Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification
Roser Morató A C , Míriam Castillo-Martín A , Marc Yeste B and Sergi Bonet A
+ Author Affiliations
- Author Affiliations
A Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
B Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Barcelona), Spain.
C Corresponding author. Email: rmoratomolet@gmail.com; roser.morato@udg.edu
Reproduction, Fertility and Development 28(7) 886-892 https://doi.org/10.1071/RD14203
Submitted: 12 June 2014 Accepted: 20 October 2014 Published: 4 December 2014
Abstract
The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24 h. Re-expansion rates were recorded at 3 and 24 h and total cell number and apoptotic cells were determined at 24 h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification–warming procedures and length of in vitro culture, as expanding and hatching–hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.
Additional keywords: cryotop, DNA fragmentation, embryo quality, expanded, post-warming survival.
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