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A. POULOS and I. G. WHITE

Summary.

The phospholipid composition of human spermatozoa and seminal plasma was examined by quantitative two-dimensional thinlayer chromatography, followed by phosphorus analysis. Sperm phospholipid comprised 28·8% phosphatidyl choline, 21·6% phosphatidyl ethanolamine, 21·4% sphingomyelin, 9·4% ethanolamine plasmalogen, 4·7% phosphatidyl serine, 2·7% choline plasmalogen, 1·9% phosphatidyl inositol and 1·6% cardiolipin.

Seminal plasma phospholipids comprised 44·0% sphingomyelin, 12·3% ethanolamine plasmalogen, 11·2% phosphatidyl serine, 8·5% phosphatidyl ethanolamine, 7·8% phosphatidyl choline, 0·8% choline plasmalogen, 0·8% cardiolipin and 1·7% phosphatidyl inositol.

Prolonged aerobic incubation of human spermatozoa in the presence or absence of glucose produced no significant alteration either in the amount of phospholipid phosphorus extracted or in the phospholipid composition of this extracted material.

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I. G. WHITE and R. G. WALES

Summary.

A comparison has been made of the physiological and chemical properties of ejaculated and epididymal semen collected from rams with a surgical fistula into one vas deferens.

The most striking difference between the spermatozoa was the resistance of epididymal cells to cold shock, which was correlated with the attachment of kinoplasmic droplets.

Other intrinsic differences between the spermatozoa were small and there was little evidence of a difference in metabolic pattern.

The two types of seminal plasma differed considerably in chemical composition, although the pH was about the same and glycerylphosphorylcholine was present in high concentration in both.

Evidence was obtained that the fructose in ejaculated ram semen enables the motility of the spermatozoa to be better maintained under anaerobic conditions than is the case with epididymal spermatozoa.

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R. G. WALES and I. G. WHITE

Summary.

The motility of dog spermatozoa is depressed by high dilution. Dog spermatozoa survived best in a chloride diluent, buffered with phosphate. Citrate, tartrate and peptone diluents were less favourable. Adding 2 to 25% prostatic fluid to diluents partially prevented the effects of dilution on dog spermatozoa, but motility was depressed by neat prostatic fluid. There was little evidence for loss of protective substances from dog spermatozoa when left overnight, or after repeated freezing and thawing. Bovine globulin, casein, egg albumin and alanine were even more effective than prostatic fluid in preventing the dilution phenomenon, and motility in their presence was as good as in concentrated suspensions. Egg albumin, casein, acacia, serine and alanine improved the motility of washed dog spermatozoa.

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R. G. WALES and I. G. WHITE

Summary.

Penicillin, streptomycin and the sulphonamides were well tolerated by dog spermatozoa. Penicillin did, however, become more toxic on storage at 37° C in neutral or alkaline solutions.

Chloromycetin and the tetracycline derivatives were much more spermicidal and greatly reduced oxygen uptake, lactic acid production and fructose utilization.

Sulphamezathine and tetracyn were superior to other antibacterials in maintaining the viability of dog spermatozoa stored at 5° C.

The toxicity of high concentrations (2000 μg/ml) of biotin was reduced by aureomycin.

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R. G. WALES and I. G. WHITE

Summary.

Data are presented on the chemical composition of the three fractions of dog semen. Sodium is the main cation present in all fractions; potassium, magnesium and calcium occur in much lower concentration. Chloride is the main anion. All fractions are characterized by low concentrations of total reducing sugar, fructose, lactic acid and citric acid. Orcinol reactive carbohydrate is present in significant amounts and much is acid insoluble and probably bound to protein.

The sperm-bearing fraction is characterized by a high concentration of total phosphorus and the acid insoluble fraction is mainly confined to the spermatozoa. The high concentration of acid soluble phosphorus in this fraction is partly due to the appreciable amounts of glyceryl-phosphorylcholine present.

Analyses of seminal carbohydrate and protein indicate inherent differences between dogs in the composition of semen.

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R. A. P. HARRISON and I. G. WHITE

Summary.

