The influence of DNA stabilizing buffer on the results of genomic bovine embryo analysis

Nowadays breeding programs involve the biopsy of preimplantation bovine embryos to determine sex (S), polled status (PS), hereditary defects (HD) and early genetic selection. Those techniques are more cost effective the higher the success rate of analyzed samples. To minimize the loss of information due to DNA degradation the obtained cells must be successfully stored in tubes prior to whole genome amplification. The objective of this study was to analyze the use of a DNA stabilizing buffer on the successful outcome of S, PS, HD determination and on the call rate after chip SNP analysis. Embryos were obtained on day 7 after insemination by superovulation of German Simmental animals with a standard protocol. Immediately after recovery, embryos were biopsied by a single operator under a mobile stereo microscope (Olympus, Japan) at 50x magnification and a steel blade mounted on a blade holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Two different biopsy media (BM) were used, (1: PBS from IMV, France or 2: 0.9% NaCl solution with 1.5% PVP). Removed cells were brought to reaction tubes by pipetting them with 0.5 µl medium in either the empty tube or in 2.5 µl TE buffer (TE buffer, 10 mM Tris, 1 mM EDTA, pH 8.0). Biopsied cells, approximately 10, were immediately used after biopsy for whole genome amplification (Repli-g Mini Kit, Qiagen, Germany) followed by PCR analysis of S and PS. HD were analyzed using a 5’-exonuclease assay. Embryos were genotyped with the Illumina Bovine 54k BeadChip. Call rates were recorded. Differences in success (%) and call rates were analyzed by proc GLM, SAS (fixed factors: BM (1+2), TE (yes/no), developmental stage of embryo, morulae (4), early blastocyst (5), blastocyst (6), and expanded blastocyst (7). In total 503 embryos were analyzed for S, PS and HD. From the total number of embryos 101 Embryos were SNP analyzed and call rates obtained. The BM had no influence on the success rate of positive analyzed S (90.7% vs. 86.0%), PS (89.4% vs. 86.9%) or HD (90.0% vs. 82.8%), P > 0.10. The stage of embryo had also no influence on the success rate of S (4= 90.1%, 5= 85.7%, 6= 87.9%, 7= 85.0%, P > 0.10), PS (4= 89.8%, 5= 85.2%, 6= 86.5, 7= 95.0%; P > 0.10), HD (4= 89.8%, 5= 85.2%, 6= 86.5%, 7= 86.7%, P > 0.10) analysis and call rate (4= 0.8939, 5= 0.8893, 6= 0.9211, 7= 0.9224, P > 0.10). Highly significant differences showed the use of TE buffer vs. no TE buffer for call rate (0.9137 vs. 0.8396, P < 0.0001), S (91.4% vs. 78.0%, P < 0.0001) and PS (90.9% vs. 79.7%, P < 0.0001) and HD (89.3% vs. 74.3%, P < 0.0001). The biopsy medium and the stage of embryo had no effect on the success rate for the analysis of S, PS and HD. The use of a DNA stabilizing TE buffer improved results by 10-15%. Therefore, we advise the use of TE buffer for storing small embryo cell samples to optimize analysis.