INFLUENCE OF LACTATE ON THE RATES OF FRUCTOLYSIS AND RESPIRATION IN RAM SPERMATOZOA

D. AMIR and H. SCHINDLER The National and University Institute of Agriculture, Rehovot, Israel (Received 3rd June 1966, revised 27th September 1966) Summary. Rates of fructolysis and oxygen uptake were measured in suspensions of ram spermatozoa containing various concentrations of lactate at different pH. It was found that lactate by itself causes a reversible depression of the metabolic rate, the magnitude of the effect being dependent on the lactate concentration.

INTRODUCTION Moore, Mayer & McKenzie (1940) observed that the motility of ram spermato¬ zoa declined during incubation of whole semen. Norman, Johnson, Porterfield & Dunbar (1958) found that, in addition to the decline of the motility, a re¬ versible inhibition of the metabolic rate of bull spermatozoa occurred after they had been kept in a coconut milk extender at room temperature. In both studies the inhibition was attributed to the increase of hydrogen ion concentra¬ tion which took place during incubation or storage. As pointed out by Mann (1964), hydrogen ion concentration is undoubtedly one of the most important factors which influence the motility and rate of metabolism of spermatozoa. However, it has been found that the presence in the medium of the lactate ion by itself may cause a reversible inhibition of the motility of bull and ram spermatozoa (Milovanov & Khabibulin, 1933; Willett & Ohms, 1958; Schindler & Amir, 1961). In view of the fact that lactate accumulates during incubation or storage of sperm suspensions in the presence of a glycolysable substance, the present study was undertaken to determine to what extent the lactate ion, in addition to its immobilizing effect, affects the metabolic rate of spermatozoa, and whether its effect is pH dependent and reversible. In order to elucidate these questions, the rates of fructolysis and oxygen uptake in suspensions of ram spermatozoa were examined as a function of pH and lactate concentration.

MATERIALS AND METHODS Semen Pooled ejaculates from rams were used. The sperm density was measured with an EEL colorimeter calibrated against haemocytometer counts. The * Contribution from The National and University Institute ofAgriculture, Rehovot, Israel, No. 726 E. 121

Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access 122 D. Amir and H. Schindler intensity of wave motion (wm) in an uncovered drop was estimated micro¬ scopically and rated from absent (0) to very turbulent (5). Only ejaculates with good wave motion and a density of at least 3-5 IO9 cells/ml were used. For the estimation of wave motion under anaerobic conditions, observation cells were used as described by Schindler & Nevo (1962). Diluents The diluent was prepared by adding 25 parts of egg-yolk to 75 parts of a buffer solution (pH 6-80) containing 2 g sodium citrate dihydrate, 2 g glycine, 0-5 g fructose and 0-25 ml of 10% citric acid/100 ml of distilled water. This solution was allowed to sediment by standing or centrifugation, and the clear supernatant fluid (pH 6-7) was used (Stower & Bud-Husaim, 1957). This diluent has the advantage that the motility of ram spermatozoa is preserved in it for a long period of time (Schindler & Amir, 1961), and that no further sedimentation of any constituent of the diluent occurs, so that the spermatozoa can easily be separated by centrifugation if it is desired to change the medium. L( + )Lactic acid (Nutritional Biochemicals Corporation, Cleveland, U.S.A.) was added to the buffer to give concentrations of lactate ranging from 0-5 to 4 mg/ml in the final diluent. Sodium hydroxide (1 n) or 10% citric acid solutions were added to the buffer, in order to give to the final diluent pH values ranging from 5-4 to 6-7. The semen was diluted to a concentration of 109 cells/ml. Oxygen uptake This was measured manometrically in duplicates by the direct method of Warburg (Umbreit, Burris & Stauffer, 1957) at 37° C; the shaking rate was 110 strokes/min. Fructolysis Fructolysis was determined by measuring the fructose disappearance and lactic acid accumulation in samples incubated at 37° C in vials filled to the top (anaerobic fructolysis) and in Warburg flasks (aerobic fructolysis). Fructose concentration before and after incubation was determined as described by Mann (1948), but using a cold solution of 10% trichloroacetic acid for deprotein- ization (Erb, Flerchinger, Ehlers & Gassner, 1956). Lactate concentration before and after incubation was determined by the enzymatic method (Barker & Britton, 1957) using Boehringer reagents (Biochemical Test Combination TC-B 15972, C. F. Boehringer and Soehne GmBH, Mannheim, Germany). Statistical analysis The results were subjected to an analysis of variance and the significance of the differences between the means of the various treatments was ascertained, using the q test (Fédérer, 1955).

