Sex and PRNP genotype determination in preimplantation caprine embryos.
The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.
INRA, UMR85 Physiologie de la Reproduction et des Comportements CNRS, Nouzilly, France. Florence.guignot@tours.inra.fr
F
F
Guignot
C
C
Perreau
C
C
Cavarroc
J-L
JL
Touzé
J-L
JL
Pougnard
F
F
Dupont
J-F
JF
Beckers
B
B
Rémy
J-M
JM
Babilliot
B
B
Bed'Hom
J M
JM
Lamorinière
P
P
Mermillod
G
G
Baril
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D004247:DNA / Q000235:genetics; D004624:Embryo Transfer; D005260:Female; D016678:Genome; D005838:Genotype; D006041:Goats; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D011328:Prions / Q000235:genetics*; D012732:Sex Determination Analysis / Q000662:veterinary*
D011328:Prions; D004247:DNA