93 BLASTOCYST PRODUCTION FROM BOVINE OOCYTES VITRIFIED IN A CLOSED (BIOSECURE) SYSTEM FOLLOWING IN VITRO MATURATION IN THE PRESENCE OR ABSENCE OF VITAMIN E

Abstract

The value of assisted reproductive technologies intended for conservation of livestock genetics ultimately will depend on their effectiveness in both sustaining gamete/embryo viability and ensuring stringent biosecurity. This study investigated bovine oocyte survival following vitrification in a sealed system prior to storage in liquid nitrogen. It also tested the effect of supplementary vitamin E on tolerance of oocytes to vitrification procedures. Healthy COCs from abattoir-derived ovaries were matured in TCM-199 supplemented with 10% v/v fetal calf serum (FCS) in the absence (control) or presence (VitE) of 100 μM α-tocopherol (Sigma, Poole, UK) in humidified atmosphere (5% CO₂ in air; 38.5°C). Between 22 and 24 h after commencement of IVM, COCs were pipetted to remove excess cumulus cells, and then equilibrated at room temperature in 7.5% DMSO plus 7.5% ethylene glycol (EG) in HEPES-buffered Holding Medium (HM; Irvine Scientific, Santa Ana, CA, USA) for 7 min before transfer to vitrification solution (15% DMSO, 15% EG, and 0.5 M sucrose in HM; Irvine Scientific). Loading of oocytes (n = 78 control and 85 VitE) into CryoTips (Irvine Scientific) and heat-sealing (each end) was achieved within 90 s, with tips then plunged into liquid nitrogen. Subsequent warming and cryoprotectant removal were at room temperature in HM with 1 M sucrose for 2 min, 0.5 M sucrose for 4 min, and HM alone for 6 min. Oocytes were allowed recover for approximately 3 h in TCM-199 with 20% FCS (5% CO₂ in air; 38.5°C), and then fertilized in vitro (single sire). After 22 h (Day 1) presumptive zygotes were transferred to SOF containing fatty acid-free BSA (4 mg mL⁻¹) and incubated for up to 8 days (5% O₂, 5% CO₂, 90% N₂; 38.5°C). Cleavage data (Day 2) and blastocyst yields (Days 7 to 9) were analyzed by chi-square test. In addition to those that were vitrified, some oocytes (n = 9 per treatment) were observed via video to permit analysis (ImageJ; NIH, USA) of volume excursions during the 7 min immediately following initial exposure to HM with 7.5% DMSO plus 7.5% EG. Data were compared using ANOVA. Overall incidence of cleavage by Day 2 was 45% (range: 36 to 51%) and 35% (31 to 43%) for control and VitE, respectively, (NS). Day 7 and total control blastocyst yields were 7.4% and 18.5%, respectively; corresponding yields for VitE were 19% and 25% (control vs. VitE, NS). Video evidence indicated that although Control oocytes invariably reached minimal volume later than VitE oocytes (30 vs. 20 s), in each case this was 52% of initial size. By 7 min, both had similar volumes, the respective means being 94% and 92% of initial size. In the present study provision of vitamin E during IVM did not significantly enhance the subsequent resilience and development of oocytes subjected to a vitrification protocol. However, this protocol achieved efficient and biosafe bovine gamete storage.

This work was funded by SEERAD; CryoTips and vitrification solutions were donated by Irvine Scientific; VCM was supported by MLC, UK.