The cause of the higher total calcium level in bovine seminal plasma than in spermatozoa is due mainly to a much higher concentration of complexed calcium in seminal plasma than in spermatozoa. The concentration of protein-bound calcium was approximately the same in spermatozoa and seminal plasma; that of ionized calcium lower in seminal plasma than in spermatozoa.
Sudden cooling (cold shock) of fresh bull semen, which irreversibly abolished motility, led to a significant increase in the concentration of total calcium in spermatozoa, and a corresponding decrease of total calcium in the seminal plasma; the effect was more marked when semen was rapidly cooled to 0°C than to 5°C. The cold-shock induced change in the distribution of calcium between spermatozoa and seminal plasma was reflected in a decreased content of ionized calcium in the seminal plasma and a simultaneously increased content of complexed and protein-bound calcium in spermatozoa. No increase of calcium in spermatozoa was observed following slow cooling under conditions when motility had not been lost irreversibly. Heat inactivation by itself had no marked effect on the calcium concentration ratio between spermatozoa and seminal plasma, but cold-shocking of spermatozoa that had first been irreversibly immobilized by heating produced an increase in the level of sperm calcium. The increase in the calcium level occurred irrespective of whether the spermatozoa had been initially motile. When spermatozoa were reversibly immobilized by treatment with formaldehyde at a low concentration, the calcium level remained unchanged, increasing significantly after cold shock, but not to the same level as in the formaldehyde-free control sample. Addition of EDTA or detergents to the semen prevented the accumulation of calcium by cold-shocked spermatozoa.