Laser assisted blastomere extrusion biopsy of in vitro produced cattle embryos-A potential high throughput, minimally invasive approach for sampling pre-morula and morula stage embryos.

ABSTRACT

Whilst adoption of in vitro production (IVP) of cattle embryos and subsequent biopsy for genetic evaluation is increasing, biopsy techniques primarily used were developed to sample in vivo-produced blastocysts. This study was conducted to develop a laser-assisted blastomere extrusion approach for rapid and minimal-invasive biopsy of IVP cattle embryos at pre-morula to morula stages of development (Day 5 or 6 post-fertilisation). Embryo development into blastocysts was not compromised when ≤3 cells were collected by blastomere extrusion on Day 5 (44.4 ± 4.4 % and 34.3 ± 4.6 %) or Day 6 (58.0 ± 4.3 % and 57.5 ± 5.3 %) post-fertilisation compared with non-biopsied control embryos. Similarly, capacity to withstand cryopreservation was not different between embryos biopsied at Day 5 and 6 post-fertilisation and control-embryos (58.8 ± 6.0 %, 63.5 ± 5.6 %, and 56.0 ± 4.8 %, respectively). When more cells were collected from embryos at Day 6 post-fertilisation (≥8 compared to ≤3 cells), subsequent embryo development was not different (63.6 ± 6.1 % and 73.1 ± 6.2 %, respectively) nor was the capacity to withstand cryopreservation (67.9 ± 9.0 % and 62.5 ± 8.7 %, respectively). For biopsies on Day 6 post-fertilization, 95 % of samples produced a PCR product; however, when compared to the whole embryo PCR results, approximately 11 % of biopsy-samples classified as being from a male embryo were from female embryos (false positive), indicating DNA contamination between samples. In conclusion, results of this study indicate laser-assisted blastomere extrusion is a time efficient and minimally invasive approach to biopsy IVP morula and pre-morula cattle embryos to facilitate genetic analysis.