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Effects of Semen Collection and Storage Temperatures on Sperm Viability and Fertility in Turkeys
Effects of Semen Collection and Storage Temperatures on Sperm Viability and Fertility in Turkeys G. W. F R I A R S AND S. C H A T T E R J E E 1 Department of Poultry Science, University of Guelph, Guelph, Ontario, Canada (Received for publication March 8, 1969)
T
H E increased and almost universal use of artificial insemination procedures for turkeys emphasises the need for semen transport and hence short term storage. Brown (1966) showed t h a t holding turkey semen for two hours at 15°C. gave consistent b u t non-significant decreases in fertility, however, Harper (1955) observed t h a t decreased fertility from semen held for more than one hour at 15°C. became more pronounced as the breeding season progressed. A review of differential staining techniques by Hackett and Macpherson (1965) included discussions of eosin nigrosin and congo red-nigrosin for the sperm of mammalian species. Reports of comparisons of staining techniques on sperm in avian species have not been found in the literature by the authors. T h e present studies were undertaken with the objectives to compare three staining techniques on avian sperm, select one of these stains to measure live:dead sperm ratios of turkey semen under different collection and storage temperatures and to evaluate the effect of these collection and storage temperatures b y a fertility trial. METHODS AND MATERIALS
Comparisons of Techniques. T h e preliminary experiment, involving the comparisons of techniques, was run on chicken semen since turkey semen was not readily available at t h a t particular time. Three 1
Present address: Department of Animal Science, University of Guelph.
stains, eosin nigrosin (Cooper and Rowell, 1958), congo red-nigrosin (Blackshaw, 1958) and bromphenol blue-nigrosin (Kamar, 1959) were compared on split semen samples using duplicate slides and two counts of 100 sperm in randomly chosen fields on each slide for each sample. Any sperm showing complete or partial staining were counted as dead. T h e stains were compared with respect to the contrast of sperm cells and background and the percentage of stained sperm transformed to arc sine -^/percentage, however, for purposes of discussion arc sine V p e r c e n t a g e means have been transformed to percentages. Experiment 1. Pooled semen was collected from 25 to 30 Large White and Large Auburn turkey males for six collections on different days spread over the breeding season. Collections were made directly into two vials immersed in thermos flasks containing water at 15°C. and 30°C. respectively, using both vials simultaneously with each male. A total of six collection-storage treatments were involved b y subjecting three samples, from each collection flask, t o eosin nigrosin stain after treatment as follows: 1) Held for less than one half hour, 2) Stored at 15°C. for four hours, 3) Stored at 30°C. for four hours. Experiment 2. Procedures in Experiment 2 were similar to those in Experiment 1 except t h a t storage durations were for five hours instead of four hours.
1434
SPERM VIABILITY AND FERTILITY
Twelve White turkey females representing each of the six treatments, were inseminated with approximately 0.25 ml. of semen. First inseminations were made on the 19th day following the shift of females from approximately an 11 to a 14 hour photoperiod per day. Subsequent inseminations were carried out on the 22nd and 29th days following the extension of the photoperiod and at two week intervals thereafter. Eggs were identified with the treatment and cage number of individual hens. Three separate hatches were made from eggs collected over three consecutive two week storage periods. Eggs were candled on the 10th day of incubation and classified as infertile or fertile. All eggs showing no evidence of fertility under the candler were broken and final classification was made by assessing an egg as fertile if blood was noted to be present in the germinal disk area. RESULTS AND DISCUSSION
The variance analysis of the data on stained sperm in chicken semen (Table 1) revealed that the mean level of stained sperm for eosin nigrosin (26.8 percent) was significantly less than for either bromphenol blue-nigrosin (37.9 percent) or T A B L E 1.—Variance analysis oj arc siney/percentages of stained sperm in chicken semen sampled on different days Sourcesf
Degrees of freedom
Mean squares
Stains (S) ft Eosin Nigrosin vs. Bromphenol blue-nigrosin ft Eosin Nigrosin vs. Congo red-nigrosin ft Bromphenol blue-nigrosin vs. Congo red-nigrosin Days (D) SXD Slides (L) w/n (SXD) Counts (C) w/n L w/n (SXD)
5 10 18 36
1
560"
1
850"
1
28 5,802" 119** 21 4
t S and D considered as fixed effects and L and C as random effects. ft Non-orthogonal comparisons. ** Significant at P<.01.
