2195
TALLOW ABSORPTION
duced by feeding unheated soybean proteins. Proc. Soc. Expt. Biol. Med. 118: 1022-1025. Gomez, M. X., and D. Polin, 1974. Influence of cholic acid on the utilization of fats in the growing chicken. Poultry Sci. 53:773-781. Renner, R., and F. W. Hill, 1960. The utilization of corn oil, lard and tallow by chickens of various ages. Poultry Sci. 39: 849-854. Serafin, J. A., andM. C. Nesheim, 1967. The influence of diet on bile and production and excretion in the chick. Proc. Cornell Nutr. Conf., 1967: 146-150. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th edition, The Iowa State University Press, Ames, Iowa. Webling, D. D'A., and E. S. Holdsworth, 1965. The effect of bile, bile acids and detergents on calcium absorption in the chick. Biochem. J. 97: 408-421. Wiggins, A. S., 1955. A study of the occurrence and relationship of the bile acids and their conjugates. Ph.D. thesis, University of London.
Fertility of Frozen Chicken Semen After Intravaginal and Intrauterine Inseminations Using Various Concentrations and Equilibration Times of Dimethylsulfoxide and a Range of Freezing and Thawing Rates R.
L.
MITCHELL AND R.
B . BUCKLAND
Department of Animal Science, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, Canada H0A 1C0 (Received for publication December 24, 1975)
ABSTRACT The use of dimethyl sulfoxide (DMSO) as a cryoprotective agent for freezing chicken semen was examined with respect to intravaginal (I.V.) and intrauterine (I.U.) inseminations. Semen diluted in DMSO was subjected to various freeze rates, equilibration times and thawing rates. Various concentrations of DMSO were examined as was the effect of diluting DMSO with phosphate buffer and Lake's solution. There was no significant (P > 0.05) effect of rate of freeze (ranging from 1.3° C to 7.2° C. per min) on fertility. However, rapid thawing in an ice bath resulted in a longer (P < 0.05) duration of fertility than thawing at 1° C. per min., but had no effect on percent fertility during duration or percent hens fertile. Equilibration time and concentration of DMSO had no significant (P > 0.05) effects on fertility. There was an interaction between insemination route and Lake's solution vs. phosphate buffer with respect to duration of fertility. It was concluded that intrauterine (I.U.) insemination resulted in superior fertility to intravaginal (I.V.) inseminations with semen frozen in DMSO. POULTRY SCIENCE 55: 2195-2200,
INTRODUCTION
P
OLGE (1949, 1951) found that if fowl semen is diluted to contain 15-20 percent glycerol, it can be frozen to -79° C. and thawed at 40° C without impairing the motility
1976
of the spermatozoa. However, insemination into hens of semen containing more than 5 percent glycerol, frozen or unfrozen, failed to result in any fertile eggs. Dimethyl sulfoxide (DMSO) has received some attention as a cryopreservative as it
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 12, 2015
and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chick to dietary fat. Poultry Sci. 36: 807-815. Duckworth, J., J. M. Naftalin and A. C. Dalgarno, 1950. Digestibility of linseed oil and mutton fat by chicks. J. Agric. Sci. 40: 39-43. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Dunnett, C. W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statistical Assoc. 50: 1096-1121. Edwards, H. M., 1962. Observations on feeding cholic acid to broilers. Poultry Sci. 41: 340-341. Eyssen, H., and P. deSomer, 1963. Toxicity of lithocolic acid for the chick. Poultry Sci. 42: 1020-1021. Fedde, M. R., P. E. Waibel and R. E. Burger, 1960. Factors affecting the absorbability of certain dietary fats in the chick. J. Nutr. 70: 447-452. Garlich, J. D., and M. C. Nesheim, 1964. The effect of sodium taurocholate on fat mal-absorption in-
2196
R. L. MITCHELL AND R. B. BUCKLAND
MATERIALS AND METHODS The freezing apparatus used was as described by Mitchell et al. (1975) and Forgrave and Baker (1975). The solutions used in this study were: (1) phosphate buffer which was composed of 16.34 g. of N a 2 H P 0 4 , 5 . 1 6 g . of^NaH 2 P0 4 , 2.4 g. of fructose, 0.004 g. of oxytetracycline dihydrate, 0.02 g. of dihydrostreptomycin sulfate, and 15.62 g. of DMSO per 100 ml. distilled water, and (2) Lake's solution (Lake, 1968). Pooled semen was collected by the massage technique into disposable culture tubes from 3-5 males (14-15 months of age) of the Ottawa Meat Control line. Two methods of insemination were used: (1) intravaginal (I.V.) in which the oviduct was everted and the semen deposited into the vaginal area and (2) intrauterine (I.U.) in which the oviduct was everted and a finger forced through the uterovaginal junction to guide the insemination straw into the uterus. The females used were 14-15 months of age, except where noted. The duration of fertility was calculated for all laying hens inseminated from day one (the
