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Lipid Phosphorus and Nucleic Acid Phosphorus Content of Bull Semen during Aerated and Nonaerated Incubation1, 2
LIPID PHOSPHORUS AND NUCLEIC ACID PHOSPHORUS CONTENT OF B U L L S E M E N D U R I N G A E R A T E D A N D N O N A E R A T E D I N C U B A T I O N 1, 2 JOAN DIXON, M. H. EHLERS, AND 1¢. :E. EI~B Department of Dairy Science, Washington State University, Pullman SU~[~ARY Seventeen samples of semen from six bulls had an average lipid phosphorus (P~,p,d) content of 0.068 rag. per milliliter and an average nucleic acid phosphorus (P,u¢,~,o .°,d) content of 0.132 rag. per milliliter. Incubation of these samples as a thin layer in Erlenmeyer flasks (aerated) or in culture tubes (nonaerated) failed to decrease significantly PHp,d or P~,,c~Lo ~L~ or to increase total acid-soluble phosphorus (P~As). Inorganic phosphorus (P,) increased with both aerated and nonaerated incubation, as previously reported with nonaerated incubation. Although the source of the increase was not established and apparently did not orginate from P l ~ or P..o,~,¢ .o~, there is no conflict, at least in nonaerated incubation, with the hypothesis of ATP breakdown.
A concept of L a r d y and Phillips (10), that the lipid phosphorus (Pnpia) content of bull semen and of washed sperm substantially decreases during storage, has not been supported in work with washed sperm of the bull (3) or of the ram (9). W i t h washed ram sperm aerobically incubated at 37 ° C. up to 6 hr., Hartree and Mann (9) noted only a slight decrease in Pnpid or in plasmalogen phosphorus (Pp, . . . . ,ogen). Plasmalogen is the chief lipid in the semen of the ram, the bull, and several other species. Bomstein and Steberl (3) did note some decrease in P,ipia with once-washed, but not twice-washed, bull sperm. According to L a r d y and Phillips (10), total acid soluble phosphorus (PwAs) increased d u r i n g 56-hr. storage of diluted bull semen at 10 ° C., to partially account for their decrease in Pl~p~a. Ehlers et al. (5), however, noted no significant increase in P~As during 3 hr., 37 ° C. incubation of bull semen. F a t t y acids were freed from lipids of ram sperm, although without liberation of phosphate, d u r i n g both anaerobic and aerobic incubations by H a r t r e e and Mann (9). These f a t t y acids were suggested as substrates for endogenous respiration, and in this sense do not contradict the report (10) that lipids are oxidizable sperm reserves. Although Hartree and Mann (9) found the bulk of plasmalogen in sperm, some was always present in the plasma of ram semen. They observed that plasmalogen decreased in sperm and increased in seminal plasma as a result of cold shock. Blackshaw and Salisbury (2) similarly reported that cold shock of bull semen resulted in passage of Plipid from sperm to plasma. Received for publication July 24, 1961. 1Scientific Paper No. 2137 Washington Agricultural Experiment Stations, Pullman, Stations Project 1344. The research was financed in part under Regional [Research and Marketing Act Section 963 Public Law 733. Project W-¢9. 2298
LIPID
2299
AND N U C L E I C A C I D P H O S P t t O t C U S IN" S E M E N
Salisbury et al. (13) noted a marked decrease of desoxyribose nucleic acid (DNA) in sperm during mildly aerobic 5 ° C. storage of bull semen in yolkcitrate. These findings were based on the Feulgen reaction and microspectroscopy. Presumably, decrease in either ribose or desoxyribose would be detected also by measurement of nucleic acid phosphorus (Pnueleicacid). The principal purpose of the present study was to observe possible decreases in Pnpid and Pnucleic acid which might account for the increase in P~ upon incubation of bull semen. Except for one trial using whole semen (10), previous work o n Pnpid changes with incubation have been with washed sperm (3, 9, 10). The reactions which might bring about an increase in P1 from Pnoid o r Pnucleicacid would probably be oxidative in nature. Minimal and maximal aeration was suggested, however, because the PI increase previously reported (5, 8) had been with minimal aeration. MATERIALS AND METHODS
Semen diluted with four parts of 2.9% sodium citrate was sampled for phosphorus fractions, fructose, and lactic acid at 0 hr., divided into aliquots designated aerated and nonaerated, and again sampled after 3 hr. at 37 ° C. Aeration consisted of incubation of the semen-citrate as a thin layer within Erlenmeyer flasks and giving a liquid surface to volume ratio of 4.71 ( S in sq" em" V in ml.
