The technique of washing tissues and cells has been widely practised in the field of enzyme chemistry, for removing natural substrates and for demonstrating important components of enzyme systems.
In the field of reproductive physiology several authors (e.g. Lardy & Phillips, 1943; Mann, 1945) have used spermatozoa washed in calcium-free Ringer’s solution for studying the endogenous metabolism of the cells and to observe the effect of added substances. A further application of the technique in this field is due to Emmens & Swyer (1948) who have used repeated washing in Baker’s solution to remove seminal plasma constituents and so presumably to simulate the effect of dilution on rabbit spermatozoa. The method of washing spermatozoa in all cases has consisted of centrifuging either the neat or diluted semen, withdrawing the supernatant and replacing it by a suitable diluent.
In this investigation, the effect of one and of two washings on the motility, oxygen uptake and aerobic glycolysis of ram, bull and rabbit spermatozoa has been studied in a sodium phosphate-fructose diluent at 37° C. The object of the experiment was twofold: first, to gain information on the extent to which mammalian spermatozoa are likely to be affected by procedures normally adopted in the preparation of cell suspensions for metabolic studies, and secondly, assuming provisionally that washing and dilution produce similar effects, to obtain evidence as to a metabolic basis for the adverse effect of dilution on mammalian spermatozoa (Salisbury, Beck, Cupps & Elliot, 1943; Emmens & Swyer, 1948; Cheng, Casida & Barrett, 1949). This should not be confused with the dilution effect described by Gray (1928), who found that the motility and respiratory rate of sea-urchin spermatozoa was increased by dilution with sea water.
MATERIALS AND METHODS
Bull and rabbit semen was collected by means of the artificial vagina, and ram semen by electrical stimulation as described by Gunn (1936). In all cases only apparently normal specimens with good motility were employed. Experiments with ram and rabbit semen were started immediately after collection and those with bull semen within 2 hr. of collection. The semen was stored during this period at about 10° C., the usual slow cooling precautions being taken to avoid cold shock.
The procedure each day was to dilute the semen (which in most cases was composed of pooled ejaculates) approximately 1 in 3 with diluent to give a volume of about 10 ml. in a graduated centrifuge tube. Duplicate 1 and 0·5 ml. aliquots of the unwashed sperm suspension were then further diluted 1 in 3 with the same diluent in Warburg flasks and small tubes respectively—the flasks being used for the measurement of oxygen uptake, and the tubes, which were shaken in the Warburg bath with the flasks, for estimations of lactic acid production and observations of motility. The remaining, unwashed sperm suspension in the centrifuge tube was then spun at 1500 r.p.m. (about 300 g.) for 10 min., the supernatant withdrawn and replaced by an equal volume of diluent. Aliquots of the once-washed sperm suspension were taken as before and the washing procedure repeated. Early experiments indicated the importance of thoroughly dispersing the spermatozoa in the diluent after centrifuging. A satisfactory method of doing this without damaging the cells was found to be by sucking the sperm up and down in a wide-bore Pasteur pipette fitted with a rubber teat. An isotonic diluent of pH 7·0 containing 0·032 M-NaH2PO4.H2O, 0·048M-Na2HPO4.12H2O, 0·040M-NaCl and 0·022 M-fructose was found to be adequate to maintain a fairly constant pH within 0·1 unit of the initial value. The diluent was freshly prepared each day by the appropriate dilution of 0·4M stock solutions of the A.R. salts, with glass-distilled water, solid fructose being added to give a concentration of 0·4% (w/v). Stock phosphate and chloride solutions were stored in a refrigerator and discarded at the first signs of mould growth. The pH of all diluents was checked with the glass electrode prior to use.
Each experiment was of 5 hr. duration, with the Warburg bath operating at 37° C. and a shaking rate of 114 strokes per minute, the amplitude of the stroke being 4 cm. Oxygen uptake and motility observations were made at hourly intervals and the lactic acid production calculated over the 5 hr. period from estimations made at the start and end of experiments. In the washing experiments all observations and analyses were made in duplicate on each ejaculate, the mean values being recorded in the tables of results.
