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Organic Acids in Fresh Bovine Semen
OI~GANIC A C I D S IN F R E S H
BOVINE SEMEN
t[. A. gAMSEY, J. R. LODGE, C. N. GRAVES, AND G. W. SALISBURY Department of Dairy Science, University of Illinois, Urbana S U ~vl~/i A R Y
An organic acid analysis of fresh bovine semen has shown that acetic, lactic, glyeolic, and citric acids are major constituents of this material. Propionic, formic, suceinic, malonie, and malic acids were found in measurable quantity in a pooled semen sample, with only a trace of fl-hydroxybutyric acid. With individual ejaculates, acetic and lactic acids were in the greatest quantity, followed by formic and propionic, fl-hydroxybutyric acid was not detected and the other acids were not determined in the single ejaculates. The concentration of these acids, with the exception of fl-hydroxybutyrie, was greater in semen than in bovine blood. Presence of these acids in semen may influence results from aerobic metabolism studies, and the technique used for determining organic acids may be useful in studying oxidative pathways.
Though several studies have been conducted to determine the ability of spermatozoa to utilize various organic acids (3, 5, 10, 11), little is known about the concentration of such acids in fresh semen. I n bovine semen, Flipse and Potter (2) observed high levels of acetic and formic acids and snmll amounts of butyric and propionic acids. I n a more extensive investigation, Scott et al. (9) reported the presence of both acetic and formic acids in bovine and rabbit semen, though acetic was the only volatile fatty acid detected in the semen of other species: ram, man, stallion, dog, and fowl. Inasnmch as fructose is the sugar of semen (6), and glycolysis usually has been regarded as being the principal source of energy for motility, the organic acids of semen have received little attention as possible sources of energy for motility and other cellular functions. I n the present study, therefore, further attention was given to the analysis of bovine semen for various organic acids through the use of a chromatographic technique (7) developed recently in this laboratory. ~[ETttODS AND :~ATERIALS Semen was collected with an artificial vagina from normal, healthy bulls. Immediately after collection, one volume of the fresh semen was mixed with seven volumes of water and deproteinized by adding one volume of 10% sodium tungstate and one volume of 0.78 N H:S04. After removing the precipitate by centrifugation, the supernatant was filtered and adjusted to ca. p H 9 with I~0H. The filtrate then was reduced to near dryness by
Received for publication June 13, 1963.
evaporation under vacuum at 55 C. The acids were separated by column chronmtography, details of which have been published (7). RESULTS
Initial observations were made on a sample of pooled semen. The organic acids in the concentrated filtrate, equivalent to 15.5 ml of semen, were first extracted with diethyl ether as described for a protein-free filtrate of blood (7), then subjected to chromatographic separation. Results of this analysis are presented in Figure 1. The concentrations of each acid, expressed as ~eq per 100 ml of semen, were as follows: butyric and higher fatty acids, trace: prop;on;c, 10; acetic, 179; formic, 24; fl-hydroxybutyric, trace; lactic, 660; suceinic, 13; malonic, 23; glycol;c, 102; malic, 14; and citric plus isocitric, 5,710. For citric acid, 0.5 ml of each fraction was added to 5.0 ml of chloroform and titrated in the usual manner. Such dilution was necessary to avoid precipitation of citrate from the titration medium. I n addition to the foregoing compounds, traces of an unidentified acid were eluted from the column just prior to malic acid. Since glycolic appeared to be one of the major acids of semen, identity of this compound was investigated further. Accordingly, the fractions of this peak were combined, the solvent evaporated, and the methyl ester prepared. When subjected to gas chromatography, this ester exhibited the same retention time as pure methyl glycolate, thus leaving little doubt as to its identity. Analyses were performed subsequently on single ejaculates, since this would have additional practical significance. Ejaculates from four different bulls were collected and depro-
1132
ACIDS
IN
SEMEN
1133
K6 r O
VW
o
3c 1 . 2 -
x 0.8 - ~ C3
¢D
ej
0.4 -~ O--
¢J
0
('~
(3
--
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E
--
=E
>-
;"
~
Z
I--'~
I~ n
0 IJ-
I
0
--J ¢4.
20
40
60
--
o
:D
80
g
I00
120
140
160
180
-
1-
o
I
--I '°.°
°O 0 4
5.0
200
I
I
I
220
240
260
I 280
FRACTION NUMBER ( 5 . 2
FIG. 1. Chromatographic bovine semen (15.5 m l ) .