1435
congo red-nigrosin (40.6 percent). The difference between mean levels of stained sperm in the samples taken on different days were highly significant (Table 1) being largely influenced by the exceedingly high counts on the second day. The interaction of days X stains is reflected in the irregularity of the differences between stains on different days (Table 2). Eosin nigrosin showed the sharpest contrast between unstained sperm and the background. The differentiation between the stained and unstained sperm was poorest in the case of congo red-nigrosin. Therefore, eosin-nigrosin was favoured as the most satisfactory of the three stains in estimating the incidence of stained sperm. Experiment 1. The error term for the variance analysis (Table 3) based on expected mean squares (e.g.-Steel and Torrie, 1960-page 214) is slides within (treatments X days), however, the mean square for this term is smaller than for counts within slides within (treatmentsXdays), that is, 14 as opposed to 134. Therefore, the a priori determined error term may be reflecting significance of small differences by chance. Accepting this possibility, the day and treatment X day effects are significant, similar to the same effects accounted for by the comparison of techniques experiment (Tables 1 and 2). The treatment degrees of freedom have been partitioned into five orthogonal contrasts (Table 3) which may be interpreted as follows: 1. The various treatments during storage of semen for four hours gave a significant increase in the percentage of stained sperm as opposed to controls counted within a half hour, from 9.3 to 28.7 percent. 2. Collection of semen at 15°C. as opposed to 30° C. did not significantly affect the incidence of stained sperm
1436
G. W. FRIARS AND S. CHATTERJEE TABLE 2.-—Means
of the arc siney/percentages of stained sperm in chicken semen samples
Eosin nigrosin
Day 1 2 3 4 5 6
30.0 72.1 16.9 19.5 27.8 20.7
Means
Bromphenol bluenigrosin
(25.0)* (90.5) ( 8.5) (11.1) (21.8) (12.5)
36.7 82.2 23.7 24.1 26.3 24.9
31.2 (26.8)
(35.7) (98.1) (16.2) (16.7) (19.6) (17.7)
38.0 (37.9)
Congo rednigrosin 29.7 87.1 24.7 26.7 41.1 28.0
(24.6) (99.7) (17.5) (20.2) (43.2) (22.0)
Means 32.1 80.5 21.8 23.4 35.1 24.5
(28.2) (97.3) (13.8) (15.8) (33.1) (17.2)
39.6 (40.6)
Arc sineVpercentages transformed to percentages in parentheses.
when the semen was evaluated within a half hour of collection, 3. Collection of semen at 15°C. as opposed to 30° C. significantly increased the incidence of stained sperm from 17.5 to 31.1 percent when the semen was held for four hours prior to insemination, 4. Holding semen for four hours at 30°C. as opposed to 15°C. significantly increased the incidence of stained sperm from 21.8 to 26.2 percent, 5. Collecting semen at one temperature did not significantly affect the incidence of stained sperm with respect TABLE 3.—Variance analysis of arc siney/percentages of stained sperm in turkey semen Source!
Degrees of freedom
Mean squares
to the order in which the shift of temperatures was made. Experiment 2. The use of the (treatments X days) mean square as an error term (Table 4) leads to robust statistical tests if the interaction of treatments and hatches is in fact not equal to zero. Accepting this possibility, the only significant effect from the five orthogonal contrasts for treatment (Table 4) was the lowering of fertility from 48.1 to 10.5 percent due to storage for five hours. The other treatment effects which were significant in the experiment involving live:
TABLE 4.—Variance analysis of arc sine\/'percentages of turkey fertility Sourcef
Treatment (T) Treatments (T) Orthogonal contraststt 1. H o s s . (Hi5+H 3 0 )/2 2. C30H0 vs. CisHo 3. 4.
