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A Comparison of Various Treatments of Fowl Semen1
A Comparison of Various Treatments of Fowl Semen1 KARL E. NESTOR AND H. M. HYRE Department of Poultry Science, West Virginia University, Morgantown, West Virginia (Received for publication August 5, 1960)
T
1 This manuscript is published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 641.
four hours storage. These workers also found that whole milk and Ringer's solution were equally effective up to four hours storage, but with more extended periods of time, the milk held the fertilizing capacity better than did Ringer's solution. Several attempts have been made to assimilate the conditions of the oviduct of the female in vitro when storing sperm. One of the most common ways attempted is by the use of diluents consisting of parts of the secretion of the oviduct. Most of these attempts have been unsuccessful. Weakley and Shaffner (1952) observed that thin egg white when used in dilutions of 1:3, 1:10 and 1:50 gave unsatisfactory results. Using the most promising concentration and combination, as was shown by motility tests, of the following egg white proteins: ovalbumin, conalbumin, ovomucoid, lysozyme and ovomucin, Wilcox and Shaffner (1956) found that a dilution (1:10) of semen and this mixture did not enhance fertilizing ability after storage for one day at 12°C. over that of the control diluted with a buffer solution. The egg white proteins were dissolved in this buffer. Similar results were obtained with milk. This manuscript contains a study of Ringer's solution, thick egg albumen and whole milk as diluents for semen at two different storage periods. PROCEDURE
Twenty-four New Hampshire pullets, which were located in two neighboring pens, were divided into eight groups of three birds each at random. Four groups were inseminated with fresh undiluted semen, semen diluted with Ringer's solution, thick egg
772
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HE ability to hold fowl semen for a period of several days and still get satisfactory fertility when used would be of utmost importance to the poultry industry. Numerous diluents have been tried for diluting fowl semen for storage and to extend its usage. Motohashi and Moritomo (1927) compared the motility of cock sperm in whole egg white diluted with distilled water, Ringer's solution, physiological salt solution (0.75%) and dextrose solution (6%). They found that motility was excellent in Ringer's solution and somewhat poorer and about equal in the diluted egg white and physiological salt solution. Poor results were obtained with the dextrose solution. The motility of the sperm was decreased by increasing the amount of distilled water to the egg white. A modified Ringer's solution, identical with the one used in this experiment, was used by Bonnier and Trulsson (1939) who found that when a mixture of 30% semen and 70% Ringer's solution was used, better fertility was obtained than with undiluted semen. When the semen was diluted one to ten, fertility was as good as that obtained with undiluted semen. A one to fifty dilution gave decidedly lower fertility. Schindler et al. (1955) used a dilution rate of one part of semen to three parts of Ringer's solution and found that fertility of the diluted semen was almost as great as that of undiluted semen when inseminated immediately after collection or at two and four hours storage, but gave significantly lower fertility than the undiluted controls after
TREATMENT OF SEMEN
of the last insemination to serve as a control for the inseminated group. The inseminating technique used was one similar to that described by Mueller (1949) except that two operators were employed rather than one. Each hen was inseminated with one-tenth ml. of semen. A second trial was run with Ringer's solution, with a composition similar to that described by Lorenz (1958). The major difference of this type of Ringer's solution from the first one used was that it contained a phosphate buffer along with the bicarbonate buffer which the first one contained. In this trial, meat-type cross females, commonly called Dominant Whites, were used. Semen used to inseminate these birds was obtained from two Barred Plymouth Rock males. The diluting and inseminating procedure was similar to that described previously. Nine females were used per group. The trial covered a period of four weeks. An experiment was conducted so as to ascertain whether or not sperm concentration could be determined accurately by the use of visual rating. The semen was collected from a group of nine Rhode Island Reds and given a visual rating between one and five according to the method of McDaniel and Craig (1959). The sperm concentration was then determined by the use of a haemocytometer using a semen dilution of one part of semen to 400 parts of a 3% NaCl solution. A value of one in the above rating indicated watery semen and five indicated semen that is dense, white and viscous. During these studies, recording of the semen volume per ejaculate of the various males on hand was a regular routine. At various times, sperm concentration determinations were made on the various breeds. The semen collection schedule was the same for all breeds. The volume of semen was determined to the nearest hundredth of one ml. by the use of a one ml. tuberculin syringe. The sperm concentration of the vari-