300
320
ML.)
separation of the organic acids in a proteln-free
teinized immediately. The protein-free filtrates were concentrated, acidified, mixed with a small quantity of silicic acid, and placed directly on the chromatographic column, thus by-passing the extraction step with ether. These analyses were stopped after lactic acid had been eluted. Results in Table 1 show measurable quantities of propionie, acetic, formic, and lactic acids. The level of formic acid was variable, though somewhat higher than that of the pooled sample in Figure 1. The level of lactic acid, however, was appreciably lower than that of the pooled sample. None of the acids found in trace quan-
filtrate of
tity in the pooled sample was found in the single ejaculates, presumably because of the smaller volume of semen used. DISCUSSION
These results confirm the presence of acetic, formic, lactic, and citric acids in fresh bovine semen and also show that a number of other acids are present. Glycolie acid appears to be a major constituent o£ bovine semen, and trace but measurable amounts of some of the citric acid cycle intermediates were found. A comparison of the concentration of acids found
T~LE1 Organic acid content of bovine semen Sample no. 11914 11915 119'16 11918 Mean
Amount of semen (ml) 4.8 5.9 9.2 5.0
Propionie --(~eq 25 30 16 28 25
Acetic
Formic
per 100 ml of s e m e n ) - - 336 119 309 265 252 108 212 244 277 184
Lacti~ 348 357 224 369 325
1134
H. A. I~AMSEY ET AL
in semen with those found in bovine blood (7), using the same technique, shows that with the exception of fi-hydroxybutyric acid semen contained higher concentrations than blood. The differences for acetic, lactic, and glycolic are marked and suggest that they aTe either produced in the nmle reproductive tract or are concentrated by the accessory glands. That fihydroxybutyric acid is found in lesser concentration in semen than in blood suggests some selective permeability. Of the acids found in semen, acetic and lactic markedly stimulate 0" consumption (3, 5, 10, 11), glycolate has been reported to be oxidized (1), but the others have not been shown to be utilized, at least to any significant extent, by bull spermatozoa (3-5, 10). This may not be true, however, for spermatozoa f r o m other species (10). The presence of these acids may p l a y a significant role in studies on the aerobic metabolism of spemuatozoa in semen or spermatozoa subjected to sen]inal plasma for a period of time prior to removal by washing. The utilization of these acids may help to explain the variable glycolytie rate reported among semen samples, since considerable variability in the concentration of' the acids was found in this study. Also, the absorption of these acids by spermatozoa and their subsequent utilization may influence results obtained f r o m washed ejaculated spermatozoa, as has been reported f o r fructose (8). The technique employed for determining organic acids in this study should prove useful for studying sonic of the oxidative pathways of spermatozoan metabolism.
ACKNOWLEDGiV[ENT
The authors thank C. L. Davis for his help in identifying glycolie acid. REFERENCES (1) FLIPSE~ R. J., AND BENSON, A. A. Catabolism of Glycine-C1~ by Washed Bovine Spermatozoa. Exptl. Cell Research, 13: 611. 1957. (2) FL~S~, R. J., AND POTT~, F. E. Presence of Volatile F a t t y Acids in Bovine Semen. Prec. Soc. Exptl. Biol. Med., 89: 432. ]955. (3) G~vEs, C. N. Ph.D. thesis, University of Illinois. 1962. (4) HUMPHP~Y, G. F., AN]) MANN, T. Studies on the Metabolism of Semen. 5. Citric Acid in Semen. Biochem. J., 44: 97. 1949. (5) LAI~D¥, H. A., HANSBN, 1~. G., AND PHILLIPS, P. H. The Metabolism of Epididymal Spermatozoa. Arch. Biochem., 6:41. 1945. (6) ~ N N , T. Fructose, A Constituent of Semen. Nature, 157: 79. 2946. (7) I~AI~S~¥, It. A. Separation of Organic Acids in Blood by Partition Chromatography. J. Dairy Sei., 46: 480. 1963. (8) SALISBURY, G. W., AN]) GRAVES, C. N. Substrate-free Epididymal-like Bovine Spermatozoa. J. Reprod. Fertil. (In press.) (9) SCOTT, T. W., WHITE, I. G., A N D A~NIS0N, E. F. Fatty Acids in Semen. Biochem. J., 78: 740. 1961. (10) SCOTT, T. W., WHITE~ 2. G., AND ANNISON, E. F. Oxidation of Short-Chain Fatty Acids (C~-Cs) by Ram, Bull, Dog and Fowl Spermatozoa. Bioehem. J., 83': 392. 1962. (11) TERNS, C. Oxidation of Exogenous Substrafes by Isolated Human Spermatozoa. Am. J. Physiol., 198: 48. 1960.
