171 LASER-ASSISTED ZONA PELLUCIDA HATCHING IN FROZEN-THAWED BOVINE EMBRYOS

Abstract

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo derived frozen-thawed bovine embryo hatching rates. In Exp. 1, direct-transfer beef cattle embryos were air-thawed for 15 s, placed in a 30°C water bath for 15 s, then held in TALP-HEPES, evaluated for stage and grade (1 = good to 3 = poor) and randomly applied to treatments. Embryos (n = 156) received either 2 or 3 symmetrical rents 40% through the outer zona surface using the XYClone diode laser (Hamilton Thorne, Beverly, MA, USA) at 90% power with a 600 μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were then cultured in vitro in CR1aa supplemented with 10% calf serum at 39°C in 5% CO² and 5% O² for 4 d. Embryo hatching rates were 47% for Treatment A and 53% for Treatment B. In Exp. 2, in vivo produced, nonsurgically collected direct-transfer Hereford embryos (n = 64) were utilized. In Exp. 3, in vivo produced nonsurgically collected glycerol frozen Brangus embryos (n= 46) were utilized. Embryos utilized in Exp. 2 and 3 were air-thawed for 15 s, placed in a 30°C water bath for 15 s, and then held in 1 M sucrose for 7 min. Embryos were then held in phosphate-buffered saline with 10% calf serum (Exp. 2) or ViGRO Holding Plus (Bioniche, Pullman, WA, USA) (Exp. 3), evaluated for stage and grade before being randomly assigned to either Treatment A or B. Embryos received either 3 symmetrical rents 40% through the outer zona surface using the XYClone laser at 90% power with a 600-μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were transferred nonsurgically (1 embryo/female) by the same technician into synchronized mixed breed recipient beef cows on Day 7 of the estrous cycle. Pregnancy status was determined at 35 days and 60 days via ultrasonography. In Exp. 2, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Also, treatment did not affect pregnancy rates at 35 or 60 days (41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively). Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Exp. 3, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Pregnancy rates at 35 and 60 days were not affected by treatment (65% and 65% for Treatment A and 76% and 59% for Treatment B, respectively). Calving rates for those recipients in Exp. 3 were not available at the time of abstract preparation. Based on the data presented herein, it does not appear that laser-assisted hatching with the XYClone laser increases the number of in vivo derived frozen-thawed embryos that hatch following in vitro culture or increase pregnancy rates after transfer to recipient cattle.