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The Effect of Different Handling Methods and Added Fructose on the Fertilizing Ability of Chicken Spermatozoa after Storage
The Effect of Different Handling Methods and Added Fructose on the Fertilizing Ability of Chicken Spermatozoa after Storage F. H. WILCOX AND C. S. SHAFFNER
Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication April 7, 1958)
HE poor success in attempts to store chicken semen in vitro is in sharp contrast to success obtained in storing chicken spermatozoa in the hen's oviduct and in storing bull semen in vitro. Work at this laboratory has provided evidence of the necessity of controlling the pH of semen during storage (Wilcox, 1958a). This can most easily be done by dilution of semen with a buffer. However, dilution of semen, even when inseminated fresh, results in lowered fertility, so that from the outset it can be anticipated that fertility will be lowered when semen is diluted. Evidence has been obtained (Wilcox, 1958b) that semen can be centrifuged and the seminal plasma replaced by a sodium phosphate buffer with little loss of fertilizing capacity. This suggests a procedure by which semen can be diluted and stored, and still be inseminated "undiluted" by centrifuging the diluted semen just before insemination and replacing the supernatant with only enough diluent to bring the solution to its original volume. By using centrifugation it should also be possible to control pH by replacing seminal plasma, which is poorly buffered, with a good buffer, and storing the sperm in that condition until insemination. Accordingly, an experiment was set up to test these methods of handling semen. Scientific Article No. A685. Contribution No. 2903 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
A third method was also attempted: dialysis of undiluted semen against a phosphate buffer containing antibiotics. The idea behind this was that toxic end-products formed by the sperm might account for loss of fertility of stored sperm; if these end-products are of low molecular weight they would be removed by dialysis, and pH would be controlled by entrance of ions of the buffer into the semen. In addition to these treatments, fructose was added in some cases at the end of the storage period just before insemination. Fructose has been shown to increase activity of chicken spermatozoa (Shaffner et al., 1941; Wilcox, unpublished) and it was desired to determine if this increased activity at the time of insemination would boost fertility. MATERIAL AND METHODS
General. Males used as the source of semen were of two breeds: a Flightless strain under development at this station and a broiler-type strain. White Leghorn females in individual batteries were used to test fertilizing ability. Semen was collected by a one-man technique described by Bogdonoff and Shaffner (1954). Semen was pooled and a group of females was inseminated with 0.1 ml. of semen immediately after collection, using a syringe with an extension tube similar to that used by Parker et al. (1942). The remaining semen was then treated by various methods and stored in a cooler at 10°C. At the end of the prescribed storage period, hens
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F. H. WILCOX AND C. S. SHAFFNER
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
without antibiotics was added to bring the solution to its volume before dilution. The sperm were then resuspended with a small stirring rod. The following variations were used: a. Aerobic conditions were employed during storage, using a container stoppered with cotton. Aerobic conditions were also used in all other treatments, except for (2b). b. Oxygen tension and availability was reduced during storage by filling the container with the diluted semen and using a rubber stopper. The term anaerobic will be used to describe this condition, even though this is somewhat inaccurate in the strict sense of the term. c. The buffer added at the end contained fructose. For this purpose an isotonic solution of fructose (60 mg./ml.) was diluted with the buffer to give 2.5 mg./ml. of fructose. 3. Semen was centrifuged at 1,200 R.C.F. for 10 min. before storage, the seminal plasma was discarded, and enough buffer with antibiotics was added to bring it to its original volume. The sperm were The solution used for dilution, replacement, or dialysis during storage was in then resuspended with a small stirring rod all cases a buffer containing 16.34 g. and stored. The following variations were Na 2 HP0 4 , 5.16 g. NaH 2 P0 4 -H 2 0 per liter used: with a pH of 7.2. It also contained 100 /*g. a. The treated semen was inseminated oxytetracycline hydrochloride and 1,000 without further additions after storage. fig. dihydrostreptomycin per ml. These b. Enough of an isotonic fructose soluantibiotics have been previously shown to tion was added after storage to provide improve fertility (Wilcox and Shorb, the desired level of fructose. 