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The Effect of Different Hydrogen Ion Concentrations During Storage and at Insemination and of Added Magnesium and Potassium on the Fertilizing Ability of Chicken Semen1
The Effect of Different Hydrogen Ion Concentrations During Storage and at Insemination and of Added Magnesium and Potassium on the Fertilizing Ability of Chicken Semen1 F. H.
WILCOX
Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication March 23, 1959)
ECENT results with mammalian • sperm have indicated that the storage time can be greatly extended by inactivating the sperm, usually by employing a diluent with a low pH, followed by reactivation by adding an alkaline solution. These results have been summarized by Willett and Ohms (1958). A new handling method for chicken semen has recently been described (Wilcox and Shaffner, 1958), and entails the dilution of semen during storage, followed by centrifugation, removal of the supernatant, and the resuspension of the sperm. This method appeared to be one that could be easily employed in an investigation in which different conditions were to be used during storage and at insemination. It has previously been established (Wilcox and Shaffner, 1957) that a wide range of pH levels can be used in storing chicken semen (from 6.5 to 8) without affecting fertility. Accordingly, experiments have been conducted in which three different pH levels (6.5, 7.2, and 7.8) have been used both during storage and at insemination in all possible combinations. This was done to determine if one pH level is optimum during storage and another one is optimum at the time of insemination. One would anticipate that conditions which maintain but slow down 1
Scientific Article No. A7S3, Contribution No. 3013 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
the sperm would be optimum during storage, but that conditions which stimulate sperm activity would be optimum at the time of insemination. It has been shown with bull semen (Salisbury and Kinney, 1957) that the pH has a marked effect on the fructolysis and lactic acid production, with increased activity at higher pH values. This is in agreement with the work already cited on activation and inactivation in which the activity is greater at alkaline pH levels. In one of the fertility trials conducted, K+ and Mg++ were added before insemination. The method employed for storing chicken semen removes nearly all of the seminal plasma and undoubtedly reduces the level of most compounds in chicken semen other than the sodium and phosphate. It appeared possible that the removal of the K+ and Mg + + might be detrimental since these are known to be involved in the metabolism of fructose. It has since been reported by Wales and White (1958) that the addition of these cations to chicken sperm washed two or four times prevents to a large extent the detrimental effect of washing on motility. EXPERIMENTAL PROCEDURE
The stock used and method of semen collection and insemination were the same as described previously (Wilcox and Shaffner, 1958). Semen was diluted 1 to 10 with a sodium phosphate buffer containing
1159
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
R
1160
F. H. WILCOX TABLE 1.-—Composition of
buffers used
5 volumes when added to the centrifuged sperm.
g./liter pH of buffer 6.49 7.22 7.78
NaH 2 P0 4 -H 2 0
RESULTS AND DISCUSSION
10.21 16.34 19.40
12.90 5.16 1.29
The use of all possible combinations of the three pH solutions revealed no difference in the optimum pH for storage and that at insemination (Table 2). This is seen most clearly and meaningfully by summarizing all three treatment groups for each pH. Even so, two pH combinations appeared to provide better fertility than the control of pH 7.2 during storage and at insemination. These were pH 7.2 during storage and pH 7.8 at insemination and also pH 7.8 at storage and pH 7.2 at insemination. These were further tested in an additional trial (Table 3), which clearly showed that the latter two combinations are not superior. These results furthermore provide additional support for the conclusion made previously (Wilcox and Shaffner, 1957) that there is little if any effect of pH on fertility between values of 6.5 and 8.0.
oxytetracycline and dihydrostreptomycin and stored aerobically at 10°C; just before insemination this diluted semen was centrifuged at 1,200 R.C.F. for 10 minutes, the supernatant discarded, and the sperm suspended in buffer containing sufficient fructose to provide a final concentration of 2 mg./ml. The composition of the sodium phosphate buffers is given in Table 1. Fructose, Mg++, and K+ were added to the phosphate buffer by adding the proper amounts of isotonic solutions to provide the desired concentration. These isotonic solutions contained 60 g. fructose/liter, 25.10 g. MgCl 2 -6H 2 0/ liter, and 13.08 g. KCl/liter. The level of K+ used (.004 M) was that found by White (1953) to prevent loss of motility upon repeated washing of ram sperm. The concentrations given in the tables are those in the resuspended semen just before insemination, for which it was assumed that the compounds in the "diluent" would be diluted from 4 volumes to
There was also no improvement in fertility upon the addition of K + and Mg + + . The findings would indicate that K + and Mg++ are present in sufficient concentration in the washed and resuspended semen. The results of Wales and White
TABLE 2.—The effect on fertility of different pH levels during storage and at insemination Trial No. 1 Hours stored
Trial No. 2
31.5 26 Percent fertility (first week after insemination)" p H at end
pH at end
pH during storage
6.5
7.2
7.8
Ave.