day after insemination) until the last fertile egg was laid up to 15 days after the insemination. The percent fertility during duration was the percentage of fertile eggs to the total eggs laid during the duration of fertility. The percent hens fertile was calculated as the percentage of laying hens inseminated which laid at least one fertile egg. The percent production over the 15 days post insemination was calculated on a hen-day basis for all hens inseminated. The general procedure for freezing, except where noted, was as outlined below. The pooled semen sample was taken from the laying house to a cold room (5° C.) and allowed to stand for 5 min. Two ml. of semen were then placed in a test tube which was put in an ice bath at — 2° C. Two ml. of phosphate buffer (without DMSO) was added and mixed. Next, 4 ml. of the phosphate buffer (with DMSO) was added in aliquots of 0.4 ml. every 30 sec. with mixing between each addition. This brought the final concentration of DMSO to 1.0 M. The diluted semen was then placed in 0.5 ml. plastic straws (United Breeders Inc., RR#5, Guelph, Ontario), by the use of a 1.0 ml. tuberculin syringe and the straws were then sealed with a thermo impulse sealer (Edwards Agri-Supply Inc., Box 65, Baraboo, Wisconsin, model JE902). The diluted semen was allowed to equilibrate for 15 min. which included the time it took to transfer the samples to the straws and to transport them from the cold room to the freezing apparatus while in the ice bath. The diluted semen was frozen at 1.3° C. per min. to -196° C. and thawed at a rate of 8.7° C. per min. until the samples reached —2° C , at which time they were placed in an ice bath ( - 2 ± 1° C.) and taken to the laying house and inseminated. For all parameters examined,0.5 ml. (one straw) of diluted semen was inseminated per hen to five or
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 12, 2015
does not need to be removed before insemination. However, Lake (1968) reported that DMSO was not as effective as glycerol in protection against freeze damage. Kurbatov et al. (1974) and Harris (1968)used DMSO with limited success. Sexton (1973, 1974, 1975a, b) has reported no deleterious effects of DMSO on metabolism and fertility of unfrozen chicken semen. The objective of the work reported here was to examine, in more detail, the use of DMSO as a cryoprotective agent for chicken semen. Semen diluted in DMSO was subjected to various freeze-rates, thaw-rates and equilibration times. Various concentrations of DMSO were examined as was the effect of diluting DMSO with phosphate buffer and Lake's solution.
2197
FERTILITY OF FROZEN SEMEN
RESULTS AND DISCUSSION Freezing Rate. The basic procedure was followed except for the freezing rates which were: (1) 1.3° C. per min., (2) 3.7° C. per min. and (3) 7.2° C. per min. Freezing rate and the interaction of freezing rate by insemination route were not significant (P > 0.05) for any of the parameters examined (Table 1). These results indicate that over the range studied freezing rate had no effect on fertility. The results agree with the work of Shaffner (1964) and Clark and Shaffner (1960), though they did not agree with those of Lake (1968) who reported that slow freezing gave the best revival of spermatozoa. Insemination route had no effect on dura-
tion of fertility or percent production, but I.V. inseminations resulted in a significant decrease in percent fertility and percent hens fertile. Block had a significant (P < 0.05) effect on duration of fertility and percent hens fertile. Rate of Thaw. The basic procedure was followed except that the rates of thaw investigated were: (1) 1.0° C. per min. and (2) direct from the liquid air into an ice bath at -2° C. and the results are presented in Table 2. Rate of thaw had no significant (P > 0.05) effect on any parameters measured except duration of fertility (P < 0.05) where going directly to the ice bath resulted in an improvement of 3 days in duration of fertility after I.U. insemination. These results are of importance, not only because of a longer duration of fertility, but also because thawing in an ice bath is faster and easier. None of the parameters measured were influenced (P > 0.05) by insemination route, block or the interaction of thaw rate by insemination route. It should be noted that I.V. insemination resulted in zero fertility in this trial. Equilibration Time. The basic procedure was followed except that the effect of allowing the semen and DMSO to equilibrate for various times (15, 45, 75 or 105 min.) before
TABLE 1.—Effects of freezing rates on fertility of semen diluted in DMSO with I. V. and I. U. inseminations 1.3 Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
I.V. 4.0 1.3 7.1 57.8 14 1
Freezing rate ( ° C . / m i n .) 7.2 3.7 3.7 Insemination route I.U. I.V. I.U. I.V. 3.0 5.5 3.5 6.5 1.45 23.5 1.6 26.6 41.6 39.2 7.1 7.1 51.2 57.2 53.3 63.9 15 16 14 15 12 1 11 1 1.3
7.2 I.U. 4.0 25.0 43.8 43.8 16 13
Standard error ±1.4 ±12.9 ±21.1 ±8.9
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six hens I.V. and to five or six hens I.U. Eggs were collected for a 15-day period and incubated within four days of collection. Fertility was based on candling after five or six days of incubation. The treatments, which were examined in separate trials, were: freezing rate, equilibration time with DMSO, thawing rate, concentration of DMSO and the effect of diluting DMSO with phosphate buffer and Lake's solution. Each trial consisted of two blocks; each block represented a separate day. Analysis of variance was used to determine the significance of block, treatment, insemination route, and the treatment by insemination route interaction in each trial.
