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The role of sample depth in storing chilled rainbow trout (Salmo gairdneri) semen under oxygen
Aquaculture, 61 (1987) 275-279 Elsevier Science Publishers B.V., Amsterdam
275 -
Printed
in The Netherlands
The Role of Sample Depth in Storing Chilled Rainbow Trout (SaZmo guirdneri) Semen under Oxygen J. STOSS, L. GERIES
and W. HOLTZ
Znstitut fiir Tierzucht und Haustiergenetik der Uniuersitiit GSttingen, Albrecht-Thaer- Weg 1, D-3400 Cattingen (Federal Republic of Germany) (Accepted
5 December
1986)
ABSTRACT Stoss, J., Geries, L. and Holtz, W., 1987. The role of sample depth in storing chilled rainbow trout (Salmo gairdneri) semen under oxygen. Aquaculture, 61: 275-279. In order to investigate the role of oxygen exposure when storing rainbow trout semen at 0’ C, three experiments were conducted. In experiment 1, semen was placed in a test tube of 14.5 mm diameter which had a vertical slit covered with latex tubing to allow repeated sampling at different levels. After 3.5 days of storage, the motility rating at and as low as 5 mm below the surface was approximately 30%. At depths of 7.5 mm or lower it was 6.5% and less, and at 17.5 mm motion had ceased. After 7 days of storage motility was between 11.8 and 12.8% in the upper 5 mm, 3.0% at 7.5 mm and 0% at 10 mm andlower. In experiment 2, storage vials of different diameters were compared with each other. In narrow vials (31.0 mm sample depth) motility ceased rapidly. In very shallow vials (2.5 mm sample depth) motility was maintained at a reasonable level until day 26. Best results were obtained with an intermediate sample depth of 6.5 mm; motility rates of more than 20% were maintained until day 31. In a third experiment it was established that evaporation is not a problem in storing rainbow trout semen as long as the gas atmosphere is moisture-saturated. In conclusion, when storing chilled semen in open vials under oxygen, sample depth should be 5-6 mm and agitation of samples should be reduced to a minimum.
INTRODUCTION
The availability of a suitable method for preserving rainbow trout semen for a period of several days could be of considerable value. In our laboratory we have attempted to devise such a technique. Biiyiikhatipoglu and Holtz (1978) found that rainbow trout semen is stored best undiluted at low temperature under aerobic conditions. Subsequent trials by Stoss et al. (1978) revealed that a temperature just above freezing is most suitable, that addition of antibiotics
0044-8486/87/$03.50
0 1987 Elsevier Science Publishers
B.V.
276
controls bacterial growth and that agitation of stored semen should be avoided. Taking these points into consideration, Stoss and Holtz (1983) obtained satisfactory fertilization results with semen stored up to 34 days. In those experiments, fertilization trials were conducted with excessive amounts of semen, and this possibly concealed the fact that only part of the sperm cells had maintained their ability to fertilize. The present experiment was conducted with the intention of further improving the technique and, in an empirical way, studying the role of oxygen in this context. MATERIAL AND METHODS
The semen used in this experiment was stripped from 3- and 4-year-old anesthetized males in the middle of the spawning season. From each stripping 4ml aliquots were kept for 2-4 h in open test tubes on crushed ice. During that period motility estimates were conducted according to the method described by Holtz et al. (1977). The criteria recorded were (1) percent motile sperm, (2) intensity of motility and (3) duration of mass motility. In order to obtain sperm-free seminal plasma, pooled semen from eight strippings with more than 70% motile sperm was spun down at 600 g for 15 min. The plasma was used to dissolve penicillin and streptomycin at concentrations of 20 000 IU and 20 000 ,ug per ml, respectively. Of this liquid 25 ~1 was added to each ml of stored semen. In experiment 1, polyethylene test tubes with internal diameter 14.5 mm and having a l-mm slit running down the side were inserted into tight-fitting latex tubing. By piercing the tubing over the slit with a 25-g hypodermic needle attached to a l-ml tuberculin syringe it was possible to sample the content of the test tube at different levels without stirring it. Each of three such test tubes was filled with semen pooled from eight strippings. The tubes were placed into a 10-l desiccator kept in the dark at 0 + 0.5 ‘C and gassed daily with moisturesaturated oxygen as described by Stoss and Holtz (1983). After 3.5 and 7.0 days of storage, motility estimates were conducted on samples of 0.01-0.02 ml that were taken at 2.5-mm intervals from the surface. Each sampling was replicated five times for each of the three tubes. Experiment 2 was conducted with pooled semen from ten males, selected, pooled and treated as described above. Aliquots of 2 ml were pipetted into glass cylinders, five each with internal diameters 32, 20 and 9 mm. The cylinders were kept at 0 ? 0.5”C in a desiccator under oxygen as described above. At 4-6day intervals motility was estimated in a drop of semen adhering to a dissecting needle after it was dipped to the bottom of the cylinder. The experiment was repeated for the 32 and 20 mm cylinders. This time motility estimation was started on day 16 and continued at 5-day intervals. In order to find out whether evaporation plays a significant role during the