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albumen and whole milk respectively. The other four groups were inseminated with semen given similar treatment except it was stored for six hours at 10°C. Semen used in this study was obtained from six New Hampshire cockerels by the method of Burrows and Quinn (1937) and pooled. A dilution ratio of three parts diluent to one part semen was used for all of the diluents. The Ringer's solution used was of the composition given by Bonnier and Trulsson (1939); however, special precautions were found necessary for desirable preparations. It was found that the salts could best be kept in solution by first cooling the distilled water to be used, mixing the ingredients separately until dissolved and then combining the resulting solutions. After the solution was prepared, it was kept cool as recommended by the above workers. The thick egg albumen was obtained from fresh eggs. The eggs used were broken on a clean porcelain tray and the thick albumen separated by means of a tuberculin syringe and placed in a test tube or beaker. Homogenized and pasteurized whole milk was purchased locally and stored with the other diluents at 10°C. The eggs collected for fertility studies were pedigreed according to hen and day of laying and set at weekly intervals. Fertility was determined on the seventh day of incubation by means of a candler emitting filtered blue light similar to the one described by King (1939). All eggs removed as infertiles were broken and examined macroscopically to detect any early dead germs which were not detected by candling. The birds were inseminated at weekly intervals for nine weeks after which the inseminations were discontinued and the birds allowed to become infertile in order to determine the duration of fertility. The cockerels were removed from a nearby pen of Barred Plymouth Rock pullets on the day
773
774
K. E. NESTOR AND H. M. HYRE TABLE 1.—Average fertility for undiluted semen and semen diluted in the various diluents % Fertility of Fertile Hens Diluent
Time of storage (hours)
Average % fertility seven days
Days after insemination
7
1 Undiluted
0 6
83.3 100. 46.6 75.
Ringer's solution
0 6
71.4 40.
58.8 55.5
Whole milk
0 6
64.7 66.6
Thick egg albumen
0 6
50. 62.5
100. 70.
93.7 76.4
72.7 50.
71.4 33.3
76.9 58.8
86.17(97)* 57.8 (128)
80.9 53.8
73.3 60.
61.1 54.5
55.5 75.
75. 60.
67.8 (115) 57.5 ( 8 0 )
47. 64.2
56.2 50.
60. 77.7
50. 12.3
47.3 46.1
46.1 64.7
52.5 (113) 51.0(88)
44.4 62.5
42.1 37.5
36.8 66.6
60. 36.3
63.1 62.5
71.4 42.8
55.0 (125) 52.5 ( 59)
ous breeds was determined by counting, as above, on a haemocytometer. Two or more determinations of sperm concentration were made on each breed. Semen was collected three times weekly from a group of six New Hampshire males over a period of approximately six months. These three weekly collections consisted of two collections at two day intervals and one collection at a three day interval. In order to determine the effect of collecting at shorter and longer intervals on the semen volume, a special collection schedule was set up in order to determine the semen volume of ejaculates collected at one, four and seven day intervals. Five successive collections from each of the males were used to obtain data for the one day interval, whereas, a single collection from each male was the basis for the data on the other two intervals. RESULTS AND DISCUSSION
The average fertility of the undiluted semen and diluted semen in the first trial can be seen in Table 1. There was a highly significant difference (p < .01) between the treatment of semen. A significant difference (p < .05) also occurred between storage times. A significant interaction was
found between semen handled in various ways and hours of storage. At the 0 hour storage period, a significant difference was found between undiluted semen and semen diluted in Ringer's solution; however, no significant difference occurred at the six hour storage period. This indicates that although the fertility of semen diluted in Ringer's solution is initially depressed, the fertility is maintained more constant during storage. These results at the 0 hour storage period were not in agreement with those of Bonnier and Trulsson (1939) and Schindler et al (1955) who found that Ringer's solution of the composition used here was equally as good or better than undiluted at this storage period. A highly significant difference was found between semen diluted in whole milk and undiluted semen and between semen diluted in thick egg albumen and undiluted at 0 hours of storage. The former result is not in agreement with the work of Schindler et al. (1955). However, in this experiment, the milk was mixed in a Waring blender, boiled for ten minutes and filtered. According to Norman and Goldberg (1960), whole milk contains a spermicidial factor which is non-specific for cock, rabbit and bull semen and which is removed by heating and filter-
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' Number of eggs on which the percentages are based.
