90 THE EFFECT OF LIPID SEGREGATION WITH OR WITHOUT ZONA PELLUCIDA LASER DRILLING ON POST-THAW EMBRYO DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

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High levels of lipid within in vitro-produced embryos during freezing can increase intracellular damage and lower production rates (Seidel 2006 Theriogenology 65, 228). The objective of this study was to determine if lipid segregation with or without laser-assisted hatching (LAH), or zona pellucida drilling of in vitro-fertilized (IVF) embryos would enhance in vitro survivability and development 24 h post-thaw. Three replicates utilizing 1179 bovine oocytes (BOMED, Madison, WI, USA) were fertilized with frozen/thawed Tuli bull semen and cultured in G1.3/G2.3 supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 of culture, grade 1 & 2 embryos were morphologically divided into 3 developmental stages: 32-cell (n = 78), compact morula (CM, n = 223), and blastocyst (n = 56). Each group was then randomly allocated to the following treatments prior to cryopreservation in 1.5 m ethylene glycol (Vigro Freeze Plus, Bioniche, Pullman, WA, USA): no treatment (control), 7.5 µg mL–1 cytochalasin B for 20 min (CB), or CB with centrifugation (16 000g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Embryos were loaded in sterile straws, frozen at 0.5�C min–1 from –6�C to –32�C, and then plunged into LN2. Frozen embryos were air-thawed for 7 s and then thawed in 35�C H2O for 10 s before being assessed for survivability. Immediately post-thaw, one-half of the CBCF and control groups were subjected to LAH, using a single laser pulse at 90% laser power for 600 µs using the XY Clone� system (Hamilton Thorne Biosciences, Beverly, MA, USA), creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured in G2.3 for 24 h and evaluated morphologically to determine survivability and development. Live/dead staining was performed by using Hoechst 33342 (2.5 µg mL–1) and propidium iodide (5 µg mL–1) under UV light. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student's t-test. Due primarily to low numbers in embryos in stages other than CM, no differences among treatments were detected. For CM, treatment means ranged from 89.6 to 95.0% and from 69.6 to 82.6% for survival and development, respectively, and no treatment differences were observed. Within the CM stage, CBCFLAH was not different from LAH, CBCF, and control (77.0 v. 71.9, 68.8, and 68.3%, respectively; P > 0.05), but showed a significantly greater percentage of live cells than CB (77.0 v. 65.5%; P < 0.05). CBCFLAH and LAH exhibited a significantly greater number of both total and live cells than control (total cells: 69.4, 69.3, and 53.0; live cells: 56.4, 54.7, and 39.3, respectively; P < 0.05). These data indicate that LAH post-thaw alone or in combination with CBCF improves both total cell number and embryo viability following cryopreservation. Financial support was provided by a grant from TAMU-CONACYT (USA-Mexico) and OvaGenix.