(C30H16+C30H30) vs. ( C I B H I 6 + C I B H 3 O ) (C16H30+C30H30) M. ( C I B H I S + C 3 O H I J )
5. (C30H30+C16H16) vs. (C30H15+C15H30) D a y s (D) TXD Slides (S) w / n ( T X D ) Counts (C) w / n S w / n ( T X D )
5 25 36
n
4,232" 18 2,040** 237** 28 168" 70** 14 134
t T and D considered as fixed effects and S and L as random effects. f t Cie—semen collected a t 15°C C30—semen collected a t 30°C. Ho—counts made within a half hour. Hie—counts made after semen was held for four hours a t 15°C. H30—counts made after semen was held for four hours a t 30°C. ** Significant a t P < . 0 1 .
Orthogonal C o n t r a s t s t t 1. H o w . (His+H!o)/2 2. C30H0vs. OsHo 3. (C30H16+C30H30) vs. (C16H16+C15H30) 4. (C16H30+C30H30) vs. (C16H16+C30H16) 5. (C30H30+C16H15) vs. (C30H16+C1SH30) Hatches (H) TXH
De S£rees freedom
536**
5 1 1 1 1 1 2 10
Mean *uare
s<
2,504** 46 1 126 2 29 27
f T and H considered as fixed effects and T X H interaction assumed equal to zero and hence this source represents random error. f t Cie—semen collected a t 15°C. C30—semen collected a t 30°C. Ho—inseminations made within a half hour. H16—inseminations made after semen was held for four hours a t 15°C. H30—inseminations made after semen was held for four hours a t 30°C. ** Significant at P < . 0 1 .
SPERM VIABILITY AND FERTILITY
dead sperm ratios were not reflected in the fertility experiment.
1437
to semen used for inseminating hens within a half hour. ACKNOWLEDGEMENTS
SUMMARY Eosin nigrosin was estimated to be a more satisfactory stain than bromphenol blue-nigrosin or Congo red-nigrosin for differentiating between the degrees of viability of sperm on the basis of trials with chicken semen, using a total of 72 counts each comprised of 100 sperm. The proportion of stained turkey sperm, using eosin nigrosin on 144 counts each comprised of 100 sperm, was increased during four hours of storage. A collection temperature of 30° C. produced a lower proportion of stained sperm than 15°C. while fewer stained sperm were encountered with a holding temperature of 15°C. as opposed to 30°C. during four hours of storage. However, the use of the same collection and storage temperatures in a fertility experiment with 84 turkey females revealed a significant drop in fertility only in the comparison of semen, stored for five hours at 15°C. and 30°C,
Support for this research was received from the Ontario Department of Agriculture and Food and the National Research Council of Canada Operating Grant A1970. The authors gratefully acknowledge the cooperation and assistance of Dr. J.W. Macpherson in conducting these studies and reviewing the manuscript. REFERENCES Blackshaw, A. W., 1958. The effect of glycerol on supra-vital staining of spermatozoa. Australian Vet. J. 34: 71-76. Brown, K. I., 1966. Some factors affecting storage of turkey semen. Ohio Res. Summ. 17, Ohio Agr. Res. and Dev. Center, Wooster, Ohio. Cooper, D. M., and J. C. Rowell, 1958. Relations between fertility, embryonic survival and some semen characteristics in the chicken. Poultry Sci. 37: 699-707. Hackett, A. J., and J. W. Macpherson, 1965. Some staining procedures for spermatozoa. A review. Can. Vet. J. 6: 55-62. Harper, J. A., 1955. The effect of holding time of turkey semen on fertilizing capacity. Poultry Sci. 34: 1289-1291.
Magnesium Nutriture of the Hen: Influence on Retention of Magnesium, Calcium, and Nitrogen, and on Ration Metabolizable Energy Value1 J. L. SELL Animal Science Department, North Dakota State University, Fargo, North Dakota 58102 (Received for publication March 10, 1969)
M
AGNESIUM (Mg) deficiency has been described for many animal species including chicks (Almquist, 1942; and Gardiner et al, 1960), ducks (Van 1 Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 186, Project H-7-26.
