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The Influence of Glycerol Equilibration Time on the Metabolism, Motility and Fertility of Frozen Chicken Spermatozoa1,2
The Influence of Glycerol Equilibration Time on the Metabolism, Motility and Fertility of Frozen Chicken Spermatozoa1'2 J. E. BROWN 3 4 AND G. C. HARRIS, JR. University of Arkansas, Fayetteville, Arkansas (Received for publication September 5, 1962)
chicken spermatozoa has been limited. Shaffner (1942) obtained 25% fertility from chicken spermatozoa that were partially dehydrated with a sugar solution prior to freezing; however, embryonic development did not proceed for more than 10 to IS hours. The motility of chicken spermatozoa was protected during freezing and thawing by the addition of 15% glycerol (Polge et ah, 1949) but fertility was nil with either frozen or nonfrozen glycerolized semen when the semen was deposited in the vagina (Smith and Polge, 1950; Polge, 1951; Allen and Bobr, 1955). However, some fertility was produced in 6 1 % of the hens when the glycerol was removed by slow dialysis (Polge, 1951). In contrast, when the glycerolized semen was deposited in the uterus, 73% and 25% fertility was obtained with nonfrozen (Allen and Bobr, 1955) and frozen (Allen, 1958) semen, respectively. Clark and Shaffner (1960) obtained 40% fertility with intrauterine insemination of semen that had been frozen and the glyc1 Published .with the approval of the Director of the Arkansas Agricultural Experiment Station. . 2 The research reported in this paper was supported in part by a grant-in-aid by the American Poultry and Hatchery Federation. 3 Present address: Hubbard Farms, Walpole, N. H. 4 Portion of a thesis presented by the senior author in partial fulfillment of the requirements for the degree of Master of Science.
erol removed after thawing by dilution and centrifugation. Research on mammalian species has established that equilibration between glycerol and semen is necessary prior to freezing for optimum fertility. However, Clark and Shaffner (1960) observed that slow cooling and long equilibration were not mandatory for freezing chicken semen. Since equilibration data for chicken semen were quite limited, this study was initiated to determine the influence of this variable on the fertilizing capacity of frozen spermatozoa. MATERIALS AND METHODS
Pooled semen was obtained from a group of White Leghorn cockerels by the abdominal massage technique of Burrows and Quinn (1937). The semen was diluted 1 to 3 (1 part semen to 2 parts diluent) at room temperature with an isotonic phosphate buffer (Wilcox, 1958). The diluted semen contained a final concentration of 8% glycerol, 200 meg. oxytetracycline, and 1,000 meg. dihydrostreptomycin sulphate per ml. The diluent was added to the semen in bulk (1 portion) or fractions (5 equal portions at 6 minute intervals) in a test tube and was mixed by gentle stirring. Semen samples were equilibrated in 5 ml. sealed glass ampules at 2°C. for various times. Following equilibration, the samples were frozen by immersion into an alcohol bath at — 79°C. and were stored at this temperature for one hour. The bath was cooled to temperature with
377
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
with freezing to - 7 9 ° C . as a SUCCESS means of in vitro preservation of
378
J. E. BROWN AND G. C. HARRIS, JR.
T A B L E 1.—Effect of equilibration time and method of glycerol addition on oxygen uptake of chicken spermatozoa frozen for one hour at -79°C. Equilibration time (inin.) Bulk
Readings (minutes) 30
60
Fractions 90
30
60
90
1.57 3.64 5.70 6.89
2.24 4.78 6.36 8.05ab
(Microliters) 1 ' 2 30 60 90 120 s ' 1
2.11 3.10 4.71 6.28a
1.76 3.92 5.89 7.57 d
2.21 4.35 5.97 7.66°
2.00 2.97 4.60 5.74bod
Oxygen uptake per 10s cells. Each value represents the mean of 3 replicate flasks. Duncan's multiple range test—means with same letter are significantly different at 5% level. < Pooled S.E.± 0.4620.