BOVINE SEMEN
t[. A. gAMSEY, J. R. LODGE, C. N. GRAVES, AND G. W. SALISBURY Department of Dairy Science, University of Illinois, Urbana S U ~vl~/i A R Y
An organic acid analysis of fresh bovine semen has shown that acetic, lactic, glyeolic, and citric acids are major constituents of this material. Propionic, formic, suceinic, malonie, and malic acids were found in measurable quantity in a pooled semen sample, with only a trace of fl-hydroxybutyric acid. With individual ejaculates, acetic and lactic acids were in the greatest quantity, followed by formic and propionic, fl-hydroxybutyric acid was not detected and the other acids were not determined in the single ejaculates. The concentration of these acids, with the exception of fl-hydroxybutyrie, was greater in semen than in bovine blood. Presence of these acids in semen may influence results from aerobic metabolism studies, and the technique used for determining organic acids may be useful in studying oxidative pathways.
Though several studies have been conducted to determine the ability of spermatozoa to utilize various organic acids (3, 5, 10, 11), little is known about the concentration of such acids in fresh semen. I n bovine semen, Flipse and Potter (2) observed high levels of acetic and formic acids and snmll amounts of butyric and propionic acids. I n a more extensive investigation, Scott et al. (9) reported the presence of both acetic and formic acids in bovine and rabbit semen, though acetic was the only volatile fatty acid detected in the semen of other species: ram, man, stallion, dog, and fowl. Inasnmch as fructose is the sugar of semen (6), and glycolysis usually has been regarded as being the principal source of energy for motility, the organic acids of semen have received little attention as possible sources of energy for motility and other cellular functions. I n the present study, therefore, further attention was given to the analysis of bovine semen for various organic acids through the use of a chromatographic technique (7) developed recently in this laboratory. ~[ETttODS AND :~ATERIALS Semen was collected with an artificial vagina from normal, healthy bulls. Immediately after collection, one volume of the fresh semen was mixed with seven volumes of water and deproteinized by adding one volume of 10% sodium tungstate and one volume of 0.78 N H:S04. After removing the precipitate by centrifugation, the supernatant was filtered and adjusted to ca. p H 9 with I~0H. The filtrate then was reduced to near dryness by
Received for publication June 13, 1963.
evaporation under vacuum at 55 C. The acids were separated by column chronmtography, details of which have been published (7). RESULTS
Initial observations were made on a sample of pooled semen. The organic acids in the concentrated filtrate, equivalent to 15.5 ml of semen, were first extracted with diethyl ether as described for a protein-free filtrate of blood (7), then subjected to chromatographic separation. Results of this analysis are presented in Figure 1. The concentrations of each acid, expressed as ~eq per 100 ml of semen, were as follows: butyric and higher fatty acids, trace: prop;on;c, 10; acetic, 179; formic, 24; fl-hydroxybutyric, trace; lactic, 660; suceinic, 13; malonic, 23; glycol;c, 102; malic, 14; and citric plus isocitric, 5,710. For citric acid, 0.5 ml of each fraction was added to 5.0 ml of chloroform and titrated in the usual manner. Such dilution was necessary to avoid precipitation of citrate from the titration medium. I n addition to the foregoing compounds, traces of an unidentified acid were eluted from the column just prior to malic acid. Since glycolic appeared to be one of the major acids of semen, identity of this compound was investigated further. Accordingly, the fractions of this peak were combined, the solvent evaporated, and the methyl ester prepared. When subjected to gas chromatography, this ester exhibited the same retention time as pure methyl glycolate, thus leaving little doubt as to its identity. Analyses were performed subsequently on single ejaculates, since this would have additional practical significance. Ejaculates from four different bulls were collected and depro-
1132
ACIDS
IN
SEMEN
1133
K6 r O
VW
o
3c 1 . 2 -
x 0.8 - ~ C3
¢D
ej
0.4 -~ O--
¢J
0
('~
(3
--
E:
E
--
=E
>-
;"
~
Z
I--'~
I~ n
0 IJ-
I
0
--J ¢4.
20
40
60
--
o
:D
80
g
I00
120
140
160
180
-
1-
o
I
--I '°.°
°O 0 4
5.0
200
I
I
I
220
240
260
I 280
FRACTION NUMBER ( 5 . 2
FIG. 1. Chromatographic bovine semen (15.5 m l ) .