-
4.71
)
.
]
Nonaerated incubation of semen-citrate, within culture tubes, had a corresponding 0.13 ratio. At each of the three samplings, 3 ml. of semen-citrate was deproteinized with 12 ml. of cold 10% trichloroacetic acid. After centrifugation and filtration, the following semen equivalent quantities (milliliters) of the filtrate were used for these determinations: fructose, 0.01; lactic acid, 0.02; PI, 0.02; P~.m 0.08; (P~+I~= inorganic + easily hydrolyzable phosphorus); and PTAs, 0.016. P,ucl~icacid was determined on 0.04 ml. of semen equivalent within the trichloroaeetic acid precipitate. Plipid and P~ot (total phosphorus) were calculated on 0.06 and 0.016 ml. of semen equivalent in separate samplings of the semen-citrate mixture. These procedures were not designed to completely partition phosphorus in semen. The fructose method was a modification (6) of Roe (12) and the lactic acid method was that of Barker and Summerson (1). Phosphorus was estimated by the method of Fiske and Subbarow (7) after suitable extraction procedures. PI, PI+H, PTAS, and PTot extractions were as previously described (5). Pnpid was extracted by an ethanol-ether method (16) and Pm,d~ie acid extracted according to Schneider (14). Plipid and P~ud~icacid fractions were then wet-washed using concentrated sulfuric and 70% perchloric acid similar to that previously described for P~As and PTot (5). Because of evaporation during the 3-hr. incubation, particularly from the Erlenmeyer flasks, the 3-hr. aerated and nonaerated values of constituents were adjusted to compensate for fluid loss. This loss was determined by the difference between P~ot before and after incubation and amounted to 8.3% in the aerated and 0.9% in the nonaerated incubations.
2300
JOAN DIXON, M. H. E H L E R S , AND R. E. EI~B
Sperm concentration was estimated with hemocytometer and motility with low-power microscopy. Statistical analysis was according to Snedecor (15). RESULTS A~TD DISCUSSIO:~ff Seventeen semen samples from six bulls provided the data of Table 1. The P~ipid value of 0.068 mg. per milliliter of semen, with 95% confidence limits of 0.059 to 0.077, compares with a previously reported 0.09 mg. mean on ejaculates of ten bulls (11) and 0.276 and 0.38 mg. (10) on single ejaculates. A Pn,id value of 0.072 mg. per 109 bull sperm was calculated from tabular data on 13 samples reported by Bomstein and Steberl (3). H a r t r e e and Mann (9) noted a F p l . . . . logen content of 0.058 rag. in one sample of bull semen. Apparently, no data are available to compare with the 0.132 mg. of Pnuc~ei¢,¢i~ in Table 1. Calculation of 95% confidence limits on PI, Pi+m PTAS, and PTot of Table 1 showed that these limits encompassed the means of a previous r e p o r t by this laboratory (5). Comparisons with other P~, PTAS, and PTot findings can be noted in this previous study (5). The failure of P~ip~ and P~uc~e~c,¢~d to decrease significantly and of PTAS to increase significantly (Table 1) indicates that P~pid and Pnu¢l~i¢~¢id do not account for the increase in P~ with either aerated or nonaerated incubation of semen. Although the present incubating conditions may not have been most conducive to P~ip~d alterations, because of low oxygen potential within the nonaerated samples and the delatively high available carbohydrate within the aerated samples, the findings arc not in harmony with the previously reported pronounced decrease in Pnp~d and the increase in P~AS upon storage of diluted whole semen (10). The data give no indication that Pli~id decreases in the presence of seminal plasma, as may be suggested by the decrease in Pnp~d of once-washed, but not twice-washed sperm, reported by Bomstein and Steberl (3). P~ and P~+r~ increased during both aerated and nonaerated semen incubation (Table 1), as has been noted for nonaerated incubation (5). No explanation is offered as to the a p p a r e n t differences between P~ and P~+H increases with aerated and nonaerated incubation (Table 1). The greater increase in P~ than in P~+I~, in the nonaerated incubation, is compatible with the suggestion (5) that increase in P~ is due partially to irreversible breakdown of A T P . Fructose utilization (0 minus 3 hr. fructose) and lactic acid accumulation (3 minus 0 hr. lactic acid), as might be expected, were considerably lower with aerated incubation, being about one-half that of nonaerated. This may be related to the differences in phosphorus metabolism, although certainly the reason is not clear. The lower rate of fructolysis in the aerated samples failed to give a higher mean percentage of sperm motility after 3 hr. of incubation (Table 1). This result supports the generally accepted concept favoring anaerobic storage of sperm. These similar mean motilities were noted, even though six samples of nonaerated semen had reached an extremely critical stage of fructose supply ( ~ 0.25 rag. per milliliter incubate) as compared with one aerated sample.
TABLE 1 Phosphorus, fructose, lactic acid, sperm concentration, and sperm motility in 17 samples of semen (six bulls) incubated 3 hr. at 37 ° C. in four parts sodium citrate buffer under aerated and nonaerated conditions Preineubation 0 hr.
Measurement Pnp,a (lipid phosphorus)
Mean Std. dev. Mean Std. dev.
P ~ I ~ ~,~ (nucleic aeidphosphorns) Acid-solublephosphorus P~ (inorganic phosphorus)
1)I+H (inorganic -k easily hydrolyzable phosphorus) PTAS (total acid-soluble phosphorus) PTot (total phosphorus) Fructose
Lactic acid
Sperm concentration Sperm motility
(rag. per ml. seme~) 0.068 0.070 0.018 0.020 0.132 0.136 0.0.36 0.036
0.069 0.021 0.125 0.034
Mean Std. dev.
0.037 0.017
0.041 0.013
0.046 0.018
Mean Std. dev.
0.056 0.022
0.062 0.024
0.060 0.023
Mean Std. dev. 1Y[ean Std. dev. Mean Std. dev.
0.49.5 0.136 0.943 0.260 6.04 1.28
0.493 0.505 0.137 0.153 ................ 3.80 1.94
2.43 1.79
i~ean Std. dev.
0.69 0.11
2.37 1.18
3.91 1.14
(× 10 ~per mL semen) Mean 1.12 Std. dev. 0.30 (per cent progressively motile) Mean 67 Std. dev. 7
Significance determined by ~~t " test.
A f t e r incubation at 37 ° C. 3 hr. 3 hr. aerated nonaerated
20 13
20 16
Remarks ~ No significant change in P~p,a with aerated or nonaerated incubation. 1~o significant change in P~1~l~ ~la with aerated or nonaerated incubation. Increase in PI ( P ~ 0.1) with aerated incubation. Increase in P~ ( P < 0.91) with nonaerated incubation. Greater increase in Pz ( P ~ 0.01) with nonaerated than aerated incubation. Increase in Pi+. (1) ~ 0.01) with aerated and nonaerated incubation. Greater increase in PI+~ ( P ~ 0.01) with aerated than nonaerated incubation. No significant change in P~As with aerated or nonaerated incubation. Decrease in fructose ( P < 0.005) with aerated and nonaerated incubation. Greater decrease in fructose ( P ~ 0.05) with nonaerated than aerated incubation. Increase in lactic acid ( P ~ 0.005) with aerated and nonaerated incubation. Greater increase in lactic acid ( P ~ 0.01) with nonaerated than aeTated incubation.
Decrease in motility ( P < 0.0,0'5) with aerated and nonaerated incubation. No significant difference in motility decrease with aerated and nonaerated incubation.
2302
JOAN DIXON, M. H. EHLERS, AND 1~. E. ERB
REFERENCES (1) BARKER, S. B., AND SUM~]~SON, W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 1961. (2) 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. (3) Box~s~r~, R. A., AN]) ST~BKa5, E. A. The Utilization of Phospholipide by Bovine Spermatozoa. Exptl. Cell. Research, 12: 254. 1957. (4) DAWSON, R. M. C., MANIT, T., A n WHITe, I. G. Glycerylphosphorylcholine and Phosphorylcholine in Semen and Their Relation to Choline. Biochem. J., 65: 627. 19'57. (5) EHL~S, M. H., DIXON, JOAn, AND ERB, R. E. Acid-Soluble Phosphorus Constituents of Bull Semen in Relation to Fructolysis and to Sperm Concentration. J. Dairy Sci., 4~: 1679. 1961. (6) EFt, R. E., ~LE~CHINGF~R, F. H., EHLERS, M. H., AND GASSN]~I~, F. X. Metabolism of Bull Semen. II. Fructolysis Relationships with Sperm Concentration and Fertility. J. Dairy Sci., 39: 326. 1956. (7) FISK~, C. H., AND SUBBA~OW, Y. The Colorimetric Determination of Phosphorus. J. Biol. Chem., 66: 375. 1925. (8) F ~ C E I N G ~ , F. H., AND ERB, R. E. Metabolism of Bull Semen. I. Inorganic and Total Phosphorus Relationships. 5. Dairy Sci., 3~8: 1028. 1955. (9) HARTR~, E. F., AND MANN, T. Plasmalogen in Ram Semen, and Its Role in Sperm Metabolism. Biochem. J., 71:423. 1959. (10) LARDY, H. A., AND PHILLIPS, P. H. The Interrelation of Oxidative and Glycolytie Processes as Sources of Energy for Bull Spermatozoa. Am. J . Physiol., 133:602. 1941. (11) MA~N, T. The Biochemistry of Semen. Methuen and Co., Ltd., London. 1954. (12) ROB, J. H. A Colorimetrie Method for the Determination of Fructose in Blood and Urine. J. Biol. Chem., 107:15. 1934. (13) SALISBURY, G. W., n~ LA TORr~, L., Bn~OE, W. J., AND L O ~ , 5. R. Effect of 50 ° C. Storage in Yolk-Citrate on ~'eulgen-Positive Material (DNA) of Sperm Heads. J. Dairy Sci., 43: 882. 1960. (14) SCHN~mF~, W. C. Phosphorus Compounds in Animal Tissues. I. Extraction and Estimation of Desoxypentose Nucleic Acid and of Pentose Nucleic Acid. 5. Biol. Chem., 161: 293. 194~5. (15) S~uEco~, G. W. Statistical Methods. 5th ed. Iowa State College Press, Ames. 1956. (16) YO~a~UR~, G. E., AND YOUNaBUr~, MA~I~ V. Phosphorus Metabolism. I. A System of Blood Phosphorus Analysis. J. Lab. Clin. Med., 16: 158. ]930.
A concept of L a r d y and Phillips (10), that the lipid phosphorus (Pnpia) content of bull semen and of washed sperm substantially decreases during storage, has not been supported in work with washed sperm of the bull (3) or of the ram (9). W i t h washed ram sperm aerobically incubated at 37 ° C. up to 6 hr., Hartree and Mann (9) noted only a slight decrease in Pnpid or in plasmalogen phosphorus (Pp, . . . . ,ogen). Plasmalogen is the chief lipid in the semen of the ram, the bull, and several other species. Bomstein and Steberl (3) did note some decrease in P,ipia with once-washed, but not twice-washed, bull sperm. According to L a r d y and Phillips (10), total acid soluble phosphorus (PwAs) increased d u r i n g 56-hr. storage of diluted bull semen at 10 ° C., to partially account for their decrease in Pl~p~a. Ehlers et al. (5), however, noted no significant increase in P~As during 3 hr., 37 ° C. incubation of bull semen. F a t t y acids were freed from lipids of ram sperm, although without liberation of phosphate, d u r i n g both anaerobic and aerobic incubations by H a r t r e e and Mann (9). These f a t t y acids were suggested as substrates for endogenous respiration, and in this sense do not contradict the report (10) that lipids are oxidizable sperm reserves. Although Hartree and Mann (9) found the bulk of plasmalogen in sperm, some was always present in the plasma of ram semen. They observed that plasmalogen decreased in sperm and increased in seminal plasma as a result of cold shock. Blackshaw and Salisbury (2) similarly reported that cold shock of bull semen resulted in passage of Plipid from sperm to plasma. Received for publication July 24, 1961. 1Scientific Paper No. 2137 Washington Agricultural Experiment Stations, Pullman, Stations Project 1344. The research was financed in part under Regional [Research and Marketing Act Section 963 Public Law 733. Project W-¢9. 2298