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TREATMENT OF SEMEN
TABLE 2.—Duration of fertility in the artificially inseminated and naturally mated groups Time of storage (hours)
Average duration of fertility* (days)
Undiluted
0 6
17 12
Ringer's solution
0 6
20 14
Whole milk
0 6
13 12
Group
Naturally mated j»roup
16
* Average number of days that fertile eggs were received from hens in each group after removal of the males or after the last insemination.
TABLE 3.—The effect of interval between collection of semen on ejaculate volume Interval Number between of collection males (days) 1 2 3 4 7
6 6 6 6 6
Total yield of semen (ml.) 23.95 200.49 117.63 6.05 6.00
Average Number volume of ejacu- of ejaculations lations (ml.) 30 215 132 6 6
0.80 0.93 0.89 1.01 1.00
the various artificially inseminated groups and the naturally mated group may be seen. The records of hens which died or were infertile or ceased production during the course of the period for studying the duration were eliminated. These factors combined made it necessary to eliminate the egg albumen group. These data show that the duration of fertility of artificially inseminated birds and naturally mated birds under similar conditions (0 hrs., undiluted) compare remarkably well. The longest duraation found was 28 days for two birds in the naturally mated group. The longest duration for any bird in the artificially inseminated groups was 26 days for a bird in the group which was inseminated with semen diluted with Ringer's and inseminated after six hours of storage. In the second fertility trial using the second form of Ringer's solution almost identical fertility was obtained with semen that was undiluted and diluted 1:3 with this solution. Using Ringer's solution as a diluent, 94 fertile eggs were obtained out of a total of 115 eggs (81.7%). Ninety fertile eggs out of a total of 112 eggs (80.4%) were obtained in the group inseminated with undiluted semen. The results of this trial when compared with the previous one indicate that there is a significant difference between different forms of Ringer's solution. Table 3 shows the results of various
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ing. The results with thick egg albumen obtained during these studies are in general agreement with the work of Weakley and Shaffner (1952) and Wilcox and Shaffner (1956), in that egg albumen is not a good diluent for cock semen. The low fertility obtained with the thick egg albumen may have been due to the rather high pH of the egg albumen. The average pH of egg albumen made by a Beckman zeromatic pH meter was 7.95. At this pH, Bogdonoff and Shaffner (1954) noticed a reduction in fertility. Since it was noted in the present study that the semen dispersed with difficulty as a result of the consistency of the thick egg albumen, improper mixing may have also been a factor. No significant difference occurred in fertility between the diluted semen and undiluted semen after six hours of storage. This was undoubtedly due to the highly significant drop in fertility encountered with the undiluted semen. As can be seen in Table 1, no definite relationship existed between the time elapsing after insemination and fertility within the seven day period following each insemination. In Table 2, the duration of fertility for
776
K. E. NESTOR AND H. M. HYRE TABLE 4.—The volume and sperm concentration of the semen of various breeds
Breed
Number of males
Number of ejaculations
Total volume collected (ml.)
Average volume of ejaculate (ml.)
New Hampshire Rhode Island Red White Leghorn Barred Plymouth Rock Dominant White
10 9 5 2 3
389 379 13 59 55
348.12 291.68 7.55 48.70 39.90
0.89 0.77 0.58 0.84 0.73
Millions/ Number of mm.3 determinations 3 94 2 4 2
5.28 3.46 4.20 3.84 4.60
eter. The above correlations are based on 93 degrees of freedom. An analysis was made of the semen yield and sperm concentration of males of the Rhode Island Red breed which gave a measurable quantity of semen on the first or second collection and those which gave a measurable quantity of semen on a later collection. There were six and three males in each group respectively. The age of these males at the start of the experiment was six months. The results of this analysis are summarized in Table 5. The early maturing males averaged 0.22 ml. more semen than the late maturing males. The sperm concentration of the two groups of males was essentially the same. SUMMARY
The production of fowl semen and its concentration was included in this study of the fertilizing power of semen that had been treated in various ways. A significant difference in fertility as found to exist between various treatments of the semen including storage time. The undiluted semen stored at 0 hours gave better fertility than any of the
TABLE 5.—The efect of maturity on semen volume and sperm concentration of Rhode Island Red males Sperm concentration Number of Collections Late maturing males Early maturing males