Reen and Pearson, 1953) and laying hens (Sell et ah, 1967). Signs of this deficiency include impaired reproduction, hypomagnesemia, anorexia and death. Despite considerable data describing clinical manifestations of Mg deficiency, little information is available concerning mineral and nitrogen balance, or utilization of di-
T
H E increased and almost universal use of artificial insemination procedures for turkeys emphasises the need for semen transport and hence short term storage. Brown (1966) showed t h a t holding turkey semen for two hours at 15°C. gave consistent b u t non-significant decreases in fertility, however, Harper (1955) observed t h a t decreased fertility from semen held for more than one hour at 15°C. became more pronounced as the breeding season progressed. A review of differential staining techniques by Hackett and Macpherson (1965) included discussions of eosin nigrosin and congo red-nigrosin for the sperm of mammalian species. Reports of comparisons of staining techniques on sperm in avian species have not been found in the literature by the authors. T h e present studies were undertaken with the objectives to compare three staining techniques on avian sperm, select one of these stains to measure live:dead sperm ratios of turkey semen under different collection and storage temperatures and to evaluate the effect of these collection and storage temperatures b y a fertility trial. METHODS AND MATERIALS
Comparisons of Techniques. T h e preliminary experiment, involving the comparisons of techniques, was run on chicken semen since turkey semen was not readily available at t h a t particular time. Three 1
Present address: Department of Animal Science, University of Guelph.
stains, eosin nigrosin (Cooper and Rowell, 1958), congo red-nigrosin (Blackshaw, 1958) and bromphenol blue-nigrosin (Kamar, 1959) were compared on split semen samples using duplicate slides and two counts of 100 sperm in randomly chosen fields on each slide for each sample. Any sperm showing complete or partial staining were counted as dead. T h e stains were compared with respect to the contrast of sperm cells and background and the percentage of stained sperm transformed to arc sine -^/percentage, however, for purposes of discussion arc sine V p e r c e n t a g e means have been transformed to percentages. Experiment 1. Pooled semen was collected from 25 to 30 Large White and Large Auburn turkey males for six collections on different days spread over the breeding season. Collections were made directly into two vials immersed in thermos flasks containing water at 15°C. and 30°C. respectively, using both vials simultaneously with each male. A total of six collection-storage treatments were involved b y subjecting three samples, from each collection flask, t o eosin nigrosin stain after treatment as follows: 1) Held for less than one half hour, 2) Stored at 15°C. for four hours, 3) Stored at 30°C. for four hours. Experiment 2. Procedures in Experiment 2 were similar to those in Experiment 1 except t h a t storage durations were for five hours instead of four hours.
1434
SPERM VIABILITY AND FERTILITY
Twelve White turkey females representing each of the six treatments, were inseminated with approximately 0.25 ml. of semen. First inseminations were made on the 19th day following the shift of females from approximately an 11 to a 14 hour photoperiod per day. Subsequent inseminations were carried out on the 22nd and 29th days following the extension of the photoperiod and at two week intervals thereafter. Eggs were identified with the treatment and cage number of individual hens. Three separate hatches were made from eggs collected over three consecutive two week storage periods. Eggs were candled on the 10th day of incubation and classified as infertile or fertile. All eggs showing no evidence of fertility under the candler were broken and final classification was made by assessing an egg as fertile if blood was noted to be present in the germinal disk area. RESULTS AND DISCUSSION
The variance analysis of the data on stained sperm in chicken semen (Table 1) revealed that the mean level of stained sperm for eosin nigrosin (26.8 percent) was significantly less than for either bromphenol blue-nigrosin (37.9 percent) or T A B L E 1.—Variance analysis oj arc siney/percentages of stained sperm in chicken semen sampled on different days Sourcesf
Degrees of freedom
Mean squares
Stains (S) ft Eosin Nigrosin vs. Bromphenol blue-nigrosin ft Eosin Nigrosin vs. Congo red-nigrosin ft Bromphenol blue-nigrosin vs. Congo red-nigrosin Days (D) SXD Slides (L) w/n (SXD) Counts (C) w/n L w/n (SXD)
5 10 18 36
1
560"
1
850"
1
28 5,802" 119** 21 4
t S and D considered as fixed effects and L and C as random effects. ft Non-orthogonal comparisons. ** Significant at P<.01.
