Ultra-low concentrations of benzimidazole derivatives can increase bull and horse semen resistance at cryopreservation and under the influence of damaging factors

One of the possible ways to improve sperm cryopreservation is to find how to increase the resistance to damaging effects of low temperatures. Here we summarize our findings on the bull and stallion semen cryoresistance as influenced by ultra-low concentrations of biologically active substances, the ethyl-1-benzimidazol-2-yl-sulfanyl, 2-ethylsulfanyl-benzimidazol-1-yl and 2-benzimidazol-1-yl-acetic acid. It was found that these substances increased survivability of bull semen during storage in lactose-citrate semen extender. The best motility and vitality of sperm after freezing and thawing was observed when sperm was diluted by extender with added 2-benzimidazol-1-yl-acetic acid in the ultra-low concentrations of 10-13 to 10-15 M. The viability of sperm to 10 % motility was 73 % higher as compared to control. Similarly, freezing equine sperm in extender supplemented with 2-benzoimidazol-1-yl-acetic acid at 10-13 M was more effective: the semen survival after freezing and thawing was 8.1 % (P < 0.01) higher than that in the control, and the intactness of acrosome was 1.9±0.63 % higher (P < 0.05). 2-Benzimidazol-1-yl-acetic acids also improved semen vitality at 40 °С when different osmolarity and after cold shock. It can be assumed that the observed phenomenon is likely due to the protective effect of 2-benzimidazole-1-yl-1-acetic acid to plasma membrane and the mitochondria membrane structure of spermatozoa. Study of respiration in bovine sperm after freezing and thawing confirmed this assumption. Indeed, dinitrophenol almost equally increased cell respiration despite the presence or absence of 2-benzimidazole-1-yl-1-acetic acid in the semen extender while succinate, which penetrates through the damaged membranes, had less stimulating effect when 2-benzimidazole-1-yl-1-acetic acid added. The studies suggested the hypothesis that benzimidazole, a biologically active substance, at ultra-low concentrations can bind to a receptor on the sperm outer membrane resulting in the cell membrane restructuring. At the same time, the changes in viscosity of water associated with the membrane proteins may occur due to hydrogen bonds between water molecules and acid residues of benzimidazole molecules. As a result stability of the membrane structures to damaging effect of varying osmotic pressure increases. Possibly crystal formation of water associated with the cell membranes is decreasing during freezing that also reduces the damaging effect.