6.5
7.2
7.8
Ave.
6.5 7.2 7.8 Ave.
48 38 22 36
43 30 46 39
40 50 29 39
43 39 32 38
61 67 50 59
70 52 81 68
49 67 46 54
60 62 59 60
Unstored, undiluted control
62
» Ave. of 10 hens and 44 eggs (range=35-52) per figure, except averages.
78
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
Na 2 HP0 4
1161
pH AND SEMEN STORAGE TABLE 3.—The effect on fertility of different pH levels during storage for 27 hours and at insemination and of added Mgv+ and K+ just before insemination Percent fertility (first week after insemination)" pH during storage
pH at insemination
7.2 7.2 7.8
7.2 7.8 7.2 Ave.
Unstored, undiluted control 0
Without K+orMg++
.004 M K+
.004 M K + +.001MMg++
Ave.
52 54 33 46
50 40 9 33
36 52 38 42
46 49 27 40
87
Ave. of 9 hens and 39 eggs (range = 31-48) per figure, except averages.
SUMMARY
Sodium phosphate buffers of pH 6.5, 7.2, and 7.8 were used in diluting chicken semen for storage and for resuspension of centrifuged semen after storage. There was no apparent effect of pH within the range employed. Addition of Mg + + and K+ just before insemination to the buffer solutions containing fructose was likewise without effect.
REFERENCES Salisbury, G. W., and W. C. Kinney, Jr., 1957. Factors influencing metabolic activity of bull spermatozoa. III. pH. J. Dairy Sci. 40: 13431349. Wales, R. G., and I. G. White, 1958. The effect of alkali metal, magnesium, and calcium ions on the motility of fowl spermatozoa. Austr. J. Biol. Sci. 11:589-597. White, I. G., 1953. The effect of potassium on the washing and dilution of mammalian spermatozoa. Austr. J. Exp. Biol. Med. Sci. 31: 193-200. Wilcox, F. H., and C. S. Shaffner, 1957. Effect of differences in salt and hydrogen ion concentration on the fertilizing ability of avian sperm. J. Appl. Physiol. 11: 429-434. Wilcox, F. H., and C. S. Shaffner, 1958. The effect of different handling methods and added fructose on the fertilizing ability of chicken spermatozoa after storage. Poultry Sci. 37: 1353-1357. Willett, E. L., and J. I. Ohms, 1958. Inactivation of spermatozoa by lactate and reactivation with alkali. J. Dairy Sci. 41: 275-280.
NEWS AND NOTES {Continued from page 1139) leave for a year. He will be conducting research with Dr. A. W. Greenwood, at the Poultry Research Centre, Edinburgh, Scotland; and also will be revising his book, Avian Physiology.
urer—R. Garrett, Salt Lake City; and SecretaryManager—C. Nail, Los Angeles. The Directors are: G. C. Bookey, Seattle; L. R. Davis, Caldwell; A. M. Jessen, Phoenix; J. Wilson, Modesto; E. R. Wyllie, San Francisco; and W. Zollezi, San Francisco. RUTGERS NOTES Dr. Paul D. Sturkie, Professor of Physiology, Department of Poultry Science, Rutgers, The State University, New Brunswick, New Jersey, will be on
NOMINATIONS—P.E.N.B. AWARD The Poultry and Egg National Board Research Achievement Award is given annually. The award has three principle objectives: (a) To recognize outstanding achievement in the fields of poultry products technology and marketing; (b) to encourage in-
(Continued on page 1181)
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
(1958) would suggest that with repeated washing, additional K+ and Mg + + would be required. Although there was found to be no influence of pH in the range employed, it would appear best to use routinely an intermediate level such as pH 7.2, which would make it less likely for the pH of diluted semen to get below pH 6.5 or above 8.0.
WILCOX
Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication March 23, 1959)
ECENT results with mammalian • sperm have indicated that the storage time can be greatly extended by inactivating the sperm, usually by employing a diluent with a low pH, followed by reactivation by adding an alkaline solution. These results have been summarized by Willett and Ohms (1958). A new handling method for chicken semen has recently been described (Wilcox and Shaffner, 1958), and entails the dilution of semen during storage, followed by centrifugation, removal of the supernatant, and the resuspension of the sperm. This method appeared to be one that could be easily employed in an investigation in which different conditions were to be used during storage and at insemination. It has previously been established (Wilcox and Shaffner, 1957) that a wide range of pH levels can be used in storing chicken semen (from 6.5 to 8) without affecting fertility. Accordingly, experiments have been conducted in which three different pH levels (6.5, 7.2, and 7.8) have been used both during storage and at insemination in all possible combinations. This was done to determine if one pH level is optimum during storage and another one is optimum at the time of insemination. One would anticipate that conditions which maintain but slow down 1
Scientific Article No. A7S3, Contribution No. 3013 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
the sperm would be optimum during storage, but that conditions which stimulate sperm activity would be optimum at the time of insemination. It has been shown with bull semen (Salisbury and Kinney, 1957) that the pH has a marked effect on the fructolysis and lactic acid production, with increased activity at higher pH values. This is in agreement with the work already cited on activation and inactivation in which the activity is greater at alkaline pH levels. In one of the fertility trials conducted, K+ and Mg++ were added before insemination. The method employed for storing chicken semen removes nearly all of the seminal plasma and undoubtedly reduces the level of most compounds in chicken semen other than the sodium and phosphate. It appeared possible that the removal of the K+ and Mg + + might be detrimental since these are known to be involved in the metabolism of fructose. It has since been reported by Wales and White (1958) that the addition of these cations to chicken sperm washed two or four times prevents to a large extent the detrimental effect of washing on motility. EXPERIMENTAL PROCEDURE
The stock used and method of semen collection and insemination were the same as described previously (Wilcox and Shaffner, 1958). Semen was diluted 1 to 10 with a sodium phosphate buffer containing
1159
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
R
1160
F. H. WILCOX TABLE 1.-—Composition of
buffers used
5 volumes when added to the centrifuged sperm.
g./liter pH of buffer 6.49 7.22 7.78
NaH 2 P0 4 -H 2 0
RESULTS AND DISCUSSION
10.21 16.34 19.40
12.90 5.16 1.29
The use of all possible combinations of the three pH solutions revealed no difference in the optimum pH for storage and that at insemination (Table 2). This is seen most clearly and meaningfully by summarizing all three treatment groups for each pH. Even so, two pH combinations appeared to provide better fertility than the control of pH 7.2 during storage and at insemination. These were pH 7.2 during storage and pH 7.8 at insemination and also pH 7.8 at storage and pH 7.2 at insemination. These were further tested in an additional trial (Table 3), which clearly showed that the latter two combinations are not superior. These results furthermore provide additional support for the conclusion made previously (Wilcox and Shaffner, 1957) that there is little if any effect of pH on fertility between values of 6.5 and 8.0.
oxytetracycline and dihydrostreptomycin and stored aerobically at 10°C; just before insemination this diluted semen was centrifuged at 1,200 R.C.F. for 10 minutes, the supernatant discarded, and the sperm suspended in buffer containing sufficient fructose to provide a final concentration of 2 mg./ml. The composition of the sodium phosphate buffers is given in Table 1. Fructose, Mg++, and K+ were added to the phosphate buffer by adding the proper amounts of isotonic solutions to provide the desired concentration. These isotonic solutions contained 60 g. fructose/liter, 25.10 g. MgCl 2 -6H 2 0/ liter, and 13.08 g. KCl/liter. The level of K+ used (.004 M) was that found by White (1953) to prevent loss of motility upon repeated washing of ram sperm. The concentrations given in the tables are those in the resuspended semen just before insemination, for which it was assumed that the compounds in the "diluent" would be diluted from 4 volumes to
There was also no improvement in fertility upon the addition of K + and Mg + + . The findings would indicate that K + and Mg++ are present in sufficient concentration in the washed and resuspended semen. The results of Wales and White
TABLE 2.—The effect on fertility of different pH levels during storage and at insemination Trial No. 1 Hours stored
Trial No. 2
31.5 26 Percent fertility (first week after insemination)" p H at end
pH at end
pH during storage
6.5
7.2
7.8
Ave.