2198
R. L. MITCHELL AND R. B. BUCKLAND
TABLE 2.—Effects
of thawing procedure on fertility of semen diluted in DMSO with I. V. and I. U. inseminations Thawing procedure l0C./min. Ice bath Insemination route I.U. I.V.
rc./min. I.V.
Standard error
I.U.
0.0
5.3
0.0
8.3
±3.1
0.0 0.0
28.1 38.1
0.0 0.0
32.9 55.5
±13.8
48.6
43.3
55.4
48.5
±7.2
16
17
16
17
0
6
0
17
Because all fertile hens were in one classification the error mean square was zero. TABLE 3.—Effects
Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
of equilibration time on fertility of semen diluted in DMSO with I. V. and I. U. inseminations Equilibration time (min •) 45 75 75 Insemination route I.V. I.U. I.V. I.U.
15
15
45
I.V.
I.U.
0.0
11.0
2.0
8.5
0.5
0.0
13.5
4.2
38.7
0.0
35.0
6.3
56.3
45.2
15 0
105
105
I.V.
I.U.
8.5
0.5
3.0
±3.0
25.0
13.1
50.0
16.6
±30.2
75.0
7.1
24.9
7.1
12.5
±17.9
58.8
61.8
61.1
54.1
56.5
47.1
±8.6
16
16
16
14
15
13
15
6
1
11
1
5
1
2
freezing was examined. The effect of equilibration time, block or the interaction of equilibration time by insemination route were not significant (P > 0.05), (Table 3). The trend, however, with I.U. inseminations seemed to be that as equilibration time increased beyond 45 min. there was a slight decline in reproductive performance. This trend, though not significant (P > 0.05) is in agreement with the work of Shaffner (1964), Harris (1968) and Lake (1968). No trend can be identified from the
Standard error
I.V. inseminations due to the low number of fertile eggs. I.V. inseminations resulted in significantly (P < 0.05) lower duration of fertility and percent hens fertile, but insemination route had no effect on percent fertility during duration or percent egg production. Concentration of Dimethyl Sulfoxide. The basic procedure was followed except that the final concentrations of DMSO examined were: 0.5, 1.0, 1.5 and 2.0 M.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 12, 2015
Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
Ice bath
2199
FERTILITY OF FROZEN SEMEN
TABLE 4.—Effects
Parameter
Concentration of DMSO (M) 1.0 1.0 1.5 1.5 Insemination route I.V. I.U. I.V. I.U.
0.5
0.5
I.V.
I.U.
0.0
5.5
0.0
0.0
0.0
0.0
6.6
0.0
0.0
0.0
30.0
0.0
65.9
49.9
60.2
2.0
2.0
I.V.
I.U.
1.0
0.0
5.0
±3.4
0.0
20.0
0.0
10.5
±10.5
0.0
0.0
33.3
0.0
12.5
±20.3
50.3
56.0
67.9
68.8
52.3
±8.5
8
10
8
10
8
9
8
9
0
4
0
0
0
2
0
4
None of the parameters measured were influenced (P > 0.05) by concentration of DMSO, insemination route or the interaction of concentration of DMSO by insemination route (Table 4). Block had a significant (P < 0.05) effect on percent production only. These results indicated that over the range studied concentration of DMSO had no effect on fertility. However, the extremely poor fertility encountered in this trial must be kept in mind when evaluating the results.
TABLE 5.—Effect
DMSO in Lake's Solution and Phosphate Buffer. The basic procedure was followed except that DMSO (15.626 g. per 100 m.) in Lake's solution was also studied and compared to DMSO in phosphate buffer. Also the females used in this trial were 7 months of age. None of the parameters measured were influenced (P > 0.05) by diluent, insemination route or block (Table 5). However, the interaction of diluent by insemination route was
of DMSO in Lake's solution and phosphate buffer (PB) on the fertility of diluted semen with I. V. and I. U. inseminations DMSO in Lake's
Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
Standard error
I.V.