1958). c. Semen was diluted 1 to 10 with bufTreatments. The following handling fer and antibiotics before it was centrifuged, in an attempt to wash the sperm methods were employed: 1. Semen was diluted 1 to 10 by mixing and remove more of the seminal plasma. an equal volume of buffer and antibiotics, No further additions were made after storfollowed by addition of sufficient buffer age. and antibiotics to give a ten-fold dilution. d. Semen was handled as in (3c) before 2. Semen was diluted 1 to 10 as in (1) storage. Enough of an isotonic fructose but after storage was centrifuged at 1,200 solution was added after storage to proR.C.F. for 10 minutes. The supernatant vide the desired level of fructose. was discarded, after which enough buffer 4. Semen was dialyzed during the were inseminated with 0.1 ml. of treated semen. The hens were assigned to each treatment by random. Hens with a hardshelled egg in the oviduct were not inseminated. From 7 to 10 hens were used per treatment, usually 9 or 10. Since these hens were used repeatedly for this purpose, two eggs were saved from each hen before insemination to confirm that the hen was infertile. Eggs laid from the 2nd through 8th day after insemination were candled after 5 days of incubation; any questionable eggs were broken out and observed macroscopically for embryonic development. Hatchability was computed as the number of fertile eggs that had hatched by the 22nd day of incubation. It took approximately 1 hour to inseminate all the hens in a given experiment, which meant that the time the semen was stored varied accordingly. Therefore, to correct for this variation, one-half of the hens designated for each treatment were inseminated first, following which the other half were inseminated in reverse order of treatments.
1355
HANDLING OF CHICKEN SEMEN
TABLE 1.—The effect of different handling conditions on fertility and hatchability thefirstweek after insemination Percent hatchability8
Percent fertility* Treatment
Fresh, undiluted Stored at 10°C. for:
Trial No.
Trial No.
1
2
3
1
2
3
85.7(49)
79.6(54)
96.2(53)
100.0(41)
81.0(42)
98.0(49)
19| hr.
24 hr.
24 hr.
19 J hr.
24 hr.
1. Diluted 1 to 10 with buffer and antibiotics1* and stored aerobically 36.4 (55) 11.5 (52) 31.7 (41)
4. Undiluted, dialyzed against buffer and antibiotics 5. Undiluted, stored aerobically
88.2 (17) 100.0 (6) 92.3 (13)
28.8(52) 50.9(57) 66.7(54) 67.9(56) 42.6(54) 50.9(53)
86.7(15) 81.1(37)
88.0(25) 95.5(22)
94.1 (34) 80.0(25)
78.7(47) 49.1(53)
79.5 (44)
91.9(37)
91.7(24)
97.1(34)
64.7(51) 37.9(58) 34.0(47)
93.9(33)
95.5(22)
93.3(15)
66.1(62) 55.8(52)
90.0(40) 96.4(28)
38.6(57)
2.4(42)
90.5 (21)
0.0 (1)
52.5(59) 42.2(45)
76.7(30)
89.5 (19)
17.6(34)
9.4(53)
100.0 (6)
80.0 (5)
2.4(42)
6.8(59)
100.0 (1)
75.0 (4)
?b ( ) =No. eggs. 100 tig. oxytetracycline hydrochloride and 1,000 fig. dihydrostreptomycin sulfate per ml. ° By centrifuging at 1,200 R.C.F. for 10 min. d 60 mg. fructose/ml.
storage period with 50 to 100 times its volume of buffer and antibiotics. The dialysate was changed once, after 16 hours of storage. RESULTS
Data for three trials on handling methods are given in Table 1. There were two handling methods that provided excellent fertility for semen stored this long (2024 hours). These were when semen was diluted 1 to 10 and concentrated before insemination and when seminal plasma was replaced by the buffer (treatments 2 and 3 above). Dialyzed semen and untreated, stored semen gave consistently poor fertility. Semen diluted 1 to 10 gave poor fertility when not concentrated before insemination. There did not appear to be any difference between anaerobic and aerobic conditions. Two discrepancies in the data are outstanding. One is the low fertility in the first trial with
semen diluted 1 to 10 and concentrated before insemination. The other is the higher fertility from semen washed before storage in Trial 2, as compared to poor fertility from this treatment in Trial 3. In 6 out of 7 comparisons the addition of fructose after storage yielded an increase in fertility when compared to the same handling method without fructose. There did not appear to be any effect of the treatments used on hatchability (Table 1). DISCUSSION
The data presented provide evidence that chicken semen can be stored in vitro for extended periods of time. Unfortunately, most of the conditions used may not be suitable for farm conditions, since centrifugation is necessary. It is of interest that these results were obtained with a diluent containing only sodium, phosphate, and antibiotics. Since removal of
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
2. Diluted 1 to 10 with buffer and antibiotics and brought to original volume0 with fresh buffer at end: a. Stored aerobically b. Stored anaerobically c. Stored aerobically with 2.5 mg. fructose/ml. in buffer at end 3. Seminal plasma replaced with buffer0 and antibiotics at start, stored aerobically: a. No fructose added at end d b. 0.035 ml. fructose solution added to 1.1 ml. semen at end c. Washed by diluting 1 to 10 with buffer and antibiotics before replacement d. Washed 0.035 ml. fructose solution*1 added to 1.1 ml. semen at end
24hr.
