Factors Influencing Metabolic Activity of Bull Spermatozoa. III. pH

FACTORS I N F L U E N C I N G METABOLIC ACTIVITY OF BULL SPERMATOZOA. III. pH G. W. S A L I S B U R Y AND W. C. K I N N E Y ,

jm

Dept. of Dairy Science, University of Illinois, UrbaT~a Effects of pH on respiration, on aerobic glycolysis of spermatozoa, and on the calculated respiratory quotients were studied. The pH levels of the saline-phosphate diluents varied from 5 to 8. Aerobic fructolysis varied several fold, and directly with pH. The oxygen consumption of the cells was not markedly influenced by pH. However, a significant pH × ejaculate interaction was found for each metabolic measure, except for the R. Q. values, of which a large part of the variance was associated with individual ejaculates. Editor.

Experiments in our laboratory have emphasized that not only are aerobic conditions frequently met with in the handling and storage of semen for artificial insemination (12), but that a wide variety of conditions influence the extent and nature of the aerobic metabolism of bovine spermatozoa. The rate of aerobic utilization of fructose by spermatozoa collected from some bulls in the summer months is significantly less than is that by spermatozoa collected from the same bulls in the winter months, though the utilization of oxygen remains nearly the same (8). Oxygen utilization per unit number of washed bull spermatozoa can be made to vary over relatively wide ranges by varying the concentration of cells (2) and by use of suspension media containing varying concentrations of phosphate, citrate, chloride (3), or bicarbonate (11) anions. The physiological age of the sperm cells appears to alter the type of substrates utilized, as reflected by the respiratory quotients observed for both human (13) and bull spermatozoa (1) in their own seminal plasma. The dependence of most enzyme systems on pH optima is well known and the interrelation of pH and respiration rate of washed bull sperm cells has been studied (6). However, the extreme p H ranges investigated usually have resulted in the use of different anions in the buffers, which might confound the observed effect of p H on the respiratory rates. Therefore, this paper reports a reappraisal of the effect of pH on the respiration. In addition, it reports the effect of p H on the aerobic glycolysis, calculated respiratory quotients, and livability at 37 ° C., of bull spermatozoa in semen diluted 1 : 4 with a diluent composed of one-half Na, K, and Mg chlorides and one-half composed of a buffer made up solely of sodium salts of phosphoric acid. Under such circumstances, the experiments show that the optimum pH for respiration ~-aries greatly from ejaculate to ejaculate, but tends to be highest at the higher p H used. The optimum pH for motility is about that for oxygen uptake, the rate of fructose utilization and of lactic acid accumulation varies directly with pH, but the calculated R.Q. values do not. At the higher pH 's Received f o r p u b l i c a t i o n J u n e 3, 1957. 1343

1344

G. w. SALISBURY I N D W. C. KINNEY, JR.

more lactic acid is produced than can be accounted for solely on the basis of fructose disappearance. I n addition, the results prove that the rate of fructose utilization is greater in the presence of the CO- evolved f r o m respiration than it is when the evolved COo is absorbed b y K O H . M ETItODS

The methods of semen collection, chemical analyses, motility estimations, p H determinations, and respiration studies have been reported (5). Respiratory quotients were determined b y the direct method of W a r b u r g (14), in which three 5-ml. W a r b u r g flasks were used for each observation; one for determinations of the initial levels of substrates in the diluted semen and of bound CO2, one containing 20% K O H in the center well for the m e a s u r e m e n t of 0~, and one containing no K O H for the measurement of evolved C02 and COo retention by the buffer. The single side a r m of two flasks contained 0.1 or 0.2 ml. 3N sulfuric acid, which was tipped into the flask contents a f t e r t e m p e r a t u r e equilibration in the one flask f r o m which initial values were obtained, and at the end of the two- or four-hour incubation period in the other flask containing no K O H . In the third flask, the reactions were stopped a few minutes later, a f t e r motility estimations and p H deternfinations were made on the flask contents. Chemical analyses were made on the contents of each flask. The semen was diluted 1 : 4 with diluents having an initial p H v a r y i n g f r o m 5 to 8. Because of the buffer capacity of the semen itself, the initial p I I values of the mixed semen and diluent did not extend over this extreme range. However, the buffers maintained p H without m a r k e d changes throughout the incubation periods (see Table 4). The diluents were composed one-half of an isotonic solution of chloride salts and one-half of an isotonic mixture of monobasie sodium phosphate, 0.17 M, and dibasic sodium phosphate, 0.13 M, combined in such proportions as to give the a p p r o p r i a t e p H . The combinations required for diluents of the stipulated p H ' s are shown (Table 1). TABLE

1

Co,mpositio~ of buffered dil~w~ts pit 0.8 g. NaC1

5

0.04 g. KC1

3.48% Na:HPO~. 7tt_O

6

(%)

7

8

2.5

20.0

71.5

96.0

97.5

80.0

28.5

4.0

1:1 0.04 g. MgCt,_,-6H~O D i s t i l l e d w a t e r to 100 ml.