LIPID
2299
AND N U C L E I C A C I D P H O S P t t O t C U S IN" S E M E N
Salisbury et al. (13) noted a marked decrease of desoxyribose nucleic acid (DNA) in sperm during mildly aerobic 5 ° C. storage of bull semen in yolkcitrate. These findings were based on the Feulgen reaction and microspectroscopy. Presumably, decrease in either ribose or desoxyribose would be detected also by measurement of nucleic acid phosphorus (Pnueleicacid). The principal purpose of the present study was to observe possible decreases in Pnpid and Pnucleic acid which might account for the increase in P~ upon incubation of bull semen. Except for one trial using whole semen (10), previous work o n Pnpid changes with incubation have been with washed sperm (3, 9, 10). The reactions which might bring about an increase in P1 from Pnoid o r Pnucleicacid would probably be oxidative in nature. Minimal and maximal aeration was suggested, however, because the PI increase previously reported (5, 8) had been with minimal aeration. MATERIALS AND METHODS
Semen diluted with four parts of 2.9% sodium citrate was sampled for phosphorus fractions, fructose, and lactic acid at 0 hr., divided into aliquots designated aerated and nonaerated, and again sampled after 3 hr. at 37 ° C. Aeration consisted of incubation of the semen-citrate as a thin layer within Erlenmeyer flasks and giving a liquid surface to volume ratio of 4.71 ( S in sq" em" V in ml.
-
4.71
)
.
]
Nonaerated incubation of semen-citrate, within culture tubes, had a corresponding 0.13 ratio. At each of the three samplings, 3 ml. of semen-citrate was deproteinized with 12 ml. of cold 10% trichloroacetic acid. After centrifugation and filtration, the following semen equivalent quantities (milliliters) of the filtrate were used for these determinations: fructose, 0.01; lactic acid, 0.02; PI, 0.02; P~.m 0.08; (P~+I~= inorganic + easily hydrolyzable phosphorus); and PTAs, 0.016. P,ucl~icacid was determined on 0.04 ml. of semen equivalent within the trichloroaeetic acid precipitate. Plipid and P~ot (total phosphorus) were calculated on 0.06 and 0.016 ml. of semen equivalent in separate samplings of the semen-citrate mixture. These procedures were not designed to completely partition phosphorus in semen. The fructose method was a modification (6) of Roe (12) and the lactic acid method was that of Barker and Summerson (1). Phosphorus was estimated by the method of Fiske and Subbarow (7) after suitable extraction procedures. PI, PI+H, PTAS, and PTot extractions were as previously described (5). Pnpid was extracted by an ethanol-ether method (16) and Pm,d~ie acid extracted according to Schneider (14). Plipid and P~ud~icacid fractions were then wet-washed using concentrated sulfuric and 70% perchloric acid similar to that previously described for P~As and PTot (5). Because of evaporation during the 3-hr. incubation, particularly from the Erlenmeyer flasks, the 3-hr. aerated and nonaerated values of constituents were adjusted to compensate for fluid loss. This loss was determined by the difference between P~ot before and after incubation and amounted to 8.3% in the aerated and 0.9% in the nonaerated incubations.