127 252
Average volTotal volume ume of ejaculate collected (ml.) (ml.) 76.63 215.05
0.63 0.85
Number of determmations
Millions/ mm.3
31 65
3.37 3.40
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intervals of semen collections. There was a slight depression in volume when collected at daily intervals. Otherwise, the interval of collecting had little influence on semen volume. The number of ejaculations collected at the 4 and 7 day intervals was inadequate for suitable comparison. The volume and sperm concentration of the semen of various breeds of males from which collection was made may be seen in Table 4. The number of determinations of sperm concentration was too small for a good comparison. There was a highly significant correlation ( r = 0 . 5 1 ) between visual rating of semen as to sperm concentration and sperm concentration as determined by a haemocytometer. McDaniel and Craig (1959) reported a highly significant correlation of 0.7SS between these two variables. These two highly significant correlations indicate that semen can be classified accurately as to sperm concentration by rating its visual density. A nonsignificant correlation (r = .10) was found between semen volume of an ejaculate and sperm concentration of that ejaculate as measured by the haemocytom-
Sperm concentration
TREATMENT OF SEMEN
REFERENCES Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity, and fertilizing capacity of chicken semen. Poultry Sci. 33 : 665-66°. Bonnier, G., and S. Trulsson, 1939. Artificial insemination of chickens with semen diluted in Ringer's solution. Proc. 7th World's Poultry Congress, Cleveland: pp. 76-79. Burrows, W. T., and J. Quinn, 1937. The collec-
tion of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. King, D. F., 1939. The detection of infertile eggs and its application to hatchery management. Alabama Agric. Exp. Sta. Cir. 82: 3-15. Lorenz, F. W., 1958. Carbohydrate metabolism of cock spermatozoa. Nature, 182: 397-398. McDaniel, G. R., and J. V. Craig, 1959. Behavior traits, semen measurements and fertility of White Leghorn males. Poultry Sci. 38: 10051014. Motohashi, H., and M. Moritomo, 1927. On the vitality of the spermatozoa of the domestic fowl in the egg white solution. Proc. 3rd World's Poultry Congress, Canada: pp 157-159. Mueller, C. D., 1949. Artificial insemination for progeny-test breeding in poultry. Poultry Sci. 28: 143-145. Norman, C , and E. Goldberg, 1960. Unpublished data. West Virginia University, Morgantown, West Virginia. Schindler, H., S. Weinstein, E. Moses and I. Gabriel, 1955. The effect of various diluents and storage times on the fertilizing capacity of cock semen. Poultry Sci. 34: 1113-1117. Weakley, C. E., I l l , and C. S. Shaffner, 1952. The fertilizing capacity of diluted chicken semen. Poultry Sci. 3 1 : 6S0-6S3.. Wilcox, F. H., and C. S. Shaffner, 1956. The effect of the major egg white proteins on the fertilizing ability of avian sperm. Poultry Sci. 35: 1179-1180.
The Storage of Chicken and Turkey Spermatozoa by Inhibition with 2-ethyl-5-methylbenzimidazole (EBM) 1 G. C. HARRIS, JR. 2 , F. H. WILCOX AND C. S. SHAFFNER Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication August 5. 1960)
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HE complete or partial reversal of the motility of chicken spermatozoa after inhibition with 2-ethyl-S-methylbenzimidazole (EMB) has been recently reported by 'Scientific Article no. A867, Contribution 3177, of Maryland Agric. Exp. Station (Department of Poultry Husbandry). 2 Present address: Department of Animal Industry and Veterinary Science, University of Arkansas, Fayetteville, Arkansas.
Blackwood and Harris (1960). The exact biochemical mechanisms interrupted are unknown but probably involve nucleic acid and/or nucleoprotein. (Tamm et al., 1953; Abbott and Dodson, 1954) The mechanism is independent of fructolysis since levels of EMB which completely inhibit motility have no effect on rate of fructose utilization in chicken semen (Blackwood and Harris, 1960). The fertility of 50-60% obtained
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diluted semen; however, the semen diluted with Ringer's solution showed the same fertility as the undiluted semen when stored at 10°C. for six hours. This study indicated Ringer's solution to be a better diluent than whole milk or egg albumen. When stored at 0 hours the semen in Ringer's solution and the undiluted semen compared favorably with natural mating for duration of fertility. A highly significant correlation of +0.S11 was found between visual rating of semen as to sperm concentration and the sperm concentration as determined by a haemocytometer. Data on the volume of semen produced per ejaculate at different collection intervals and by different breeds is presented.
