157 A WATER-SOLUBLE VITAMIN E ANALOGUE (TROLOX) IMPROVES OVINE EMBRYO DEVELOPMENT DURING SERUM-FREE CULTURE IN THE PRESENCE OF DOCOSAHEXAENOIC ACID (C22:6n–3)
T.G. McEvoy A , A. Reis A , M. Ewen A , G.J. McCallum A and J.A. Rooke AScottish Agricultural College, Sustainable Livestock Systems Group, Aberdeen, UK. email: t.mcevoy@ab.sac.ac.uk
Reproduction, Fertility and Development 16(2) 200-201 https://doi.org/10.1071/RDv16n1Ab157
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
Vitamin E (α-tocopherol) supplementation of culture media can safeguard embryo development in vitro but, as it is fat-soluble, usually serum must be present. This study investigated whether Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma, St. Louis, MO, USA), a water-soluble vitamin E analogue reported to scavenge peroxyl radicals in a manner similar to that of its parent compound, could provide antioxidant protection for ovine embryos in serum-free culture conditions in the presence or absence of supplementary docosahexaenoic acid (DHA, C22:6n–3), a polyunsaturated fatty acid found in vulnerable membranes. Abattoir-derived oocytes were matured and fertilized in vitro (IVF = Day 0). On Day 1, cleaved eggs were assigned to culture (5% CO2, 5% O2, 90% N2; 38.5°C) to the blastocyst stage in synthetic oviductal fluid plus amino acids (SOF, n = 15 replicates, mean ± SEM = 231 ± 6 per replicate) supplemented with 0.4% w/v fatty acid-free BSA in the absence (SBSA) or presence of (SBSAD) DHA or the same media with 200 μM Trolox (SBSAT, SBSADT). Respective DHA concentrations (μg/mL; mean ± SEM) in these media were 0.0, 0.5 ± 0.12, 0.0 and 0.7 ± 0.27. Blastocyst lipids were separated into polar and neutral fractions (solid-phase extraction, aminopropyl silica columns), and fatty acid composition of each fraction was determined by gas chromatography (splitless method). Using a 2-way factorial design, blastocyst yields (Generalized Linear Model; binomial distribution) and fatty acid profiles (ANOVA) were analyzed. Days 6 + 7 blastocyst yields were 37 ± 1.5%, 12 ± 0.7%, 56 ± 2.6% and 34 ± 2.1% (mean ± SEM) for SBSA, SBSAD, SBSAT and SBSADT, respectively (+DHA effect, P < 0.001; +Trolox effect, P < 0.001; Interaction, P < 0.001). Corresponding cell counts (86 ± 4, n = 64; 72 ± 4, n = 19; 89 ± 3, n = 103; 79 ± 3, n = 62) were affected only by presence of DHA (P < 0.01). Fatty acid content in neutral lipids (ng/blastocyst) was 64 ± 3, 73 ± 2, 66 ± 5 and 66 ± 5 and, in polar lipids, 47 ± 3, 56 ± 3, 46 ± 3 and 51 ± 7, for SBSA, SBSAD, SBSAT and SBSADT respectively (NS). These data indicate that addition of Trolox to DHA-supplemented culture medium improved production of blastocysts. Presence of DHA, however, was not associated with alterations in neutral and polar lipid content or fatty acid composition of the blastocysts. The fact that Trolox enhanced blastocyst yields in the absence of supplementary DHA indicates that conventional media may benefit from provision of this antioxidant. Funded by SEERAD; AR supported by Portuguese Ministry of Science and Technology.
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