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F. H. WILCOX AND C. S. SHAFFNER
Under the conditions of this experiment, dialysis of semen yielded disappointing results. A possible explanation for this may be inhibition of the enzymes involved in fructolysis by dialysis (Baldwin, 1952). The low fertility reported above after storage of undiluted semen was also observed by Warren and Gish (1945), but fertility somewhat higher than this was found by Schindler et al. (1955). Some of this reduction in fertility can be accounted for by inadequate control of pH and bacterial count, since replacement with a buffer and antibiotics greatly improved fertility. SUMMARY
Chicken semen was stored 20-24 hrs. at 10°C. using various methods of handling with a phosphate buffer containing antibiotics. Fertility of eggs laid the first week after insemination was low with untreated semen and dialyzed semen. Fertility was improved by use of a ten-fold dilution,
The authors are indebted to Chas. Pfizer and Co., Inc., New York, New York, for the oxytetracycline hydrochloride. REFERENCES Baldwin, E., 1952. Dynamic Aspects of Biochemistry, 2nd edition. Cambridge: Cambridge University Press. 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: 665-669. Parker, J. E., F. F. McKenzie and H. L. Kempster, 1942. Fertility in the male domestic fowl. Missouri Agr. Exp. Sta. Res. Bui. 347: 1-50. 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. Shaffner, C. S., E. W. Henderson and E. G. Card, 1941. Viability of spermatozoa of the chicken under various environmental conditions. Poultry Sci. 20: 259-265. Warren, D. C, and C. L. Gish, 1943. The value of artificial insemination in poultry breeding work. Poultry Sci. 22: 108-117. Wilcox, F. H., 1958a. Changes in the pH of semen
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
the seminal plasma and replacement with and was further improved by concentratthis diluent did not appear to be harmful, ing the diluted solution to its original volit is questionable whether added nutri- ume just before insemination by using ents are necessary during storage. centrifugation. A similar degree of imIt is noteworthy that the addition of provement was obtained with semen in fructose at insemination increases fer- which the seminal plasma was replaced tility, since no beneficial effect has been by an equal volume of the diluent before observed in a limited number of experi- storage. Inconsistent results were obments in which fructose was added before tained with semen diluted ten-fold and storage. This suggests that one set of con- then replaced by a volume of diluent equal ditions may be optimum during storage, to that of the seminal plasma. In the latand that a different set may be optimum ter three treatments, fertility was further at insemination. One would expect that increased by the addition just before inconditions which slow down sperm metab- semination of sufficient fructose to yield olism without harming the sperm would a concentration of 2 mg./ml. There was be best during storage, whereas conditions little difference in the fertility from sperm which stimulate sperm activity would be stored aerobically and anaerobically. best at insemination. Apparently dilution Hatchability was unaffected by the treatbefore storage, and centrifugation just be- ments used. fore insemination, offers a means by which different conditions may be employed. ACKNOWLEDGEMENT
HANDLING OF CHICKEN SEMEN
of the domestic cock as affected by temperature and frequency of collection. Poultry Sci. 37: 444-449. Wilcox, F. H., 1958b. The effect of dilution and concentration of chicken semen on fertility. Poultry
1357
Sci. 37: 1357-1362. Wilcox, F. H., and M. S. Shorb, 1958. The effect of antibiotics on bacteria in semen and on motility and fertilizing ability of chicken spermatozoa, Am. J. Vet. Res. 19: 945-949.
