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Carbon Dioxide Fixation by Bovine Semen, Washed Spermatozoa, and Seminal Plasma1
Carbon Dioxide Fixation by Bovine Semen, Washed Spermatozoa, and Seminal Plasma T. J. SEXTON and R. J. FLIPSE
Department of Dairy Science, The Pennsylvania State University University Park 16802 to study possible mechanisms for COs incorporation by washed spermatozoa.
Abstract
Radioactive carbon dioxide, released by acidifying NaH14COa in a side arm of a W a r b u r g flask, was incorporated by whole semen, washed spermatozoa, and seminal plasma. When the incubation mixture was fractionated into supernatant, total lipids, nucleic acids, and protein, radioactivity was recovered in both the supernatant and total lipid fractions of whole semen but only in the supernatant of washed spermatozoa. Fixation of 14C0 2 by washed spermatozoa was significantly less in KrebsRinger-phosphate buffer, p H 7.4, than in Krebs-Ringer buffer, p H 7.4. When pyruvate was not added, there was little or no CO, fixation by washed spermatozoa; however, substantial x4C02 incorporation occurred in the presence of 15m~ pyruvate. Increasing pyruvate above 15m~ resulted in no significant additional increase in CO 2 fixation. Malic enzyme activity was found in acetone powder extracts of washed bovine spermatozoa, and N A D P + and MnC12 were required for maximal activity.
Experimental
Introduction
Incorporation of carbon dioxide into organic compounds by animal tissues has been established (6, 13, 15). Carbon dioxide plays an important role in the metabolism of spermatozoa; in the presence of COo there was a marked stimulation of respiration (4), aerobic glycolysis (12), and fructolysis (7) by bull spermatozoa. However, the mechanism of action of C02 on the metabolism of bovine spermatozoa is obscure. The objectives of this investigation were to measure carbon dioxide incorporation by bovine semen, washed spermatozoa, and seminal plasma, to study the effects of pyruvate and phosphate on CO2 fixation by washed spermatozoa, and Received for publication October 12, 1970. 1 Authorized for publication as Paper 3851 in the Journal Series of the Pennsylvania Agricultural Experiment Station. Supported in part by Public Health Service Grant HD 00039-06.
Procedure
Semen was collected from six bulls on a roufine schedule by an artificial vagina. Following semen collection, volume, percent motility, and sperm cell concentration were determined. A n aliquot of pooled first ejaculates from three bulls was centrifuged and the seminal plasma was removed. Spermatozoa were washed twice and resuspended in, depending on the experiment, Krebs-Ringer [ ( K R ) , 2m• phosphate] or Krebs-Ringer-phosphate [ ( K R - P ) , 20ram phosphate] buffer, pI-I 7.4 (14). W a r b u r g flasks had one single side arm and one double side arm (14). Buffer, sodium pyruvate (20/zmoles), except in the pyruvate concentration study, and either whole semen (109 cells per 0.5 ml), washed spermatozoa (109 cells per 0.5 ml), or seminal plasma (0.5 ml) were placed in the main chamber of the flask, final volume, 1.0 ml. The double side arm was prepared by adding 0.6 ml of 2 ~¢ I-IC1 to the smaller sac and 20 ~Ci (5.75 t~moles) of NaHx4CO3 to the larger sac and 0.1 ml of 6 ~ HC1 was added to the single side arm. A f t e r 5 rain of equilibration in a 37 C water bath, the 1~C02 was released into the gas phase of the flask by tipping 2 N HC1 into the radioactive bicarbonate. Incubation at 37 C was continued for 60 rain with constant agitation (84 cycles per rain); then the reaction was stopped by tipping 6 N HC1 into the incubation mixture. Potassium hydroxide (20% w/v) was added with a hypodermic needle through the self-sealing membrane o£ a rubber stopper into the center well and an additional 60 min at 37 C was allowed for absorption of COe. The incubation mixture was transferred to a centrifuge tube, centrifuged at 3,000 × g for 10 min, and the supernatant removed. Aliquots were then dried and assayed for radioactivity with a thin-window gas flow counter (counting efficiency, 25%). The residue was further fraetionated into the total lipids, nucleic acids, and protein according to the procedure described by Cohen (1). Boiled controls (95 C for 5 min) were included in all experi-
417
418
SEXTON
AND
TABLE 1. Total incorporation of 14CO2 by whole semen, washed spermatozoa, and seminal plasma. a Total cpm per incubation flask
Preparation tested
Control b
Active
Whole semen
638 ± 150
13,227~± 3,533
Washed spermatozoa
464 ± 119
4,188~± 1,080
Seminalplasnm
170 ±
1,566~±
47
457
Mean and standard deviation for six determinations. b Preparation tested was boiled at 95 C for 5 rain before incubation. Means differ significantly from each other (P < 0.05). meats. Analysis of variance evaluated treatment effects. Results
Significant amounts (P < 0.05) of 14C02 were incorporated by the three preparations tested (Table 1). Incorporation into whole semen was approximately three times that in washed spermatozoa and eight times that in seminal plasma. Significant amounts (P < 0.05) of radioactivity were recovered in the supernatant fraction of both whole semen and washed spermatozoa and in the total lipid fraction of whole semen (Table 2). Relative incorporation into supernatant, total lipids, nucleic acids, and protein was about the same in whole semen (84, 9, 6, and 1%) and in washed spermatozoa (85, 5, 8, and 2 % ) , but the distribution was somewhat different in seminal plasma (29, 37, 32, and 2%).