RESULTS The influence of lactate concentration in media of different pH on sperm motility and on the rates offructolysis and oxygen uptake The influence of lactate concentration was measured in samples containing

Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access Effect of lactate on sperm metabolism 123 0 to 4 mg lactate/ml diluent, adjusted to pH 6-7 (Experiment A) or to pH 6 (Experiment B). As can be seen from Table 1, Exp. A, the rate of both anaerobic and aerobic fructolysis decreased with increasing concentrations of lactate. However, the rate of oxygen uptake was not affected by lactate concentrations of up to 2-0 mg/ml and it was depressed only slightly by a lactate concentration of 4 mg/ml diluent. In Exp. (Table 1) the influence of lactate concentration in the medium at pH 6-0 was compared with that at pH 6-7. As expected, the rate of fructolysis was altogether lower at pH 6-0 than at pH 6-7, and it was further depressed by the addition of lactate. The respiratory rate was not significantly affected by Table 1 influence of lactate on the rates of sperm fructolysis and oxygen uptake (six replicates)

Anaerobic incubation Aerobic incubation Lactate pH concentration of Fructose Lactic acid Oxygen uptake Fructose Lactic acid in the diluent the disappearance accumulation pH* (µ O2/10' disappearance accumulation pm (mg/ml) diluent (ßglIO9 cellsIhr) cellsjhr) (ßgl 9 cellsß0 min) Experiment A 0 6-7 1266"{ 1030" 5-9 150-1« 923" 664» 6-3 0-5 6-7 1097" 979" 5-9 144-3° 824" 536" 6-4 1-0 6-7 983e 710" 5-9 148-0° 736° 377" 6-4 2-0 6-7 891" 743" 60 144-5« 581" 356e 6-4 4-0 6-7 769" 580" 6-0 129-9" 455e 230« 6-5 Experiment 0 6-7 1178« 798« 5-9 153-1« 6-3 1-0 6-7 1056" 629« 5-9 146-1° 6-4 2-0 6-7 872e 5516e 5-9 142-6° 6-4 0 6-0 881e 5366e 5-7 145-7° 60 1-0 6-0 727° 426e" 5-7 134-0° 6-1 2-0 6-0 609e 354° 5-7 130-5" 6-1

* Measured at the end of 60 min incubation. t Measured at the end of 90 min incubation. J Any two mean values not marked with a common superscript—a, b, e, d or e—differ significantly (P<0-05). Comparisons to be made in vertical rows of each experiment. the low pH in the lactate-free diluent, but it was reduced significantly, although only slightly, by the addition of lactate at that low pH. It can also be seen from both experiments that the addition of different amounts of lactate did not influence the pH (as determined at the end of the incubation period). A depressing effect of lactate was also noticed in aerobically incubated samples with final pH of 6-4 to 6-5. Hence it can be concluded that the lactate ion by itself exerts a depressing effect on the metabolic rate. The wave motion of the aerobically incubated samples, as estimated by microscopic observation, did not appear to be influenced either by lactate concentration or by pH. In order to observe the influence of lactate concentration on sperm motility under anaerobic conditions, aliquots of semen with an initial wave motion intensity (wm) of 5 were diluted with lactate-free and lactate-containing

Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access 124 D. Amir and H. Schindler diluents (2-0 mg lactate/ml) at pH 6-7 and 6 in observation cells at 37° C. The wave motion was estimated after incubation for 1 hr. The WM-values were as follows: without lactate, pH 6-7, wm = 4; without lactate, pH 6 , wm = 3; with lactate, pH 6-7, wm = 3; with lactate, pH 6 , wm = 2. Reversibility of the depressing effect of lactate In order to establish the lactate concentration and pH which would cause immediate inhibition of motility the following preliminary experiment was carried out. Ram semen was diluted to a concentration of 109 cells/ml in the lactate-free diluent at pH 6-7, 6 , 5-6 and 5-4, respectively, and in the diluent Table 2 rates of anaerobic fructolysis and oxygen uptake by spermatozoa at 37°c measured first in the presence of lactate (1st hr of incubation) and then after removal of lactate (2nd hr of incubation) (three replicates)

2nd hr of incubation (after 1st hr of incubation substitution of the medium) Lactate in diluent (mg/ml) 0 0 2-0 0 0 0 Initial pH of the sperm suspension 6-5 5-5 5-5 6-4 6-3 6-3 Experiment A Fructose disappearance (ßgjlO9 cells/hr) 1352°í 673" 343e 1211° 1200° 1117° Lactic acid accumulation (/ig/109 cells/hr) 1039° 569° 175" 1039° 1071° 973« Final pH of the suspension* 5-8 5-3 5-3 5-8 5-7 5-¡ Experiment Oxygen uptake (ßl O2/109 cells/hr) 146-7° 130-0° 77-4e 121-5° 122-7° 130-8° Final pH of the suspension! 6-4 5-6 5-5 6-4 6-2 6-3

* Measured at the end of 60 min incubation. t Measured at the end of 90 min incubation. j Any two mean values not marked with a common superscript—a, b or c—differ significantly (P<0-05). Comparisons to be made in horizontal rows within each incubation period.

containing 2-0 mg lactate/ml at pH ranging from 6 to 5-4 with 0-1 unit inter¬ vals. The wave motion in an uncovered drop was thereafter immediately in¬ spected. In the lactate-free diluent, no inhibition ofmotility was apparent at any pH in the above-stated range, whereas in the lactate-containing diluent, motility was impaired at pH 5-6 and it was almost completely inhibited atpH 5-4. By substituting the medium with the lactate-free diluent at pH 6-7 full motility was restored even after 1 hr incubation at 37° C; the inhibition was thus shown to be completely reversible. In subsequent experiments, measurements were made of the rates ofanaerobic fructolysis and oxygen uptake during 1 hr in semen samples diluted with the lactate-containing diluent at pH 5-4, and in split samples diluted with the lactate-free diluent at pH 6-7 and 5-4. Thereafter, the media in all samples were substituted with the lactate-free diluent at pH 6-7, and the metabolic rates again measured during an additional hour of incubation. It was found (Table 2) that during the 1st hr period, i.e. in the presence of lactate, both the fructolysis

Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access Effect of lactate on sperm metabolism 125 and respiratory rates were considerably lower than in the control samples containing the lactate-free diluents. However, during the 2nd hr of incubation, that is after substitution of the media with the lactate-free diluent, all samples exhibited similar metabolic rates. Thus, the depressing effect of lactate on the rates of sperm metabolism is completely reversible. Influence of storage, and of supernatant fluid recoveredfrom stored sperm suspensions This was examined in two experiments, Exps. A and B. In Exp. A, diluted semen was stored for 12 days at 4° C, after which time the spermatozoa were almost immotile. The rate of fructolysis of these spermatozoa was low when examined at 37° C, but the respiration was less depressed. After substitution of the medium with fresh diluent, the motility and the rates of fructolysis and oxygen uptake were only partly restored (Table 3, Exp. A). Table 3 rates of anaerobic fructolysis and oxygen uptake at 37°c in fresh and stored semen which had been diluted either with fresh diluent or with supernatant fluid recovered from stored sperm suspensions (six replicates)