A study was made of three methods of washing bull, boar and ram spermatozoa, using as criteria contamination with free-lying cytoplasmic droplets, sperm motility and leakage of glucose-phosphate isomerase and hexokinase into the medium. The methods used were: washing in a Ringer-fructose-phosphate medium, washing in a hypoosmotic phosphate medium and washing with balanced media in the cold after slow cooling and storage overnight at 4° C in a milk diluent. Only by using the last method was it possible to remove adequately the freelying cytoplasmic droplets ; this method also caused least damage to the sperm suspensions.

The problems of washing spermatozoa with respect to cell damage and leakage of enzymes are discussed in the light of the present findings. The evidence suggests that glucose-phosphate isomerase and hexokinase leak from the cytoplasmic droplets as well as from the spermatozoa during washing. Hypo-osmotic media appear to cause an immediate and marked release of these enzymes from the droplets.

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P. J. QUINN and I. G. WHITE

Summary.

Cold-shocking and deep-freezing bull and ram semen caused an influx of sodium to and an efflux of potassium and magnesium from the spermatozoa. The movement of these cations was accentuated when the semen was diluted 1 : 8 in sodium phosphate buffers. Calcium is actively accumulated in the spermatozoa of these two species after cold-shocking.

No significant changes in the cation content of human, dog, rabbit or fowl spermatozoa were found after cold-shocking. There was no great disturbance of the cation status of human and fowl spermatozoa, even after deep-freezing.

Lecithin prevented calcium accumulation in bull and ram spermatozoa on cold-shocking, and glycerol also had this effect on cold-shocked bull spermatozoa. An egg-yolk-citrate/glycerol-fructose diluent protected bull and ram spermatozoa to a considerable extent from the influx of sodium and the efflux of potassium that occurs after deep-freezing to −79°C.

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J. C. Rodger and I. G. White

Department of Veterinary Physiology, University of Sydney, N.S.W. 2006, Australia

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R. A. P. HARRISON and I. G. WHITE

Summary.

A method has been developed for the simultaneous isolation of spermatozoa and cytoplasmic droplets from bull, boar and ram semen. The glycolytic enzymes, hexokinase (HK), glucose phosphate isomerase (GPI) and lactate dehydrogenase (LDH) were demonstrated in both types of particle: on a per particle basis compared with spermatozoa, droplets contained similar quantities of GPI, less HK and only one-tenth the amount of LDH. The HK was partly bound while the GPI was entirely soluble in both. The LDH was partly bound in spermatozoa but soluble in droplets.

Suspensions of spermatozoa and cytoplasmic droplets were subjected to sudden cooling, freezing or hypo-osmotic shock, and leakage of GPI, HK and LDH into the extracellular medium was measured. Both spermatozoa and droplets released glycolytic enzymes during the treatments, but the droplets were more fragile than the spermatozoa. The proportion of loss varied between enzymes, as well as between droplet and spermatozoon; considerable GPI and little HK was released from both, whereas much LDH was lost from droplets but hardly any from spermatozoa.

The activities of these three glycolytic enzymes were also measured in seminal plasma from bull, boar and ram. The ratios of the activities found were compared with those in droplets or spermatozoa from the same species. It is suggested that much of the glycolytic enzyme activity in seminal plasma arises from disintegrated cytoplasmic droplets rather than from spermatozoa.

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J. C. RODGER and I. G. WHITE

Summary.

Total carbohydrate, glucose, fructose and resorcinol-reactive material were estimated in homogenates of the prostates of brush-tailed possum (Trichosurus vulpecula) and red kangaroo (Megaleia rufa). Fructose was virtually absent from the gland in both species. Glucose occurred in quite considerable quantities in the posterior segment of the prostate of M. rufa (92·2 mg/100 g) and to a lesser extent in the small anterior segment of T. vulpecula (22·4 mg/100 g). The posterior prostate of T. vulpecula contained an unidentified material which reacted with the resorcinol reagent as fructose (51·2 mg/100 g) but did not appear to be fructose or a commonly occurring ketose.