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Fertility of Chickens Inseminated Intraperitoneally with Semen Preserved in Liquid Nitrogen1
Fertility of Chickens Inseminated Intraperitoneally with Semen Preserved in Liquid Nitrogen 1 G. C. HARRIS, JR. Department of Animal Sciences, University of Arkansas, Payelteville, Arkansas 72701 (Received for publication February 27, 1967)
INTRODUCTION
I
1 The research reported in this paper was supported in part by a grant-in-aid from the American Poultry and Hatchery Federation.
tors have reported that glycerol removal was necessary to obtain fertility (Polge, 1951; Clark and Shaffner, 1960). The purpose of this study was to determine the effects of insemination method, cooling rate, sample volume, equilibration time, and egg yolk concentration of the diluent on fertility. In these experiments, dimethylsulfoxide (DMSO) and polyvinylpyrrolidone (PVP), rather than glycerol, were used as the protective agents for the spermatozoa during freezing (Table 1). EXPERIMENTAL PROCEDURE
Pooled semen was obtained from 24 New Hampshire males by the abdominal massage technique (Burrows and Quinn, 1937). The pooled sample of semen was diluted 1 to 4 with non-yolk diluents in two additions of equal volume at 15-minute intervals. All samples were either immediately frozen or further equilibrated at 2°C. for periods up to 24 hours before freezing. Samples were frozen using a Linde BF 3-1 Biological Freezer at cooling rates from 1°C. to 6.7°C. per minute. Temperature was continuously recorded for a single sample during each cooling cycle. All samples were transferred directly to a liquid nitrogen refrigerator ( —196°C.) when sample temperature had declined to — 80°C. The storage periods at — 196°C. were for 3 or 12 days. All samples were rapidly thawed at 41°C. immediately before insemination. Three replicate samples with a total volume of 0.4 ml. or 1.2 ml. of diluted semen per sample were frozen per treatment.
384
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NTRAPERITONEAL insemination, the deposition of fresh semen in the peritoneal cavity in the area of the infundibulum, has resulted in fertilization in the chicken (Kosin, 1944; Van Drimmelen, 1945; Grigg and Skaller, 1958; Brown et al., 1963) and pigeon (Van Drimmelen, 1951). In earlier work at this laboratory, the intraperitoneal method of insemination was used for frozen chicken semen (Brown et al., 1963) and the best fertility obtained was 28%. Data are limited on the effects of equilibration and cooling rate before freezing on the survival of chicken spermatozoa. Polge et al. (1949) observed that slowly cooled fowl spermatozoa regained full motility upon thawing. No spermatozoa survived rapid freezing to — 79°C. Clark and Shaffner (1960) reported that slow cooling with long equilibration was not necessary when glycerol was added to chicken spermatozoa suspended in phosphate buffer. Brown and Harris (1963) noted that chicken semen equilibrated in a glycerol diluent for 1.5 hours at 2°C. had higher oxygen uptake, motility, and fertility when compared to 1.0 hour and 0.5 hour equilibrations. Equilibration before freezing reduced the harmful effects of glycerol on fertilizing capacity. The removal of glycerol before insemination was not required for fertilization. Other investiga-
385
LIQUID NITROGEN PRESERVED SEMEN TABLE 1.—Composition of diluents Standard solutions Dimethyl sulfoxide6 (ml.)
Distilled water (ml.)
Total volume (ml.)
3.00 3.00
.40 .40
0.00 0.00
0.00 3.20
6.60 3.40
15.00 15.00
5.00 5.00
3.00 3.00
.40 .40
1.00 1.00
0.00 3.20
5.60 2.40
15.00 15.00
3a b
5.00 5.00
3.00 3.00
.40 .40
2.00 2.00
2.00 3.20
4.60 1.40
15.00 15.00
4a b
5.00 5.00
3.00 3.00
.40 .40
3.00 3.00
0.00 3.20
3.60 0.40
15.00 15.00
Monosodium glutamate 1 (ml.)
