Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts.
To establish a protocol for production of bovine in-vitro produced (IVP) blastocysts that were sex-identified and cryopreserved, we examined the sexing efficiency and accuracy of Day-3 and Day-4 embryos by polymerase chain reaction (PCR), the development of the biopsied embryos into Day-7 blastocysts and the freezability of these blastocysts by vitrification in gel-loading tips. One or two blastomeres were isolated from IVP embryos at either the 8-cell or 16-cell stage (Days 3 and 4, respectively) by a pressing-out method, and were then subjected to primer extension preamplification (PEP)-PCR. The successful sex-identification rate of biopsied samples amplified, purified and analyzed for sex by a second PCR (88.9%) was higher than that of those amplified and analyzed without purification (32.0%). Developmental rates into Day-7 blastocysts of biopsied embryos (Day-3, 65.5%; Day-4, 70.8%) were similar to those of non-biopsied control embryos (Day-3, 74.5%; Day-4, 65.1%). Total cell numbers and the inner cell mass (ICM) ratio of blastocysts derived from biopsied embryos were also comparable with those of control embryos. Blastocysts were vitrified-warmed in the presence of 20% DMSO, 20% ethylene glycol and 0.6M sucrose using gel-loading tips as containers. The proportions of biopsied blastocysts that were hatched or hatching rates after warming were high, regardless of the biopsy time (Day-3, 94.1%; Day-4, 91.9%), similar to the rates for control blastocysts (Day-3, 97.5%; Day-4, 96.9%). In conclusion, a protocol that allows sexing of Day-3 and Day-4 bovine embryos without compromising either the developmental ability to the blastocyst stage or freezability of Day-7 blastocysts was developed.
Tominaga
Keiichiro
K
Hyogo Prefectural Institute for Agriculture, Forestry and Fisheries, 1533 Minamino-oka, Befu, Kasai, Hyogo 679-0198, Japan. tomi-@nike.eonet.ne.jp
Hamada
Yukako
Y
D000818:Animals; D001706:Biopsy / Q000662:veterinary; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002417:Cattle / Q000196:embryology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006358:Hot Temperature; D008297:Male; D016133:Polymerase Chain Reaction; D012732:Sex Determination Analysis / Q000379:methods / Q000662:veterinary*; D013395:Sucrose
D013395:Sucrose; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide