55 EFFECT OF DIMETHYL SULFOXIDE SUPPLEMENTATION ON BOVINE IN VITRO EMBRYO DEVELOPMENT

Abstract

Many techniques for in vitro production of embryos frequently make use of dimethyl sulfoxide (DMSO) as a solvent for compounds that have little or no solubility in water. DMSO is also used as a cryoprotectant. Based on its high glass-forming characteristics, it is also essential for vitrification. Although it is known that high concentrations of DMSO could be embryo toxic, less attention has been focused on whether an effect could be detected from the small concentrations present in culture media when used as a vehicle. Earlier studies deemed concentrations of up to 0.4% in in vitro maturation and 0.1% in in vitro culture (IVC) as safe with regards to morphological criteria. In the present study, bovine in vitro-produced embryos employing standard protocols were exposed to the following DMSO concentrations during IVC: 0% (control), 0.05%, 0.10%, 0.15%, 0.20%, and 0.25%. At Day 8 cleavage and developmental rates were recorded. The morphological quality of expanded Day 8 blastocyst was assessed with determination of the total cell number, live-dead (live-dead ratio), and TUNEL staining (apoptotic index). Fat accumulation was analysed by red-oil staining. Cleavage and developmental rates did not differ (P > 0.05) between embryos of the various groups. Mean cleavage and development rates (%) averaged 58.3 ± 10.6 and 28.4 ± 9.2 (0%), 59.5 ± 11.5 and 26.1 ± 7.4 (0.05%), 57.6 ± 6.6 and 21.7 ± 7.1 (0.10%), 58.1 ± 7.8 and 27.8 ± 5.6 (0.15%), 56.6 ± 7.3 and 24.5 ± 7.0 (0.20%), 56.3 ± 10.9 and 23.5 ± 9.9 (0.25%). Total cell numbers were similar [123.9 ± 26.9 (0%), 115.3 ± 21.5 (0.05%), 114.8 ± 22.3 (0.10%), 125.4 ± 22.4 (0.15%), 122.9 ± 24.7 (0.20%), 115.9 ± 19.7 (0.25%)]; P > 0.05. The live/dead cell ratio was significantly higher (P ≤ 0.05) in those embryos derived from the 0.1% group (40.1 ± 23.1) than that from embryos of the other groups [22.6 ± 13.5 (0%), 23.4 ± 10.4 (0.05%), 24.2 ± 14.6 (0.15%), 22.7 ± 14.0 (0.20%), 20.3 ± 9.9 (0.25%)]. Apoptotic cells were significantly lower in embryos exposed to 0.10% and 0.20% DMSO than in those of other groups, and the number of apoptotic cells in embryos of 0.05% was also slightly lower compared with those of the control group (P = 0.08). Apoptotic index tended to be lower in embryos out of the groups supplemented with 0.1% and 0.2% DMSO compared with those of the control group (0%: 3.8 ± 1.6; 0.05%: 2.6 ± 1.6; 0.10%: 2.3 ± 1.8; 0.15%: 3.2 ± 1.5; 0.20%: 2.2 ± 1.5; 0.25%: 3.1 ± 1.7; P = 0.09; P = 0.06). Fat accumulation was significant higher (P ≤ 0.05) in embryos stemming from the group supplemented with 0.15% DMSO (0%: 6617 ± 2703 µm²; 0.05%: 7346 ± 1981 µm²; 0.10%: 6976 ± 1848 µm²; 0.15%: 9301 ± 1703 µm²; 0.20%: 8675 ± 2271 µm²; 0.25%: 8301 ± 2711 µm²). These findings show that DMSO concentrations of 0.10% and 0.20% used during in vitro culture influence the quality of embryos at the morphological level. However, further analyses to verify these results at the molecular level via RT-qPCR are still needed.

The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.