Antibiotics (ml.)
la b
5.00 5.00
2a b
2
1
Monosodium glutamate (100 mg./ml.). Oxytetracycline hydrochloride (2 mg./ml.) and dihydrostreptomycin sulfate (10 mg,/ml.), supplied by Chas. Pfizer & Co. Laboratories, Dallas, Texas. 3 Polyvinylpyrrolidone, Type PVP-kl5, (100 mg./ml.), General Aniline and Film Corporation, New York N. Y. 4 One-week-old turkey yolk. 6 Dimethylsulfoxide, K and K Laboratories, Inc., Plainview, N . Y. 2
Five White Leghorn hens were inseminated per treatment with 0.2 ml. of diluted semen. This dosage was equivalent to 0.05 ml. undiluted semen. The hens were inseminated by either the intraperitoneal or intravaginal method. Fertility data were obtained from eggs laid the 2nd and 8th day after insemination. After 7 days of incubation, fertility was determined by candling. The embryos were not considered fertile unless normal blood vessel development was observed. Hatchability was recorded on the 22nd day of incubation. Data were analyzed by analysis of variance after transformation percentages had been converted to arc sine values (Steel and Torrie, 1960). RESULTS AND DISCUSSION
In three replicate trials, intraperitoneal (IP) insemination gave significantly higher (P<.01) fertility than intravaginal (IV) insemination (Tables 2 and 3). No significant differences in fertility were noted due to cooling rate, volume of di-
luted semen frozen per sample, or equilibration time. The only significant interaction was between equilibration time and insemination method (P<.05). Semen that was equilibrated for 24 hours fertilized only 2% of the hens with IV insemination; whereas, 45% of the hens were fertilized by IP insemination. The removal of the protective agents prior to insemination was not required for normal embryonic development. The average hatch ability of fertile eggs was 92% and 84% from hens that were inseminated by the IP and IV methods, respectively. Data are not available on the rate of cooling with liquid nitrogen before and after freezing chicken spermatozoa. The cooling rates within various temperature ranges for optimum survival of bull spermatozoa have been studied using liquid nitrogen. Bruemmer et al. (1963) reported that bull spermatozoa survival was highest with cooling rates of 1°C. to 5°C. per minute between 0°C. and — 10°C. Fertility data were compared from several trials using the same diluent for cool-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
PVP-K153 (ml.)
Turkey egg yolk4 (ml.)
Diluent No.
386
G. C. HARRIS, J R . TABLE 2.—Effect
Treatment no.
3 4
Cooling rate (°C. per min.)
Equilibration time (hrs.)
Fertility of total eggs laid during days 2-6 inc. after single insemination
Volume of diluted semen frozen per sample (ml.) -
:IP
2
IV 3
Trials
Trials
1
2
3
Mean 4
1
Percent fertility 38 20 24 35
2
3
Mean 4
0 0
0 0
7 12
1 2
0.5 0.5
1.0 1.0
0.4 1.2
75 11
25 63
9 0
3 4
0.5 0.5
6.0 6.0
0.4 1.2
14 55
6 24
42 11
24 35
35 5
6 16
42 15
27 11
5 6
24.0 24.0
1.0 1.0
0.4 1.2
37 24
33 20
6 23
32 19
0 0
0 0
0 5
0 2
7 8
24.0 24.0
6.0 6.0
0.4 1.2
67 75
47 0
13 29
44 41
0 0
0 0
0 0
0 0
Diluted with Diluent 1 and stored 3 days at -196°C. IP=Intraperitoneal Insemination. IV=Intravaginal Insemination. Standard error of mean +8.42.
ing rates between 1.2°C. to 6.7°C. per minute (Table 3). The lowest average fertility was with cooling rates of 1.2°C. per TABLE 3.—Analysis of variance showing mean squares and "F" values for fertility data of Table 2 Source
d.f.
M.S.
Total Replicates Equilibration time (A) Cooling rate (B) Volume of diluted semen frozen (C) Insemination method (D) AXB AXC AXD BXC BXD CXD AXBXC AXBXD AXCXD BXBXD AXBXCXD Error
47 2
708.43
3.33*
1 1
597.57 246.65
2.81 NNSS 1.16
1
59.36
0.28 N S
1 1 1 1 1 1 1 1 1 1 1 1 30
5,414.34 59.19 9.53 1,109.40 11.24 33.24 26.27 56.02 450.99 48.33 151.03 245.46 212.82
25.44** NS 0.28 NS 0.04 5.21* 0.05 N S 0.16 NS 0.12 NS 0.26 NS 2.12 NS 0.23 N S 0.71 N S 1.15NS
* Significant at .05 level. ** Significant at .01 level. NS Nonsignificant.