1435
congo red-nigrosin (40.6 percent). The difference between mean levels of stained sperm in the samples taken on different days were highly significant (Table 1) being largely influenced by the exceedingly high counts on the second day. The interaction of days X stains is reflected in the irregularity of the differences between stains on different days (Table 2). Eosin nigrosin showed the sharpest contrast between unstained sperm and the background. The differentiation between the stained and unstained sperm was poorest in the case of congo red-nigrosin. Therefore, eosin-nigrosin was favoured as the most satisfactory of the three stains in estimating the incidence of stained sperm. Experiment 1. The error term for the variance analysis (Table 3) based on expected mean squares (e.g.-Steel and Torrie, 1960-page 214) is slides within (treatments X days), however, the mean square for this term is smaller than for counts within slides within (treatmentsXdays), that is, 14 as opposed to 134. Therefore, the a priori determined error term may be reflecting significance of small differences by chance. Accepting this possibility, the day and treatment X day effects are significant, similar to the same effects accounted for by the comparison of techniques experiment (Tables 1 and 2). The treatment degrees of freedom have been partitioned into five orthogonal contrasts (Table 3) which may be interpreted as follows: 1. The various treatments during storage of semen for four hours gave a significant increase in the percentage of stained sperm as opposed to controls counted within a half hour, from 9.3 to 28.7 percent. 2. Collection of semen at 15°C. as opposed to 30° C. did not significantly affect the incidence of stained sperm
1436
G. W. FRIARS AND S. CHATTERJEE TABLE 2.-—Means
of the arc siney/percentages of stained sperm in chicken semen samples
Eosin nigrosin
Day 1 2 3 4 5 6
30.0 72.1 16.9 19.5 27.8 20.7
Means
Bromphenol bluenigrosin
(25.0)* (90.5) ( 8.5) (11.1) (21.8) (12.5)
36.7 82.2 23.7 24.1 26.3 24.9
31.2 (26.8)
(35.7) (98.1) (16.2) (16.7) (19.6) (17.7)
38.0 (37.9)
Congo rednigrosin 29.7 87.1 24.7 26.7 41.1 28.0
(24.6) (99.7) (17.5) (20.2) (43.2) (22.0)
Means 32.1 80.5 21.8 23.4 35.1 24.5
(28.2) (97.3) (13.8) (15.8) (33.1) (17.2)
39.6 (40.6)
Arc sineVpercentages transformed to percentages in parentheses.
when the semen was evaluated within a half hour of collection, 3. Collection of semen at 15°C. as opposed to 30° C. significantly increased the incidence of stained sperm from 17.5 to 31.1 percent when the semen was held for four hours prior to insemination, 4. Holding semen for four hours at 30°C. as opposed to 15°C. significantly increased the incidence of stained sperm from 21.8 to 26.2 percent, 5. Collecting semen at one temperature did not significantly affect the incidence of stained sperm with respect TABLE 3.—Variance analysis of arc siney/percentages of stained sperm in turkey semen Source!
Degrees of freedom
Mean squares
to the order in which the shift of temperatures was made. Experiment 2. The use of the (treatments X days) mean square as an error term (Table 4) leads to robust statistical tests if the interaction of treatments and hatches is in fact not equal to zero. Accepting this possibility, the only significant effect from the five orthogonal contrasts for treatment (Table 4) was the lowering of fertility from 48.1 to 10.5 percent due to storage for five hours. The other treatment effects which were significant in the experiment involving live:
TABLE 4.—Variance analysis of arc sine\/'percentages of turkey fertility Sourcef
Treatment (T) Treatments (T) Orthogonal contraststt 1. H o s s . (Hi5+H 3 0 )/2 2. C30H0 vs. CisHo 3. 4.
