The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD(+) and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP(+) and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100x[NAD(+)/(NAD(+)+NADH)], without altering the total content of NAD(+)+NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25 degrees C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25 degrees C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD(+)+NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ;Cold shock', resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14 degrees C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.