pieces of solid carbon dioxide. The semen was thawed in a thermos bottle containing water with the temperature adjusted at 20° C , and the samples were held at this temperature until completely liquid. Motility was scored subjectively from 0 to 4, with four representing optimum motility, as described by Smith (1949). In the metabolism studies, the oxygen uptake was measured at 37°C. with a Warburg respiration apparatus according to Umbreit el al. (1949). The fertility and hatchability data were obtained from eggs produced by White Leghorn hens maintained in individual cages. Five to ten hens per treatment were inseminated by the intravaginal method (Wilcox, 1958) or the intrauterine method (Allen and Bobr, 1955) with 0.1 ml. of undiluted, fresh semen for the control, or 0.3 ml. of diluted semen per hen for the treatments, respectively. A 6.0 or 14.5 cm. glass tube extension was connected to a 1 ml. tuberculin syringe for the intravaginal and intrauterine techniques, respectively. The fertility and hatchability data represents eggs produced during the 2nd through the 8th day after insemination. The eggs were collected twice daily and were held at 55°F. until incubated. The fertility was determined by candling after 5 to 7 days of incubation. Hatch-
RESULTS AND DISCUSSION
The total oxygen uptake of chicken spermatozoa that had been frozen (Table 1) was increased with longer equilibration. Comparisons of the means for 120 minute oxygen uptake showed that 90 minute equilibration gave significantly higher uptake than 30 minute equilibration when glycerol was added in fractions. However, the differences in oxygen uptake between 60 and 90 minute equilibration times were non-significant. No significant differences in oxygen uptake were noted between equilibration times when the glycerol was added in bulk. An analysis of variance of these data showed that the differences in oxygen uptake between the two methods of glycerol addition were non-significant. Similar nonsignificant differences between these two methods were observed in two fertility trials (Table 2) which was in agreement with Clark and Shaffner (1960) in motility studies of unfrozen glycerolized chicken semen. In contrast, Sherman (1957) obtained significantly higher motilT A B L E 2.—Effect of glycerol equilibration time method of glycerol addition on fertility and hatchability of chicken spermatozoa frozen for one hour at —79°C. Method of addition Equilibration time (min.)
Bulk
2
Mean 1 Percent
30 60 90 Fresh control 1
Percent hatchability of Trials fertile 1 eggs 2 Mean
Fractions
Trials 1
and
1 fertility
13 4 15
14 2 34
13.5 3.0 24.5
6 23 12
16 14 13
72
93
82.5
-
-
10.5 18.5 12.5
85 95 97
-
91
Mean of 20 hens and 98 eggs (range 95-101).
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
1 2 3
ability was taken on the 22nd day of incubation. Percentages were converted to angles by arcsin transformation and were analyzed according to Steel and Torrie (1960).
379
FROZEN CHICKEN SPERMATOZOA
In two trials, a wider range in equilibration time was studied. The fertility data (Table 3) indicates that slightly higher fertility was obtained by using a 90minute equilibration time, but this increase in fertility was nonsignificant. The optimum equilibration time varied between trials which indicated a difference between semen samples. The hatchability was increased by equilibration; however, the number of fertile eggs involved was small. A 90-minute glycerol
TABLE 3.—Effect
of glycerol equilibration time on
motility, fertility and hatchability of chicken spermatozoa frozen for one hour at -7fi°Cl Percent fertility
Equilibra-
- Motility score
Trials
Percent hatchability of fertile eggs
time (min.)
1
2
Mean 2
10 30 60 90 120 150
7 0 8 29 23 19
8 43 27 20 19 4
7.5 21.5 17.5 24.5 21.0 1K5
2.0 2.5 3.3 3.8 3.0 3.0
67 75 78 87 83 100
54
74
64.0
4.0
89
Fresh control 1
Diluent containing glycerol was added to semen in fractions (for equilibration times less than 30 min.; added in 5 equal time intervals). 2 Mean of 10 hens and 54 eggs (range 46-61).
