136 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS SUBMITTED TO LASER ASSISTED HATCHING ON DAY 6 AND DAY 7 OF EMBRYO CULTURE

In vitro production (IVP) of embryos is a valuable tool in bovine assisted reproduction. IVP embryos show lower pregnancy rates when compared to in vivo produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is believed to be one contributing factor. The aim of this study was to compare the ability of IVP embryos to hatch and develop in vitro after being submitted to laser assisted hatching (LAH) at two different time periods of embryo culture: 144 h (Day 6) and 168 h (Day 7). In vitro maturated oocytes from slaughterhouse ovaries (Ovitra Biotechnologies, Amarillo, TX, USA) were fertilized with frozen/thawed semen from two different bulls (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL–1 of BSA (Vitrolife, Englewood, CA, USA) at 38.5°C, 5% CO2, 5% O2, 5% N2, in humidified atmosphere. On Day 6 post-fertilization, all viable embryos (n = 251), from three replicates, were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA) and divided into four treatment groups: laser assisted hatching Day 6 and Day 7 (LAHD6 and LAHD7), control Day 6 and Day 7 (CD6 and CD7). The groups LAHD7 and CD7 were immediately placed in G2.5 and returned to the incubator until Day 7. Embryos from groups LAHD6 and CD6 were placed in G2.5 in separate wells of a four well dish and covered with 300 μL of mineral oil. Embryos from LAHD6 group had one third to one half of the external edge of the ZP exposed to a laser beam, using XY Clone® (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) laser system, with a pulse strength of 90% and a pulse length of 600 μs. The group CD6 was submitted to the same conditions, but did not receive the laser treatment. On Day 7, the experiment was repeated for embryos belonging to groups LAHD7 and CD7. Embryos from all groups were cultured in vitro and evaluated on Day 8 and Day 9 for stage of development. On Day 9, a random sample of embryos from each treatment group (n = 48) was stained with Hoechst 33342 (2.5 μg mL–1) and evaluated under UV light to determine the total number of cells. The number of hatched blastocysts was not different (chi-square, p > 0.05) among the groups on Day 9 of culture (LAHD6 = 49/66, CD6 = 38/61, LAHD7 = 42/59, CD7 = 47/65; 74, 62, 71 and 72%, respectively). However, on Day 8 of culture, LAHD6 showed a higher number (p < 0.05) of hatched blastocysts (40/66, 60%), when compared to group CD6 (26/61, 42%). There was no difference between groups LAHD7 (33/59, 55%) and CD7 (31/65, 47%) on Day 8. Comparison of the total number of cells showed no difference (Student’s t-test, p > 0.05) among the groups (LAHD6 = 154.8 ± 12.2, CD6 = 184.4 ± 19.5, LAHD7 = 170.4 ± 15.8, CD7 = 162.5 ± 14.6), indicating that LAH does not have a detrimental effect on mean cell production throughout embryo development in vitro. These data show that LAH on Day 6 of culture improves in vitro hatching rates on Day 8, while LAH on Day 7 shows no improvement on either Day 8 or 9. Kathy Bradley, Hamilton Thorne Biosciences, Inc.