Oxygen uptake was measured by the direct Warburg technique described by Umbreit, Burris & Stauffer (1949) using 0·2 ml. of 20% (w/v) KOH for the absorption of C02. Flasks of 22-35 ml. volume were used in some early experiments, but these were subsequently replaced by smaller ones of 12-16 ml. capacity in order to increase the sensitivity of the readings. Both types were calibrated in duplicate using mercury (Dixon, 1943). Agreement between duplicate calibrations was good, the mean between-duplicate difference for thirty-five flasks being 0·14 ± 0·15% of the mean volume, indicating that the contribution of calibration to the overall error of the Warburg technique is negligible. The calibration of the flasks and the accuracy of the manometric technique was further checked by setting up flasks containing a mixture of 1 ml. of 5 % (v/v) H2SO4 and 1 ml. of 0·1 vol. H2O2 in the body of the flask with 0·1 N-KMnO4 in a side-arm. After equilibration the permanganate was tipped into the acidified peroxide and the gas production recorded after i hr. Experiments on two different days, involving twelve and ten flasks respectively, gave readings of 196·2 ±5·1 and 191·8 ± 5·3 µl. There was no evidence of bias with respect to any one manometer. Standard deviations of a similar order were obtained in preliminary experiments in which the oxygen uptake of ram spermatozoa was measured. Thus in two experiments, each of 1 hr. duration and each involving ten flasks, the mean oxygen uptakes were 49·0 ±4·5 and 77·0 + 4·5 µl. respectively. The corresponding standard errors are 1·4 and 1·5 µ1., which compare favourably with a mean standard error of 3·9 µl. quoted by Comstock, Green, Winters & Nordskog (1943) for four trials with ram spermatozoa, each involving six flasks.
Lactic acid was estimated by the method of Barker & Summerson (1941). Duplicate analyses, on the same lactic acid sample, in most cases did not differ by more than 0·20 µg., which is similar to the accuracy stated by Umbreit et al. (1949). Three tests, each involving duplicate estimations on at least eight different concentrations of standard lithium lactate, indicated that the colour development obeyed the Beer-Lambert law over a final lactate concentration of 0-8 µg. The stability of the colour was found to be good, duplicate tests made on nine equispaced different lactate concentrations over the above range showing no significant variation in transmission during a 3 hr. period. A check on the spectral-transmission graph in which duplicate readings were made on three different days on 2 µg. lactate standards showed that the wave-length of maximum light absorption was 570 mµ. The wave-length of maximum absorption according to the originators of the method is 565 mµ. As a routine procedure six estimations on four different concentrations of standard lithium lactate were made with each batch of unknowns and a new calibration curve constructed on each occasion. An analysis of variance, using the within-duplicate mean square as the test variance, showed that there was no significant variation either between slopes or between positions of the curves.
Motility observations were made according to the system of Emmens (1947), full motility being scored as 4 and complete immotility as zero.
Spermatozoa counts, using an improved Neubauer haemocytometer under high power, were made on duplicate samples taken at the appropriate stage during the washing procedure and diluted with 3% (w/v) sodium chloride. The difference between duplicate counts on sixty-four semen samples exceeded twice the theoretical standard error on twelve occasions; thus any departure from expectation in the spermatozoa counting was not serious.
Samples were taken from the Warburg flasks at the end of some experiments for bacterial counts. These samples were sealed in phials and stored in solid carbon dioxide until the actual counts were made. The technique adopted for counting bacteria was a direct one, similar to that for spermatozoa, except that phasecontrast illumination was used.
The effect of washing once was studied on four pooled ejaculates from each of the three species, and the effect of washing twice on eight pooled ejaculates from rams and four each from bulls and rabbits.
In the analyses of variance for the oxygen uptake, lactic acid production and motility data, the figure for the total 5 hr. period has been used as the unit observation.
The results of the experiments with the once- and twice-washed ram spermatozoa have been analysed separately because of unequal numbers of replicates, whilst with the bull and rabbit data, where both groups have equal numbers of replicates, they have been analysed together. In each case the interaction mean square has been used as the error term.