Diluent DMSO in DMSO in Lake's PB Insemination route I.U. I.V.
DMSO in PB I.U.
Standard error
3.5
1.0
0.0
5.5
±1.3
8.6 15.4
16.6 16.6
0.0 0.0
11.1 30.0
±11.5 ±12.0
75.4
66.6
75.6
76.6
±11.2
14
15
14
16
2
1
0
4
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Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
of DMSO concentration on fertility of diluted semen with I. V. and I. U. inseminations
2200
R. L. MITCHELL AND R. B. BUCKLAND
significant (P < 0.05) with respect to duration of fertility. This interaction does suggest a superiority of Lake's solution for I. V. inseminations and a superiority of phosphate buffer for I.U. inseminations. Intravaginal vs. Intrauterine
Inseminations.
The improved fertility from I.U. insemination is in agreement with the results of Etches et al. (1974) who showed that spermatozoa of lower fertilizing capacity showed a greater inseminations than spermatozoa of a higher fertilizing capacity. Also, these results agree with the work of Harris (1968) who reported higher
fertility
of
frozen
semen
from
intraperitoneal inseminations than intravaginal insemination in the chicken. In view of the cumbersomeness of I.U. insemination and the very poor fertility obtained for all I.V. inseminations, it was concluded that DMSO was, under the conditions studied, not a satisfactory cryopreservative for chicken semen.
REFERENCES Clark, C. E., and C. S. Shaffner, 1960. The fertilizing capacity of frozen chicken sperm and the influence of related in vitro processes. Poultry Sci. 39: 12131220. Etches, R. J., R. B. Buckland and R. O. Hawes, 1974. The effect of the genes for rosecomb and Polydactyly on sperm transport in the hen's oviduct. Poultry Sci. 53: 422-424. Forgrave, L., and R. D. Baker, 1975. Cryopreservation
NEWS AND NOTES (Continued from page 2175) sional career as a poultry scientist he made outstanding contributions to poultry genetics and breeding. His research has included genetic coloration in ducks; growth in turkeys; major gene genetics; genetics of growth and reproduction in egg-and meat-type chickens; hormonal fattening in turkeys and chickens;
genetics of hormone responses; physiological effects of the surplus yolk syndrome and chromosomal aberrations in broilers; and body size, egg size, and egg production interrelationships in egg- and broilertype chickens. A Canadian by birth, he received B.A. and B.S.A.
(Continued on page 2227)
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improvement in fertility after upper tract
of mammalian embryos in Macdonald freeze-thaw apparatus. Abstr. 25th Ann. Meet. Canadian Soc. Animal Sci. p. 18. Harris, G. C , 1968. Fertility of chickens inseminated intraperitoneally with semen preserved in liquid nitrogen. Poultry Sci. 47: 384-388. Kurbatov, A. D., L. E. Narubina and Bi. I. Ivanov, 1974. A test of some cryoprotectants in the freezing of cock semen. Animal Breeding Abstracts, 42 (5): 222. Lake, P. E., 1968. Observations on freezing fowl spermatozoa in liquid nitrogen. VI Cong. Inter. Reprod. Anim. Insem. Artif., Vol. 11. Mitchell, R. L., R. B. Buckland, L. Forgrave and R. D. Baker, 1975. A simple controlled rate freezing apparatus as applied to freezing poultry semen. Poultry Sci. 54: 1796. Polge, C , A. U. Smith and A. S. Parks, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Polge. C , 1951. Functional survival of fowl spermatozoa after freezing at -79° C. Nature, 167: 949-950. Sexton, T. J., 1973. Effect of various cryoprotective agents on the viability and reproductive efficiency of chicken spermatozoa. Poultry Sci. 52: 1353-1357. Sexton, T. J., 1974. Comparison of various cryoprotective agents on washed chicken spermatozoa. 4. Metabolism and release of glutamic-oxalacetic transaminase. Poultry Sci. 53: 284-287. Sexton, T. J., 1975a. Relationship of the method and temperature of cryoprotective agents to the fertilizing capacity of cooled chicken spermatozoa. Poultry Sci. 54: 845-848. Sexton, T. J., 1975b. Comparison of various cryoprotective agents on washed chicken spermatozoa. 5. Effect of glucose, sucrose and polyvinylpyrrolidone. Poultry Sci. 54: 1297-1299. Shaffner, C. S., 1964. Observations on freezing chicken semen. V. Congresso Internazionale per la riproduzione animale et la fecondazione artificiale. Trento, 6-13.