The Effect of Dilution and Concentration of Chicken Semen on Fertility* WILCOX
(Received for publication April 7, 1958)
I
N ARTIFICIAL insemination of poultry it would be of considerable practical value if semen could be diluted without a decrease in fertility. Any resulting decrease could be the result of either (1) a reduction in sperm concentration and/or sperm number or (2) harmful effects from the diluent used. In investigations on this subject with chickens and turkeys, two general approaches have been used. These have been repeated insemination and a single insemination of each bird. As regards investigations in which repeated insemination was used, Bonnier and Trulsson (1939) inseminated hens 4 to 7 times per week and found little difference in fertility from undiluted semen versus semen diluted 1 to 3 and 1 to 10 with a modified Ringer's solution; there was, however, a decline in fertility from semen diluted 1 to 50. Gordon and Phillips (1951) inseminated hens weekly and obtained better results when semen was diluted 1 to 2 rather than 1 to 5 with Ringer's solution. Gilbreath and Davis (1949) inseminated turkey hens biweekly and found a marked reduction in the fertility from diluted semen, whether * Scientific article No. A684. Contribution No. 2902 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
diluted 1 to 10,1 to 20, 1 to 50, or 1 to 100 with physiological saline. As regards investigations where a single insemination was used, Munro (1938) found fertility to be reduced progressively as the dilution rate was increased from 1 to 2 to 1 to 128 with Milanov's solution and seminal plasma. When the number of sperm inseminated was kept constant by increasing the volume used, the decline in fertility with semen diluted with seminal plasma was much less pronounced. Weakley and Shaffner (1952) found fertility to be little affected when diluted 1 to 3 or 1 to 10 with seminal plasma, but at higher dilution rates to be adversely affected. Van Tienhoven and Steel (1957) diluted turkey semen as much as 1 to 16 with Tyrode's solution, milk, and blood serum, and concluded that there were no significant differences due to dilution rate. However, their data did indicate a tendency for fertility to decline with increasing dilution rate. Rowell and Cooper (1957) found fertility to decrease markedly as the dilution rate of chicken semen was increased from 1 to 2 to 1 to 4.38. Since osmotic pressure was an uncontrolled variable in this experiment, interpretation of the results is difficult. To date the research on this has involved reducing
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
F. H.
Department of Poultry Husbandry, University of Maryland, College Park, Maryland
Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication April 7, 1958)
HE poor success in attempts to store chicken semen in vitro is in sharp contrast to success obtained in storing chicken spermatozoa in the hen's oviduct and in storing bull semen in vitro. Work at this laboratory has provided evidence of the necessity of controlling the pH of semen during storage (Wilcox, 1958a). This can most easily be done by dilution of semen with a buffer. However, dilution of semen, even when inseminated fresh, results in lowered fertility, so that from the outset it can be anticipated that fertility will be lowered when semen is diluted. Evidence has been obtained (Wilcox, 1958b) that semen can be centrifuged and the seminal plasma replaced by a sodium phosphate buffer with little loss of fertilizing capacity. This suggests a procedure by which semen can be diluted and stored, and still be inseminated "undiluted" by centrifuging the diluted semen just before insemination and replacing the supernatant with only enough diluent to bring the solution to its original volume. By using centrifugation it should also be possible to control pH by replacing seminal plasma, which is poorly buffered, with a good buffer, and storing the sperm in that condition until insemination. Accordingly, an experiment was set up to test these methods of handling semen. Scientific Article No. A685. Contribution No. 2903 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
A third method was also attempted: dialysis of undiluted semen against a phosphate buffer containing antibiotics. The idea behind this was that toxic end-products formed by the sperm might account for loss of fertility of stored sperm; if these end-products are of low molecular weight they would be removed by dialysis, and pH would be controlled by entrance of ions of the buffer into the semen. In addition to these treatments, fructose was added in some cases at the end of the storage period just before insemination. Fructose has been shown to increase activity of chicken spermatozoa (Shaffner et al., 1941; Wilcox, unpublished) and it was desired to determine if this increased activity at the time of insemination would boost fertility. MATERIAL AND METHODS
