLAPSE IMPROVES SURVIVAL RATES OF PORCINE BLASTOCYSTS AFTER VITRIFICATION

Abstract

Cryopreservation of porcine blastocysts is regarded as a demanding task because of the high chilling sensitivity and low survival rates. Ultrarapid vitrification techniques combined with the previous artificial induction of collapse of the blastocoel have been successfully applied in mouse and human. The purpose of our work was to use the Cryotop (Kitasato Supply Co., Tokyo, Japan) vitrification method combined with induced blastocyst collapse for cryopreservation of porcine blastocysts produced with parthenogenetic activation of in vitro-matured oocytes. A total of 1200 slaughterhouse-derived porcine oocytes were matured for 41–42 h. Zona-intact or zona-free oocytes (after pronase digestion) were activated with a single electric impulse of 63 or 43 V cm^–1, respectively, and incubated in 5 μg mL^–1 cytochalasin B and 10 μg mL^–1 cycloheximide for 4 h (Du et al. 2007 Cryobiology 54, 181–187). Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O₂, and 90% N₂ and maximum humidity. On Day 6, collapse was induced manually in approximately half of the blastocysts with the use of a fire-polished holding pipette and injection pipette (Cook Australia, Brisbane, Queensland, Australia). Ten min after the manipulation, collapsed and control (not manipulated) blastocysts were vitrified with the Cryotop, and then warmed and diluted in 1, 0.5, and 0 m sucrose, respectively (Kuwayama et al. 2005, Reprod. BioMed. Online 11, 300–308). Reexpansion of blastocysts was evaluated under a stereomicroscope after 16–18 h of culture under conditions described above. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical calculations; values with P < 0.05 were regarded as significant. Reexpansion rates of blastocysts are shown in Table 1. Our results indicate that manually induced collapse of the blastocoel increases reexpansion rates of both zona-intact and zona-free porcine blastocysts after Cryotop vitrification. The combination of these simple techniques may help to improve the efficiency of cryopreservation of pig embryos for both experimental and practical applications.

Table 1. Reexpansion rates after vitrification of blastocysts with or without induced blastocoel collapse

The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen, and ClausWillemoes for their help and excellent technical assistance.