140 UTILIZATION OF LASER-ASSISTED HATCHING TO IMPROVE THE PREGNANCY RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS

Abstract

In vitro-produced (IVP) bovine embryos are known to produce a lower pregnancy rate when compared to conventional in vivo-produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is thought to be one contributing factor. This study was designed to evaluate the utilization of a microscope objective-mounted laser to cut the ZP to assist hatching prior to transfer into the recipient. Preliminary data were acquired to evaluate the effect of laser treatment on in vitro development and blastomere survival following treatment. In six replicates, bovine oocytes were in vitro-matured, fertilized, and cultured as per standard laboratory procedures (TransOva Genetics, Sioux Center, IA, USA). On Days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 treatment groups: no treatment (Control; n = 63), sham ZP cut (Sham; n = 68), or ZP cut (Cut; n = 70). Control embryos were immediately returned to the incubator following selection. Sham embryos were exposed to all conditions as Cut except laser-assisted hatching. The XYClone^® system is a 300-mW, class 1 laser that emits a 3.5-µm beam at a wavelength of 1480 nm (Hamilton Thorne Biosciences, Beverly, MA, USA). This laser was used to produce the Cut group, using a pulse strength of 90% and pulse length of 600 µs. Embryos were returned to culture until Day 8 when rates of embryonic development and the percentage of live cells were determined. Chi-square was used to analyze all data. No significant effect of treatment or day of exposure was noted in either the total number of developing embryos or the ratio of live cells in each embryo. Mean live cells ranged from 89 to 96% across all treatments regardless of day of treatment. To investigate IVP embryo viability after laser-assisted hatching, commercially produced embryos (TransOva Genetics, Sioux Center, IA, USA) were randomly divided into two groups on the day of transfer, Control or Cut. The ZP of treated embryos were cut with slightly reduced laser exposure of 80% pulse strength and pulse length of 500 µs on Day 7, immediately prior to transfer into estrus-synchronized recipients. Pregnancy rates were determined via ultrasonagraphy at Day 30 (n = 337) and, due to the commercial nature of this project, only a subset of the Day 30 pregnant cows was checked at Day 60 (n = 289). The 30-day pregnancy rates were 49.2% and 54.1% for Control (n = 189) and Cut (n = 148) embryos, respectively, and were not statistically different (P > 0.05). However, at Day 60, the pregnancy rates for the Control (45.7%; n = 166) and Cut groups (57.7%; n = 123) were statistically different (P < 0.05). These results demonstrate that laser-assisted hatching using the XYClone system can improve 60-day pregnancy rates for in vitro-produced embryos.