777
T
1 This manuscript is published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 641.
four hours storage. These workers also found that whole milk and Ringer's solution were equally effective up to four hours storage, but with more extended periods of time, the milk held the fertilizing capacity better than did Ringer's solution. Several attempts have been made to assimilate the conditions of the oviduct of the female in vitro when storing sperm. One of the most common ways attempted is by the use of diluents consisting of parts of the secretion of the oviduct. Most of these attempts have been unsuccessful. Weakley and Shaffner (1952) observed that thin egg white when used in dilutions of 1:3, 1:10 and 1:50 gave unsatisfactory results. Using the most promising concentration and combination, as was shown by motility tests, of the following egg white proteins: ovalbumin, conalbumin, ovomucoid, lysozyme and ovomucin, Wilcox and Shaffner (1956) found that a dilution (1:10) of semen and this mixture did not enhance fertilizing ability after storage for one day at 12°C. over that of the control diluted with a buffer solution. The egg white proteins were dissolved in this buffer. Similar results were obtained with milk. This manuscript contains a study of Ringer's solution, thick egg albumen and whole milk as diluents for semen at two different storage periods. PROCEDURE
Twenty-four New Hampshire pullets, which were located in two neighboring pens, were divided into eight groups of three birds each at random. Four groups were inseminated with fresh undiluted semen, semen diluted with Ringer's solution, thick egg
772
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
HE ability to hold fowl semen for a period of several days and still get satisfactory fertility when used would be of utmost importance to the poultry industry. Numerous diluents have been tried for diluting fowl semen for storage and to extend its usage. Motohashi and Moritomo (1927) compared the motility of cock sperm in whole egg white diluted with distilled water, Ringer's solution, physiological salt solution (0.75%) and dextrose solution (6%). They found that motility was excellent in Ringer's solution and somewhat poorer and about equal in the diluted egg white and physiological salt solution. Poor results were obtained with the dextrose solution. The motility of the sperm was decreased by increasing the amount of distilled water to the egg white. A modified Ringer's solution, identical with the one used in this experiment, was used by Bonnier and Trulsson (1939) who found that when a mixture of 30% semen and 70% Ringer's solution was used, better fertility was obtained than with undiluted semen. When the semen was diluted one to ten, fertility was as good as that obtained with undiluted semen. A one to fifty dilution gave decidedly lower fertility. Schindler et al. (1955) used a dilution rate of one part of semen to three parts of Ringer's solution and found that fertility of the diluted semen was almost as great as that of undiluted semen when inseminated immediately after collection or at two and four hours storage, but gave significantly lower fertility than the undiluted controls after
TREATMENT OF SEMEN
of the last insemination to serve as a control for the inseminated group. The inseminating technique used was one similar to that described by Mueller (1949) except that two operators were employed rather than one. Each hen was inseminated with one-tenth ml. of semen. A second trial was run with Ringer's solution, with a composition similar to that described by Lorenz (1958). The major difference of this type of Ringer's solution from the first one used was that it contained a phosphate buffer along with the bicarbonate buffer which the first one contained. In this trial, meat-type cross females, commonly called Dominant Whites, were used. Semen used to inseminate these birds was obtained from two Barred Plymouth Rock males. The diluting and inseminating procedure was similar to that described previously. Nine females were used per group. The trial covered a period of four weeks. An experiment was conducted so as to ascertain whether or not sperm concentration could be determined accurately by the use of visual rating. The semen was collected from a group of nine Rhode Island Reds and given a visual rating between one and five according to the method of McDaniel and Craig (1959). The sperm concentration was then determined by the use of a haemocytometer using a semen dilution of one part of semen to 400 parts of a 3% NaCl solution. A value of one in the above rating indicated watery semen and five indicated semen that is dense, white and viscous. During these studies, recording of the semen volume per ejaculate of the various males on hand was a regular routine. At various times, sperm concentration determinations were made on the various breeds. The semen collection schedule was the same for all breeds. The volume of semen was determined to the nearest hundredth of one ml. by the use of a one ml. tuberculin syringe. The sperm concentration of the vari-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
