The effect of glycerol on the viability, mitochondrial function and acrosomal integrity of bovine spermatozoa

Eight bull ejaculates were split to evaluate the effects of glycerol on sperm organelle function. Although glycerol protects sperm membranes during cryopreservation, preliminary data suggested that glycerol was detrimental to sperm organelles to varying degrees. To assess the compartmental effects, three organelle-specific fluorophores were used to analyze with (G+) and without glycerol (G-) in spermatozoa stored for 24h at 5 degrees C in an egg-yolk-based extender. The mitochondrial probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide (JC-I) was used to examine the level of mitochondrial metabolic function by it's discrimination between high (J-aggregate staining, red-orange) and low membrane potentials (JC-I monomeric staining, green); while acosomal reacted spermatozoa were identified using fluorescein-labelled lectin from arachis hypogaea, PNA-FITC. The proportions of living and dead spermatozoa were determined by staining with the combination of SYBR-14 and propidium iodide (PI). Split-plot analysis of variance revealed that within bulls, glycerol altered the proportions of sperm staining with each organelle-specific fluorophore to varying degrees. The proportion of spermatozoa labelled with SYBR-14, indicating intact plasmalemmae, were not affected by the addition of glycerol (p = 0.11). Although the total proportion of JC-1-labeled spermatozoa were similar in G- and G+ samples (p = 0.90), the presence of glycerol decreased the proportion of spermatozoa that exhibited J-aggregate staining (p < 0.01) while producing an increase in monomeric staining (p = 0.02). The proportions of acrosome-reacted spermatozoa, however, were greater in the G- samples than in the G+ samples as indicated by PNA-FITC (p = 0.03). These findings suggest that mitochondria, acrosomes and the plasmalemmae of unfrozen spermatozoa vary in their response to the addition of the cryoprotectant glycerol.