142 DONOR CONTRIBUTION HAS A LARGER IMPACT ON HSP70.1 EXPRESSION IN BOVINE EMBRYOS THAN THE CUMULUS REMOVAL TECHNIQUE

Abstract

The impact of embryo manipulation and donor contribution on molecular markers was investigated in the bovine model. Transcript expression analysis was performed on Holstein 2-cell embryos 24 h after they underwent one of two cumulus removal techniques. Oocytes from each of 5 abattoir-obtained Holstein ovaries were fertilized with semen of an individual Holstein bull, yielding families of 4, 5, 18, 25, and 28 zygotes. Cumulus cells from zygotes within each family were removed 24 h post-fertilization by either repetitive pipetting with a 135-µm pipette or by manipulation in a microfluidic chamber with a 30-µm aperture. Levels of the stress response transcript, HSP70.1, were used to evaluate cumulus removal technique in the 5 full sib families of developing embryos. Embryonic HSP70.1 cDNA from differentially treated full siblings was analyzed by qPCR and the resultant C_T values were normalized to 18S rRNA, the reference gene, to account for potential differences in cDNA levels in each reaction. The data were analyzed by using the GLM procedure in SAS (SAS Institute, Inc., Cary, NC, USA), testing for treatment and family effects. HSP70.1 did not differ between microfluidically treated and pipetted embryos (P = 0.45). However, comparison of HSP70.1 expression across families was different (P < 0.001). These results indicate that pooling of genetically unrelated embryos may skew transcript expression data and influence research conclusions. Additionally, the question of culture conditions on embryo vitality needs to be evaluated without donor health status or heredity being additional factors.