F ratio
minute and 6.7°C. per minute. This indicates that the cooling rate requirements before freezing for chicken spermatozoa are similar to other species. The highest fertility (59%) was obtained with a cooling rate of 5.9°C. Thus, a cooling rate of 6°C. per minute was used in all later trials. With IP insemination, 50% of the hens were fertile, and the average hatchability of fertile eggs was 93%. Fresh chicken egg yolk has been widely used in citrate diluents to protect bull spermatozoa against temperature shock during freezing (Willett and Salisbury, 1942). The addition of fresh egg yolk to the diluent has resulted in a makred reduction in the fertility of stored nonfrozen spermatozoa of the chicken (Wilcox, 1960) and turkey (Bajpai and Brown, 1963). Preliminary storage experiments at 2°C. for 24 hours showed that chicken egg yolk was more toxic to chicken spermatozoa than turkey egg yolk. Chicken spermatozoa stored in a diluent containing the yolk from turkey eggs one-week-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
1 2
of equilibration, cooling rate and sample volume on fertility of semen inseminated by two methods1
387
LIQUID NITROGEN PRESERVED SEMEN
TABLE 4.—Effect of cooling rate on fertility1
2
Treatment Cooling rate (°C. per min.) No.
Fertility of total eggs laid during days 2-6 incl. after single insemination Trials 1 2
1.2 1.9 3.9 5.1 5.9 6.7
0 29 73 33 73 14
33 14 23 42 55 12
3
4
5
6 Mean'
Percent fertility 33 75 25 9 33 46 35 71 57 14 — — 39 40 — — 50 — — — 17 44 — —
T A B L E 5.—Effect of equilibration yolk level on fertility1
Fertility of total eggs laid during days 2-8 Total in cl aft er Treatment Diluent equilibration Y°JH' . - . f! n 8 le 1 No. No. time S™1 insemination (hrs.) (%) Trials
29 38 42 39 59 22
Mean3
1
Percent fertility 33 55 44 41 58 31 25 13 20 30 42 50 44 23 13 9 9
0 6.7 13.3 20.0 0 6.7 13.3 20.0
1 Cooling rate of 6°C. per minute; stored for 12 days at -196°C; IP insemination; 0.4 ml. diluted semen per sample. 2 Percent turkey egg yolk in diluent. 3 Standard error of mean ± 6.60.
terial multiplication appeared to become a major problem with the addition of egg yolk and extended equilibration. T h e yolk diluent was compared to the non-yolk diluent with equilibration times up to 24 hours (Table 6). The fertility of eggs fertilized with spermatozoa equilibrated in the non-yolk diluent was significantly higher ( P < . 0 5 ) for T r e a t m e n t 1 (0.5 hour equilibration) than T r e a t m e n t 3 (3.0 hours equilibration). Table 5 shows t h a t higher fertility was obtained with sperm equilibrated in the yolk diluent for 0.5 hour than with sperm equilibrated for
TABLE 6.—Effect of equilibration time and addition of yolk on fertility1 Fertility of total eggs laid during day!3 2-6 incl . after single insemination Trials Treatment no.
1 2 3 4 5 6
Equilibration time (hrs.)
0.5 1.5 3.0 6.0 12.0 24.0
Mean 2
4
3
1
2
No Yolk3
No Yolk3
No Yolk3
Yolk4
No Yolk3
Yolk4
No Yolk3
Yolk4
39 25 50 59 50 41
50 60 0 27 0 0
73 16 11 32 50 33
73 11 13 50 11 21
40 29 18 24 36 18
80 17 47 30 56 17
51 33 20 36 34 23
77 14 30 40 34 19
1 Cooling rate of 6°C. per minute; equilibration temperature 30°C. for 0.5 hour then held at 2°C; stored for 212 days at — 196°C; IP insemination; 0.4 ml diluted semen per sample. Standard error of mean + 6.79. 3 Diluent 1. 4 Diluent 2 (6.7% yolk).
