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The effects of cold shock and freeze-thawing on the release of total phospholipid phosphorus and of specific phospholipids from ram, bull, and boar spermatozoa are examined. Species differences are apparent, both in the absolute amount of total phospholipids released and in the conditions required to effect a loss of individual phospholipids. The phosphoglycerides most affected by the temperature treatment are choline plasmalogen, phosphatidyl choline, and phosphat idyl ethanolamine. The loss of phospholipids is most specific in boar spermatozoa. Bull spermatozoa suffer a greater overall and more general breakdown of phospholipids, indicating disruption of the cell structure. This is in contrast to the more specific phospholipid losses from ram and boar spermatozoa, which may indicate a more localized region of damage and release of material, possibly acrosomal. The loss of phospholipid may be correlated with the species differences in the fertility of the frozen spermatozoa.

Recent work has established the composition of phospholipids and phospholipid\x=req-\ bound fatty acid esters and aldehydes in ram, bull, boar, human and rabbit spermatozoa (

Lardy & Phillips (1941a,b) postulated that the endogenous phospholipids are used as a source of oxidizable energy by bull spermatozoa in the absence of sugar. Their results were not confirmed by the later work of Bomstein & Steberl (1957) with bull spermatozoa, or by with human spermatozoa. Hartree & Mann (1959, 1961 examined the effect of aerobic and anaerobic incubation of washed ram spermatozoa on the utilization of endogenous phospholipids and their results, though different from those of Lardy & Phillips (1941a,b), supported the latter's original hypothesis. Scott & Dawson (1968) briefly examined the effect of incubation on the phospholipids of washed ram spermatozoa and did not detect the same degree of plasmalogen loss as did Hartree & Mann (1961). The latter authors also found that incubation resulted in the release of phospholipid-bound fatty acid esters, specifically myristic and palmitic acids. However, in view of the established presence of large amounts of docosahexaenoic acid in ram spermatozoa (Dott & Dingle, 1968;Poulos, Darin-Bennett & White, 1973), it was decided to re-examine the effects of incubation on the phospholipids and phospholipid-bound fatty acids.Washed suspensions of spermatozoa from Merino rams were prepared accord¬ ing to the method described by Hartree & Mann (1961). The semen was pro¬ cessed within 60 min of collection, during which time the samples were kept at 20°C and only those displaying good initial motility were used. Triplicate aliquots were removed for counting in a haemocytometer and the spermatozoa incubated at 37°C for 3 or 4 hr at a mean concentration of 7-5 IO8 cells/ml. An analysis of the fructose concentration by the method of Mann (1948) of the prepared sperm suspensions revealed a fructose content of less than 2 µg| l. Sperm suspensions contained streptomycin (1500 units/ml) and penicillin (7300 units/ml) and samples were incubated in both test-tubes and conical flasks, with constant shaking. To ensure fully anaerobic conditions, oxygen-free Krebs-Ringer solution was used for anaerobic experiments, the samples were flushed with nitrogen and incubated in ground-glass stoppered containers. After incubation, the phospholipids were extracted from the spermatozoa and the phospholipid fatty acid esters were prepared and estimated as described in Poulos et al. (1973). The composition of the phospholipids was determined after thin-layer chromatography. Plates were developed in single dimension in 543