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T. O'SHEA

Summary.

The amount of acetate accumulated from lactate by ram spermatozoa during incubation at 37° C was increased by storage of semen at 5° C and by incubating washed cells in diluents without added potassium and magnesium ions.

When ram semen was incubated shortly after ejaculation, 70% of the acetate accumulated was intracellular. After storage overnight at − 79° C, only 10% of the acetate accumulated during incubation was intracellular.

The intracellular acetate was not extracted from the cell by hexane: butanol mixtures but was removed by extraction with 2·% perchloric acid.

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T. O'SHEA and R. G. WALES

Summary.

The metabolism of specifically labelled lactate by ram spermatozoa was studied under various experimental conditions. Increasing the rate at which diluted ram semen was cooled to 5° C caused a greater decrease in the formation of labelled carbon dioxide from [2-14C]lactate than from [1-14C]lactate. Changes in the amount of lactate present did not affect the relative amounts of carbon dioxide formed from carbon atoms one and two of lactate.

Washing ram spermatozoa, that had been recently ejaculated, decreased the amount of labelled carbon dioxide formed from [2-14C] lactate relative to that formed from [1-14C]lactate, whereas the reverse effect occurred on washing spermatozoa that had been stored in test tubes at 37° C for 3 hr.

When washed ram spermatozoa were incubated with lactate as substrate, labelled acetic acid accumulated from [2-14C]lactate but not from [1-14C]lactate, both in the absence and in the presence of added potassium chloride (1 mm) and magnesium chloride (2 mm).

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T. O'SHEA and R. G. WALES

Summary.

The metabolism of sorbitol and fructose by ram spermatozoa has been examined under both aerobic and anaerobic conditions. Isotopically labelled substrates were used to measure substrate oxidation under aerobic conditions or substrate dismutation under anaerobic conditions.

In combination, fructose was used preferentially and, when little or no sorbitol was present at the beginning of incubation, some of the fructose disappearing accumulated as the polyol. The remaining fructose utilized, but not oxidized or dismutated, was quantitatively converted to lactate. When sorbitol was the only substrate in the diluent, it was not utilized under anaerobic conditions and a large part of the sorbitol utilized under aerobic conditions was oxidized. Under the latter conditions, the rate of utilization of the polyol was related to its concentration. It appears that this substrate is made available to the cell by passive diffusion and that this process plays a part in limiting the availability of sorbitol to the cell.

The addition of 10-4 m 2 : 4 dinitrophenol increased oxygen uptake and substrate oxidation when fructose was present in the diluent. With sorbitol as substrate, dinitrophenol decreased substrate oxidation. Under anaerobic conditions, dinitrophenol decreased both sorbitol accumulation from fructose and dismutation of fructose. It is suggested that the dismutation reaction may be linked with the reduction of fructose to sorbitol to balance nad : nadh ratios.

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T. O'SHEA and R. G. WALES

Summary.

The relative contributions of fructose, lactate, acetate, citrate and inositol to the aerobic metabolism of ram spermatozoa have been examined in various combinations of these substrates. By using isotopically labelled compounds it was shown that fructose and lactate were important substrates oxidized by ram spermatozoa incubated in diluents containing fructose and low levels of other metabolites. Although the addition of sodium lactate to a spermatozoal suspension caused no change in the amount of fructose converted to lactate, there was a reduction in the amount of fructose oxidized to carbon dioxide.

At the levels of acetate and citrate occurring in semen, their effect on the oxidation of fructose by ram spermatozoa was small. Higher concentrations of these compounds, however, caused a decrease in fructose oxidation. The addition of citrate and acetate to a fructosecontaining diluent did not affect the amount of fructose utilized by ram spermatozoa. While acetate had no effect on the accumulation of lactate from fructose, the presence of the high levels of citrate decreased the amount of lactate produced.

Thus fructose and lactate are the main metabolites for unwashed ram semen incubated under aerobic conditions in a diluent containing fructose. In these circumstances, a quantitative measure of the oxidation of these substrates, coupled with an estimation of fructolysis, should give a good measure of the metabolism of the cell.

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T. O'SHEA and R. G. WALES

Summary.

Potassium (16 to 64 mm) adversely affected ram spermatozoa during cooling from 30 to 5° C, while magnesium was beneficial. The effect of potassium was independent of the rate of chilling or the dilution of semen between 1 in 10 and 1 in 20, but it was decreased by increasing the sodium content of the diluent and was increased by the presence of phosphate. The addition of potassium after chilling did not affect subsequent motility.

