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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
A comparison of one and fifteen minutes' equilibration in the technique of preserving fowl spermatozoa at subzero temperatures a
a
M. Watanabe , K. Ashizawa & T. Terada
a
a
Department of Animal Husbandry, Faculty of Fisheries and Animal Husbandry , Hiroshima University , Fukuyama, Japan Published online: 08 Nov 2007.
To cite this article: M. Watanabe , K. Ashizawa & T. Terada (1975) A comparison of one and fifteen minutes' equilibration in the technique of preserving fowl spermatozoa at subzero temperatures, British Poultry Science, 16:5, 535-539, DOI: 10.1080/00071667508416223 To link to this article: http://dx.doi.org/10.1080/00071667508416223
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
Br. Poult. Sci., 16: 535-539. 1975
Longman: printed in Great Britain
RESEARCH NOTE
A COMPARISON OF ONE AND FIFTEEN MINUTES' EQUILIBRATION IN THE TECHNIQUE OF PRESERVING FOWL SPERMATOZOA AT SUBZERO TEMPERATURES Downloaded by [New York University] at 06:17 10 May 2015
M. WATANABE, K. ASHIZAWA AND T. TERADA Department of Animal Husbandry, Faculty of Fisheries and Animal Husbandry, Hiroshima University, Fukuyama, Japan Received for publication 11th October 1974
1. There was no difference in the fertilising potential of thawed fowl semen whether equilibrated for 1 min or 15 min in a glycerolised medium prior to freezing. INTRODUCTION
In the deep-freezing preservation of spermatozoa of various animals, the time of equilibration with glycerol has been as much as 12 h in the bull (Polge and Rowson, 1952; Saroff and Mixner, 1955; Graham and Bayley, 1957; O'Dell and Almquist, 1957) and several hours in the stallion (Nagase et al., 1966; Bielanski et al., 1970; Platov et al., 1971). Berndtson and Foote (1972) and Jondet (1972) recently reported that equilibrations in glycerol for shorter than a few minutes did not affect the fertilising ability of bovine spermatozoa. Nishikawa (1972, 1974) also found no significant differences in the survival of stallion sperm after freezing with previous glycerol equilibration times of 18 to 32 s and 1 to 2 h. The present study was designed to compare an equilibration time of 1 min with 15 min that was used by Watanabe (1972) who reported improved results in the long term preservation of fowl semen. MATERIALS AND METHODS
Pooled semen was obtained from three White Leghorn cockerels (7 to 9 months old) by abdominal massage between 07.00 and 08.00 h. Immediately after collection, the pooled semen was diluted 4-fold with dilutor, such that there was 7% glycerol concentration in the final diluted semen sample, and kept at 5 °C for 1 or 15 min. The composition of the dilutor was 5% C6H12O6 (85 parts) and fresh egg yolk (15 parts by volume) (Watanabe, 1967). During these equilibration times, the diluted semen sub-samples were dispensed into 1 ml straw ampoules and sealed. 16/5—H
535
Downloaded by [New York University] at 06:17 10 May 2015
O OS O
TABLE I Comparison with the motility of spermatozoa and the abnormal spermatozoa treated by two equilibration times
Trial I
Freezing period (days)
Glycerol equilibration time (min)
Motility of undiluted spermatozoa
1
95 95
15 1
95 95
15
Motility of spermatozoa after thawing
III
14
15 1
95 95
IV
14
15
95 95
15 1
VI
15 1
A
f
\
15 min
1 min
k
47-5
S N > 82-5
1
% abnormal spermatozoa
15 min
82-5
II
Mean % abnormal spermatozoa in the thawed semen after storage for 7, 14 and 21 d
Mean % motile spermatozoa in the thawed semen after storage for 7, 14 and 21 d
62-5
85
13-2
95 95 75-°
52-5
14-8
15-2
8o-o*
54-2'
13-0*
13-4*
95 95
Average * Significant at Po-5 level.