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
0.5 0.5 0.5 0.5 1.5 1.5 1.5 1.5
1 Diluted with Diluent 1; stored for an average of 12 days at 2— 196°C; IP insemination; 0.4 ml. diluted semen per sample. Cooling rate represents a linear temperature change from 30°C. until frozen. 8 Standard error of the mean ± 6.65.
old gave higher fertility than spermatozoa stored in a fresh yolk diluent. Based on these findings, the yolk of one-week-old turkey eggs was used in the yolk diluents for freezing studies on chicken spermatozoa. T h e effects of turkey egg yolk level in the diluent and equilibration time on fertility of chicken spermatozoa after freezing were studied. Highest fertilities, 5 8 % (Table 5) and 7 7 % (Table 6), were obtained with 0.5 hour equilibration and 6.7% yolk in the diluent. The fertility was lowered when the yolk level was increased above 6.7% and the equilibration period extended beyond 0.5 hour (Table 5). Bac-
time and
388
G. C. HARRIS, JR.
1.5, 3.0, 12.0 and 24.0 hours. Six hours of equilibration gave lower fertility, but the differences were non-significant. The average hatchability of fertile eggs for all treatments was 92% (Tables 4 and 5). In this experiment, 47% of the hens were fertile. SUMMARY
ACKNOWLED GMENT
The author wishes to express his appreciation to Chas. Pfizer & Co., Dallas, Texas for supplying the antibiotics used in this study. REFERENCES Bajpai, P. K., and K. I. Brown, 1963. The effect of some diluents on semen characteristics of turkeys. Poultry Sci. 42: 882-888. Brown, J. E., and G. C. Harris, Jr., 1963. The influence of glycerol equilibration time on the metabolism, motility and fertility of frozen chicken spermatozoa. Poultry Sci. 42: 383-386.
JULY 21-26. SIXTH INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, PARIS, FRANCE.
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The effects of cooling rate, equilibration time, and addition of turkey egg yolk to a glutamate diluent on the fertility of frozen chicken spermatozoa were studied. Diluted chicken spermatozoa were cooled with liquid nitrogen at linear cooling rates between 1°C. and 6.7°C. per minute from 30°C. until frozen. Semen samples were stored for periods up to 12 days in liquid nitrogen ( - 1 9 6 ° C ) . Highest fertility (77%) was obtained with intraperitoneally inseminated semen that was equilibrated 0.5 hour in a turkey egg yolk diluent and cooled at a rate of 6°C. per minute. Fertility of spermatozoa diluted with egg yolk diluent was significantly reduced by longer equilibration periods. The hatchability of fertile eggs for all trials was 91.4%.
Brown, J. E., G. C. Harris, Jr. and T. D. Hobbs, 1963. Effect of intraperitoneal insemination on egg production and fertilizing capacity of fresh and frozen chicken sperm. Poultry Sci. 42: 810-815. Bruemmer, J. H., R. W. Eddy and W. J. Duryea, 1963. Temperature control in the low temperature preservation of spermatozoa. J. Cell. Comp. Physiol. 62: 113-117. Burrows, W. H., and J. P. Quinn, 1937. Collection of spermatozoa from domestic fowl and turkey. Poultry Sci. 16: 19-24. Clark, C. E., and S. S. Shaffner, 1960. The fertilizing capacity of frozen chicken sperm and the influence of related in vitro processes. Poultry Sci. 39: 1213-1220. Grigg, G. W., and F. Skaller, 1958. A case of sterility in the hen. Poultry Sci. 37: 954-955. Kosin, I. L., 1944. Some aspects of the biological action of X-rays on cock spermatozoa. Physiol. Zool. 17:289-319. Polge, C , 1951. Functional survival of fowl spermatozoa after freezing at — 79°C. Nature, 167: 949-950. Polge, C , A. V. Smith and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, McGraw-Hill Book Co., New York. Van Drimmelen, G. C , 1945. Intraperitoneal insemination of birds. J. South African Vet. Med. Assoc. 16: 1-6. Van Drimmelen, G. C , 1951. Artificial insemination of birds by the intraperitoneal route. Onderstepoort J. Vet. Res. Suppl. No. 1: 212. Wilcox, F. H., 1960. Effect on fertility of temperature, handling methods, Lake's solution, and the addition of egg white, egg yolk, and sugars to the diluent used in storing chicken semen. Poultry Sci. 39: 459-467. Willett, E. L., and G. W. Salisbury, 1942. The effect of various dilutors, cooling rate, temperature of storage, and some other factors on livability of spermatozoa in stored samples of bull semen. Cornell Univ. Agr. Expt. Sta. Mem. 249.