FLIPSE
As shown in Table 3, a significant incorporation of radioactive C02 occurred in washed bovine spermatozoa suspended in either K R or K R - P buffer at p t t 7.4. However, total fixation of 1 4 C 0 2 by washed spermatozoa was significantly less in K R - P than in KR. The effect of various concentrations (0, 15, 30, 60m~r) of pyruvate on 14C02 incorporation was nleasured in washed spermatozoa (KR, p H 7.4). W i t h no pyruvate added to the system, little 14C02 incorporation took place, 842 ± 191 cpm per incubation flask. Concentrations of 15, 30, and 60m~ pyruvate all significantly increased 14CO2 incorporation, 4,037 ± 510, 4,941 ± 471, and 4,033 ± 490 cpm per incubation flask. But above 15mM there was little evidence of additional response. Malie enzyme activity was measured by the procedure of Ochoa (10). Malic enzyme activity, 33.7m/~moles N A D P H formed per milligram of protein, was determined in acetone powder extracts (40 sag p e r ml) of washed bovine spermatozoa. I n the absence of N A D P + and MnCls, malie enzyme activity was reduced by 66 and 100%, respectively. Discussion
These investigations have demonstrated that CO2 fixation occurs in bovine spermatozoa. The results are consistent with the theory that bicarbonate, which is the sole sperm-stimulating component in oviduct fluid, may be responsible for the increase in respiration of capaeitated spermatozoa compared to ejaculated spermatozoa (3) by providing an anaplerotic means for the replenishment of oxaloacetate (via malate) that is required for the operation of the citric acid cycle. Therefore, the ability of spermatozoa to fix CO2 may be important in the capacitation process. In agreement with O'Shea and Wales (11) we found more carbon dioxide fixation occurring
TABLE 2. Total incorporation of l~CO 2 into the supernatant, total lipids, nucleic acids, and protein of whole semen, washed spermatozoa, and seminal plasma, a Total cpm p e r incubation flask Whole semen
Washed spermatozoa
Fraction
Control h
Active
Control b
Active
Supernatant Total lipids Nucleic acids Protein
224±100 330±151 0± 0 8 4 ± 29
10,985~±2,178 ],593~± 450 692 ± 259 198 ± 167
336±107 8 0 ± 45 0± 0 4 8 ± 22
4,331~±1,665 310 ± 355 ± 188 ±
a Mean and standard deviation of 6 determinations. b Preparation tested was boiled at 95 C for 5 rain before incubation. Significantly different from control (P < 0.05). JO~JI~NAL O~ DAIRY SCIEk'ffOEVOL. 54, NO. 3
Seminal plasma ControP' Active
244 101 50
] 7 0 ± 2 7 421±343 0 ± 0 337±242 0 ± 0 265± 69 0± 0 1 9 ± 57
SEMEN CARBON DIOXIDE
TABLE 3. Effect of added phosphate on total incorporation of 14C02 by washed bovine spermatozoa, a Total cpm per incubation flask Treatment Control b Active
K R buffer 714 ± 217
4~396~±
968
K R - P buffer 324 --+ 191
2,252~'~±160
Mean and standard deviation of four determinations. b Washed spermatozoa were boiled at 95 C for 5 rain before incubation. ~:~Significantly different from each other and from the respective control (P < 0.05). in semen than in washed spermatozoa and seminal plasma. They concluded that this was presumably due to endogenous substrates in whole semen acting as metabolic traps. Most of the radioactivity was incorporated into the supernatant fraction of both whole semen and washed spermatozoa. This finding suggests that the majority of CO 2 fixation reactions in spermatozoa are involved with connecting the glycolytie pathway to the oxidative system. Mounib and Eisan (9) reported that ahnost all of the 14C0, incorporation by washed cod spermatozoa occurred in the supernatant. Of the three preparations only whole semen incorporated a