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RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.



Article << Previous     |     Next >>   Contents Vol 22(1)

117 EFFECT OF DIFFERENT CRYOPRESERVATION METHODS ON THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

H. Stinshoff A, K. Brüning A, A. Hanstedt A, D. Müller A, S. Wilkening A and C. Wrenzycki A

A University of Veterinary Medicine Hannover, Clinic for Cattle, Hannover, Germany;
B University of Veterinary Medicine Hannover, Reproductive Medicine Unit, Hannover, Germany
   

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Abstract

In vitro production (IVP) of bovine embryos has been greatly improved over the last couple of years. However, only one-third of the total number of embryos transferred worldwide are of in vitro origin. The IVP embryos still show remarkable differences compared with their in vivo-derived counterparts (i.e. bovine embryos produced in vitro are more sensitive to cryopreservation). So far, vitrification seems to be the most promising method to cryopreserve in vitro-produced bovine embryos. The aim of this study was to determine the effect of 2 different cryopreservation methods on the quality of in vitro-produced bovine embryos at the molecular level using a sensitive RT-qPCR assay. Bovine blastocysts were produced using abattoir ovaries and a standard protocol for IVP (Wrenzycki et al. 2001). They were randomly vitrified employing PBS plus ethylene glycol and DMSO or cryopreserved using a programmable freezer and 1.5 M ethylene glycol. After thawing, embryos from both groups were cultured for 48 h. After 24 h of culture re-expansion rates were documented, and after 48 h hatching rates were documented. After hatching, blastocysts were stored at -80°C for subsequent RT-qPCR analysis. The following gene transcripts known to play important roles during preimplantation development were analyzed: HSP70, GLUT-1, GLUT-3, E-CAD, ZO-1, DNMT3a, IFNτ, DCII. Re-expansion rates were 74.7% (68/91) and 75.0% (87/116) for vitrified and conventionally cryopreserved blastocysts, and 57.1% (52/91) and 55.2% (64/116) of re-expanded embryos hatched. The relative abundances of HSP70, GLUT-1, and ZO-1 transcripts were significantly affected in both groups of cryopreservation compared with the control group (hatched blastocysts without cryopreservation). Conventional cryopreservation had a significant effect on the amount of GLUT-3, DNMT3a, and IFNτ mRNA, whereas vitrification significantly affected DCII transcripts. E-CAD mRNA expression was similar in all groups of embryos. These results suggest that not only the cryopreservation process itself but also the method used to freeze the embryos had a significant influence on the mRNA expression of developmentally important genes in hatched bovine blastocysts.


The support of the H.W. Schaumann Stiftung (Germany) and Gynemed Medizinprodukte GmbH & Co. KG (Germany) is gratefully acknowledged.

Reproduction, Fertility and Development 22(5305) 217–217   http://dx.doi.org/10.1071/RDv22n1Ab117
Published online: 08 December 2009




 
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