INTRODUCTION
I
1 The research reported in this paper was supported in part by a grant-in-aid from the American Poultry and Hatchery Federation.
tors have reported that glycerol removal was necessary to obtain fertility (Polge, 1951; Clark and Shaffner, 1960). The purpose of this study was to determine the effects of insemination method, cooling rate, sample volume, equilibration time, and egg yolk concentration of the diluent on fertility. In these experiments, dimethylsulfoxide (DMSO) and polyvinylpyrrolidone (PVP), rather than glycerol, were used as the protective agents for the spermatozoa during freezing (Table 1). EXPERIMENTAL PROCEDURE
Pooled semen was obtained from 24 New Hampshire males by the abdominal massage technique (Burrows and Quinn, 1937). The pooled sample of semen was diluted 1 to 4 with non-yolk diluents in two additions of equal volume at 15-minute intervals. All samples were either immediately frozen or further equilibrated at 2°C. for periods up to 24 hours before freezing. Samples were frozen using a Linde BF 3-1 Biological Freezer at cooling rates from 1°C. to 6.7°C. per minute. Temperature was continuously recorded for a single sample during each cooling cycle. All samples were transferred directly to a liquid nitrogen refrigerator ( —196°C.) when sample temperature had declined to — 80°C. The storage periods at — 196°C. were for 3 or 12 days. All samples were rapidly thawed at 41°C. immediately before insemination. Three replicate samples with a total volume of 0.4 ml. or 1.2 ml. of diluted semen per sample were frozen per treatment.
384
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
NTRAPERITONEAL insemination, the deposition of fresh semen in the peritoneal cavity in the area of the infundibulum, has resulted in fertilization in the chicken (Kosin, 1944; Van Drimmelen, 1945; Grigg and Skaller, 1958; Brown et al., 1963) and pigeon (Van Drimmelen, 1951). In earlier work at this laboratory, the intraperitoneal method of insemination was used for frozen chicken semen (Brown et al., 1963) and the best fertility obtained was 28%. Data are limited on the effects of equilibration and cooling rate before freezing on the survival of chicken spermatozoa. Polge et al. (1949) observed that slowly cooled fowl spermatozoa regained full motility upon thawing. No spermatozoa survived rapid freezing to — 79°C. Clark and Shaffner (1960) reported that slow cooling with long equilibration was not necessary when glycerol was added to chicken spermatozoa suspended in phosphate buffer. Brown and Harris (1963) noted that chicken semen equilibrated in a glycerol diluent for 1.5 hours at 2°C. had higher oxygen uptake, motility, and fertility when compared to 1.0 hour and 0.5 hour equilibrations. Equilibration before freezing reduced the harmful effects of glycerol on fertilizing capacity. The removal of glycerol before insemination was not required for fertilization. Other investiga-
385
LIQUID NITROGEN PRESERVED SEMEN TABLE 1.—Composition of diluents Standard solutions Dimethyl sulfoxide6 (ml.)
Distilled water (ml.)
Total volume (ml.)
3.00 3.00
.40 .40
0.00 0.00
0.00 3.20
6.60 3.40
15.00 15.00
5.00 5.00
3.00 3.00
.40 .40
1.00 1.00
0.00 3.20
5.60 2.40
15.00 15.00
3a b
5.00 5.00
3.00 3.00
.40 .40
2.00 2.00
2.00 3.20
4.60 1.40
15.00 15.00
4a b
5.00 5.00
3.00 3.00
.40 .40
3.00 3.00
0.00 3.20
3.60 0.40
15.00 15.00
Monosodium glutamate 1 (ml.)