General. Males used as the source of semen were of two breeds: a Flightless strain under development at this station and a broiler-type strain. White Leghorn females in individual batteries were used to test fertilizing ability. Semen was collected by a one-man technique described by Bogdonoff and Shaffner (1954). Semen was pooled and a group of females was inseminated with 0.1 ml. of semen immediately after collection, using a syringe with an extension tube similar to that used by Parker et al. (1942). The remaining semen was then treated by various methods and stored in a cooler at 10°C. At the end of the prescribed storage period, hens
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Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
T
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F. H. WILCOX AND C. S. SHAFFNER
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
without antibiotics was added to bring the solution to its volume before dilution. The sperm were then resuspended with a small stirring rod. The following variations were used: a. Aerobic conditions were employed during storage, using a container stoppered with cotton. Aerobic conditions were also used in all other treatments, except for (2b). b. Oxygen tension and availability was reduced during storage by filling the container with the diluted semen and using a rubber stopper. The term anaerobic will be used to describe this condition, even though this is somewhat inaccurate in the strict sense of the term. c. The buffer added at the end contained fructose. For this purpose an isotonic solution of fructose (60 mg./ml.) was diluted with the buffer to give 2.5 mg./ml. of fructose. 3. Semen was centrifuged at 1,200 R.C.F. for 10 min. before storage, the seminal plasma was discarded, and enough buffer with antibiotics was added to bring it to its original volume. The sperm were The solution used for dilution, replacement, or dialysis during storage was in then resuspended with a small stirring rod all cases a buffer containing 16.34 g. and stored. The following variations were Na 2 HP0 4 , 5.16 g. NaH 2 P0 4 -H 2 0 per liter used: with a pH of 7.2. It also contained 100 /*g. a. The treated semen was inseminated oxytetracycline hydrochloride and 1,000 without further additions after storage. fig. dihydrostreptomycin per ml. These b. Enough of an isotonic fructose soluantibiotics have been previously shown to tion was added after storage to provide improve fertility (Wilcox and Shorb, the desired level of fructose. 1958). c. Semen was diluted 1 to 10 with bufTreatments. The following handling fer and antibiotics before it was centrifuged, in an attempt to wash the sperm methods were employed: 1. Semen was diluted 1 to 10 by mixing and remove more of the seminal plasma. an equal volume of buffer and antibiotics, No further additions were made after storfollowed by addition of sufficient buffer age. and antibiotics to give a ten-fold dilution. d. Semen was handled as in (3c) before 2. Semen was diluted 1 to 10 as in (1) storage. Enough of an isotonic fructose but after storage was centrifuged at 1,200 solution was added after storage to proR.C.F. for 10 minutes. The supernatant vide the desired level of fructose. was discarded, after which enough buffer 4. Semen was dialyzed during the were inseminated with 0.1 ml. of treated semen. The hens were assigned to each treatment by random. Hens with a hardshelled egg in the oviduct were not inseminated. From 7 to 10 hens were used per treatment, usually 9 or 10. Since these hens were used repeatedly for this purpose, two eggs were saved from each hen before insemination to confirm that the hen was infertile. Eggs laid from the 2nd through 8th day after insemination were candled after 5 days of incubation; any questionable eggs were broken out and observed macroscopically for embryonic development. Hatchability was computed as the number of fertile eggs that had hatched by the 22nd day of incubation. It took approximately 1 hour to inseminate all the hens in a given experiment, which meant that the time the semen was stored varied accordingly. Therefore, to correct for this variation, one-half of the hens designated for each treatment were inseminated first, following which the other half were inseminated in reverse order of treatments.
1355
HANDLING OF CHICKEN SEMEN
TABLE 1.—The effect of different handling conditions on fertility and hatchability thefirstweek after insemination Percent hatchability8
Percent fertility* Treatment
Fresh, undiluted Stored at 10°C. for:
Trial No.
Trial No.
1
2
3
1
2
3
85.7(49)
79.6(54)
96.2(53)
100.0(41)
81.0(42)
98.0(49)
19| hr.
24 hr.
24 hr.
19 J hr.
24 hr.
