Evaluation of DNA amplification kits for genomic analysis of cell biopsies from in vitro produced bovine embryos

Cattle producers are continually looking for ways to increase the number of genetically superior animals, while at the same time reducing production costs. A genetic screen of in vitro produced embryos for traits of economic importance, prior to transfer, would greatly benefit genetic selection and reduce costs. This technique involves removal of a few cells from the growing embryo at the morula or blastocyst stage. The limitations of whole genome analysis (WGA) of embryo biopsies is to balance the number of cells that can be removed without compromising the embryo and obtaining sufficient amounts of DNA. With the development of molecular biological techniques such as DNA amplification, these limitations are no longer an issue. The objectives of this project were to evaluate: 1) the effects of biopsy and vitrification in a 2x2 factorial design on embryo re-expansion, and 2) the call rates of DNA from embryo biopsies that were amplified with commercially available DNA amplification kits. In experiment 1, at day 7 of in vitro production, bovine embryos either not manipulated (C) or subjected to embryo biopsy (B), in which around 10 cells from the trophoblast layer were removed. Both groups were either placed back into culture for 18 hrs (Fresh: C/F and Biopsied/Fresh: B/F) or cryopreserved by vitrification (C/V and B/V). Vitrified embryos were then thawed and placed into culture for 24 hrs. Re-expansion was evaluated in all groups. In experiment 2, DNA from biopsied embryos was amplified by GenomePlex Single Cell WGA (Sigma), REPLI-g Single Cell (Qiagen) or Illustra Single Cell GenomiPhi (Ge Healthcare) kits. Whole genome analysis was performed on the Geneseek Genomic Profiler LD (GGP-LD) for Indicus-35,000 SNP Platform (Neogen). One-way ANOVA test was utilized to determine significance (P<0.05) for re-expansion and call rates and differences in means was determined by Tukey`s. Re-expansion of B/F embryos (93.3+4.6%) was no different than C/F group (100%). Re-expansion of B/V embryos (75.0+6.7%) was lower than C/F, with C/V embryos having the lowest re-expansion rates (52.2+ 15.5%). There was a significant difference in call rates for DNA amplified using the REPLI-g (86.6+5.2%) and GenomiPhi (59.9+7.8%; P<0.05) kits. The GenomePlex kit (82.7+2.1%) was not different than the other kits. These results suggest that whole genome analysis of embryo biopsies is possible with DNA amplification without compromising embryo survival.