Antibiotics (ml.)
la b
5.00 5.00
2a b
2
1
Monosodium glutamate (100 mg./ml.). Oxytetracycline hydrochloride (2 mg./ml.) and dihydrostreptomycin sulfate (10 mg,/ml.), supplied by Chas. Pfizer & Co. Laboratories, Dallas, Texas. 3 Polyvinylpyrrolidone, Type PVP-kl5, (100 mg./ml.), General Aniline and Film Corporation, New York N. Y. 4 One-week-old turkey yolk. 6 Dimethylsulfoxide, K and K Laboratories, Inc., Plainview, N . Y. 2
Five White Leghorn hens were inseminated per treatment with 0.2 ml. of diluted semen. This dosage was equivalent to 0.05 ml. undiluted semen. The hens were inseminated by either the intraperitoneal or intravaginal method. Fertility data were obtained from eggs laid the 2nd and 8th day after insemination. After 7 days of incubation, fertility was determined by candling. The embryos were not considered fertile unless normal blood vessel development was observed. Hatchability was recorded on the 22nd day of incubation. Data were analyzed by analysis of variance after transformation percentages had been converted to arc sine values (Steel and Torrie, 1960). RESULTS AND DISCUSSION
In three replicate trials, intraperitoneal (IP) insemination gave significantly higher (P<.01) fertility than intravaginal (IV) insemination (Tables 2 and 3). No significant differences in fertility were noted due to cooling rate, volume of di-
luted semen frozen per sample, or equilibration time. The only significant interaction was between equilibration time and insemination method (P<.05). Semen that was equilibrated for 24 hours fertilized only 2% of the hens with IV insemination; whereas, 45% of the hens were fertilized by IP insemination. The removal of the protective agents prior to insemination was not required for normal embryonic development. The average hatch ability of fertile eggs was 92% and 84% from hens that were inseminated by the IP and IV methods, respectively. Data are not available on the rate of cooling with liquid nitrogen before and after freezing chicken spermatozoa. The cooling rates within various temperature ranges for optimum survival of bull spermatozoa have been studied using liquid nitrogen. Bruemmer et al. (1963) reported that bull spermatozoa survival was highest with cooling rates of 1°C. to 5°C. per minute between 0°C. and — 10°C. Fertility data were compared from several trials using the same diluent for cool-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
PVP-K153 (ml.)
Turkey egg yolk4 (ml.)
Diluent No.
386
G. C. HARRIS, J R . TABLE 2.—Effect
Treatment no.
3 4
Cooling rate (°C. per min.)
Equilibration time (hrs.)
Fertility of total eggs laid during days 2-6 inc. after single insemination
Volume of diluted semen frozen per sample (ml.) -
:IP
2
IV 3
Trials
Trials
1
2
3
Mean 4
1
Percent fertility 38 20 24 35
2
3
Mean 4
0 0
0 0
7 12
1 2
0.5 0.5
1.0 1.0
0.4 1.2
75 11
25 63
9 0
3 4
0.5 0.5
6.0 6.0
0.4 1.2
14 55
6 24
42 11
24 35
35 5
6 16
42 15
27 11
5 6
24.0 24.0
1.0 1.0
0.4 1.2
37 24
33 20
6 23
32 19
0 0
0 0
0 5
0 2
7 8
24.0 24.0
6.0 6.0
0.4 1.2
67 75
47 0
13 29
44 41
0 0
0 0
0 0
0 0
Diluted with Diluent 1 and stored 3 days at -196°C. IP=Intraperitoneal Insemination. IV=Intravaginal Insemination. Standard error of mean +8.42.
ing rates between 1.2°C. to 6.7°C. per minute (Table 3). The lowest average fertility was with cooling rates of 1.2°C. per TABLE 3.—Analysis of variance showing mean squares and "F" values for fertility data of Table 2 Source
d.f.
M.S.
Total Replicates Equilibration time (A) Cooling rate (B) Volume of diluted semen frozen (C) Insemination method (D) AXB AXC AXD BXC BXD CXD AXBXC AXBXD AXCXD BXBXD AXBXCXD Error
47 2
708.43
3.33*
1 1
597.57 246.65
2.81 NNSS 1.16
1
59.36
0.28 N S
1 1 1 1 1 1 1 1 1 1 1 1 30
5,414.34 59.19 9.53 1,109.40 11.24 33.24 26.27 56.02 450.99 48.33 151.03 245.46 212.82
25.44** NS 0.28 NS 0.04 5.21* 0.05 N S 0.16 NS 0.12 NS 0.26 NS 2.12 NS 0.23 N S 0.71 N S 1.15NS
* Significant at .05 level. ** Significant at .01 level. NS Nonsignificant.