Downloaded by [New York University] at 06:17 10 May 2015
EQUILIBRATION IN FREEZING FOWL SEMEN
537
After the appropriate equilibration times the semen samples were subjected to prefreezing in the vapour of liquid nitrogen (about — 110 °C to — 120 °C) for 2-5 min and then stored in liquid nitrogen. After each of 7, 14 and 21 d storage, duplicate sub-samples subjected to each of the equilibration times were thawed by standing the straw ampoules in water at 5 °C for 5 min and then at 20 °C for another 5 min and the motility of spermatozoa and the percentage of morphologically abnormal spermatozoa, including neck-bent ones, were estimated microscopically by observing approximately 500 spermatozoa in each case. Motility was scored using a scale of five points ( + + + , + + , + , ± , —). Motility and morphologically abnormal spermatozoa for each of the two equilibration times after different storage periods were thus examined in 12 subsamples of the original pooled semen (see Table 1). For morphological studies portions of the semen sub-samples were fixed with formalin and stained with carbolfuchsin-eosin solution. The composition of carbol-fuchsin-eosin solution is as follows: 96% alcohol, 25 ml; saturated eosin solution, 25 ml; carbol-fuchsin solution, 50 ml. The fertility of frozen semen stored for 11 to 14 d was also examined by inseminating artificially 22 White Leghorn hens (7 to 8 months old); 10 hens with semen equilibrated for 15 min and 12 hens with semen equilibrated for 1 min. RESULTS AND DISCUSSION
The motility of spermatozoa which had been stored in liquid nitrogen for 7, 14 and 21 d subsequent to being equilibrated for 15 and 1 min is shown in Table 1. The mean percentage of motile spermatozoa in thawed semen after storage for 7, r4 and 21 d and which had been equilibrated for 15 min was 80%; each percentage for 7, 14 and 21 d was 82*5, 82#5 and 75% respectively. They were over + + on the scale. The mean percentage with 1 min equilibration was 54-2%; percentages for 7, 14 and 21 d were 47-5, 62-5 and 52-5% respectively. Thus, there was a significant difference between the two equilibration treatments (P
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
A comparison of one and fifteen minutes' equilibration in the technique of preserving fowl spermatozoa at subzero temperatures a
a
M. Watanabe , K. Ashizawa & T. Terada
a
a
Department of Animal Husbandry, Faculty of Fisheries and Animal Husbandry , Hiroshima University , Fukuyama, Japan Published online: 08 Nov 2007.
To cite this article: M. Watanabe , K. Ashizawa & T. Terada (1975) A comparison of one and fifteen minutes' equilibration in the technique of preserving fowl spermatozoa at subzero temperatures, British Poultry Science, 16:5, 535-539, DOI: 10.1080/00071667508416223 To link to this article: http://dx.doi.org/10.1080/00071667508416223
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
Br. Poult. Sci., 16: 535-539. 1975
Longman: printed in Great Britain
RESEARCH NOTE
A COMPARISON OF ONE AND FIFTEEN MINUTES' EQUILIBRATION IN THE TECHNIQUE OF PRESERVING FOWL SPERMATOZOA AT SUBZERO TEMPERATURES Downloaded by [New York University] at 06:17 10 May 2015
M. WATANABE, K. ASHIZAWA AND T. TERADA Department of Animal Husbandry, Faculty of Fisheries and Animal Husbandry, Hiroshima University, Fukuyama, Japan Received for publication 11th October 1974
1. There was no difference in the fertilising potential of thawed fowl semen whether equilibrated for 1 min or 15 min in a glycerolised medium prior to freezing. INTRODUCTION