albumen and whole milk respectively. The other four groups were inseminated with semen given similar treatment except it was stored for six hours at 10°C. Semen used in this study was obtained from six New Hampshire cockerels by the method of Burrows and Quinn (1937) and pooled. A dilution ratio of three parts diluent to one part semen was used for all of the diluents. The Ringer's solution used was of the composition given by Bonnier and Trulsson (1939); however, special precautions were found necessary for desirable preparations. It was found that the salts could best be kept in solution by first cooling the distilled water to be used, mixing the ingredients separately until dissolved and then combining the resulting solutions. After the solution was prepared, it was kept cool as recommended by the above workers. The thick egg albumen was obtained from fresh eggs. The eggs used were broken on a clean porcelain tray and the thick albumen separated by means of a tuberculin syringe and placed in a test tube or beaker. Homogenized and pasteurized whole milk was purchased locally and stored with the other diluents at 10°C. The eggs collected for fertility studies were pedigreed according to hen and day of laying and set at weekly intervals. Fertility was determined on the seventh day of incubation by means of a candler emitting filtered blue light similar to the one described by King (1939). All eggs removed as infertiles were broken and examined macroscopically to detect any early dead germs which were not detected by candling. The birds were inseminated at weekly intervals for nine weeks after which the inseminations were discontinued and the birds allowed to become infertile in order to determine the duration of fertility. The cockerels were removed from a nearby pen of Barred Plymouth Rock pullets on the day
773
774
K. E. NESTOR AND H. M. HYRE TABLE 1.—Average fertility for undiluted semen and semen diluted in the various diluents % Fertility of Fertile Hens Diluent
Time of storage (hours)
Average % fertility seven days
Days after insemination
7
1 Undiluted
0 6
83.3 100. 46.6 75.
Ringer's solution
0 6
71.4 40.
58.8 55.5
Whole milk
0 6
64.7 66.6
Thick egg albumen
0 6
50. 62.5
100. 70.
93.7 76.4
72.7 50.
71.4 33.3
76.9 58.8
86.17(97)* 57.8 (128)
80.9 53.8
73.3 60.
61.1 54.5
55.5 75.
75. 60.
67.8 (115) 57.5 ( 8 0 )
47. 64.2
56.2 50.
60. 77.7
50. 12.3
47.3 46.1
46.1 64.7
52.5 (113) 51.0(88)
44.4 62.5
42.1 37.5
36.8 66.6
60. 36.3
63.1 62.5
71.4 42.8
55.0 (125) 52.5 ( 59)
ous breeds was determined by counting, as above, on a haemocytometer. Two or more determinations of sperm concentration were made on each breed. Semen was collected three times weekly from a group of six New Hampshire males over a period of approximately six months. These three weekly collections consisted of two collections at two day intervals and one collection at a three day interval. In order to determine the effect of collecting at shorter and longer intervals on the semen volume, a special collection schedule was set up in order to determine the semen volume of ejaculates collected at one, four and seven day intervals. Five successive collections from each of the males were used to obtain data for the one day interval, whereas, a single collection from each male was the basis for the data on the other two intervals. RESULTS AND DISCUSSION
The average fertility of the undiluted semen and diluted semen in the first trial can be seen in Table 1. There was a highly significant difference (p < .01) between the treatment of semen. A significant difference (p < .05) also occurred between storage times. A significant interaction was
found between semen handled in various ways and hours of storage. At the 0 hour storage period, a significant difference was found between undiluted semen and semen diluted in Ringer's solution; however, no significant difference occurred at the six hour storage period. This indicates that although the fertility of semen diluted in Ringer's solution is initially depressed, the fertility is maintained more constant during storage. These results at the 0 hour storage period were not in agreement with those of Bonnier and Trulsson (1939) and Schindler et al (1955) who found that Ringer's solution of the composition used here was equally as good or better than undiluted at this storage period. A highly significant difference was found between semen diluted in whole milk and undiluted semen and between semen diluted in thick egg albumen and undiluted at 0 hours of storage. The former result is not in agreement with the work of Schindler et al. (1955). However, in this experiment, the milk was mixed in a Waring blender, boiled for ten minutes and filtered. According to Norman and Goldberg (1960), whole milk contains a spermicidial factor which is non-specific for cock, rabbit and bull semen and which is removed by heating and filter-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
' Number of eggs on which the percentages are based.
