Animals; Biopsy; Cryopreservation; DNA Primers/genetics; Embryo Transfer/methods; Embryo, Mammalian/embryology; Genotype; Immunity, Innate/genetics; Prions/genetics; Scrapie/genetics/immunology; Sex Determination (Analysis); Sheep
Abstract :
[en] The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Guignot, F.; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Baril, Gerard; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Dupont, Francis; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Cognié, Yves; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Folch, Jose; Centro de Investigacion y Tecnologia Agroalimentaria (CITA), Zaragoza, Spain
Alabart, Jose Luis; Centro de Investigacion y Tecnologıa Agroalimentaria (CITA), Zaragoza, Spain
Poulin, Naty; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Beckers, Jean-François ; Université de Liège - ULiège > Département de sciences fonctionnelles > Physiologie de la reproduction
Bed'hom, Bertrand; INRA, AgroParisTech, UMR1236 Génétique et Diversité Animales, Jouy en Josas, France
Babilliot, Jean-Marc; LABOGENA, Unité Ovicap, Jouy en Josas, France
Mermillod, Pascal; UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France
Language :
English
Title :
Determination of sex and scrapie resistance genotype in preimplantation ovine embryos.
Publication date :
2009
Journal title :
Molecular Reproduction and Development
ISSN :
1040-452X
eISSN :
1098-2795
Publisher :
Wiley Liss, Inc., New York, United States - New York
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