300
320
ML.)
separation of the organic acids in a proteln-free
teinized immediately. The protein-free filtrates were concentrated, acidified, mixed with a small quantity of silicic acid, and placed directly on the chromatographic column, thus by-passing the extraction step with ether. These analyses were stopped after lactic acid had been eluted. Results in Table 1 show measurable quantities of propionie, acetic, formic, and lactic acids. The level of formic acid was variable, though somewhat higher than that of the pooled sample in Figure 1. The level of lactic acid, however, was appreciably lower than that of the pooled sample. None of the acids found in trace quan-
filtrate of
tity in the pooled sample was found in the single ejaculates, presumably because of the smaller volume of semen used. DISCUSSION
These results confirm the presence of acetic, formic, lactic, and citric acids in fresh bovine semen and also show that a number of other acids are present. Glycolie acid appears to be a major constituent o£ bovine semen, and trace but measurable amounts of some of the citric acid cycle intermediates were found. A comparison of the concentration of acids found
T~LE1 Organic acid content of bovine semen Sample no. 11914 11915 119'16 11918 Mean
Amount of semen (ml) 4.8 5.9 9.2 5.0
Propionie --(~eq 25 30 16 28 25
Acetic
Formic
per 100 ml of s e m e n ) - - 336 119 309 265 252 108 212 244 277 184
Lacti~ 348 357 224 369 325
1134
H. A. I~AMSEY ET AL
in semen with those found in bovine blood (7), using the same technique, shows that with the exception of fi-hydroxybutyric acid semen contained higher concentrations than blood. The differences for acetic, lactic, and glycolic are marked and suggest that they aTe either produced in the nmle reproductive tract or are concentrated by the accessory glands. That fihydroxybutyric acid is found in lesser concentration in semen than in blood suggests some selective permeability. Of the acids found in semen, acetic and lactic markedly stimulate 0" consumption (3, 5, 10, 11), glycolate has been reported to be oxidized (1), but the others have not been shown to be utilized, at least to any significant extent, by bull spermatozoa (3-5, 10). This may not be true, however, for spermatozoa f r o m other species (10). The presence of these acids may p l a y a significant role in studies on the aerobic metabolism of spemuatozoa in semen or spermatozoa subjected to sen]inal plasma for a period of time prior to removal by washing. The utilization of these acids may help to explain the variable glycolytie rate reported among semen samples, since considerable variability in the concentration of' the acids was found in this study. Also, the absorption of these acids by spermatozoa and their subsequent utilization may influence results obtained f r o m washed ejaculated spermatozoa, as has been reported f o r fructose (8). The technique employed for determining organic acids in this study should prove useful for studying sonic of the oxidative pathways of spermatozoan metabolism.
ACKNOWLEDGiV[ENT
The authors thank C. L. Davis for his help in identifying glycolie acid. REFERENCES (1) FLIPSE~ R. J., AND BENSON, A. A. Catabolism of Glycine-C1~ by Washed Bovine Spermatozoa. Exptl. Cell Research, 13: 611. 1957. (2) FL~S~, R. J., AND POTT~, F. E. Presence of Volatile F a t t y Acids in Bovine Semen. Prec. Soc. Exptl. Biol. Med., 89: 432. ]955. (3) G~vEs, C. N. Ph.D. thesis, University of Illinois. 1962. (4) HUMPHP~Y, G. F., AN]) MANN, T. Studies on the Metabolism of Semen. 5. Citric Acid in Semen. Biochem. J., 44: 97. 1949. (5) LAI~D¥, H. A., HANSBN, 1~. G., AND PHILLIPS, P. H. The Metabolism of Epididymal Spermatozoa. Arch. Biochem., 6:41. 1945. (6) ~ N N , T. Fructose, A Constituent of Semen. Nature, 157: 79. 2946. (7) I~AI~S~¥, It. A. Separation of Organic Acids in Blood by Partition Chromatography. J. Dairy Sei., 46: 480. 1963. (8) SALISBURY, G. W., AN]) GRAVES, C. N. Substrate-free Epididymal-like Bovine Spermatozoa. J. Reprod. Fertil. (In press.) (9) SCOTT, T. W., WHITE, I. G., A N D A~NIS0N, E. F. Fatty Acids in Semen. Biochem. J., 78: 740. 1961. (10) SCOTT, T. W., WHITE~ 2. G., AND ANNISON, E. F. Oxidation of Short-Chain Fatty Acids (C~-Cs) by Ram, Bull, Dog and Fowl Spermatozoa. Bioehem. J., 83': 392. 1962. (11) TERNS, C. Oxidation of Exogenous Substrafes by Isolated Human Spermatozoa. Am. J. Physiol., 198: 48. 1960.