775
TREATMENT OF SEMEN
TABLE 2.—Duration of fertility in the artificially inseminated and naturally mated groups Time of storage (hours)
Average duration of fertility* (days)
Undiluted
0 6
17 12
Ringer's solution
0 6
20 14
Whole milk
0 6
13 12
Group
Naturally mated j»roup
16
* Average number of days that fertile eggs were received from hens in each group after removal of the males or after the last insemination.
TABLE 3.—The effect of interval between collection of semen on ejaculate volume Interval Number between of collection males (days) 1 2 3 4 7
6 6 6 6 6
Total yield of semen (ml.) 23.95 200.49 117.63 6.05 6.00
Average Number volume of ejacu- of ejaculations lations (ml.) 30 215 132 6 6
0.80 0.93 0.89 1.01 1.00
the various artificially inseminated groups and the naturally mated group may be seen. The records of hens which died or were infertile or ceased production during the course of the period for studying the duration were eliminated. These factors combined made it necessary to eliminate the egg albumen group. These data show that the duration of fertility of artificially inseminated birds and naturally mated birds under similar conditions (0 hrs., undiluted) compare remarkably well. The longest duraation found was 28 days for two birds in the naturally mated group. The longest duration for any bird in the artificially inseminated groups was 26 days for a bird in the group which was inseminated with semen diluted with Ringer's and inseminated after six hours of storage. In the second fertility trial using the second form of Ringer's solution almost identical fertility was obtained with semen that was undiluted and diluted 1:3 with this solution. Using Ringer's solution as a diluent, 94 fertile eggs were obtained out of a total of 115 eggs (81.7%). Ninety fertile eggs out of a total of 112 eggs (80.4%) were obtained in the group inseminated with undiluted semen. The results of this trial when compared with the previous one indicate that there is a significant difference between different forms of Ringer's solution. Table 3 shows the results of various
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
ing. The results with thick egg albumen obtained during these studies are in general agreement with the work of Weakley and Shaffner (1952) and Wilcox and Shaffner (1956), in that egg albumen is not a good diluent for cock semen. The low fertility obtained with the thick egg albumen may have been due to the rather high pH of the egg albumen. The average pH of egg albumen made by a Beckman zeromatic pH meter was 7.95. At this pH, Bogdonoff and Shaffner (1954) noticed a reduction in fertility. Since it was noted in the present study that the semen dispersed with difficulty as a result of the consistency of the thick egg albumen, improper mixing may have also been a factor. No significant difference occurred in fertility between the diluted semen and undiluted semen after six hours of storage. This was undoubtedly due to the highly significant drop in fertility encountered with the undiluted semen. As can be seen in Table 1, no definite relationship existed between the time elapsing after insemination and fertility within the seven day period following each insemination. In Table 2, the duration of fertility for
776
K. E. NESTOR AND H. M. HYRE TABLE 4.—The volume and sperm concentration of the semen of various breeds
Breed
Number of males
Number of ejaculations
Total volume collected (ml.)
Average volume of ejaculate (ml.)
New Hampshire Rhode Island Red White Leghorn Barred Plymouth Rock Dominant White
10 9 5 2 3
389 379 13 59 55
348.12 291.68 7.55 48.70 39.90
0.89 0.77 0.58 0.84 0.73
Millions/ Number of mm.3 determinations 3 94 2 4 2
5.28 3.46 4.20 3.84 4.60
eter. The above correlations are based on 93 degrees of freedom. An analysis was made of the semen yield and sperm concentration of males of the Rhode Island Red breed which gave a measurable quantity of semen on the first or second collection and those which gave a measurable quantity of semen on a later collection. There were six and three males in each group respectively. The age of these males at the start of the experiment was six months. The results of this analysis are summarized in Table 5. The early maturing males averaged 0.22 ml. more semen than the late maturing males. The sperm concentration of the two groups of males was essentially the same. SUMMARY
The production of fowl semen and its concentration was included in this study of the fertilizing power of semen that had been treated in various ways. A significant difference in fertility as found to exist between various treatments of the semen including storage time. The undiluted semen stored at 0 hours gave better fertility than any of the
TABLE 5.—The efect of maturity on semen volume and sperm concentration of Rhode Island Red males Sperm concentration Number of Collections Late maturing males Early maturing males