TALLOW ABSORPTION
duced by feeding unheated soybean proteins. Proc. Soc. Expt. Biol. Med. 118: 1022-1025. Gomez, M. X., and D. Polin, 1974. Influence of cholic acid on the utilization of fats in the growing chicken. Poultry Sci. 53:773-781. Renner, R., and F. W. Hill, 1960. The utilization of corn oil, lard and tallow by chickens of various ages. Poultry Sci. 39: 849-854. Serafin, J. A., andM. C. Nesheim, 1967. The influence of diet on bile and production and excretion in the chick. Proc. Cornell Nutr. Conf., 1967: 146-150. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th edition, The Iowa State University Press, Ames, Iowa. Webling, D. D'A., and E. S. Holdsworth, 1965. The effect of bile, bile acids and detergents on calcium absorption in the chick. Biochem. J. 97: 408-421. Wiggins, A. S., 1955. A study of the occurrence and relationship of the bile acids and their conjugates. Ph.D. thesis, University of London.
Fertility of Frozen Chicken Semen After Intravaginal and Intrauterine Inseminations Using Various Concentrations and Equilibration Times of Dimethylsulfoxide and a Range of Freezing and Thawing Rates R.
L.
MITCHELL AND R.
B . BUCKLAND
Department of Animal Science, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, Canada H0A 1C0 (Received for publication December 24, 1975)
ABSTRACT The use of dimethyl sulfoxide (DMSO) as a cryoprotective agent for freezing chicken semen was examined with respect to intravaginal (I.V.) and intrauterine (I.U.) inseminations. Semen diluted in DMSO was subjected to various freeze rates, equilibration times and thawing rates. Various concentrations of DMSO were examined as was the effect of diluting DMSO with phosphate buffer and Lake's solution. There was no significant (P > 0.05) effect of rate of freeze (ranging from 1.3° C to 7.2° C. per min) on fertility. However, rapid thawing in an ice bath resulted in a longer (P < 0.05) duration of fertility than thawing at 1° C. per min., but had no effect on percent fertility during duration or percent hens fertile. Equilibration time and concentration of DMSO had no significant (P > 0.05) effects on fertility. There was an interaction between insemination route and Lake's solution vs. phosphate buffer with respect to duration of fertility. It was concluded that intrauterine (I.U.) insemination resulted in superior fertility to intravaginal (I.V.) inseminations with semen frozen in DMSO. POULTRY SCIENCE 55: 2195-2200,
INTRODUCTION
P
OLGE (1949, 1951) found that if fowl semen is diluted to contain 15-20 percent glycerol, it can be frozen to -79° C. and thawed at 40° C without impairing the motility
1976
of the spermatozoa. However, insemination into hens of semen containing more than 5 percent glycerol, frozen or unfrozen, failed to result in any fertile eggs. Dimethyl sulfoxide (DMSO) has received some attention as a cryopreservative as it
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 12, 2015
and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chick to dietary fat. Poultry Sci. 36: 807-815. Duckworth, J., J. M. Naftalin and A. C. Dalgarno, 1950. Digestibility of linseed oil and mutton fat by chicks. J. Agric. Sci. 40: 39-43. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Dunnett, C. W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statistical Assoc. 50: 1096-1121. Edwards, H. M., 1962. Observations on feeding cholic acid to broilers. Poultry Sci. 41: 340-341. Eyssen, H., and P. deSomer, 1963. Toxicity of lithocolic acid for the chick. Poultry Sci. 42: 1020-1021. Fedde, M. R., P. E. Waibel and R. E. Burger, 1960. Factors affecting the absorbability of certain dietary fats in the chick. J. Nutr. 70: 447-452. Garlich, J. D., and M. C. Nesheim, 1964. The effect of sodium taurocholate on fat mal-absorption in-
2196
R. L. MITCHELL AND R. B. BUCKLAND
MATERIALS AND METHODS The freezing apparatus used was as described by Mitchell et al. (1975) and Forgrave and Baker (1975). The solutions used in this study were: (1) phosphate buffer which was composed of 16.34 g. of N a 2 H P 0 4 , 5 . 1 6 g . of^NaH 2 P0 4 , 2.4 g. of fructose, 0.004 g. of oxytetracycline dihydrate, 0.02 g. of dihydrostreptomycin sulfate, and 15.62 g. of DMSO per 100 ml. distilled water, and (2) Lake's solution (Lake, 1968). Pooled semen was collected by the massage technique into disposable culture tubes from 3-5 males (14-15 months of age) of the Ottawa Meat Control line. Two methods of insemination were used: (1) intravaginal (I.V.) in which the oviduct was everted and the semen deposited into the vaginal area and (2) intrauterine (I.U.) in which the oviduct was everted and a finger forced through the uterovaginal junction to guide the insemination straw into the uterus. The females used were 14-15 months of age, except where noted. The duration of fertility was calculated for all laying hens inseminated from day one (the
day after insemination) until the last fertile egg was laid up to 15 days after the insemination. The percent fertility during duration was the percentage of fertile eggs to the total eggs laid during the duration of fertility. The percent hens fertile was calculated as the percentage of laying hens inseminated which laid at least one fertile egg. The percent production over the 15 days post insemination was calculated on a hen-day basis for all hens inseminated. The general procedure for freezing, except where noted, was as outlined below. The pooled semen sample was taken from the laying house to a cold room (5° C.) and allowed to stand for 5 min. Two ml. of semen were then placed in a test tube which was put in an ice bath at — 2° C. Two ml. of phosphate buffer (without DMSO) was added and mixed. Next, 4 ml. of the phosphate buffer (with DMSO) was added in aliquots of 0.4 ml. every 30 sec. with mixing between each addition. This brought the final concentration of DMSO to 1.0 M. The diluted semen was then placed in 0.5 ml. plastic straws (United Breeders Inc., RR#5, Guelph, Ontario), by the use of a 1.0 ml. tuberculin syringe and the straws were then sealed with a thermo impulse sealer (Edwards Agri-Supply Inc., Box 65, Baraboo, Wisconsin, model JE902). The diluted semen was allowed to equilibrate for 15 min. which included the time it took to transfer the samples to the straws and to transport them from the cold room to the freezing apparatus while in the ice bath. The diluted semen was frozen at 1.3° C. per min. to -196° C. and thawed at a rate of 8.7° C. per min. until the samples reached —2° C , at which time they were placed in an ice bath ( - 2 ± 1° C.) and taken to the laying house and inseminated. For all parameters examined,0.5 ml. (one straw) of diluted semen was inseminated per hen to five or
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 12, 2015
does not need to be removed before insemination. However, Lake (1968) reported that DMSO was not as effective as glycerol in protection against freeze damage. Kurbatov et al. (1974) and Harris (1968)used DMSO with limited success. Sexton (1973, 1974, 1975a, b) has reported no deleterious effects of DMSO on metabolism and fertility of unfrozen chicken semen. The objective of the work reported here was to examine, in more detail, the use of DMSO as a cryoprotective agent for chicken semen. Semen diluted in DMSO was subjected to various freeze-rates, thaw-rates and equilibration times. Various concentrations of DMSO were examined as was the effect of diluting DMSO with phosphate buffer and Lake's solution.
2197
FERTILITY OF FROZEN SEMEN
RESULTS AND DISCUSSION Freezing Rate. The basic procedure was followed except for the freezing rates which were: (1) 1.3° C. per min., (2) 3.7° C. per min. and (3) 7.2° C. per min. Freezing rate and the interaction of freezing rate by insemination route were not significant (P > 0.05) for any of the parameters examined (Table 1). These results indicate that over the range studied freezing rate had no effect on fertility. The results agree with the work of Shaffner (1964) and Clark and Shaffner (1960), though they did not agree with those of Lake (1968) who reported that slow freezing gave the best revival of spermatozoa. Insemination route had no effect on dura-
tion of fertility or percent production, but I.V. inseminations resulted in a significant decrease in percent fertility and percent hens fertile. Block had a significant (P < 0.05) effect on duration of fertility and percent hens fertile. Rate of Thaw. The basic procedure was followed except that the rates of thaw investigated were: (1) 1.0° C. per min. and (2) direct from the liquid air into an ice bath at -2° C. and the results are presented in Table 2. Rate of thaw had no significant (P > 0.05) effect on any parameters measured except duration of fertility (P < 0.05) where going directly to the ice bath resulted in an improvement of 3 days in duration of fertility after I.U. insemination. These results are of importance, not only because of a longer duration of fertility, but also because thawing in an ice bath is faster and easier. None of the parameters measured were influenced (P > 0.05) by insemination route, block or the interaction of thaw rate by insemination route. It should be noted that I.V. insemination resulted in zero fertility in this trial. Equilibration Time. The basic procedure was followed except that the effect of allowing the semen and DMSO to equilibrate for various times (15, 45, 75 or 105 min.) before
TABLE 1.—Effects of freezing rates on fertility of semen diluted in DMSO with I. V. and I. U. inseminations 1.3 Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
I.V. 4.0 1.3 7.1 57.8 14 1
Freezing rate ( ° C . / m i n .) 7.2 3.7 3.7 Insemination route I.U. I.V. I.U. I.V. 3.0 5.5 3.5 6.5 1.45 23.5 1.6 26.6 41.6 39.2 7.1 7.1 51.2 57.2 53.3 63.9 15 16 14 15 12 1 11 1 1.3
7.2 I.U. 4.0 25.0 43.8 43.8 16 13
Standard error ±1.4 ±12.9 ±21.1 ±8.9
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six hens I.V. and to five or six hens I.U. Eggs were collected for a 15-day period and incubated within four days of collection. Fertility was based on candling after five or six days of incubation. The treatments, which were examined in separate trials, were: freezing rate, equilibration time with DMSO, thawing rate, concentration of DMSO and the effect of diluting DMSO with phosphate buffer and Lake's solution. Each trial consisted of two blocks; each block represented a separate day. Analysis of variance was used to determine the significance of block, treatment, insemination route, and the treatment by insemination route interaction in each trial.