F ratio
minute and 6.7°C. per minute. This indicates that the cooling rate requirements before freezing for chicken spermatozoa are similar to other species. The highest fertility (59%) was obtained with a cooling rate of 5.9°C. Thus, a cooling rate of 6°C. per minute was used in all later trials. With IP insemination, 50% of the hens were fertile, and the average hatchability of fertile eggs was 93%. Fresh chicken egg yolk has been widely used in citrate diluents to protect bull spermatozoa against temperature shock during freezing (Willett and Salisbury, 1942). The addition of fresh egg yolk to the diluent has resulted in a makred reduction in the fertility of stored nonfrozen spermatozoa of the chicken (Wilcox, 1960) and turkey (Bajpai and Brown, 1963). Preliminary storage experiments at 2°C. for 24 hours showed that chicken egg yolk was more toxic to chicken spermatozoa than turkey egg yolk. Chicken spermatozoa stored in a diluent containing the yolk from turkey eggs one-week-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 8, 2015
1 2
of equilibration, cooling rate and sample volume on fertility of semen inseminated by two methods1
387
LIQUID NITROGEN PRESERVED SEMEN
TABLE 4.—Effect of cooling rate on fertility1
2
Treatment Cooling rate (°C. per min.) No.
Fertility of total eggs laid during days 2-6 incl. after single insemination Trials 1 2
1.2 1.9 3.9 5.1 5.9 6.7
0 29 73 33 73 14
33 14 23 42 55 12
3
4
5
6 Mean'
Percent fertility 33 75 25 9 33 46 35 71 57 14 — — 39 40 — — 50 — — — 17 44 — —
T A B L E 5.—Effect of equilibration yolk level on fertility1
Fertility of total eggs laid during days 2-8 Total in cl aft er Treatment Diluent equilibration Y°JH' . - . f! n 8 le 1 No. No. time S™1 insemination (hrs.) (%) Trials
29 38 42 39 59 22
Mean3
1
Percent fertility 33 55 44 41 58 31 25 13 20 30 42 50 44 23 13 9 9
0 6.7 13.3 20.0 0 6.7 13.3 20.0
1 Cooling rate of 6°C. per minute; stored for 12 days at -196°C; IP insemination; 0.4 ml. diluted semen per sample. 2 Percent turkey egg yolk in diluent. 3 Standard error of mean ± 6.60.
terial multiplication appeared to become a major problem with the addition of egg yolk and extended equilibration. T h e yolk diluent was compared to the non-yolk diluent with equilibration times up to 24 hours (Table 6). The fertility of eggs fertilized with spermatozoa equilibrated in the non-yolk diluent was significantly higher ( P < . 0 5 ) for T r e a t m e n t 1 (0.5 hour equilibration) than T r e a t m e n t 3 (3.0 hours equilibration). Table 5 shows t h a t higher fertility was obtained with sperm equilibrated in the yolk diluent for 0.5 hour than with sperm equilibrated for
TABLE 6.—Effect of equilibration time and addition of yolk on fertility1 Fertility of total eggs laid during day!3 2-6 incl . after single insemination Trials Treatment no.
1 2 3 4 5 6
Equilibration time (hrs.)
0.5 1.5 3.0 6.0 12.0 24.0
Mean 2
4
3
1
2
No Yolk3
No Yolk3
No Yolk3
Yolk4
No Yolk3
Yolk4
No Yolk3
Yolk4
39 25 50 59 50 41
50 60 0 27 0 0
73 16 11 32 50 33
73 11 13 50 11 21
40 29 18 24 36 18
80 17 47 30 56 17
51 33 20 36 34 23
77 14 30 40 34 19
1 Cooling rate of 6°C. per minute; equilibration temperature 30°C. for 0.5 hour then held at 2°C; stored for 212 days at — 196°C; IP insemination; 0.4 ml diluted semen per sample. Standard error of mean + 6.79. 3 Diluent 1. 4 Diluent 2 (6.7% yolk).
