120 Laser-assisted bovine embryo biopsy: efficiency of the whole-genome amplification and embryo development of in vivo bovine embryos in a selected breeding program

In order to drive genetic improvement of cattle, in vivo embryo biopsy with the microblade method has been used since 1990 in our selected breeding program with acceptable results in terms of pregnancy rate. However, new biopsy should be processed to decrease embryos damage and then improve its viability. The aim of this study was to evaluate the impact of laser-assisted biopsy of in vivo-produced embryos on single-nucleotide polymorphism (SNP) call rate (measure of quality for SNP to be used in genomic evaluation), survival rate of post-thawing cryopreserved embryos cultured for 48 h, and pregnancy rate, in comparison with a conventional microblade method. For the laser biopsy procedure, a hole in the zona pellucida of Day 7 in vivo-produced embryos (n = 955) was created by two laser pulses of 3.7 ms (Octax Laser-Germany), through which a biopsy pipette was inserted to aspirate 3–8 trophoblast cells. For microblade biopsy, embryos (n = 2993) were immobilised by a holding pipette and a steel blade was used to cut 3–10 trophoblast cells. Collected cells were stored at −20°C until whole-genome amplification (WGA). DNA extraction and WGA were performed using the REPLI-g Single Cell Kit (Qiagen). Biopsied embryos were frozen using ethylene glycol (1.5 M) plus sucrose (0.1 M). For the biopsied embryos discarded from a genetic-selection program (laser = 48; microblade = 88), the survival rate was evaluated after post-thawing embryo culture for 48 h. For the embryos with a genetic interest, the pregnancy rate was evaluated after direct transfer of frozen-thawed embryos into Day 7-synchronised heifers and first-parity cow recipients (laser = 78; microblade = 1,322) under farm conditions. Call rate was slightly (difference of only 2%) but significantly decreased for laser-assisted biopsy compared with microblade one (Table 1). The biopsied embryo survival rate 48 h after in vitro culture was higher with the laser method compared with microblade (chi-2, P < 0.05). The pregnancy rate after transfer of embryos submitted to laser-assisted biopsy was not significantly different from embryos biopsied by microblade procedure or from nonbiopsied embryos. By contrast, the pregnancy rate of embryos biopsied by microblade was significantly lower than that of nonbiopsied embryos (Fischer exact, P < 0.001). In conclusion, our data indicate that laser-assisted biopsy of in vivo-produced bovine embryos is an efficient and minimally invasive method to facilitate genetic analysis compared with microblade procedure, and it can be used efficiently to obtain genetic material for WGA. It also reduces damage caused to the embryo, as attested by higher embryo survival rate after in vitro culture and results in equivalent pregnancy rate compared with that obtained for nonbiopsied embryos.

Table 1. Call rate (CR), embryo survival rate after in vitro culture for 48 h and pregnancy rate of frozen-thawed bovine embryos biopsied by laser and microblade methods