127 252
Average volTotal volume ume of ejaculate collected (ml.) (ml.) 76.63 215.05
0.63 0.85
Number of determmations
Millions/ mm.3
31 65
3.37 3.40
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intervals of semen collections. There was a slight depression in volume when collected at daily intervals. Otherwise, the interval of collecting had little influence on semen volume. The number of ejaculations collected at the 4 and 7 day intervals was inadequate for suitable comparison. The volume and sperm concentration of the semen of various breeds of males from which collection was made may be seen in Table 4. The number of determinations of sperm concentration was too small for a good comparison. There was a highly significant correlation ( r = 0 . 5 1 ) between visual rating of semen as to sperm concentration and sperm concentration as determined by a haemocytometer. McDaniel and Craig (1959) reported a highly significant correlation of 0.7SS between these two variables. These two highly significant correlations indicate that semen can be classified accurately as to sperm concentration by rating its visual density. A nonsignificant correlation (r = .10) was found between semen volume of an ejaculate and sperm concentration of that ejaculate as measured by the haemocytom-
Sperm concentration
TREATMENT OF SEMEN
REFERENCES Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity, and fertilizing capacity of chicken semen. Poultry Sci. 33 : 665-66°. Bonnier, G., and S. Trulsson, 1939. Artificial insemination of chickens with semen diluted in Ringer's solution. Proc. 7th World's Poultry Congress, Cleveland: pp. 76-79. Burrows, W. T., and J. Quinn, 1937. The collec-
tion of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. King, D. F., 1939. The detection of infertile eggs and its application to hatchery management. Alabama Agric. Exp. Sta. Cir. 82: 3-15. Lorenz, F. W., 1958. Carbohydrate metabolism of cock spermatozoa. Nature, 182: 397-398. McDaniel, G. R., and J. V. Craig, 1959. Behavior traits, semen measurements and fertility of White Leghorn males. Poultry Sci. 38: 10051014. Motohashi, H., and M. Moritomo, 1927. On the vitality of the spermatozoa of the domestic fowl in the egg white solution. Proc. 3rd World's Poultry Congress, Canada: pp 157-159. Mueller, C. D., 1949. Artificial insemination for progeny-test breeding in poultry. Poultry Sci. 28: 143-145. Norman, C , and E. Goldberg, 1960. Unpublished data. West Virginia University, Morgantown, West Virginia. Schindler, H., S. Weinstein, E. Moses and I. Gabriel, 1955. The effect of various diluents and storage times on the fertilizing capacity of cock semen. Poultry Sci. 34: 1113-1117. Weakley, C. E., I l l , and C. S. Shaffner, 1952. The fertilizing capacity of diluted chicken semen. Poultry Sci. 3 1 : 6S0-6S3.. Wilcox, F. H., and C. S. Shaffner, 1956. The effect of the major egg white proteins on the fertilizing ability of avian sperm. Poultry Sci. 35: 1179-1180.
The Storage of Chicken and Turkey Spermatozoa by Inhibition with 2-ethyl-5-methylbenzimidazole (EBM) 1 G. C. HARRIS, JR. 2 , F. H. WILCOX AND C. S. SHAFFNER Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication August 5. 1960)
T
HE complete or partial reversal of the motility of chicken spermatozoa after inhibition with 2-ethyl-S-methylbenzimidazole (EMB) has been recently reported by 'Scientific Article no. A867, Contribution 3177, of Maryland Agric. Exp. Station (Department of Poultry Husbandry). 2 Present address: Department of Animal Industry and Veterinary Science, University of Arkansas, Fayetteville, Arkansas.
Blackwood and Harris (1960). The exact biochemical mechanisms interrupted are unknown but probably involve nucleic acid and/or nucleoprotein. (Tamm et al., 1953; Abbott and Dodson, 1954) The mechanism is independent of fructolysis since levels of EMB which completely inhibit motility have no effect on rate of fructose utilization in chicken semen (Blackwood and Harris, 1960). The fertility of 50-60% obtained
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diluted semen; however, the semen diluted with Ringer's solution showed the same fertility as the undiluted semen when stored at 10°C. for six hours. This study indicated Ringer's solution to be a better diluent than whole milk or egg albumen. When stored at 0 hours the semen in Ringer's solution and the undiluted semen compared favorably with natural mating for duration of fertility. A highly significant correlation of +0.S11 was found between visual rating of semen as to sperm concentration and the sperm concentration as determined by a haemocytometer. Data on the volume of semen produced per ejaculate at different collection intervals and by different breeds is presented.
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