(C30H16+C30H30) vs. ( C I B H I 6 + C I B H 3 O ) (C16H30+C30H30) M. ( C I B H I S + C 3 O H I J )
5. (C30H30+C16H16) vs. (C30H15+C15H30) D a y s (D) TXD Slides (S) w / n ( T X D ) Counts (C) w / n S w / n ( T X D )
5 25 36
n
4,232" 18 2,040** 237** 28 168" 70** 14 134
t T and D considered as fixed effects and S and L as random effects. f t Cie—semen collected a t 15°C C30—semen collected a t 30°C. Ho—counts made within a half hour. Hie—counts made after semen was held for four hours a t 15°C. H30—counts made after semen was held for four hours a t 30°C. ** Significant a t P < . 0 1 .
Orthogonal C o n t r a s t s t t 1. H o w . (His+H!o)/2 2. C30H0vs. OsHo 3. (C30H16+C30H30) vs. (C16H16+C15H30) 4. (C16H30+C30H30) vs. (C16H16+C30H16) 5. (C30H30+C16H15) vs. (C30H16+C1SH30) Hatches (H) TXH
De S£rees freedom
536**
5 1 1 1 1 1 2 10
Mean *uare
s<
2,504** 46 1 126 2 29 27
f T and H considered as fixed effects and T X H interaction assumed equal to zero and hence this source represents random error. f t Cie—semen collected a t 15°C. C30—semen collected a t 30°C. Ho—inseminations made within a half hour. H16—inseminations made after semen was held for four hours a t 15°C. H30—inseminations made after semen was held for four hours a t 30°C. ** Significant at P < . 0 1 .
SPERM VIABILITY AND FERTILITY
dead sperm ratios were not reflected in the fertility experiment.
1437
to semen used for inseminating hens within a half hour. ACKNOWLEDGEMENTS
SUMMARY Eosin nigrosin was estimated to be a more satisfactory stain than bromphenol blue-nigrosin or Congo red-nigrosin for differentiating between the degrees of viability of sperm on the basis of trials with chicken semen, using a total of 72 counts each comprised of 100 sperm. The proportion of stained turkey sperm, using eosin nigrosin on 144 counts each comprised of 100 sperm, was increased during four hours of storage. A collection temperature of 30° C. produced a lower proportion of stained sperm than 15°C. while fewer stained sperm were encountered with a holding temperature of 15°C. as opposed to 30°C. during four hours of storage. However, the use of the same collection and storage temperatures in a fertility experiment with 84 turkey females revealed a significant drop in fertility only in the comparison of semen, stored for five hours at 15°C. and 30°C,
Support for this research was received from the Ontario Department of Agriculture and Food and the National Research Council of Canada Operating Grant A1970. The authors gratefully acknowledge the cooperation and assistance of Dr. J.W. Macpherson in conducting these studies and reviewing the manuscript. REFERENCES Blackshaw, A. W., 1958. The effect of glycerol on supra-vital staining of spermatozoa. Australian Vet. J. 34: 71-76. Brown, K. I., 1966. Some factors affecting storage of turkey semen. Ohio Res. Summ. 17, Ohio Agr. Res. and Dev. Center, Wooster, Ohio. Cooper, D. M., and J. C. Rowell, 1958. Relations between fertility, embryonic survival and some semen characteristics in the chicken. Poultry Sci. 37: 699-707. Hackett, A. J., and J. W. Macpherson, 1965. Some staining procedures for spermatozoa. A review. Can. Vet. J. 6: 55-62. Harper, J. A., 1955. The effect of holding time of turkey semen on fertilizing capacity. Poultry Sci. 34: 1289-1291.
Magnesium Nutriture of the Hen: Influence on Retention of Magnesium, Calcium, and Nitrogen, and on Ration Metabolizable Energy Value1 J. L. SELL Animal Science Department, North Dakota State University, Fargo, North Dakota 58102 (Received for publication March 10, 1969)
M
AGNESIUM (Mg) deficiency has been described for many animal species including chicks (Almquist, 1942; and Gardiner et al, 1960), ducks (Van 1 Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 186, Project H-7-26.
Reen and Pearson, 1953) and laying hens (Sell et ah, 1967). Signs of this deficiency include impaired reproduction, hypomagnesemia, anorexia and death. Despite considerable data describing clinical manifestations of Mg deficiency, little information is available concerning mineral and nitrogen balance, or utilization of di-