significant amount of 14C0~ into total lipids. These data indicate that seminal plasma may contribute substrates or cofactors for lipid synthesis or both. Several factors including pyruvate concentration (2) apparently influence the rate of carbon dioxide fixation in some animal tissues. In our study carbon dioxide fixation by washed spermatozoa was depressed in the absence of pyruvate. K r a f t and Lodge (5) reported that pyruvate was required for anaerobic carbon dioxide uptake by bovine spermatozoa. The rate of 14C02 fixation by washed spermatozoa was reduced with a 10-fold increase in phosphate concentration (2 to 20m~) of the diluent. Mann noted (8) that phosphate concentrations in the range of 15 to 80m~ depress metabolism. Malic enzyme activity was demonstrated in acetone powder extracts of bovine spermatozoa. Mounib and Eisan (9) reported similar results in cod sperm. One possible mechanism for fixation of C0u with pyruvate by bovine spermatozoa is by way of the malie enzyme pathway which could provide a mechanism for the replenishment of oxaloacetate for the operation
FIXATION
419
of citric acid cycle and for the synthesis of amino acids. However, the possibility cannot be excluded that other pathways such as pyrurate carboxylase may be involved in CO2 fixation by bovine spermatozoa. References
(1) Cohen, S. 1963. The metabolism of 14CO 2 during amphibian development. J. Biol. Chem., 211: 337. (2) Crane, R. K., and E. G. Ball. 1951. Factors affecting the fixation of 14CO 2 by aninm] tissues. J. Biol. Chem., 188: 819. (3) Hamner, C. E., and W. L. Williams. 1965. Composition of rabbit oviduct secretions. Fertil. and Steril., 16: 170. (4) Humphrey, G. F., and T. Mann. 1949. Studies on the metabolism of semen. V. Citric acid in semen. Bioehem. J., 44: 97. (5) Kraft, L. I:I., and J. R. Lodge. 1969. Anaerobic carbon dioxide uptake by bovine spermatozoa, sonifled cell suspensions, and supernatant from sonifled cells. J. Dairy Set., 52: 920. (6) Krebs, H. A., and L. V. Eggleston. 1940. Biological synthesis of oxa]oaeetic acid from pyruvic acid and carbon dioxide. Biochem. J., 34: 1383. (7) Lodge, J. R., and G. W. Salisbury. 1963. Factors influencing metabolie activity of bull spermatozoa VL Metabolic CO2 and fructose. J. Dairy Sci., 46: 140. (8) Mann, T. 1964. The Biochemistry of Semen and of the Male Reproductive Tract. John Wiley and Sons, Inc., New York. p. 346. (9) Mounib, M. S., and J. S. Eisan. 1968. Carbon dioxide fixation by spermatozoa of cod. Comp. Biochem. Physiol., 25: 703. (10) Ochoa, S. 1953. In Methods in Enzymology (ed. by Colowick, S., and N. Kaplan). Vol. 1. Academic Press, New York. p. 739-753. (11) O'Shea, T., and R. G. Wales. 1967. Fixation of carbon dioxide by ram spermatozoa. J. Reprod. Fert., 14: 333. (12) Salisbury, G. W., and W. C. Kinney. 1957. Factors influencing metabolic activity of bull spermatozoa. I I L pit. J. Dairy Set., 40 : 1343. (13) Solomon, A. I(., B. Vennesland, F. W. Klemperer, J. M. Buchanan, and A. B. Hastings. 1941. The participation of carbon dioxide in the carbohydrate cycle. J. Biol. Chem., 140 : 171. (14) Umbreit, W. W., R. I-I. Burris, and J. F. Stauffer. 1 9 5 7 . Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minnesota. p. 149. (15) Wood, If. G., C. H. Werkman, A. Hemingway, and A. O. Nier. 1942. Fixation of carbon dioxide by pigeon liver in the dissimilation of pyruvic acid. J. Biol. Chem., 142 : 31. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 3
Department of Dairy Science, The Pennsylvania State University University Park 16802 to study possible mechanisms for COs incorporation by washed spermatozoa.
