87 VITRIFICATION OF BOVINE OOCYTES TREATED WITH CHOLESTEROL-LOADED METHYL-β-CYCLODEXTRIN
G. Horvath A B , L. Solti A and G. Seidel BA Faculty of Veterinary Science, Szent Istvan University, Budapest, Hungary
B Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80521, USA. Email: gseidel@colostate.edu
Reproduction, Fertility and Development 17(2) 193-194 https://doi.org/10.1071/RDv17n2Ab87
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Addition of cholesterol-loaded cyclodextrin (CLC) can increase sperm cryosurvival (Purdy et al. 2000 Cryobiology 48, 36–45). The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with CLC prior to vitrification. Slaughterhouse-derived cumulus oocyte complexes were matured in a chemically defined medium with fatty acid-free BSA and hormones for 21 h followed by partial cumulus removal with 100 U/mL hyaluronidase and gentle pipetting. For an additional hour, oocytes were placed into maturation medium supplemented with 0.5% PVA instead of BSA with or without 2.5 mg/mL CLC. At 22 h after the start of maturation, oocytes were transferred to handling media containing 20% FCS or 0.5% PVA in TCM-199 + HEPES (HTCM-199). Oocytes with approximately 3 layers of cumulus were vitrified in two steps. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP, or 20% FCS, in HTCM-199) for 30 s, then exposed to VS2 (20% EG, 20% DMSO, 6% PVP, or 20% FCS, 0.48 M galactose in HTCM-199) for 25 s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise through 0.5, 0.25, 0.125, and 0 M galactose in HTCM-199 + 20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, fertilized with semen from 3 bulls, 3 replicates each, and cultured according to standard procedures (Zhang et al. 2003 Theriogenology 60, 1657–1663). For each replicate, 30 oocytes were assigned to the following treatments: A: chemically-defined media with PVA for the last hour of maturation, handling and vitrification; B: same as A except CLC treatment, for 1 h before vitrification; C: chemically defined media for maturation, but with 20% FCS for HM, VS1 and VS2. Data were analyzed by ANOVA. CLC treatment resulted in higher cleavage rates and 8- to 16-cell embryo production, but not higher blastocyst (bl) production (Table 1). Non-vitrified oocytes developed better than vitrified ones (means: cleavage, 76%; 8- to 16-cell, 64%; bl D8, 21%; bl D9, 24%). Further studies with vitrification of cholesterol-loaded cyclodextrin-treated oocytes and chemically defined media are warranted.
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