1. Diluted 1 to 10 with buffer and antibiotics1* and stored aerobically 36.4 (55) 11.5 (52) 31.7 (41)
4. Undiluted, dialyzed against buffer and antibiotics 5. Undiluted, stored aerobically
88.2 (17) 100.0 (6) 92.3 (13)
28.8(52) 50.9(57) 66.7(54) 67.9(56) 42.6(54) 50.9(53)
86.7(15) 81.1(37)
88.0(25) 95.5(22)
94.1 (34) 80.0(25)
78.7(47) 49.1(53)
79.5 (44)
91.9(37)
91.7(24)
97.1(34)
64.7(51) 37.9(58) 34.0(47)
93.9(33)
95.5(22)
93.3(15)
66.1(62) 55.8(52)
90.0(40) 96.4(28)
38.6(57)
2.4(42)
90.5 (21)
0.0 (1)
52.5(59) 42.2(45)
76.7(30)
89.5 (19)
17.6(34)
9.4(53)
100.0 (6)
80.0 (5)
2.4(42)
6.8(59)
100.0 (1)
75.0 (4)
?b ( ) =No. eggs. 100 tig. oxytetracycline hydrochloride and 1,000 fig. dihydrostreptomycin sulfate per ml. ° By centrifuging at 1,200 R.C.F. for 10 min. d 60 mg. fructose/ml.
storage period with 50 to 100 times its volume of buffer and antibiotics. The dialysate was changed once, after 16 hours of storage. RESULTS
Data for three trials on handling methods are given in Table 1. There were two handling methods that provided excellent fertility for semen stored this long (2024 hours). These were when semen was diluted 1 to 10 and concentrated before insemination and when seminal plasma was replaced by the buffer (treatments 2 and 3 above). Dialyzed semen and untreated, stored semen gave consistently poor fertility. Semen diluted 1 to 10 gave poor fertility when not concentrated before insemination. There did not appear to be any difference between anaerobic and aerobic conditions. Two discrepancies in the data are outstanding. One is the low fertility in the first trial with
semen diluted 1 to 10 and concentrated before insemination. The other is the higher fertility from semen washed before storage in Trial 2, as compared to poor fertility from this treatment in Trial 3. In 6 out of 7 comparisons the addition of fructose after storage yielded an increase in fertility when compared to the same handling method without fructose. There did not appear to be any effect of the treatments used on hatchability (Table 1). DISCUSSION
The data presented provide evidence that chicken semen can be stored in vitro for extended periods of time. Unfortunately, most of the conditions used may not be suitable for farm conditions, since centrifugation is necessary. It is of interest that these results were obtained with a diluent containing only sodium, phosphate, and antibiotics. Since removal of
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
2. Diluted 1 to 10 with buffer and antibiotics and brought to original volume0 with fresh buffer at end: a. Stored aerobically b. Stored anaerobically c. Stored aerobically with 2.5 mg. fructose/ml. in buffer at end 3. Seminal plasma replaced with buffer0 and antibiotics at start, stored aerobically: a. No fructose added at end d b. 0.035 ml. fructose solution added to 1.1 ml. semen at end c. Washed by diluting 1 to 10 with buffer and antibiotics before replacement d. Washed 0.035 ml. fructose solution*1 added to 1.1 ml. semen at end
24hr.
1356
F. H. WILCOX AND C. S. SHAFFNER
Under the conditions of this experiment, dialysis of semen yielded disappointing results. A possible explanation for this may be inhibition of the enzymes involved in fructolysis by dialysis (Baldwin, 1952). The low fertility reported above after storage of undiluted semen was also observed by Warren and Gish (1945), but fertility somewhat higher than this was found by Schindler et al. (1955). Some of this reduction in fertility can be accounted for by inadequate control of pH and bacterial count, since replacement with a buffer and antibiotics greatly improved fertility. SUMMARY
Chicken semen was stored 20-24 hrs. at 10°C. using various methods of handling with a phosphate buffer containing antibiotics. Fertility of eggs laid the first week after insemination was low with untreated semen and dialyzed semen. Fertility was improved by use of a ten-fold dilution,
The authors are indebted to Chas. Pfizer and Co., Inc., New York, New York, for the oxytetracycline hydrochloride. REFERENCES Baldwin, E., 1952. Dynamic Aspects of Biochemistry, 2nd edition. Cambridge: Cambridge University Press. 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: 665-669. Parker, J. E., F. F. McKenzie and H. L. Kempster, 1942. Fertility in the male domestic fowl. Missouri Agr. Exp. Sta. Res. Bui. 347: 1-50. 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. Shaffner, C. S., E. W. Henderson and E. G. Card, 1941. Viability of spermatozoa of the chicken under various environmental conditions. Poultry Sci. 20: 259-265. Warren, D. C, and C. L. Gish, 1943. The value of artificial insemination in poultry breeding work. Poultry Sci. 22: 108-117. Wilcox, F. H., 1958a. Changes in the pH of semen
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on April 26, 2015