Abstract
Radioactive carbon dioxide, released by acidifying NaH14COa in a side arm of a W a r b u r g flask, was incorporated by whole semen, washed spermatozoa, and seminal plasma. When the incubation mixture was fractionated into supernatant, total lipids, nucleic acids, and protein, radioactivity was recovered in both the supernatant and total lipid fractions of whole semen but only in the supernatant of washed spermatozoa. Fixation of 14C0 2 by washed spermatozoa was significantly less in KrebsRinger-phosphate buffer, p H 7.4, than in Krebs-Ringer buffer, p H 7.4. When pyruvate was not added, there was little or no CO, fixation by washed spermatozoa; however, substantial x4C02 incorporation occurred in the presence of 15m~ pyruvate. Increasing pyruvate above 15m~ resulted in no significant additional increase in CO 2 fixation. Malic enzyme activity was found in acetone powder extracts of washed bovine spermatozoa, and N A D P + and MnC12 were required for maximal activity.
Experimental
Introduction
Incorporation of carbon dioxide into organic compounds by animal tissues has been established (6, 13, 15). Carbon dioxide plays an important role in the metabolism of spermatozoa; in the presence of COo there was a marked stimulation of respiration (4), aerobic glycolysis (12), and fructolysis (7) by bull spermatozoa. However, the mechanism of action of C02 on the metabolism of bovine spermatozoa is obscure. The objectives of this investigation were to measure carbon dioxide incorporation by bovine semen, washed spermatozoa, and seminal plasma, to study the effects of pyruvate and phosphate on CO2 fixation by washed spermatozoa, and Received for publication October 12, 1970. 1 Authorized for publication as Paper 3851 in the Journal Series of the Pennsylvania Agricultural Experiment Station. Supported in part by Public Health Service Grant HD 00039-06.
Procedure
Semen was collected from six bulls on a roufine schedule by an artificial vagina. Following semen collection, volume, percent motility, and sperm cell concentration were determined. A n aliquot of pooled first ejaculates from three bulls was centrifuged and the seminal plasma was removed. Spermatozoa were washed twice and resuspended in, depending on the experiment, Krebs-Ringer [ ( K R ) , 2m• phosphate] or Krebs-Ringer-phosphate [ ( K R - P ) , 20ram phosphate] buffer, pI-I 7.4 (14). W a r b u r g flasks had one single side arm and one double side arm (14). Buffer, sodium pyruvate (20/zmoles), except in the pyruvate concentration study, and either whole semen (109 cells per 0.5 ml), washed spermatozoa (109 cells per 0.5 ml), or seminal plasma (0.5 ml) were placed in the main chamber of the flask, final volume, 1.0 ml. The double side arm was prepared by adding 0.6 ml of 2 ~¢ I-IC1 to the smaller sac and 20 ~Ci (5.75 t~moles) of NaHx4CO3 to the larger sac and 0.1 ml of 6 ~ HC1 was added to the single side arm. A f t e r 5 rain of equilibration in a 37 C water bath, the 1~C02 was released into the gas phase of the flask by tipping 2 N HC1 into the radioactive bicarbonate. Incubation at 37 C was continued for 60 rain with constant agitation (84 cycles per rain); then the reaction was stopped by tipping 6 N HC1 into the incubation mixture. Potassium hydroxide (20% w/v) was added with a hypodermic needle through the self-sealing membrane o£ a rubber stopper into the center well and an additional 60 min at 37 C was allowed for absorption of COe. The incubation mixture was transferred to a centrifuge tube, centrifuged at 3,000 × g for 10 min, and the supernatant removed. Aliquots were then dried and assayed for radioactivity with a thin-window gas flow counter (counting efficiency, 25%). The residue was further fraetionated into the total lipids, nucleic acids, and protein according to the procedure described by Cohen (1). Boiled controls (95 C for 5 min) were included in all experi-
417
418
SEXTON
AND
TABLE 1. Total incorporation of 14CO2 by whole semen, washed spermatozoa, and seminal plasma. a Total cpm per incubation flask
Preparation tested
Control b
Active
Whole semen
638 ± 150
13,227~± 3,533
Washed spermatozoa
464 ± 119
4,188~± 1,080
Seminalplasnm
170 ±
1,566~±
47
457
Mean and standard deviation for six determinations. b Preparation tested was boiled at 95 C for 5 rain before incubation. Means differ significantly from each other (P < 0.05). meats. Analysis of variance evaluated treatment effects. Results
Significant amounts (P < 0.05) of 14C02 were incorporated by the three preparations tested (Table 1). Incorporation into whole semen was approximately three times that in washed spermatozoa and eight times that in seminal plasma. Significant amounts (P < 0.05) of radioactivity were recovered in the supernatant fraction of both whole semen and washed spermatozoa and in the total lipid fraction of whole semen (Table 2). Relative incorporation into supernatant, total lipids, nucleic acids, and protein was about the same in whole semen (84, 9, 6, and 1%) and in washed spermatozoa (85, 5, 8, and 2 % ) , but the distribution was somewhat different in seminal plasma (29, 37, 32, and 2%).