2300
JOAN DIXON, M. H. E H L E R S , AND R. E. EI~B
Sperm concentration was estimated with hemocytometer and motility with low-power microscopy. Statistical analysis was according to Snedecor (15). RESULTS A~TD DISCUSSIO:~ff Seventeen semen samples from six bulls provided the data of Table 1. The P~ipid value of 0.068 mg. per milliliter of semen, with 95% confidence limits of 0.059 to 0.077, compares with a previously reported 0.09 mg. mean on ejaculates of ten bulls (11) and 0.276 and 0.38 mg. (10) on single ejaculates. A Pn,id value of 0.072 mg. per 109 bull sperm was calculated from tabular data on 13 samples reported by Bomstein and Steberl (3). H a r t r e e and Mann (9) noted a F p l . . . . logen content of 0.058 rag. in one sample of bull semen. Apparently, no data are available to compare with the 0.132 mg. of Pnuc~ei¢,¢i~ in Table 1. Calculation of 95% confidence limits on PI, Pi+m PTAS, and PTot of Table 1 showed that these limits encompassed the means of a previous r e p o r t by this laboratory (5). Comparisons with other P~, PTAS, and PTot findings can be noted in this previous study (5). The failure of P~ip~ and P~uc~e~c,¢~d to decrease significantly and of PTAS to increase significantly (Table 1) indicates that P~pid and Pnu¢l~i¢~¢id do not account for the increase in P~ with either aerated or nonaerated incubation of semen. Although the present incubating conditions may not have been most conducive to P~ip~d alterations, because of low oxygen potential within the nonaerated samples and the delatively high available carbohydrate within the aerated samples, the findings arc not in harmony with the previously reported pronounced decrease in Pnp~d and the increase in P~AS upon storage of diluted whole semen (10). The data give no indication that Pli~id decreases in the presence of seminal plasma, as may be suggested by the decrease in Pnp~d of once-washed, but not twice-washed sperm, reported by Bomstein and Steberl (3). P~ and P~+r~ increased during both aerated and nonaerated semen incubation (Table 1), as has been noted for nonaerated incubation (5). No explanation is offered as to the a p p a r e n t differences between P~ and P~+H increases with aerated and nonaerated incubation (Table 1). The greater increase in P~ than in P~+I~, in the nonaerated incubation, is compatible with the suggestion (5) that increase in P~ is due partially to irreversible breakdown of A T P . Fructose utilization (0 minus 3 hr. fructose) and lactic acid accumulation (3 minus 0 hr. lactic acid), as might be expected, were considerably lower with aerated incubation, being about one-half that of nonaerated. This may be related to the differences in phosphorus metabolism, although certainly the reason is not clear. The lower rate of fructolysis in the aerated samples failed to give a higher mean percentage of sperm motility after 3 hr. of incubation (Table 1). This result supports the generally accepted concept favoring anaerobic storage of sperm. These similar mean motilities were noted, even though six samples of nonaerated semen had reached an extremely critical stage of fructose supply ( ~ 0.25 rag. per milliliter incubate) as compared with one aerated sample.
TABLE 1 Phosphorus, fructose, lactic acid, sperm concentration, and sperm motility in 17 samples of semen (six bulls) incubated 3 hr. at 37 ° C. in four parts sodium citrate buffer under aerated and nonaerated conditions Preineubation 0 hr.
Measurement Pnp,a (lipid phosphorus)
Mean Std. dev. Mean Std. dev.
P ~ I ~ ~,~ (nucleic aeidphosphorns) Acid-solublephosphorus P~ (inorganic phosphorus)
1)I+H (inorganic -k easily hydrolyzable phosphorus) PTAS (total acid-soluble phosphorus) PTot (total phosphorus) Fructose
Lactic acid
Sperm concentration Sperm motility
(rag. per ml. seme~) 0.068 0.070 0.018 0.020 0.132 0.136 0.0.36 0.036
0.069 0.021 0.125 0.034
Mean Std. dev.
0.037 0.017
0.041 0.013
0.046 0.018
Mean Std. dev.
0.056 0.022
0.062 0.024
0.060 0.023
Mean Std. dev. 1Y[ean Std. dev. Mean Std. dev.
0.49.5 0.136 0.943 0.260 6.04 1.28
0.493 0.505 0.137 0.153 ................ 3.80 1.94
2.43 1.79
i~ean Std. dev.
0.69 0.11
2.37 1.18
3.91 1.14
(× 10 ~per mL semen) Mean 1.12 Std. dev. 0.30 (per cent progressively motile) Mean 67 Std. dev. 7
Significance determined by ~~t " test.