the seminal plasma and replacement with and was further improved by concentratthis diluent did not appear to be harmful, ing the diluted solution to its original volit is questionable whether added nutri- ume just before insemination by using ents are necessary during storage. centrifugation. A similar degree of imIt is noteworthy that the addition of provement was obtained with semen in fructose at insemination increases fer- which the seminal plasma was replaced tility, since no beneficial effect has been by an equal volume of the diluent before observed in a limited number of experi- storage. Inconsistent results were obments in which fructose was added before tained with semen diluted ten-fold and storage. This suggests that one set of con- then replaced by a volume of diluent equal ditions may be optimum during storage, to that of the seminal plasma. In the latand that a different set may be optimum ter three treatments, fertility was further at insemination. One would expect that increased by the addition just before inconditions which slow down sperm metab- semination of sufficient fructose to yield olism without harming the sperm would a concentration of 2 mg./ml. There was be best during storage, whereas conditions little difference in the fertility from sperm which stimulate sperm activity would be stored aerobically and anaerobically. best at insemination. Apparently dilution Hatchability was unaffected by the treatbefore storage, and centrifugation just be- ments used. fore insemination, offers a means by which different conditions may be employed. ACKNOWLEDGEMENT
HANDLING OF CHICKEN SEMEN
of the domestic cock as affected by temperature and frequency of collection. Poultry Sci. 37: 444-449. Wilcox, F. H., 1958b. The effect of dilution and concentration of chicken semen on fertility. Poultry
1357
Sci. 37: 1357-1362. Wilcox, F. H., and M. S. Shorb, 1958. The effect of antibiotics on bacteria in semen and on motility and fertilizing ability of chicken spermatozoa, Am. J. Vet. Res. 19: 945-949.
The Effect of Dilution and Concentration of Chicken Semen on Fertility* WILCOX
(Received for publication April 7, 1958)
I
N ARTIFICIAL insemination of poultry it would be of considerable practical value if semen could be diluted without a decrease in fertility. Any resulting decrease could be the result of either (1) a reduction in sperm concentration and/or sperm number or (2) harmful effects from the diluent used. In investigations on this subject with chickens and turkeys, two general approaches have been used. These have been repeated insemination and a single insemination of each bird. As regards investigations in which repeated insemination was used, Bonnier and Trulsson (1939) inseminated hens 4 to 7 times per week and found little difference in fertility from undiluted semen versus semen diluted 1 to 3 and 1 to 10 with a modified Ringer's solution; there was, however, a decline in fertility from semen diluted 1 to 50. Gordon and Phillips (1951) inseminated hens weekly and obtained better results when semen was diluted 1 to 2 rather than 1 to 5 with Ringer's solution. Gilbreath and Davis (1949) inseminated turkey hens biweekly and found a marked reduction in the fertility from diluted semen, whether * Scientific article No. A684. Contribution No. 2902 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry).
diluted 1 to 10,1 to 20, 1 to 50, or 1 to 100 with physiological saline. As regards investigations where a single insemination was used, Munro (1938) found fertility to be reduced progressively as the dilution rate was increased from 1 to 2 to 1 to 128 with Milanov's solution and seminal plasma. When the number of sperm inseminated was kept constant by increasing the volume used, the decline in fertility with semen diluted with seminal plasma was much less pronounced. Weakley and Shaffner (1952) found fertility to be little affected when diluted 1 to 3 or 1 to 10 with seminal plasma, but at higher dilution rates to be adversely affected. Van Tienhoven and Steel (1957) diluted turkey semen as much as 1 to 16 with Tyrode's solution, milk, and blood serum, and concluded that there were no significant differences due to dilution rate. However, their data did indicate a tendency for fertility to decline with increasing dilution rate. Rowell and Cooper (1957) found fertility to decrease markedly as the dilution rate of chicken semen was increased from 1 to 2 to 1 to 4.38. Since osmotic pressure was an uncontrolled variable in this experiment, interpretation of the results is difficult. To date the research on this has involved reducing
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F. H.
Department of Poultry Husbandry, University of Maryland, College Park, Maryland