6.5
7.2
7.8
Ave.
6.5 7.2 7.8 Ave.
48 38 22 36
43 30 46 39
40 50 29 39
43 39 32 38
61 67 50 59
70 52 81 68
49 67 46 54
60 62 59 60
Unstored, undiluted control
62
» Ave. of 10 hens and 44 eggs (range=35-52) per figure, except averages.
78
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
Na 2 HP0 4
1161
pH AND SEMEN STORAGE TABLE 3.—The effect on fertility of different pH levels during storage for 27 hours and at insemination and of added Mgv+ and K+ just before insemination Percent fertility (first week after insemination)" pH during storage
pH at insemination
7.2 7.2 7.8
7.2 7.8 7.2 Ave.
Unstored, undiluted control 0
Without K+orMg++
.004 M K+
.004 M K + +.001MMg++
Ave.
52 54 33 46
50 40 9 33
36 52 38 42
46 49 27 40
87
Ave. of 9 hens and 39 eggs (range = 31-48) per figure, except averages.
SUMMARY
Sodium phosphate buffers of pH 6.5, 7.2, and 7.8 were used in diluting chicken semen for storage and for resuspension of centrifuged semen after storage. There was no apparent effect of pH within the range employed. Addition of Mg + + and K+ just before insemination to the buffer solutions containing fructose was likewise without effect.
REFERENCES Salisbury, G. W., and W. C. Kinney, Jr., 1957. Factors influencing metabolic activity of bull spermatozoa. III. pH. J. Dairy Sci. 40: 13431349. Wales, R. G., and I. G. White, 1958. The effect of alkali metal, magnesium, and calcium ions on the motility of fowl spermatozoa. Austr. J. Biol. Sci. 11:589-597. White, I. G., 1953. The effect of potassium on the washing and dilution of mammalian spermatozoa. Austr. J. Exp. Biol. Med. Sci. 31: 193-200. Wilcox, F. H., and C. S. Shaffner, 1957. Effect of differences in salt and hydrogen ion concentration on the fertilizing ability of avian sperm. J. Appl. Physiol. 11: 429-434. Wilcox, F. H., and C. S. Shaffner, 1958. The effect of different handling methods and added fructose on the fertilizing ability of chicken spermatozoa after storage. Poultry Sci. 37: 1353-1357. Willett, E. L., and J. I. Ohms, 1958. Inactivation of spermatozoa by lactate and reactivation with alkali. J. Dairy Sci. 41: 275-280.
NEWS AND NOTES {Continued from page 1139) leave for a year. He will be conducting research with Dr. A. W. Greenwood, at the Poultry Research Centre, Edinburgh, Scotland; and also will be revising his book, Avian Physiology.
urer—R. Garrett, Salt Lake City; and SecretaryManager—C. Nail, Los Angeles. The Directors are: G. C. Bookey, Seattle; L. R. Davis, Caldwell; A. M. Jessen, Phoenix; J. Wilson, Modesto; E. R. Wyllie, San Francisco; and W. Zollezi, San Francisco. RUTGERS NOTES Dr. Paul D. Sturkie, Professor of Physiology, Department of Poultry Science, Rutgers, The State University, New Brunswick, New Jersey, will be on
NOMINATIONS—P.E.N.B. AWARD The Poultry and Egg National Board Research Achievement Award is given annually. The award has three principle objectives: (a) To recognize outstanding achievement in the fields of poultry products technology and marketing; (b) to encourage in-
(Continued on page 1181)
Downloaded from http://ps.oxfordjournals.org/ at New York University on March 13, 2016
(1958) would suggest that with repeated washing, additional K+ and Mg + + would be required. Although there was found to be no influence of pH in the range employed, it would appear best to use routinely an intermediate level such as pH 7.2, which would make it less likely for the pH of diluted semen to get below pH 6.5 or above 8.0.