2.35% NaH~PO~'H.~O

The molarity of the phosphate ion in these final solutions varied from 0.084 at p H 5 to 0.066 at p H 8. This is somewhat higher than the level of 0.05 M phosphate, earlier f o u n d to be the m i n i m u m level inhibitory of respiration (10), but is much lower than the level found to ha v e a m a x i n m m depression on respiration (3).

ACTIVITY OF BULL SPERMATOZOA,

1345

III.

RESULTS

The first experiment was designed to determine the livability at 37 ° C. of spermatozoa suspended at seven different p H levels under two conditions. I n one, 1.0 ml. of diluted semen was placed in 10 × 75 ram. test tubes, resulting in only p a r t i a l l y aerobic conditions, for the surface area to volume ratio was only 0.5 (12) and the tubes were not shaken during incubation. I n the other condition, the 1.0 ml. of diluted semen was placed in 5.0-ml. W a r b u r g flasks and the contents shaken at the rate of 110 strokes per minute, resulting in complete aeration and a surface area to volume ratio somewhat greater t h a n the measured ratio of 1.71 at the gas-liquid interface. The initial p H of the nine ejaculates averaged 6.4. The mean cell count per t r e a t m e n t was about 200 × 106 cells per ml. The respiration measurements were terminated at the end of two hours, when final motility and p H estimations were made. The time required to complete these procedures made it impossible to terminate the test-tube experiments at the same time. A r b i t r a r i l y , it was decided to determine motility at one-half hour intervals and to terminate these studies when the motility reached zero for any p I I level. The cessation of motility invariably occurred first in the tubes diluted with the p H 5 diluent, and the time interval varied f r o m 7.5 to 9.5 hr. This arbit r a r y decision rendered statistical analysis of the combined results impossible. TABLE 2

Livabilit:!! o f s p e r m a t o z o a at 37 ° C, in 10 × 75 ram. test t~lbes, a~d i~t Warb~u'g flasks wader air ( m e a n of ~line ejaculates f o r each vab~e) p i t of added diluent

M o t i l i t y a f t e r "~ 7.5 to 9.5 hr.

(%) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Undiluted semen a t 37 ° C. A t room temp .

I n 5-ml. W a r b u r g flasks

In unshaken test tubes 1~inal pH

(rate)

'2 6 10 21 32 29

0.0 0.1 0.2 0.4 1.0 2.1 1.5

5.26 5.60 6.03 6,52 6.94 7.70 7,46

12 47

0.3 2.4

-5,40

(I

Motility a f t e r 2 hr.

(¢/c)

(rate)

14 16 2'2 29 36 34 32

0.7 0.7 0.8 1.6 1.8 2.1 2.0

Fbml pH

-Zo2 b

5.58 5.86 6.05 6.51 6.99 7.41 7.66

11.2 12.2 12.0 13.2 12.9 11.9 12.6

" M o t i l i t y e s t i m a t e s recorded when m o t i l i t y reache d zero for a n y one t r e a t m e n t ( p K 5 in every ease), b _Zoe=M. 0_o u p t a k e / l O s c e l l s / h r .

The mean data are shown (Table 2). The cells retained their motility better under the conditions of restricted respiration in the small storage tubes. U n d e r both procedures, the motility was maintained better at the higher p H ' s . I n the diluent buffered at p i t 7.5, the motility at the end of 7.5 to 9.5 hr. in the test tubes was nearly the same as in the completely aerated flasks a f t e r only two hours. In comparison with that p H as a s t a n d a r d for each procedure, the aerated cells in the ~Varburg flasks at the low p H ' s lived b e t t e r t h a n did those in the

1346

G, W. SALISBURY AND W. C. KINNEY, JR.