2198
R. L. MITCHELL AND R. B. BUCKLAND
TABLE 2.—Effects
of thawing procedure on fertility of semen diluted in DMSO with I. V. and I. U. inseminations Thawing procedure l0C./min. Ice bath Insemination route I.U. I.V.
rc./min. I.V.
Standard error
I.U.
0.0
5.3
0.0
8.3
±3.1
0.0 0.0
28.1 38.1
0.0 0.0
32.9 55.5
±13.8
48.6
43.3
55.4
48.5
±7.2
16
17
16
17
0
6
0
17
Because all fertile hens were in one classification the error mean square was zero. TABLE 3.—Effects
Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
of equilibration time on fertility of semen diluted in DMSO with I. V. and I. U. inseminations Equilibration time (min •) 45 75 75 Insemination route I.V. I.U. I.V. I.U.
15
15
45
I.V.
I.U.
0.0
11.0
2.0
8.5
0.5
0.0
13.5
4.2
38.7
0.0
35.0
6.3
56.3
45.2
15 0
105
105
I.V.
I.U.
8.5
0.5
3.0
±3.0
25.0
13.1
50.0
16.6
±30.2
75.0
7.1
24.9
7.1
12.5
±17.9
58.8
61.8
61.1
54.1
56.5
47.1
±8.6
16
16
16
14
15
13
15
6
1
11
1
5
1
2
freezing was examined. The effect of equilibration time, block or the interaction of equilibration time by insemination route were not significant (P > 0.05), (Table 3). The trend, however, with I.U. inseminations seemed to be that as equilibration time increased beyond 45 min. there was a slight decline in reproductive performance. This trend, though not significant (P > 0.05) is in agreement with the work of Shaffner (1964), Harris (1968) and Lake (1968). No trend can be identified from the
Standard error
I.V. inseminations due to the low number of fertile eggs. I.V. inseminations resulted in significantly (P < 0.05) lower duration of fertility and percent hens fertile, but insemination route had no effect on percent fertility during duration or percent egg production. Concentration of Dimethyl Sulfoxide. The basic procedure was followed except that the final concentrations of DMSO examined were: 0.5, 1.0, 1.5 and 2.0 M.
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Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
Ice bath
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FERTILITY OF FROZEN SEMEN
TABLE 4.—Effects
Parameter
Concentration of DMSO (M) 1.0 1.0 1.5 1.5 Insemination route I.V. I.U. I.V. I.U.
0.5
0.5
I.V.
I.U.
0.0
5.5
0.0
0.0
0.0
0.0
6.6
0.0
0.0
0.0
30.0
0.0
65.9
49.9
60.2
2.0
2.0
I.V.
I.U.
1.0
0.0
5.0
±3.4
0.0
20.0
0.0
10.5
±10.5
0.0
0.0
33.3
0.0
12.5
±20.3
50.3
56.0
67.9
68.8
52.3
±8.5
8
10
8
10
8
9
8
9
0
4
0
0
0
2
0
4
None of the parameters measured were influenced (P > 0.05) by concentration of DMSO, insemination route or the interaction of concentration of DMSO by insemination route (Table 4). Block had a significant (P < 0.05) effect on percent production only. These results indicated that over the range studied concentration of DMSO had no effect on fertility. However, the extremely poor fertility encountered in this trial must be kept in mind when evaluating the results.
TABLE 5.—Effect
DMSO in Lake's Solution and Phosphate Buffer. The basic procedure was followed except that DMSO (15.626 g. per 100 m.) in Lake's solution was also studied and compared to DMSO in phosphate buffer. Also the females used in this trial were 7 months of age. None of the parameters measured were influenced (P > 0.05) by diluent, insemination route or block (Table 5). However, the interaction of diluent by insemination route was
of DMSO in Lake's solution and phosphate buffer (PB) on the fertility of diluted semen with I. V. and I. U. inseminations DMSO in Lake's
Parameter Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
Standard error
I.V.
