Protective Effect of Sucrose and Antioxidants on Cryopreservation of Sperm Motility and DNA Integrity in C57BL/6 Mice

Currently the most popular mouse sperm freezing medium is R18SM3+MTG containing 18% raffinose, 3% skim milk, and 0.5 mM monothioglycerol (MTG), but there is no information available about whether MTG and other antioxidants can cryoprotect mouse sperm DNA integrity. It is also uncertain if sucrose can be used successfully for sperm cryopreservation. In this report we compared the cryoprotective effects of sucrose and raffinose, as well as the antioxidants MTG, reduced glutathione (GSH), and quercetin on sperm motility, DNA integrity, and fertility in the C57BL/6J mouse strain. Results show that: (1) 10% sucrose in the presence of 3% skim milk and 0.5 mM MTG (S10SM3+MTG) was as effective as R18SM3+MTG ( p > 0.05) in cryopreserving sperm motility (21.0% ± 4.0% vs. 19.0% ± 3.6%), DNA integrity (8.1% ± 1.5% vs. 9.0% ± 1.5% TUNEL positive), fertilization rate (48.3% ± 7.5% vs. 45.0% ± 7.9%), and pup birth rate (36.7% ± 10.0% vs. 37.7% ± 3.5%); (2) Supplementation of freezing medium with MTG (0.5 mM), GSH (0.5 mM), or quercetin (25 μM) had a significant ( p < 0.05) cryoprotective effect on sperm motility recovery rate compared to that of controls (MTG, 32.7% ± 10.7% vs. 19.4% ± 4.2%; GSH, 34.3% ± 3.7% vs. 24.5% ± 1.8%; quercetin, 36.3% ± 3.3% vs. 25.1% ± 3.6%); and (3) Cryopreservation significantly increased sperm DNA fragmentation level (16.4% ± 2.3% in R18SM3 and 14.6% ± 2.5% in S10SM3) compared to that of fresh sperm (3.0% ± 2.0%); however, supplementation with MTG (0.5 mM), GSH (0.5 mM), or quercetin (25 μM) significantly ( p < 0.05) decreased the sperm DNA fragmentation level (MTG, 8.1% ± 1.5%; GSH, 9.0% ± 1.5%; and quercetin, 8.3% ± 1.3%). It was concluded that sucrose can be used as effectively as raffinose for mouse sperm cryopreservation, and supplementation of MTG, GSH, or quercetin at an appropriate concentration can help cryoprotect sperm motility and DNA integrity.