A f t e r incubation at 37 ° C. 3 hr. 3 hr. aerated nonaerated
20 13
20 16
Remarks ~ No significant change in P~p,a with aerated or nonaerated incubation. 1~o significant change in P~1~l~ ~la with aerated or nonaerated incubation. Increase in PI ( P ~ 0.1) with aerated incubation. Increase in P~ ( P < 0.91) with nonaerated incubation. Greater increase in Pz ( P ~ 0.01) with nonaerated than aerated incubation. Increase in Pi+. (1) ~ 0.01) with aerated and nonaerated incubation. Greater increase in PI+~ ( P ~ 0.01) with aerated than nonaerated incubation. No significant change in P~As with aerated or nonaerated incubation. Decrease in fructose ( P < 0.005) with aerated and nonaerated incubation. Greater decrease in fructose ( P ~ 0.05) with nonaerated than aerated incubation. Increase in lactic acid ( P ~ 0.005) with aerated and nonaerated incubation. Greater increase in lactic acid ( P ~ 0.01) with nonaerated than aeTated incubation.
Decrease in motility ( P < 0.0,0'5) with aerated and nonaerated incubation. No significant difference in motility decrease with aerated and nonaerated incubation.
2302
JOAN DIXON, M. H. EHLERS, AND 1~. E. ERB
REFERENCES (1) BARKER, S. B., AND SUM~]~SON, W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 1961. (2) 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. (3) Box~s~r~, R. A., AN]) ST~BKa5, E. A. The Utilization of Phospholipide by Bovine Spermatozoa. Exptl. Cell. Research, 12: 254. 1957. (4) DAWSON, R. M. C., MANIT, T., A n WHITe, I. G. Glycerylphosphorylcholine and Phosphorylcholine in Semen and Their Relation to Choline. Biochem. J., 65: 627. 19'57. (5) EHL~S, M. H., DIXON, JOAn, AND ERB, R. E. Acid-Soluble Phosphorus Constituents of Bull Semen in Relation to Fructolysis and to Sperm Concentration. J. Dairy Sci., 4~: 1679. 1961. (6) EFt, R. E., ~LE~CHINGF~R, F. H., EHLERS, M. H., AND GASSN]~I~, F. X. Metabolism of Bull Semen. II. Fructolysis Relationships with Sperm Concentration and Fertility. J. Dairy Sci., 39: 326. 1956. (7) FISK~, C. H., AND SUBBA~OW, Y. The Colorimetric Determination of Phosphorus. J. Biol. Chem., 66: 375. 1925. (8) F ~ C E I N G ~ , F. H., AND ERB, R. E. Metabolism of Bull Semen. I. Inorganic and Total Phosphorus Relationships. 5. Dairy Sci., 3~8: 1028. 1955. (9) HARTR~, E. F., AND MANN, T. Plasmalogen in Ram Semen, and Its Role in Sperm Metabolism. Biochem. J., 71:423. 1959. (10) LARDY, H. A., AND PHILLIPS, P. H. The Interrelation of Oxidative and Glycolytie Processes as Sources of Energy for Bull Spermatozoa. Am. J . Physiol., 133:602. 1941. (11) MA~N, T. The Biochemistry of Semen. Methuen and Co., Ltd., London. 1954. (12) ROB, J. H. A Colorimetrie Method for the Determination of Fructose in Blood and Urine. J. Biol. Chem., 107:15. 1934. (13) SALISBURY, G. W., n~ LA TORr~, L., Bn~OE, W. J., AND L O ~ , 5. R. Effect of 50 ° C. Storage in Yolk-Citrate on ~'eulgen-Positive Material (DNA) of Sperm Heads. J. Dairy Sci., 43: 882. 1960. (14) SCHN~mF~, W. C. Phosphorus Compounds in Animal Tissues. I. Extraction and Estimation of Desoxypentose Nucleic Acid and of Pentose Nucleic Acid. 5. Biol. Chem., 161: 293. 194~5. (15) S~uEco~, G. W. Statistical Methods. 5th ed. Iowa State College Press, Ames. 1956. (16) YO~a~UR~, G. E., AND YOUNaBUr~, MA~I~ V. Phosphorus Metabolism. I. A System of Blood Phosphorus Analysis. J. Lab. Clin. Med., 16: 158. ]930.