High potassium levels during chilling were detrimental to washed ram spermatozoa. Low levels, on the other hand, helped to maintain the motility of washed cells during the period of storage. The addition of seminal plasma did not modify the effect of potassium on washed cells.

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R. G. WALES and T. O'SHEA

Summary.

The metabolism of semen in the presence and in the absence of carbon dioxide was examined using isotopically labelled substrates. When carbon dioxide was present, both oxygen consumption and carbon dioxide formation were determined in the one manometric flask. Respired carbon dioxide had no effect on oxygen uptake, substrate oxidation or aerobic fructolysis of ram spermatozoa. The addition of low levels of potassium and magnesium ions to the diluent stimulated the metabolism of washed ram spermatozoa, but there was still no effect of carbon dioxide on aerobic metabolism. Incubation of ram spermatozoa in an atmosphere containing 3·8% carbon dioxide increased the amount of lactate accumulated but had no effect on oxygen uptake or substrate oxidation. The necessity to distinguish between the effects of pH and carbon dioxide has been shown. In the presence of added fructose, the respiratory quotient of ram spermatozoa was approximately unity but was 0·78 for washed semen incubated in phosphate-buffered saline without added substrate.

The presence of respired carbon dioxide during incubation of bull spermatozoa had no effect on oxygen uptake or substrate oxidation but increased lactate accumulation. With fructose as substrate, incubation of bull spermatozoa in phosphate-buffered saline and saline gave respiratory quotients of 0·97 and 0·83 respectively.

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T. O'SHEA and R. G. WALES

As the effect of carbon dioxide on mammalian spermatozoa is of current interest (Lodge & Salisbury, 1963; Hammer & Williams, 1964; VanDemark, Koyama & Lodge, 1965; Wales & O'Shea, 1966; Wales & Restall, 1966), the possibility of its fixation by ram spermatozoa has been investigated.

Aliquots (0·4 ml) of ram semen or twice-washed spermatozoa (5·4 × 108 cells/ flask) were incubated at 37° C with [14C]sodium bicarbonate (1 μmole and 25 μc) in phosphate-buffered saline containing 1 mm-potassium chloride, 2mm-magnesium chloride, 10 mm-sodium Pyruvate, 30 mg/100 ml penicillin and 50 mg/100 ml streptomycin. After 15 or 60 min, 0·5 ml of the incubated semen was removed and stored at -15° C for subsequent chromatography. The remaining 0·5 ml was acidified (0·1 ml of 6 n-H2

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R. G. WALES, L. MARTIN and T. O'SHEA

Summary.

Adult does were inseminated with a constant number of spermatozoa in different volumes of diluent or with varying numbers of spermatozoa in a constant volume. The number of spermatozoa required for maximum fertility was 1 × 106; 50% of does littered when inseminated with 1 to 2 × 105 cells and inseminations with 2 to 4 × 104 cells were sterile. Even when the number of spermatozoa approached the minimum for fertility, increased dilution rates did not decrease the numbers of does kindling. Some litters were obtained with semen diluted 1 in 10,000 with calcium-free Krebs'-Ringer phosphate.

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T. O'Shea, J.-L. Dacheux and M. Paquignon

Summary. Boar spermatozoa incorporated more [14C]glycerol into lipid when incubated with 200 mm- than with 25 mm-glycerol. Measurements were made of the metabolism of spermatozoa while they were being prepared for frozen storage. [14C]Glucose was converted to CO2 and lipid while the cells were cooling to 15°C. Glycerol was added at 15°C and during further cooling to 5°C glucose metabolism was greatly reduced but [14C]glycerol was converted to CO2 and lipid. Under aerobic conditions spermatozoa accumulated lactate while cooling from 30 to 15°C and from 15 to 5°C. With essentially anaerobic conditions, although more lactate was accumulated this occurred only while the cells were cooling from 30 to 15°C, and no further accumulation could be detected during cooling from 15 to 5°C. When boar spermatozoa were incubated at 37°C after storage in liquid nitrogen, metabolism of glycerol was greater than metabolism of glucose. It is suggested that this preferential use of glycerol during cooling and after storage may be one facet of its cryoprotective function. After storage, boar spermatozoa incorporated relatively less [14C]stearic and [14C]palmitic acids into phospholipids (especially phosphatidyl choline) than did freshly collected cells. Caffeine stimulated the oxygen uptake of freshly collected and thawed cells.

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J. L. Dacheux, T. O'Shea and M. Paquignon

Summary. Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.