Determination of sex and scrapie resistance genotype in preimplantation ovine embryos.
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.
Guignot
Florence
F
UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France. guignot@tours.inra.fr
Baril
Gerard
G
Dupont
Francis
F
Cognie
Yves
Y
Folch
Jose
J
Alabart
Jose Luis
JL
Poulin
Naty
N
Beckers
Jean-Francois
JF
Bed'hom
Bertrand
B
Babilliot
Jean-Marc
JM
Mermillod
Pascal
P
D000818:Animals; D001706:Biopsy; D015925:Cryopreservation; D017931:DNA Primers / Q000235:genetics; D004624:Embryo Transfer / Q000379:methods; D004622:Embryo, Mammalian / Q000196:embryology*; D005838:Genotype; D007113:Immunity, Innate / Q000235:genetics*; D011328:Prions / Q000235:genetics*; D012608:Scrapie / Q000235:genetics* / Q000276:immunology; D012732:Sex Determination Analysis; D012756:Sheep; D016019:Survival Analysis
D017931:DNA Primers; D011328:Prions