Effect of different levels of l-Glutamine and glycerol on freezing of ram spermatozoa

Abstract

Two experiments were designed to evaluate the protective effect of l-Glutamine against freezing damages on spermatozoa by using four rams. In Experiment 1, l-Glutamine from 20 to 80mM was evaluated as a supplement for a conventional freezing medium for ram spermatozoa (Tris–20% egg yolk–7% glycerol). Semen samples (n=20) were diluted with a medium containing 0, 20, 40, 60 or 80mM l-Glutamine and cryopreserved using a standard protocol. At 6h after thawing, the sperm viability was lowest in 20 (28.88%) and 0 (25.92%) mM l-Glutamine (P<0.05). At 3h, functional membrane integrity was highest in 40 (43.69%), 60 (42.13%) and 80 (45.74%) mM l-Glutamine (P<0.05). Sperm motility was highest in the treatments of 40 (29.32%), 60 (29.32%) and 80 (30.29%) mM l-Glutamine (P<0.05). In Experiment 2, the goal was to determine the interaction between l-Glutamine and glycerol on ram sperm freezing. Ejaculates (n=20) were diluted by Tris–egg yolk (20%), added 3 (G3), 5 (G5) or 7% (G7) glycerol and 0 (GL0), 40 (GL40) or 80 (GL80) mM l-Glutamine and thereafter aliquots were frozen. An interaction was observed between l-Glutamine and glycerol on sperm viability (P<0.05). In the presence of 7% glycerol, sperm viability was highest in G7GL80. There was no difference between G5GL40 and G5GL80 on sperm viability (P<0.05). There was an interaction between l-Glutamin and incubation time on functional membrane integrity (P<0.05). At 6h, functional membrane integrity was highest in 40 (35.18%) and 80 (41.97%) mM l-Glutamine (P<0.05). The main effect of l-Glutamine showed that post-thaw sperm motility was lowest in 0mM l-Glutamine (21.62%; P<0.05). Therefore, combination of 7% glycerol and 80mM l-Glutamine or 5% glycerol and 40–80mM l-Glutamine may be useful in the freezing process of ram sperm.