Washing once had little effect, but washing twice depressed motility (P<0·05). Ejaculates varied significantly in performance.
No significant effect was seen on washing, nor did significant differences occur between ejaculates.
Again washing had no significant effect, but highly significant variation occurred between ejaculates.
Washing once had no significant effect, but washing twice reduced the total oxygen consumption (P<0 ·01). Ejaculates did not vary significantly.
No significant effect was seen on washing, but highly significant variation occurred between ejaculates.
Results for both the once- and twice-washed groups were each less than the unwashed control (P<0 ·05). No significant variation occurred between ejaculates.
Lactic acid production
Washing once had no significant effect, but washing twice reduced lactic acid production (P <0 ·01). Significant variation occurred between ejaculates in the experiments on twice-washed ram spermatozoa but not in those on once-washed suspensions.
Again the production of lactic acid was decreased on washing twice (P<0 ·01) but not after a single washing. Highly significant variation occurred between ejaculates.
Although the overall between-treatment variance does not test as significantly different from the interaction mean square, it is nevertheless clear from the analyses that lactic acid production was also depressed on washing rabbit spermatozoa twice (P < 0 ·05). Ejaculates did not differ significantly.
Comparison of metabolic data
The ZO2 values obtained in these experiments over the first hour with ram spermatozoa vary from 8 ·9 to 19 ·6 with a mean of 13 ·6 (Table 5), and tend to be lower than those of Lardy, Winchester & Phillips (1945) who recorded values between 11·0 and 46 ·0. The divergence might be due not only to animal variation but also to differences in diluents, the one used here being much simpler in composition than the Ringer-glucose-phosphate of Lardy & Phillips (1943). It would be interesting, therefore, to study the effect on the oxygen uptake of those ions present in Ringer’s solution but absent from the diluent used here.
The ZO2 (µ.1. O2/108 spermatozoa/hr.) values obtained in these experiments over the first hour with bull spermatozoa vary from 8·4 to 18·9 with a mean of 13·3 (Table 6) and are thus similar to those for the ram. Values recorded in the literature for the ZO2 of bull spermatozoa vary considerably. Thus Lardy & Phillips (1943) from a study of nineteen samples obtained a ZO2 range from 16·1 to 29·8, whilst subsequent studies on a large number of other bulls (Ghosh, Casida & Lardy, 1949) gave figures mostly between 3·0 and 10·0, the highest value being 12·6. The cause of the considerable variation in the respiratory activity of bull spermatozoa has not been studied ; one possible factor, however, might be differences in the thyroid status of the animals. It has been demonstrated by Schultze & Davis (1948) and confirmed by Maqsood (1950) that the oxygen uptake of bull spermatozoa, at least in vitro, is increased by treatment with thyroxine. An extension of this work to include studies of the in vivo effect of thyroxine on the oxygen consumption of spermatozoa is clearly desirable.
The ZO2 values obtained in these experiments over the first hour with rabbit spermatozoa vary from 4·6 to 10·0 with a mean of 7·1 (Table 7) compared with a mean figure of 10·9 reported by Lardy & Phillips (1943) and values between 4 and 28 calculated from the data of Carter (1932).
The mean aerobic lactic acid production [161 µg./5 hr. (Table 9)] by twice-washed ram spermatozoa in these experiments is less than half the values quoted by Mann & Lutwak-Mann (1948), whilst those for unwashed and once-washed bull spermatozoa [439 and 441 µg./5 hr. respectively (Table 9)] are about twice those reported by Henle & Zittle (1942) and Lardy & Phillips (1941). There appear to be no figures in the literature for the lactic acid production of rabbit spermatozoa, but the observations made here indicate that its metabolism, like that of the ram and bull, is highly glycolytic.
