306 Embryo Analysis in Field Moet: High Success Rate After Overnight Culture of Microblade-Biopsied Cattle Embryos

Abstract

Embryo storage before embryo transfer is a necessity in field MOET to enable complex embryo analysis. Cryopreservation of biopsied embryos may reduce pregnancy rates to unacceptable levels, below 40% (Shea BF 1999 Theriogenology 51, 841–854). Our primary objective was to investigate the effect of short-term storage in overnight culture on the pregnancy rate of microblade-biopsied and sexed cattle embryos. A specific aim was to apply embryo storage in culture to marker-assisted selection of MOET embryos. Day 6.5 embryos were produced using standard superovulatory, AI, and embryo flushing procedures. Embryo donors were lactating cows of top genetic merit. Embryos were transported in straws in Holding medium (ICPbio, Auckland, New Zealand) to the laboratory and individually biopsied by a microblade. Biopsied embryos were cultured overnight in individual oil-overlaid 20-μL drops of Medium 199 with glutamax-1 (GIBCOTM, Baisley, UK) containing 0.25 mM sodium pyruvate, antibiotics, and 4 mg mL−1 fatty acid-free albumin. The biopsies were lysed in proteinase K and analyzed for sex with the BOV-Y/kappa-casein PCR method (Peura et al. 1991 Theriogenology 35, 547–555). Overnight-cultured grade I–II female embryos were transferred into the uteri of heat-synchronized recipients (Day 7.5). Pregnancy was confirmed by rectal palpation at 2–3 months after ET. Grade I-II male embryos as well as embryos of unknown sex were frozen in ethylene glycol, stored in liquid nitrogen, thawed, and cultured overnight for estimation of re-expansion rate and cell counts. In total, 74 embryos of eight donors were overnight-cultured and sexed. The success rate of the sexing method was 95%. Of the successfully analyzed embryos, 41% (29 of 70) were females and 59% (59 of 70) males. The survival rate of microblade-biopsied overnight-cultured embryos was 99% (73 of 74); 73% of the surviving embryos were of grade I, 23% of grade II, and 4% of grade III. Sixteen of the 27 (59%) grade I–II female embryos transferred resulted in pregnancy. Forty-two grade I–II embryos were frozen and 36 (86%) re-expanded after thawing and overnight culture. Twenty-four of the re-expanded embryos (67%) were of grade I. The re-expanded embryos had on average 73 cells (range 43–114). In conclusion, overnight culture of microblade-biopsied cattle embryos does not compromise embryo viability, resulting in high pregnancy rate and post-thaw re-expansion rate. The method can be utilized as a short-term embryo storage in the field MOET scheme and it will be applied in marker-assisted selection of MOET embryos for genotypes associated with milk production. The study was supported by the HAKA-Top breeding animals from North-Savo-project.