FLIPSE
As shown in Table 3, a significant incorporation of radioactive C02 occurred in washed bovine spermatozoa suspended in either K R or K R - P buffer at p t t 7.4. However, total fixation of 1 4 C 0 2 by washed spermatozoa was significantly less in K R - P than in KR. The effect of various concentrations (0, 15, 30, 60m~r) of pyruvate on 14C02 incorporation was nleasured in washed spermatozoa (KR, p H 7.4). W i t h no pyruvate added to the system, little 14C02 incorporation took place, 842 ± 191 cpm per incubation flask. Concentrations of 15, 30, and 60m~ pyruvate all significantly increased 14CO2 incorporation, 4,037 ± 510, 4,941 ± 471, and 4,033 ± 490 cpm per incubation flask. But above 15mM there was little evidence of additional response. Malie enzyme activity was measured by the procedure of Ochoa (10). Malic enzyme activity, 33.7m/~moles N A D P H formed per milligram of protein, was determined in acetone powder extracts (40 sag p e r ml) of washed bovine spermatozoa. I n the absence of N A D P + and MnCls, malie enzyme activity was reduced by 66 and 100%, respectively. Discussion
These investigations have demonstrated that CO2 fixation occurs in bovine spermatozoa. The results are consistent with the theory that bicarbonate, which is the sole sperm-stimulating component in oviduct fluid, may be responsible for the increase in respiration of capaeitated spermatozoa compared to ejaculated spermatozoa (3) by providing an anaplerotic means for the replenishment of oxaloacetate (via malate) that is required for the operation of the citric acid cycle. Therefore, the ability of spermatozoa to fix CO2 may be important in the capacitation process. In agreement with O'Shea and Wales (11) we found more carbon dioxide fixation occurring
TABLE 2. Total incorporation of l~CO 2 into the supernatant, total lipids, nucleic acids, and protein of whole semen, washed spermatozoa, and seminal plasma, a Total cpm p e r incubation flask Whole semen
Washed spermatozoa
Fraction
Control h
Active
Control b
Active
Supernatant Total lipids Nucleic acids Protein
224±100 330±151 0± 0 8 4 ± 29
10,985~±2,178 ],593~± 450 692 ± 259 198 ± 167
336±107 8 0 ± 45 0± 0 4 8 ± 22
4,331~±1,665 310 ± 355 ± 188 ±
a Mean and standard deviation of 6 determinations. b Preparation tested was boiled at 95 C for 5 rain before incubation. Significantly different from control (P < 0.05). JO~JI~NAL O~ DAIRY SCIEk'ffOEVOL. 54, NO. 3
Seminal plasma ControP' Active
244 101 50
] 7 0 ± 2 7 421±343 0 ± 0 337±242 0 ± 0 265± 69 0± 0 1 9 ± 57
SEMEN CARBON DIOXIDE
TABLE 3. Effect of added phosphate on total incorporation of 14C02 by washed bovine spermatozoa, a Total cpm per incubation flask Treatment Control b Active
K R buffer 714 ± 217
4~396~±
968
K R - P buffer 324 --+ 191
2,252~'~±160
Mean and standard deviation of four determinations. b Washed spermatozoa were boiled at 95 C for 5 rain before incubation. ~:~Significantly different from each other and from the respective control (P < 0.05). in semen than in washed spermatozoa and seminal plasma. They concluded that this was presumably due to endogenous substrates in whole semen acting as metabolic traps. Most of the radioactivity was incorporated into the supernatant fraction of both whole semen and washed spermatozoa. This finding suggests that the majority of CO 2 fixation reactions in spermatozoa are involved with connecting the glycolytie pathway to the oxidative system. Mounib and Eisan (9) reported that ahnost all of the 14C0, incorporation by washed cod spermatozoa occurred in