THE INFLUENCE OF CRYOPRESERVATION ON PARAMETERS OF ENERGETIC METABOLISM AND MOTILITY OF FOWL SPERMATOZOA

The objective of this study was to estimate the effect of cryopreserva tion on the main pathways of energetic metabolism and motility of fowl spermatozoa. Sperm diluted 1:5 with the cryoprotective medium contain ing ethylene glycol (1.4 M final concentration) was frozen at the rate of 2-3 degrees C/min to - 25 degrees C with a pause on the plateau of crystallization and then at an exponentially increasing rate to - 196 degrees C. The frozen sperm was thawed in two successive water baths at 0 and at 41 degrees C. After cryopreservation, the rate of radioact ive glucose oxidation to (CO2)-C-14 slightly decreased, the rate of la beled glutamate oxidation remained unchanged, and the rate of labeled succinate oxidation increased two-fold. After freeze-thawing, the rate s of endogenous respiration with and without 2,4-dinitrophenol decreas ed; the oxidation rate of exogenous succinate in the presence of 2,4-d initrophenol, rotenone, and digitonin slightly decreased; and the rate of respiration in the presence of ascorbate, N,N,N',N'- tetramethyl-p -phenylenediamine, antimycin A, 2,4-dinitrophenol, and digitonin did n ot differ from that seen in control. Sperm respiration was highly sens itive to rotenone; antimycin A and cyanide blocked oxygen consumption completely. Succinate, added after 2,4-dinitrophenol and rotenone, sti mulated respiration of thawed spermatozoa, which indicated plasma memb rane damage. The addition of exogenous malate in the presence of 2,4-d initrophenol and digitonin restored the respiration rate of thawed spe rmatozoa to that of unfrozen cells. The rate of respiration of thawed spermatozoa with oligomycin was higher than that of control cells. 2,4 -dinitrophenol stimulated respiration under these conditions. Cryopres ervation caused a decrease in the percentage of progressively moving c ells and the percentage of beating flagella before and after the succe ssive treatment with the medium containing Triton X-100 and ATP. The d ata obtained suggest that cryopreservation decreased the overall flow through glycolysis, the citric acid cycle, and the respiratory chain, affected oxidative phosphorylation, injured plasma membranes, and dama ged the motility apparatus of spermatozoa; however, the respiratory ch ain and tricarboxylic acid cycle retained a high degree of functional integrity. (C) 1994 Academic Press, Inc.