equilibration time gave highest average motility and fertility. However, the differences in fertility between the six equilibration times were non-significant. It appears that further studies will be required to establish the optimum equilibration time for chicken spermatozoa. In addition, further improvements in the intrauterine insemination technique will be necessary for optimum fertility and egg production. In these studies, a decline in egg.production was noted from the 2nd to 4th days after insemination with the intrauterine method. The average time between collection and insemination of the fresh semen was 67 minutes which could partially account for the reduced fertility of the control in some trials. SUMMARY
The effects of glycerol equilibration on the oxygen uptake, motility, fertility and hatchability of frozen chicken spermatozoa, stored at — 79°C. for one hour, were studied. The removal of glycerol was apparently unnecessary since the intrauterine insemination of glycerolized chicken spermatozoa that were equilibrated prior to freezing gave normal embryonic development. No fertility was obtained when the glycerol was removed by centrifuga-
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
ity of bull spermatozoa after freezing by the addition of glycerol in five steps as compared with one step. The fertility was highly variable, which was partially due to the inexperience of the inseminator with the intrauterine technique in these two trials, but equilibration improved the livability of the embryos. In preliminary trials with non-equilibrated semen and the glycerol added in one step, only one or two embryos were observed by candling at 5 days. Equilibration increased the post-thawing metabolism of the sperm cells, thus indicating a higher rate of survival after freezing which was also reflected in fertility. Equlibration before freezing reduced the harmful effects of glycerol on the fertilizing capacity, thus the removal of glycerol may not be necessary before insemination. In contrast, glycerol was removed by dialysis (Polge, 1951) and dilution followed by centrifugation (Clark and Shaffner, 1960). These investigators theorized that the removal of glycerol was necessary for optimum fertility. In preliminary trials, no fertility was obtained with either non-equilibrated or equilibrated samples when the glycerol was removed by centrifugation. However, these results may have been due to the time required for processing and transporting the semen to the farm (approximately 30 minutes after thawing).
380
J. E. BROWN AND G. C. HARRIS, JR.
ACKNOWLEDGMENT
The authors wish to express their appreciation to T. D. Hobbs for his technical assistance. REFERENCES Allen, T. E., 1958. The storage of fowl semen at low temperature. Proc. Austr. Soc. Anim. Prod. 2: 118-119. Allen, T. E., and L. W. Bobr, 1955. The fertility of fowl spermatozoa in glycerol diluents after intra-
uterine insemination. Poultry Sci. 34: 1167-1169. 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 C. S. Shaffner, 1960. The fertilizing capacity of frozen chicken semen and the influence of related in vitro processes. Poultry Sci. 39: 1213-1219. Polge, C , 1951. Functional survival of fowl spermatozoa after freezing at —79CC. Nature, 167: 949-950. Polge, C , A. U. Smith and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Shaffner, C. S., 1942. Longevity of fowl spermatozoa in frozen condition. Science, 96: 377. Sherman, J. K., 1957. Freezing and freeze-drying of bull spermatozoa. Amer. J. Physiol. 190: 281-286. Smith, A. U., 1949. The control of bacterial growth in fowl semen. J. Agric. Sci. 39:194-200. Smith, A. U., and C. Polge, 1950. Survival of spermatozoa at low temperatures. Nature, 166: 668. Steel, R. G. D., and J. D. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Umbriet, W. W., R. H. Burris and J. F. Stauffer, 1949. Manometric Techniques and Tissue Metabolism. 2nd ed. Burgess Publ. Co., Minneapolis. Wilcox, F. H., 1958. Changes in the pH of semen of the domestic cock as affected by temperature and frequency of collection. Poultry Sci. 37: 444-449.
Lipids in Chick Urine: The Influence of Dietary Rapeseed Oil J. L. SELL AND J. A. MCKIRDY Animal Science Department, University of Manitoba, Winnipeg, Canada (Received for publication September 5, 1962)
T
HE lipid content of chicken urine and the manner in which it may be influenced by dietary lipid level has been a point for conjecture for some time. Edwards et al. (1960) reported that the lipid content of urine obtained from adult chickens consuming a practical ration was 12.2% of the dry matter. These
workers also found evidence indicating that the hen's diet may greatly influence lipid content of the urine. Mehring et al. (1961) found an average of 1.33% of lipid material in the dry matter of urine collected from 8-week-old chickens fed a practical ration, and presented data showing that urinary lipid excretion was
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
tion of equilibrated and non-equilibrated samples. The optimum equilibration time varied markedly between semen samples. Semen samples, equilibrated at 2°C. for 90 minutes and frozen for one hour at — 79°C, had significantly higher postthawing oxygen uptake, highest motility and slightly higher fertility when compared to 30 and 60 minute equilibration periods. The hatchability was adequate near this equilibration time; however, the differences in fertility and hatchability were non-significant. The addition of glycerol to the semen in 1 portion or 5 equal portions before freezing did not affect the oxygen uptake or fertility.