the supernatant. Of the three preparations only whole semen incorporated a significant amount of 14C0~ into total lipids. These data indicate that seminal plasma may contribute substrates or cofactors for lipid synthesis or both. Several factors including pyruvate concentration (2) apparently influence the rate of carbon dioxide fixation in some animal tissues. In our study carbon dioxide fixation by washed spermatozoa was depressed in the absence of pyruvate. K r a f t and Lodge (5) reported that pyruvate was required for anaerobic carbon dioxide uptake by bovine spermatozoa. The rate of 14C02 fixation by washed spermatozoa was reduced with a 10-fold increase in phosphate concentration (2 to 20m~) of the diluent. Mann noted (8) that phosphate concentrations in the range of 15 to 80m~ depress metabolism. Malic enzyme activity was demonstrated in acetone powder extracts of bovine spermatozoa. Mounib and Eisan (9) reported similar results in cod sperm. One possible mechanism for fixation of C0u with pyruvate by bovine spermatozoa is by way of the malie enzyme pathway which could provide a mechanism for the replenishment of oxaloacetate for the operation
FIXATION
419
of citric acid cycle and for the synthesis of amino acids. However, the possibility cannot be excluded that other pathways such as pyrurate carboxylase may be involved in CO2 fixation by bovine spermatozoa. References
(1) Cohen, S. 1963. The metabolism of 14CO 2 during amphibian development. J. Biol. Chem., 211: 337. (2) Crane, R. K., and E. G. Ball. 1951. Factors affecting the fixation of 14CO 2 by aninm] tissues. J. Biol. Chem., 188: 819. (3) Hamner, C. E., and W. L. Williams. 1965. Composition of rabbit oviduct secretions. Fertil. and Steril., 16: 170. (4) Humphrey, G. F., and T. Mann. 1949. Studies on the metabolism of semen. V. Citric acid in semen. Bioehem. J., 44: 97. (5) Kraft, L. I:I., and J. R. Lodge. 1969. Anaerobic carbon dioxide uptake by bovine spermatozoa, sonifled cell suspensions, and supernatant from sonifled cells. J. Dairy Set., 52: 920. (6) Krebs, H. A., and L. V. Eggleston. 1940. Biological synthesis of oxa]oaeetic acid from pyruvic acid and carbon dioxide. Biochem. J., 34: 1383. (7) Lodge, J. R., and G. W. Salisbury. 1963. Factors influencing metabolie activity of bull spermatozoa VL Metabolic CO2 and fructose. J. Dairy Sci., 46: 140. (8) Mann, T. 1964. The Biochemistry of Semen and of the Male Reproductive Tract. John Wiley and Sons, Inc., New York. p. 346. (9) Mounib, M. S., and J. S. Eisan. 1968. Carbon dioxide fixation by spermatozoa of cod. Comp. Biochem. Physiol., 25: 703. (10) Ochoa, S. 1953. In Methods in Enzymology (ed. by Colowick, S., and N. Kaplan). Vol. 1. Academic Press, New York. p. 739-753. (11) O'Shea, T., and R. G. Wales. 1967. Fixation of carbon dioxide by ram spermatozoa. J. Reprod. Fert., 14: 333. (12) Salisbury, G. W., and W. C. Kinney. 1957. Factors influencing metabolic activity of bull spermatozoa. I I L pit. J. Dairy Set., 40 : 1343. (13) Solomon, A. I(., B. Vennesland, F. W. Klemperer, J. M. Buchanan, and A. B. Hastings. 1941. The participation of carbon dioxide in the carbohydrate cycle. J. Biol. Chem., 140 : 171. (14) Umbreit, W. W., R. I-I. Burris, and J. F. Stauffer. 1 9 5 7 . Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minnesota. p. 149. (15) Wood, If. G., C. H. Werkman, A. Hemingway, and A. O. Nier. 1942. Fixation of carbon dioxide by pigeon liver in the dissimilation of pyruvic acid. J. Biol. Chem., 142 : 31. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 3