In these experiments significant between-ejaculate variation occurred in motility, oxygen uptake and lactic acid production. Since pooled ejaculates were used from different combinations of animals on different days it is impossible to assess from the data presented the relative magnitude of day-to-day variation within individual animals compared to day-to-day variation due to the use of different animals on different days. Other observations made in this laboratory on individual ejaculates, however, indicate that both types of variation in all three species may be considerable so far as motility is concerned. The observations of other workers would indicate that this is also true for oxygen uptake and lactic acid production. Thus Lardy, Winchester & Phillips (1945) working with ram spermatozoa, and Romijn (1950) with bull, record considerable differences in ZO2, not only between rams but also between samples taken from the same rams on different days, while Comstock’s (1939) observations are similar for lactic acid production in the ram.
In almost all experiments (Tables 1, 2 and 3) there was a decline in motility during the 5 hr. period in which observations were made; the spermatozoa of all three species were, however, usually quite active at the end.
During the course of most experiments there was also a decline in ZO2 (Tables 5, 6 and 7), although there was no striking correlation between decline in motility and falling off in oxygen consumption. This is, perhaps, not unexpected, since energy for motility would presumably be available not only from oxidative processes but also from glycolysis—a process which actively takes place in ram, bull and rabbit spermatozoa even under the aerobic conditions of these experiments (Table 9). Further factors mitigating against any close correlation between oxygen uptake and motility are that the motility observations are of a subjective nature and not necessarily linearly related to, or having the precision of, the measurements of oxygen consumption made by the manometric method.
Towards the end of some experiments on the ram (ejaculates 1, 4 and 7 in Table 5) and the rabbit (ejaculate 1 in Table 7) the oxygen consumption rose rather than fell. This was thought to be due either to the proliferation of bacteria with high respiratory activity or else to the liberation of some metabolic regulator possibly related to that of Lardy, Ghosh & Plaut (1949). Bacterial contamination was tested by experiments on unwashed ram spermatozoa in which streptomycin was added at a final concentration of 1 mg./ml. of spermatozoa suspension. Tables 11 and 12 show the results of such an experiment done on an ejaculate in triplicate and Table 13 the analysis of variance. Significant variation in total oxygen uptake occurred between the control and streptomycin-treated groups, and it is clear from the data that the difference is due to the suppression by streptomycin of the rise in ZO2 seen in the control group during the latter hours of the experiment. This is taken as evidence that the phenomenon is due to bacteria. The streptomycin was innocuous to the spermatozoa, since neither the ZO2 over the first hour (Table 11) nor the total motility score (Table 12) were significantly affected by it.
Direct bacterial counts (Table 5) made on some flask contents at the end of a 5 hr. run indicated that bacteria were present in large numbers even when there was no very great increase in oxygen consumption. No doubt the type of organisms present is as important as the total bacterial count in determining whether or not the effect on oxygen uptake will occur, since different bacterial species are known to vary widely in their oxygen requirements. The sources of bacterial contamination in these experiments are multiple and could include the urogenital tract of the animal, the artificial vagina, the glassware and the diluents. Since it is virtually impossible to achieve asepsis in these experiments, it would appear unwise to prolong them beyond 3 hr., which is in keeping with the experience of Tosic & Walton (1950). The first 3 hr. probably represent a fairly true estimate of the respiration of the actual spermatozoa; thereafter the results are likely to be complicated by bacterial growth making analysis and interpretation more difficult. Tyler & Rothschild (1951) in prolonged experiments on sea-urchin spermatozoa encountered a similar increase in oxygen uptake which, by the use of penicillin, they showed was due to the proliferation of bacteria in the sea water.
Effect of washing
The results reported here show that the spermatozoa of the ram, bull and rabbit can be washed once without any significant decrease in motility, but that on washing twice motility may be adversely affected. These findings are in agreement with those of Mann (1945), who states that, as a rule, the washing of spermatozoa should not be repeated more than once, although he does not give any data nor explain the nature of the damage observed.
Tables 5 and 7 indicate that the significant variation in total oxygen uptake seen between washings for the ram and rabbit (Table 8) is mainly due to increases in the ZO2 occurring in the unwashed group in the latter stages of some experiments. The effect of washing is, in fact, somewhat similar to that of streptomycin, i.e. it tends to reduce the terminal rise in oxygen consumption without influencing the initial oxygen uptake. This is in keeping with the belief that the terminal increase in oxygen uptake is due to bacteria, since washing significantly reduces the bacterial count of the spermatozoa suspensions (Tables 5 and 14). The fact that washing had little, or no, effect on oxygen uptake during the early hours of the experiment suggests that the respiration of the spermatozoa themselves was not affected by this treatment.