chicken spermatozoa has been limited. Shaffner (1942) obtained 25% fertility from chicken spermatozoa that were partially dehydrated with a sugar solution prior to freezing; however, embryonic development did not proceed for more than 10 to IS hours. The motility of chicken spermatozoa was protected during freezing and thawing by the addition of 15% glycerol (Polge et ah, 1949) but fertility was nil with either frozen or nonfrozen glycerolized semen when the semen was deposited in the vagina (Smith and Polge, 1950; Polge, 1951; Allen and Bobr, 1955). However, some fertility was produced in 6 1 % of the hens when the glycerol was removed by slow dialysis (Polge, 1951). In contrast, when the glycerolized semen was deposited in the uterus, 73% and 25% fertility was obtained with nonfrozen (Allen and Bobr, 1955) and frozen (Allen, 1958) semen, respectively. Clark and Shaffner (1960) obtained 40% fertility with intrauterine insemination of semen that had been frozen and the glyc1 Published .with the approval of the Director of the Arkansas Agricultural Experiment Station. . 2 The research reported in this paper was supported in part by a grant-in-aid by the American Poultry and Hatchery Federation. 3 Present address: Hubbard Farms, Walpole, N. H. 4 Portion of a thesis presented by the senior author in partial fulfillment of the requirements for the degree of Master of Science.
erol removed after thawing by dilution and centrifugation. Research on mammalian species has established that equilibration between glycerol and semen is necessary prior to freezing for optimum fertility. However, Clark and Shaffner (1960) observed that slow cooling and long equilibration were not mandatory for freezing chicken semen. Since equilibration data for chicken semen were quite limited, this study was initiated to determine the influence of this variable on the fertilizing capacity of frozen spermatozoa. MATERIALS AND METHODS
Pooled semen was obtained from a group of White Leghorn cockerels by the abdominal massage technique of Burrows and Quinn (1937). The semen was diluted 1 to 3 (1 part semen to 2 parts diluent) at room temperature with an isotonic phosphate buffer (Wilcox, 1958). The diluted semen contained a final concentration of 8% glycerol, 200 meg. oxytetracycline, and 1,000 meg. dihydrostreptomycin sulphate per ml. The diluent was added to the semen in bulk (1 portion) or fractions (5 equal portions at 6 minute intervals) in a test tube and was mixed by gentle stirring. Semen samples were equilibrated in 5 ml. sealed glass ampules at 2°C. for various times. Following equilibration, the samples were frozen by immersion into an alcohol bath at — 79°C. and were stored at this temperature for one hour. The bath was cooled to temperature with
377
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
with freezing to - 7 9 ° C . as a SUCCESS means of in vitro preservation of
378
J. E. BROWN AND G. C. HARRIS, JR.
T A B L E 1.—Effect of equilibration time and method of glycerol addition on oxygen uptake of chicken spermatozoa frozen for one hour at -79°C. Equilibration time (inin.) Bulk
Readings (minutes) 30
60
Fractions 90
30
60
90
1.57 3.64 5.70 6.89
2.24 4.78 6.36 8.05ab
(Microliters) 1 ' 2 30 60 90 120 s ' 1
2.11 3.10 4.71 6.28a
1.76 3.92 5.89 7.57 d
2.21 4.35 5.97 7.66°
2.00 2.97 4.60 5.74bod
Oxygen uptake per 10s cells. Each value represents the mean of 3 replicate flasks. Duncan's multiple range test—means with same letter are significantly different at 5% level. < Pooled S.E.± 0.4620.