test tubes at the low pH's, presumably as a result of the maintenance of a somewhat higher pH. Though the mean p H for optimum oxygen consumption was at 6.5, the highest respiration rate for one ejaculate was at p H 8.0, and for another it was at p H 5.0, resulting in a highly significant ejaculate × p H interaction. Carbon dioxide production and R. Q. The second experiment, confined to four ejaculates, was conducted to determine the amount of CO2 evolved in relation to oxygen consumption. Because of the volumes of semen required for the CO.- measurements, the n u m b e r of p H levels was reduced to four, buffers of p H 5, 6, 7, and 8 only being used. The results are shown (Table 3). TABLE 3

Effect of pH on ~7~otility and respiration of dil.uted spermatozoa (v~ean of fo~lr ejaculates) Added buffer

Final pH

(pI~) 5.0 6.0 7.0 8.0

F i n a l motility

(%) 5.61 6.16 6.89 7.28

33 35 43 53

(rate) 0.9 1.0 1.8 2.5

Oxygen uptake

-Zo~ 7.2 7.1 10.8 13.4

Carbon dioxide production

]~.Q.

(+Zco~) 5.8 5.9 8.4 10.8

0.80 0.83 0.78 0.81

Here optimmn livability, maximum oxygen uptake, and CO2 produetion resulted from the diluent buffered at p H 8.0. The differences shown in 02 uptake and CO2 production at the four p H ' s are statistieally significant, but of the I~. Q. values none differs significantly from the over-all mean of 0.8. Finally, an experiment was conducted to include not only 02 uptake and C02 production, but to determine the initial p H of the semen-diluter mixtures and the fructose utilization and the lactic aeid accumulation in each flask. In this fashion, one might obtain inferential information on the validity of R. Q. estimates, for the basic assumption of the direct technique of W a r b n r g is that respiration proceeds in exactly the same way in each of the flasks used. In this experiment, nine ejaculates were used to determine the individual values at the end of two hours, and three others provided data during a four-hour incubation. In this latter series, enough semen was available to include undiluted whole semen in the study. The first nine ejaculates had an initial p H average of 6.55, with a concentration giving 225 milliou cells per ml. in the buffer-diluted semen; whereas, that of the three samples was an average initial p H of 5.94 and a concentration yielding a final eount of 340 million cells per ml. on dilution. The initial and final p H and motility of the spermatozoa after the incubation are given (Table 4). The data on 02 and C02 exchange, R. Q., and aerobic fruetolysis are presented as Z values, or the exchange per 100 × 106 cells per hour (in Table 5). While the concentration of spermatozoa found in the semen samples used in this experiment varied widely and influenced the absolute Z values for any one ejaculate, the comparative ~'alues for the treatments are valid, eaeh ejaculate having been used for all treatments.

ACTIVITY OF B U L L

SPERMATOZOA.

liI.

1347

TABLE 4 Effect of p H on initial and final p H of tl~e mixttlre, a~td the final ,motility of the spermatozoa after aerobic inc~¢batio~ at 27 ° C.

Added diluent (pH)

pH diluted semen (Initial)

Final motility

(Final)

(%)

(rate)