Whilst the effect of washing on oxygen consumption in these experiments is most probably dependent upon washing out bacteria, the significant reduction in lactic acid production seen in the ram, bull and rabbit (Table 10) on washing is believed to be a direct effect of the spermatozoa. That this is so is indicated by the streptomycin experiment (Table 12) in which no significant difference in lactic acid production is seen between the control and the antibiotic-treated group (Table 13). This evidence has been added to in other experiments in this laboratory on ram and bull spermatozoa, in which it has been shown that washing significantly reduces lactic acid production during the first hour of incubation, when bacteria do not materially influence the results.
The decreased production of lactic acid on washing spermatozoa is in accordance with the classical observations on the ease with which the glycolytic components of tissues and cells, e.g. muscle and yeast, can be extracted in aqueous solution (Baldwin, 1949). The nature of the important components of the glycolytic cycle which might be removed in the washing of spermatozoa is a matter for speculation.
If they are the less complex ones, e.g. ions or coenzymes, then it should be possible to prevent the adverse effect of washing on glycolysis by including these substances in the diluent. If, on the other hand, the washing involves the loss of substances of high molecular weight, e.g. enzymes, the process might be more difficult to prevent. It should be noted in this regard that large molecules such as hyaluronidase (Swyer, 1947) and cytochrome c (Mann, 1951) are known to pass readily from the spermatozoan cell.
The relationship between the repetitive washing and the dilution of spermatozoa has been discussed by Emmens & Swyer (1948) who concluded that they are analogous phenomena. If this is so then one might expect from these results to find a loss of glycolytic power on severe dilution of spermatozoa and, as with the washing effect, to prevent it by the fortification of the diluting medium with those components lost from the cell. The effectiveness of such diverse agents as gum arabic, starch, glycogen and serum proteins in mitigating the effect of dilution (Emmens & Swyer, 1948) might then be to prevent the leakage of the glycolytic components from the spermatozoa.
Work being undertaken in this laboratory to elucidate the nature of the essential substances leached from spermatozoa on washing and dilution would indicate that potassium is at least one important factor.
The motility, oxygen uptake and aerobic glycolysis of unwashed, once- and twice-washed ram, bull and rabbit spermatozoa have been studied in a sodium phosphate-fructose diluent over a 5 hr. period at 37° C.
The mean ZO2 values obtained over the first hour for ram, bull and rabbit spermatozoa were 13·6, 13·3 and 7·1 respectively, and the corresponding mean total lactic acid production for each of these species over the 5 hr. period was 297, 439 and 413 µg./108 cells. Significant differences in oxygen uptake, lactic acid production and motility occurred between pooled ejaculates.
There was a decline in motility in almost all experiments, and a similar decline in oxygen consumption during the early hours. Towards the end of some experiments on unwashed ram and rabbit spermatozoa there was a rise in oxvgen uptake which was shown to be due to bacterial contamination.
Washing once had no significant effect on motility, but washing twice adversely affected the motility of ram spermatozoa.
Significant decreases in total oxygen uptake occurred on washing ram spermatozoa twice and on washing rabbit spermatozoa both once and twice. This is believed to be due to the removal of bacteria.
Washing once had no significant effect on total lactic acid production, but it was significantly reduced on washing ram, bull and rabbit spermatozoa twice. This effect is believed to be associated with the spermatozoa themselves.
The author wishes to acknowledge his indebtedness to Prof. C. W. Emmens for his interest and advice; to Mr H. Beatty of Glenfield Research Station and Mr T. Wallace of the Camden Park Estate for the collection of bull semen; to Mr A. W. Blackshaw for the collection of ram and rabbit semen; and to Mr R. M. Penn for his valuable technical assistance.
This work was assisted by Research Grants from the Commonwealth Bank of Australia and the Commonwealth Research Grant.