43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

Abstract

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL^–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique.

This work was supported by the Clinical Equine ICSI Program, Texas A&M University.