5.0 6.0 7.0 8.0

2-hour incubation (9 5.63 6.13 6.97 7.53

ejaculates) 5.71 6.15 6,96 7.55

38 31 36 48

0.9 1.1 2.1 2.7

5.0 6.0 7.0 8.0 Semen only

4-hour incubation (3 5.46 5.93 6.81 7.32 5.94

ejaculates) 5.51 5.95 6.81 7.28 6.27

17 37 37 43 37

0.2 0.4 1.3 1.8 1.7

The 02 c o n s u m e d a n d COo p r o d u c e d at p H 5 a n d p H 8 were s i g n i f i c a n t l y different ( P = < .05) t h a n those values for p H ' s 6 a n d 7. F o r the fructose consumed, the differences a m o n g p H levels were h i g h l y s i g n i f i c a n t ( P = < .01), a n d a h i g h l y s i g n i f i c a n t p H × e j a c u l a t e i n t e r a c t i o n ( P = < .01) was o b t a i n e d . The s t a t i s t i c a l significance of these sources of v a r i a t i o n i n the lactic acid a c c u m u l a t i o n was of the sanlc o r d e r of p r o b a b i l i t y . T h e p r e s e n c e or absence of K O H i n the c e n t e r wells of the flasks and, hence, the C0e t e n s i o n i n the gaseous phase, h a d a h i g h l y s i g n i f i c a n t ( P = < .01) effect on b o t h the f r u c t o s e u t i l i z a t i o n a n d lactic acid a c c u m u l a t i o n , b o t h b e i n g g r e a t e r i n the p r e s e n c e of C02. A p l o t of the d a t a of f r u c t o s e u t i l i z a t i o n a n d lactic acid a c c u m u l a t i o n for those flasks i n w h i c h 0,- c o n s u m p t i o n was m e a s u r e d , a n d w h i c h c o n t a i n e d K 0 H so t h a t r e s p i r a t i o n took place i n the a b s e n c e of CO~, shows t h a t the m e a n s fell o n e s s e n t i a l l y s t r a i g h t lines, the l i n e a r r e g r e s s i o n coefficient b e i n g h i g h l y sign i f i c a n t i n each case. A t p H 7 a n d 8, more lactic acid a c c u m u l a t e d t h a n f r u c t o s e was utilized. T h e r e was n o effect of p H on the ]%. Q. v a l u e s o b t a i n e d . However, t h e r e was g r e a t v a r i a t i o n i n m e a n 1~. Q. v a l u e s f r o m e j a c u l a t e to ejaculate, the p r o b a b i l i t y b e i n g < .001 t h a t the differences were due to chance. TABLE 5 Effect of p H on respiration,, R.Q. values, and aerobic fructolysis at 37 ° C. (mean of 19 observations)

pH added diluent

5 6 7 8 Semen~ only

--Zo ~

+Zoo~

(~l.)

(~l.)

7.47 6.65 6.79 8.30

6.50 5.59 5.31 6.96

6.65

6.90

I~.Q.

-Z

Fructose

Z+ Lactic Acid

-C02

+CO~

--CO:

+C02

(~g,)

(~g.)

(~g.)

(~g.)

0.87 0.84 0.78 0.84

37.7 62.3 74.3 113.7

57.7 82.7 179.3 230.7

12.0 63.3 144.7 203.7

34.3 121.0 223.0 224.7

1.04

55.0

60.8

--12.5

--10.8

Z = metabolic activity/10s cells/hr. b Three ejaculates only.

1348

,.

-w. S A L I S B U R Y AND W. e. K I N N E Y , J R .

DISCUSSION

The data published here make it clear t h a t variations in p H have a f a r greater and a more standard effect on the rate of fruetolysis t h a n they do on the respiration rate of spermatozoa in diluted semen. The mean respiration tended to be highest at the higher p H levels and at levels higher than t h a t reported b y L a r d y and Phillips (6) as o p t i m u m for washed bull spermatozoa. However, the variability exhibited b y the individual ejaculates in the p H at which m a x i m u m oxygen uptake occurred was striking. Such differences in respiration rate a p p e a r to be due to variations in the mineral components of the seminal plasma (9). The steadily increasing rate of aerobic fructolysis observed with increasing pH, f r o m a p p r o x i m a t e l y 5.5' through 7.5, reflects the increased motility observed at the end of the incubation period. Many investigators have observed this effect of p I I on motility, b u t the manifold increase in the rate of fructolys.is over the p H range used had not heretofore been reported. One might infer from these data that decreasing the p H of semen would depress fructolysis and thus the motility of spermatozoa, and p e r m i t of longer livability on storage. However, with the phosphate buffers here used, no evidence to substantiate this was found. The sperm cells did not live so well at the low p H , and showed no indication of an ability to revive from the treatment. The consistent observation that more lactic acid was produced t h a n fructose was utilized, during incubation at p H ' s above 6, p a r t i c u l a r l y in flasks where the COe evolved f r o m respiration was absorbed by K O H , has been observed before in our laboratory, tIowever, the experimental conditions under which this phenomenon was observed have varied widely, and no indisputable evidence of its occurrence u n d e r essentially normal metabolic conditions has been available until now (4). The data indicate that semen provides a s11bstrate for lactic acid formation by bovine spermatozoa, a p a r t f r o m that provided b y seminal fructose. I t is t e m p t i n g to speculate t h a t the unknown substrate will be found in the ~-glycerophosphocholine (7, 15) component of semen. However, White (15), while s t u d y i n g the oxidation b y bull spermatozoa of glycerol, and the f o r m a t i o n of fructose and subsequent formation of lactic acid in the process, was unable to demonstrate utilization of glycerophosphat.e b y bull spermatozoa. The R.Q. of diluted semen appears to be a function of the semen sample itself, for 59% of the total variaDee of R.Q. was associated with ejaculates. Because of the effect of COo evolved f r o m respiration, on the metabolic activity of bovine spermatozoa, the direct method of W a r b u r g (1~) is not adequate as a means of determining the R.Q. of semen, and more refined techniques must be used. SUMMARY

Bull semen was diluted 1 : 4 with a solution containing Na, K, and Mg chlorides, and the buffer composed entirely of Na phosphates combined to provide a p H range f r o m 5 to 8. p H per se did not influence the r e s p i r a t o r y quotients obtained, nor was there a marked effect of pI{ on oxygen uptake of the sperm cells,

ACTIVITY

OF

BULL

SPERMATOZOA.

though it tended to be highest at the higher pH ence markedly

aerobic fructolysis;

levels.