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0.5 0.5 0.5 0.5 1.5 1.5 1.5 1.5
1 Diluted with Diluent 1; stored for an average of 12 days at 2— 196°C; IP insemination; 0.4 ml. diluted semen per sample. Cooling rate represents a linear temperature change from 30°C. until frozen. 8 Standard error of the mean ± 6.65.
old gave higher fertility than spermatozoa stored in a fresh yolk diluent. Based on these findings, the yolk of one-week-old turkey eggs was used in the yolk diluents for freezing studies on chicken spermatozoa. T h e effects of turkey egg yolk level in the diluent and equilibration time on fertility of chicken spermatozoa after freezing were studied. Highest fertilities, 5 8 % (Table 5) and 7 7 % (Table 6), were obtained with 0.5 hour equilibration and 6.7% yolk in the diluent. The fertility was lowered when the yolk level was increased above 6.7% and the equilibration period extended beyond 0.5 hour (Table 5). Bac-
time and
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G. C. HARRIS, JR.
1.5, 3.0, 12.0 and 24.0 hours. Six hours of equilibration gave lower fertility, but the differences were non-significant. The average hatchability of fertile eggs for all treatments was 92% (Tables 4 and 5). In this experiment, 47% of the hens were fertile. SUMMARY
ACKNOWLED GMENT
The author wishes to express his appreciation to Chas. Pfizer & Co., Dallas, Texas for supplying the antibiotics used in this study. REFERENCES Bajpai, P. K., and K. I. Brown, 1963. The effect of some diluents on semen characteristics of turkeys. Poultry Sci. 42: 882-888. Brown, J. E., and G. C. Harris, Jr., 1963. The influence of glycerol equilibration time on the metabolism, motility and fertility of frozen chicken spermatozoa. Poultry Sci. 42: 383-386.
JULY 21-26. SIXTH INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, PARIS, FRANCE.
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The effects of cooling rate, equilibration time, and addition of turkey egg yolk to a glutamate diluent on the fertility of frozen chicken spermatozoa were studied. Diluted chicken spermatozoa were cooled with liquid nitrogen at linear cooling rates between 1°C. and 6.7°C. per minute from 30°C. until frozen. Semen samples were stored for periods up to 12 days in liquid nitrogen ( - 1 9 6 ° C ) . Highest fertility (77%) was obtained with intraperitoneally inseminated semen that was equilibrated 0.5 hour in a turkey egg yolk diluent and cooled at a rate of 6°C. per minute. Fertility of spermatozoa diluted with egg yolk diluent was significantly reduced by longer equilibration periods. The hatchability of fertile eggs for all trials was 91.4%.
Brown, J. E., G. C. Harris, Jr. and T. D. Hobbs, 1963. Effect of intraperitoneal insemination on egg production and fertilizing capacity of fresh and frozen chicken sperm. Poultry Sci. 42: 810-815. Bruemmer, J. H., R. W. Eddy and W. J. Duryea, 1963. Temperature control in the low temperature preservation of spermatozoa. J. Cell. Comp. Physiol. 62: 113-117. Burrows, W. H., and J. P. Quinn, 1937. Collection of spermatozoa from domestic fowl and turkey. Poultry Sci. 16: 19-24. Clark, C. E., and S. S. Shaffner, 1960. The fertilizing capacity of frozen chicken sperm and the influence of related in vitro processes. Poultry Sci. 39: 1213-1220. Grigg, G. W., and F. Skaller, 1958. A case of sterility in the hen. Poultry Sci. 37: 954-955. Kosin, I. L., 1944. Some aspects of the biological action of X-rays on cock spermatozoa. Physiol. Zool. 17:289-319. Polge, C , 1951. Functional survival of fowl spermatozoa after freezing at — 79°C. Nature, 167: 949-950. Polge, C , A. V. Smith and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, McGraw-Hill Book Co., New York. Van Drimmelen, G. C , 1945. Intraperitoneal insemination of birds. J. South African Vet. Med. Assoc. 16: 1-6. Van Drimmelen, G. C , 1951. Artificial insemination of birds by the intraperitoneal route. Onderstepoort J. Vet. Res. Suppl. No. 1: 212. Wilcox, F. H., 1960. Effect on fertility of temperature, handling methods, Lake's solution, and the addition of egg white, egg yolk, and sugars to the diluent used in storing chicken semen. Poultry Sci. 39: 459-467. Willett, E. L., and G. W. Salisbury, 1942. The effect of various dilutors, cooling rate, temperature of storage, and some other factors on livability of spermatozoa in stored samples of bull semen. Cornell Univ. Agr. Expt. Sta. Mem. 249.