Diluent DMSO in DMSO in Lake's PB Insemination route I.U. I.V.
DMSO in PB I.U.
Standard error
3.5
1.0
0.0
5.5
±1.3
8.6 15.4
16.6 16.6
0.0 0.0
11.1 30.0
±11.5 ±12.0
75.4
66.6
75.6
76.6
±11.2
14
15
14
16
2
1
0
4
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Duration of fertility (days) Percent fertility during duration Percent hens fertile Percent production (15 days) Number of hens inseminated Number of fertile eggs
of DMSO concentration on fertility of diluted semen with I. V. and I. U. inseminations
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R. L. MITCHELL AND R. B. BUCKLAND
significant (P < 0.05) with respect to duration of fertility. This interaction does suggest a superiority of Lake's solution for I. V. inseminations and a superiority of phosphate buffer for I.U. inseminations. Intravaginal vs. Intrauterine
Inseminations.
The improved fertility from I.U. insemination is in agreement with the results of Etches et al. (1974) who showed that spermatozoa of lower fertilizing capacity showed a greater inseminations than spermatozoa of a higher fertilizing capacity. Also, these results agree with the work of Harris (1968) who reported higher
fertility
of
frozen
semen
from
intraperitoneal inseminations than intravaginal insemination in the chicken. In view of the cumbersomeness of I.U. insemination and the very poor fertility obtained for all I.V. inseminations, it was concluded that DMSO was, under the conditions studied, not a satisfactory cryopreservative for chicken semen.
REFERENCES Clark, C. E., and C. S. Shaffner, 1960. The fertilizing capacity of frozen chicken sperm and the influence of related in vitro processes. Poultry Sci. 39: 12131220. Etches, R. J., R. B. Buckland and R. O. Hawes, 1974. The effect of the genes for rosecomb and Polydactyly on sperm transport in the hen's oviduct. Poultry Sci. 53: 422-424. Forgrave, L., and R. D. Baker, 1975. Cryopreservation
NEWS AND NOTES (Continued from page 2175) sional career as a poultry scientist he made outstanding contributions to poultry genetics and breeding. His research has included genetic coloration in ducks; growth in turkeys; major gene genetics; genetics of growth and reproduction in egg-and meat-type chickens; hormonal fattening in turkeys and chickens;
genetics of hormone responses; physiological effects of the surplus yolk syndrome and chromosomal aberrations in broilers; and body size, egg size, and egg production interrelationships in egg- and broilertype chickens. A Canadian by birth, he received B.A. and B.S.A.
(Continued on page 2227)
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improvement in fertility after upper tract
of mammalian embryos in Macdonald freeze-thaw apparatus. Abstr. 25th Ann. Meet. Canadian Soc. Animal Sci. p. 18. Harris, G. C , 1968. Fertility of chickens inseminated intraperitoneally with semen preserved in liquid nitrogen. Poultry Sci. 47: 384-388. Kurbatov, A. D., L. E. Narubina and Bi. I. Ivanov, 1974. A test of some cryoprotectants in the freezing of cock semen. Animal Breeding Abstracts, 42 (5): 222. Lake, P. E., 1968. Observations on freezing fowl spermatozoa in liquid nitrogen. VI Cong. Inter. Reprod. Anim. Insem. Artif., Vol. 11. Mitchell, R. L., R. B. Buckland, L. Forgrave and R. D. Baker, 1975. A simple controlled rate freezing apparatus as applied to freezing poultry semen. Poultry Sci. 54: 1796. Polge, C , A. U. Smith and A. S. Parks, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Polge. C , 1951. Functional survival of fowl spermatozoa after freezing at -79° C. Nature, 167: 949-950. Sexton, T. J., 1973. Effect of various cryoprotective agents on the viability and reproductive efficiency of chicken spermatozoa. Poultry Sci. 52: 1353-1357. Sexton, T. J., 1974. Comparison of various cryoprotective agents on washed chicken spermatozoa. 4. Metabolism and release of glutamic-oxalacetic transaminase. Poultry Sci. 53: 284-287. Sexton, T. J., 1975a. Relationship of the method and temperature of cryoprotective agents to the fertilizing capacity of cooled chicken spermatozoa. Poultry Sci. 54: 845-848. Sexton, T. J., 1975b. Comparison of various cryoprotective agents on washed chicken spermatozoa. 5. Effect of glucose, sucrose and polyvinylpyrrolidone. Poultry Sci. 54: 1297-1299. Shaffner, C. S., 1964. Observations on freezing chicken semen. V. Congresso Internazionale per la riproduzione animale et la fecondazione artificiale. Trento, 6-13.