1349

However, pH

the higher the pH, the greater

the greater

the lactic acid produced

significant

pH × ejaculate

in excess of fructose

interaction

was

found

measures, except for the R.Q. values, a large part associated with the individual

III.

for

utilization. each

of

of the variance

did influ-

the rate and the

A highly metabolic

of which was

ejaculates. REFERENCES

(1) BAKBP~, F. N., VANDEMARK, ~ . L., SALISBUF~Y, G. W., AND VANTIENHOVEN, A. The Relation of O_~Uptake to Respiratory Quotients of Bull Semel~ in Citrate-Glucose Buffer. J. A n i ~ a l Sci., 11: 788. 1952. (2) BISHOP, M. W. ~I., AND SALISBUI~Y, G. W. Effect of Sperm Concentration on the Oxygen Uptake of Bull Semen. A.m.J. Physiol., lS0: 107. 1955. (3) BISHOP, M. W. H., AND SAbISBURY, G. W. Effect of Dilution with Saline and Phosphate Solutions on Oxygen Uptake of Bull Semen. Am. J. Physiol., 181: 114. 1955. (4) BLACKSHAW, A. W., AND SALISBURY, G. W. Factors Influencing Metabolic Activity of Bull Spermatozoa. II. Cold Shock and Its Prevention. J. Dairy Sci., 40: 1099. 1957. (5) BLACKSHAW, A. W., SALISBUI~Y, G. W., AND VANDEMARK, ,~T. L. Factors Influencing Metabolic Activity of Bull Spermatozoa. I. 37, 21, and 5 ° C. ,/. Dairy Sci., 40: 1093. 1957. (6) LAP~DY,H. A., AND PHILLIPS, P. H. Effect of p H and Certain Electrolytes on the Metabolism of Ejaculated Spermatozoa. An~. J. Physiol., 138: 741. 1943. (7) LUNDQUIST, F. Studies on the Biochemistry of H u m a n Semen. 1. The N a t u r a l Substrafe of Prostatic Phosphatase. Acta Physiol. Scand., 13: 322. 1947. (8) NAKABAYASI~II,N. T., AN]) SALISBURY, G. W. Variations Observed in the Aerobic Fructolysis of Semen. Proc. I I I r d Intern. Congr. Ani~ual Reprodttction, Sec. I : 28. 1956. (9) SALISBURY, G. W., A~D Cm~GLE, R. G. Freezing Point Depressions and Mineral Levels of Fluids of the Ruminant Male Reproductive Tract. Proc. I I I r d Intern. Congr. A.ni~nal Reproduction, Sec. I: 25, 1956. (10) SALISBURY, G. W., AND NAKABAYAStII, N. T. Effect of Phosphate and Chloride Ions on Aerobic Metabolism of Bull Spermatozoa. J. Exptl. Biol., 34: 52. 1957. (11) SALISBURY, G. W., AND NAKABAYASHI, N. T. Influence of Bicarbonate Ions on the Metabolic Activity of Spermatozoa. (Unpublished results.) (12) SALISBUaY, G. W., AND SHARMA, U. D. Effect of Surface Area-to-Volume Ratios in Storage Tubes on Oxygenagon of Diluted Bull Semen. J. Dairy Sci., 40: 677. 1957. (13) SHEWWLES,L. B. The Respiration of Human Spermatozoa and Their Response to Various Gases and Low Temperatures. Am. J. Physiol., 128: 408. 1940. (14) UMBRF~IT,W. W., BUP~RIS, R. H., AND STAUPFER, J. F. Manometric Techniques and Tissue Metabolis~n. 2nd ed. Burgess Pub]. Co., Minneapolis. 1949. (15) W~I~z, I. G. Metabolism of Glycerol and Similar Compounds by Bull Spermatozoa. A~n. J. Physiol., (in press).