In the deep-freezing preservation of spermatozoa of various animals, the time of equilibration with glycerol has been as much as 12 h in the bull (Polge and Rowson, 1952; Saroff and Mixner, 1955; Graham and Bayley, 1957; O'Dell and Almquist, 1957) and several hours in the stallion (Nagase et al., 1966; Bielanski et al., 1970; Platov et al., 1971). Berndtson and Foote (1972) and Jondet (1972) recently reported that equilibrations in glycerol for shorter than a few minutes did not affect the fertilising ability of bovine spermatozoa. Nishikawa (1972, 1974) also found no significant differences in the survival of stallion sperm after freezing with previous glycerol equilibration times of 18 to 32 s and 1 to 2 h. The present study was designed to compare an equilibration time of 1 min with 15 min that was used by Watanabe (1972) who reported improved results in the long term preservation of fowl semen. MATERIALS AND METHODS
Pooled semen was obtained from three White Leghorn cockerels (7 to 9 months old) by abdominal massage between 07.00 and 08.00 h. Immediately after collection, the pooled semen was diluted 4-fold with dilutor, such that there was 7% glycerol concentration in the final diluted semen sample, and kept at 5 °C for 1 or 15 min. The composition of the dilutor was 5% C6H12O6 (85 parts) and fresh egg yolk (15 parts by volume) (Watanabe, 1967). During these equilibration times, the diluted semen sub-samples were dispensed into 1 ml straw ampoules and sealed. 16/5—H
535
Downloaded by [New York University] at 06:17 10 May 2015
O OS O
TABLE I Comparison with the motility of spermatozoa and the abnormal spermatozoa treated by two equilibration times
Trial I
Freezing period (days)
Glycerol equilibration time (min)
Motility of undiluted spermatozoa
1
95 95
15 1
95 95
15
Motility of spermatozoa after thawing
III
14
15 1
95 95
IV
14
15
95 95
15 1
VI
15 1
A
f
\
15 min
1 min
k
47-5
S N > 82-5
1
% abnormal spermatozoa
15 min
82-5
II
Mean % abnormal spermatozoa in the thawed semen after storage for 7, 14 and 21 d
Mean % motile spermatozoa in the thawed semen after storage for 7, 14 and 21 d
62-5
85
13-2
95 95 75-°
52-5
14-8
15-2
8o-o*
54-2'
13-0*
13-4*
95 95
Average * Significant at Po-5 level.
Downloaded by [New York University] at 06:17 10 May 2015
EQUILIBRATION IN FREEZING FOWL SEMEN
537
After the appropriate equilibration times the semen samples were subjected to prefreezing in the vapour of liquid nitrogen (about — 110 °C to — 120 °C) for 2-5 min and then stored in liquid nitrogen. After each of 7, 14 and 21 d storage, duplicate sub-samples subjected to each of the equilibration times were thawed by standing the straw ampoules in water at 5 °C for 5 min and then at 20 °C for another 5 min and the motility of spermatozoa and the percentage of morphologically abnormal spermatozoa, including neck-bent ones, were estimated microscopically by observing approximately 500 spermatozoa in each case. Motility was scored using a scale of five points ( + + + , + + , + , ± , —). Motility and morphologically abnormal spermatozoa for each of the two equilibration times after different storage periods were thus examined in 12 subsamples of the original pooled semen (see Table 1). For morphological studies portions of the semen sub-samples were fixed with formalin and stained with carbolfuchsin-eosin solution. The composition of carbol-fuchsin-eosin solution is as follows: 96% alcohol, 25 ml; saturated eosin solution, 25 ml; carbol-fuchsin solution, 50 ml. The fertility of frozen semen stored for 11 to 14 d was also examined by inseminating artificially 22 White Leghorn hens (7 to 8 months old); 10 hens with semen equilibrated for 15 min and 12 hens with semen equilibrated for 1 min. RESULTS AND DISCUSSION
The motility of spermatozoa which had been stored in liquid nitrogen for 7, 14 and 21 d subsequent to being equilibrated for 15 and 1 min is shown in Table 1. The mean percentage of motile spermatozoa in thawed semen after storage for 7, r4 and 21 d and which had been equilibrated for 15 min was 80%; each percentage for 7, 14 and 21 d was 82*5, 82#5 and 75% respectively. They were over + + on the scale. The mean percentage with 1 min equilibration was 54-2%; percentages for 7, 14 and 21 d were 47-5, 62-5 and 52-5% respectively. Thus, there was a significant difference between the two equilibration treatments (P