pieces of solid carbon dioxide. The semen was thawed in a thermos bottle containing water with the temperature adjusted at 20° C , and the samples were held at this temperature until completely liquid. Motility was scored subjectively from 0 to 4, with four representing optimum motility, as described by Smith (1949). In the metabolism studies, the oxygen uptake was measured at 37°C. with a Warburg respiration apparatus according to Umbreit el al. (1949). The fertility and hatchability data were obtained from eggs produced by White Leghorn hens maintained in individual cages. Five to ten hens per treatment were inseminated by the intravaginal method (Wilcox, 1958) or the intrauterine method (Allen and Bobr, 1955) with 0.1 ml. of undiluted, fresh semen for the control, or 0.3 ml. of diluted semen per hen for the treatments, respectively. A 6.0 or 14.5 cm. glass tube extension was connected to a 1 ml. tuberculin syringe for the intravaginal and intrauterine techniques, respectively. The fertility and hatchability data represents eggs produced during the 2nd through the 8th day after insemination. The eggs were collected twice daily and were held at 55°F. until incubated. The fertility was determined by candling after 5 to 7 days of incubation. Hatch-
RESULTS AND DISCUSSION
The total oxygen uptake of chicken spermatozoa that had been frozen (Table 1) was increased with longer equilibration. Comparisons of the means for 120 minute oxygen uptake showed that 90 minute equilibration gave significantly higher uptake than 30 minute equilibration when glycerol was added in fractions. However, the differences in oxygen uptake between 60 and 90 minute equilibration times were non-significant. No significant differences in oxygen uptake were noted between equilibration times when the glycerol was added in bulk. An analysis of variance of these data showed that the differences in oxygen uptake between the two methods of glycerol addition were non-significant. Similar nonsignificant differences between these two methods were observed in two fertility trials (Table 2) which was in agreement with Clark and Shaffner (1960) in motility studies of unfrozen glycerolized chicken semen. In contrast, Sherman (1957) obtained significantly higher motilT A B L E 2.—Effect of glycerol equilibration time method of glycerol addition on fertility and hatchability of chicken spermatozoa frozen for one hour at —79°C. Method of addition Equilibration time (min.)
Bulk
2
Mean 1 Percent
30 60 90 Fresh control 1
Percent hatchability of Trials fertile 1 eggs 2 Mean
Fractions
Trials 1
and
1 fertility
13 4 15
14 2 34
13.5 3.0 24.5
6 23 12
16 14 13
72
93
82.5
-
-
10.5 18.5 12.5
85 95 97
-
91
Mean of 20 hens and 98 eggs (range 95-101).
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
1 2 3
ability was taken on the 22nd day of incubation. Percentages were converted to angles by arcsin transformation and were analyzed according to Steel and Torrie (1960).
379
FROZEN CHICKEN SPERMATOZOA
In two trials, a wider range in equilibration time was studied. The fertility data (Table 3) indicates that slightly higher fertility was obtained by using a 90minute equilibration time, but this increase in fertility was nonsignificant. The optimum equilibration time varied between trials which indicated a difference between semen samples. The hatchability was increased by equilibration; however, the number of fertile eggs involved was small. A 90-minute glycerol
TABLE 3.—Effect
of glycerol equilibration time on
motility, fertility and hatchability of chicken spermatozoa frozen for one hour at -7fi°Cl Percent fertility
Equilibra-
- Motility score
Trials
Percent hatchability of fertile eggs
time (min.)
1
2
Mean 2
10 30 60 90 120 150
7 0 8 29 23 19
8 43 27 20 19 4
7.5 21.5 17.5 24.5 21.0 1K5
2.0 2.5 3.3 3.8 3.0 3.0
67 75 78 87 83 100
54
74
64.0
4.0
89
Fresh control 1
Diluent containing glycerol was added to semen in fractions (for equilibration times less than 30 min.; added in 5 equal time intervals). 2 Mean of 10 hens and 54 eggs (range 46-61).
