Effect of Amino Acids on Goat Cauda Epididymal Sperm Cryopreservation Using a Chemically Defined Model System

Abstract

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25°C min−1 to 5°C, 5°C min−1 to −20°C, and 20°C min−1 to −100°C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 ± 2% and 19 ± 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100–150 mM) cryopreserved approx. 11–17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0–5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7–10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100–150 mM to 20–70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.