Anaerobic incubation Aerobic incubation Sperm Treatment of semen motility Fructose Lactic acid (WM*) disappearance accumulation Oxygen uptake (ßgllO9 cells¡hr) (ßl O2/109 cells/hr) Experiment A Fresh, diluted 1824°t 1036« 154-8« After 12 days storage at 4° C 484° 380* 119-3° After 12 days storage at 4° C and substitution of the medium with fresh diluent 1117e 663 e 129-5e Experiment Fresh, diluted 1785« 1175" 141-1« Fresh, with 1-3 to 1-4 mg lactate per ml diluent (pH 6-2) 1138" 722° 127-3° Fresh, diluted with supernatant fluid re¬ covered from stored diluted semen (pH 6-2) 1251° 743» 128-6°

* wm, Wave motion rating of the samples from zero to very turbulent (5). t Any two mean values not marked with a common superscript—a, b or c—differ significantly (,P<0-05). Comparisons to be made in vertical rows of each experiment. In Exp. B, fresh ram semen was divided into three samples; one was diluted with fresh diluent; the second with a medium which had been recovered from stored sperm suspensions and the third with fresh diluent containing the same concentration of lactate and the same pH as the medium from the stored sperm suspensions. It was found (Table 3, Exp. B) that semen diluted with the medium recovered from stored sperm suspensions or with the lactate-containing diluent exhibited lower metabolic rates than semen which had been diluted with the lactate-free diluent.

DISCUSSION The results of this study show that lactate ions as such can exert a depressing effect on the motility and metabolic rate of ram spermatozoa. This effect is not

Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access 126 D. Amir and H. Schindler necessarily due to the acidity caused by lactic acid. Only when the pH in the diluent had been lowered, the influence of the pH is added to that of the lactate ions. The fact that the decreased motility and metabolic rates of the stored spermatozoa were only partly restored by substitution of the medium with fresh diluent, indicates that the total decline is due in part to irreversible changes in the spermatozoa, i.e. senescence, and in part to changes in the medium (lactate accumulation and pH decrease). In the past, the depression of motility and metabolic rate of ram and bull spermatozoa, which occurs during incubation or storage, was attributed by Moore et al. (1940) and by Norman et al. (1958) to the increased hydrogen ion concentration. However, our experiments lead us to conclude that this effect is the result of combined action of lactate and pH. The effect of lactate on the anaerobic as well as on the aerobic fructolysis is much more pronounced than that on the oxygen uptake. Under anaerobic conditions, when fructolysis is the only source of energy for sperm motility, wave motion is also strongly affected by the presence of lactate. In contrast, under aerobic conditions at pH 6-0 or above, the respiratory rate, being sufficiently high in spite of the presence of lactate, can yield enough energy for strong wave motion; under such conditions no differences in the wave motion were observed between samples diluted with lactate-free diluents and those diluted with lactate-containing diluents. However, since the visual estimation method used to determine the intensity of the wave motion is one which did not allow us to distinguish between slightly different intensities, such differences may have occurred and remained unnoticed. This seems possible because, according to Van Duijn & Rikmenspoel (1960), decreased pH alone affects the velocity of the spermatozoa.

ACKNOWLEDGMENTS The valuable suggestions of Dr A. Nevo in the preparation of the manuscript and the assistance of Miss O. Kempenich are gratefully acknowledged.

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Downloaded from Bioscientifica.com at 09/26/2021 09:11:30PM via free access Effect of lactate on sperm metabolism 127 Stower, J. & Bud-Husaim, P. (1957) Conception rates with bull semen diluted in a glycerolized glycine-egg yolk-fructose buffer. J. agrie. Sci,, Camb. 49, 220. Umbreit, W. W., Burris, R. ., & Stauffer, J. F. (1957) Manometric techniques. Burgess Publishing Co., Minneapolis. Van Duijn, C. & Rikmenspoel, R. (1960) The mean velocity and velocity distribution of normal bull spermatozoa at different hydrogen-ion concentrations, derived from photo-electric measure¬ ments. J. agrie. Sci., Camb. 54, 300. Willet, E. L. & Ohms, J. I. (1958) Inactivation of spermatozoa by lactate and reactivation with alkali. J. Dairy Sci. 41, 275.

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