equilibration time gave highest average motility and fertility. However, the differences in fertility between the six equilibration times were non-significant. It appears that further studies will be required to establish the optimum equilibration time for chicken spermatozoa. In addition, further improvements in the intrauterine insemination technique will be necessary for optimum fertility and egg production. In these studies, a decline in egg.production was noted from the 2nd to 4th days after insemination with the intrauterine method. The average time between collection and insemination of the fresh semen was 67 minutes which could partially account for the reduced fertility of the control in some trials. SUMMARY
The effects of glycerol equilibration on the oxygen uptake, motility, fertility and hatchability of frozen chicken spermatozoa, stored at — 79°C. for one hour, were studied. The removal of glycerol was apparently unnecessary since the intrauterine insemination of glycerolized chicken spermatozoa that were equilibrated prior to freezing gave normal embryonic development. No fertility was obtained when the glycerol was removed by centrifuga-
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
ity of bull spermatozoa after freezing by the addition of glycerol in five steps as compared with one step. The fertility was highly variable, which was partially due to the inexperience of the inseminator with the intrauterine technique in these two trials, but equilibration improved the livability of the embryos. In preliminary trials with non-equilibrated semen and the glycerol added in one step, only one or two embryos were observed by candling at 5 days. Equilibration increased the post-thawing metabolism of the sperm cells, thus indicating a higher rate of survival after freezing which was also reflected in fertility. Equlibration before freezing reduced the harmful effects of glycerol on the fertilizing capacity, thus the removal of glycerol may not be necessary before insemination. In contrast, glycerol was removed by dialysis (Polge, 1951) and dilution followed by centrifugation (Clark and Shaffner, 1960). These investigators theorized that the removal of glycerol was necessary for optimum fertility. In preliminary trials, no fertility was obtained with either non-equilibrated or equilibrated samples when the glycerol was removed by centrifugation. However, these results may have been due to the time required for processing and transporting the semen to the farm (approximately 30 minutes after thawing).
380
J. E. BROWN AND G. C. HARRIS, JR.
ACKNOWLEDGMENT
The authors wish to express their appreciation to T. D. Hobbs for his technical assistance. REFERENCES Allen, T. E., 1958. The storage of fowl semen at low temperature. Proc. Austr. Soc. Anim. Prod. 2: 118-119. Allen, T. E., and L. W. Bobr, 1955. The fertility of fowl spermatozoa in glycerol diluents after intra-
uterine insemination. Poultry Sci. 34: 1167-1169. 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 C. S. Shaffner, 1960. The fertilizing capacity of frozen chicken semen and the influence of related in vitro processes. Poultry Sci. 39: 1213-1219. Polge, C , 1951. Functional survival of fowl spermatozoa after freezing at —79CC. Nature, 167: 949-950. Polge, C , A. U. Smith and A. S. Parkes, 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666. Shaffner, C. S., 1942. Longevity of fowl spermatozoa in frozen condition. Science, 96: 377. Sherman, J. K., 1957. Freezing and freeze-drying of bull spermatozoa. Amer. J. Physiol. 190: 281-286. Smith, A. U., 1949. The control of bacterial growth in fowl semen. J. Agric. Sci. 39:194-200. Smith, A. U., and C. Polge, 1950. Survival of spermatozoa at low temperatures. Nature, 166: 668. Steel, R. G. D., and J. D. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Umbriet, W. W., R. H. Burris and J. F. Stauffer, 1949. Manometric Techniques and Tissue Metabolism. 2nd ed. Burgess Publ. Co., Minneapolis. Wilcox, F. H., 1958. Changes in the pH of semen of the domestic cock as affected by temperature and frequency of collection. Poultry Sci. 37: 444-449.
Lipids in Chick Urine: The Influence of Dietary Rapeseed Oil J. L. SELL AND J. A. MCKIRDY Animal Science Department, University of Manitoba, Winnipeg, Canada (Received for publication September 5, 1962)
T
HE lipid content of chicken urine and the manner in which it may be influenced by dietary lipid level has been a point for conjecture for some time. Edwards et al. (1960) reported that the lipid content of urine obtained from adult chickens consuming a practical ration was 12.2% of the dry matter. These
workers also found evidence indicating that the hen's diet may greatly influence lipid content of the urine. Mehring et al. (1961) found an average of 1.33% of lipid material in the dry matter of urine collected from 8-week-old chickens fed a practical ration, and presented data showing that urinary lipid excretion was
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on April 19, 2015
tion of equilibrated and non-equilibrated samples. The optimum equilibration time varied markedly between semen samples. Semen samples, equilibrated at 2°C. for 90 minutes and frozen for one hour at — 79°C, had significantly higher postthawing oxygen uptake, highest motility and slightly higher fertility when compared to 30 and 60 minute equilibration periods. The hatchability was adequate near this equilibration time; however, the differences in fertility and hatchability were non-significant. The addition of glycerol to the semen in 1 portion or 5 equal portions before freezing did not affect the oxygen uptake or fertility.