277 TABLE 1 Motility of sperm obtained from different levels below the surface of semen stored in test tubes after 3.5 and 7.0 days: each mean represents 15 estimates (3 tubes, 5 replications/tube)
(mm)
Motility score (o/o) f SD
Intensity of motility (Oto5) x SD
(s) f
SD
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5
28.0 31.0 31.0 6.5 2.5 2.0 2.6 0.0
9.9 10.7 6.8 5.1 3.0 2.8 2.6 0.0
3.8 3.7 3.5 3.0 2.8 3.0 4.2 3.5
1.3 0.9 0.9 1.0 0.9 1.1 0.4 0.7
16.2 15.0 16.0 14.7 16.1 14.3 15.4 0.0
3.6 1.9 1.7 1.6 0.9 1.5 1.8 0.0
11.0 11.0 12.8 3.0 0.0
7.5 7.0 2.8 2.2 0.0
4.3 4.1 4.1 3.6 3.6
0.6 0.9 0.5 0.6 0.5
15.6 15.8 13.3 14.7 0.0
1.9 2.5 1.0 1.8 0.0
Storage time
Sampling depth
(days ) 3.5
7.0
0
2.5 5.0 7.5 10.0
Mass motility
storage period, experiment 3 was conducted. Pooled fresh semen from seven males was distributed to 20 vials of 22 mm internal diameter as 2-ml aliquots. Ten vials were placed into each of two desiccators, one of which was gassed with dry oxygen, the other with moisture-saturated oxygen. Gassing took place twice daily, otherwise conditions were as mentioned above. Vials were weighed at 4-day intervals until day 24. RESULTS
As shown in Table 1, motility in the 36-mm semen column decreased from the surface toward the bottom of the tube. Beyond 5 mm from the surface, motility test ratings were extremely low, reaching zero at 17.5 mm within 3.5 days and at 10 mm within 7 days. As long as cells were activated upon dilution, intensity and duration of motility were essentially unaffected (Table 1) . The concentration of spermatozoa near the surface of the semen column decreased with time, presumably as a result of sedimentation. The results of the first part of experiment 2 indicated that motility in the narrow vials (sample depth 31 mm, surface 64 mm2) had ceased by day 4 and that frequent sampling was disadvantageous. When the experiment was repeated with the other two types of vials (sample depth/surface area being 6.5 mm/314 mm2 and 2.5 mm/804 mm’, respectively) which were left untouched for 16 days, the motility rating at first sampling had dropped from 80% to 67%
in the 2.5mm vials and to 37% in the 6.5mm vials. Thereafter the decline in motility continued gradually, the zero point being reached by day 31 in the 2.5 mm vials and by day 41 in the 6.5-mm vials. As observed in experiment 1, intensity and duration of motility were maintained at a constant level as long as cells remained motile. Experiment 3 indicated that with semen that was gassed with dry oxygen twice daily there was a gradual weight loss, amounting to 1.25% by day 24. When using moisturized oxygen, no weight loss was recorded. DISCUSSION
The results of experiments 1 and 2 indicate that oxygen supply to sperm cells plays a decisive role in storing rainbow trout semen. In experiment 1 most cells sampled at more than 5 mm from the interphase between semen and oxygen had perished within 3.5 days. This observation supports the opinion of Terner and Korsh (1963) and Mounib (1967)) who suggest that metabolism of rainbow trout spermatozoa is primarily aerobic. The short survival time of sperm in very shallow semen samples (2.5 mm) with large surface area (804 mm’), on the other hand, suggests that in the presence of an abundance of oxygen the limited supply of reducible substances (Holtz et al., 1979) is exhausted rather rapidly, particularly since rainbow trout semen has a very high sperm density (5 to 20 x log/ml). It appears that in deep semen samples with small surface area, the perishing cells at the bottom of the container exert a detrimental effect on those located close to the surface, because in these, too, motility ceased within a rather short period. Better survival rates in samples that were left undisturbed until day 16 in comparison to those sampled throughout the storage period confirm earlier findings by Stoss et al. (1977) who showed that agitation of samples was detrimental to sperm survival. The result of experiment 3 suggests that, under the storage conditions applied in this experiment, there is essentially no evaporation loss. It may be concluded from this investigation that, when rainbow trout semen is stored at 0” C in a moisture-saturated oxygen atmosphere, it should be kept in uncapped vials filled to a level of 5-6 mm and not be stirred until it is used to fertilize eggs.
REFERENCES Biiyiikhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. Holtz, W., Stoss, J. and Biiyiikhatipoglu, S., 1977. Beobachtungen zur Aktivierbarkeit von Forellensperma mit Fruchtwasser, Bachwasser und destilliertem Wasser. Zuchthygiene, 12: 82-88.
219 Holtz, W., Btiyukhatipoglu, S., Stoss, J., Oldings, B. and Langholz, H.-J., 1979. Preservation of trout spermatozoa for varying periods. In: T.V.R. Pillay and W.A. Dill (Editors), Advances in Aquaculture. Fishing News Books, Famham, Great Britain, pp. 141-142. Mounib, M.S., 1967. Metabolism of pyruvate, acetate and glyoxylate by fish sperm. Comp. Biochem. Physiol., 20: 987-992. Stoss, J. and Holtz, W., 1983. Successful storage of chilled rainbow trout (Salmo guirdneri) spermatozoa for up to 34 days. Aquaculture, 31: 269-274. Stoss, J., Biiyiikhatipoglu, S. and Holtz, W., 1977. Short-term and cryopreservation of trout sperm. Symposium on Reproductive Physiology of Fish, 19-20 September 1977, Paimpont, France. Stoss, J., Btiyiikhatipoglu, S. and Holtz, W., 1978. Short-term and cryopreservation of rainbow trout (Salmoguirdneri Richardson) sperm. Ann. Biol. Anim. Biochim. Biophys., 18: 1077-1082. Terner, C. and Korsh, G., 1963. The oxidative metabolism of pyruvate, acetate and glucose in isolated fish spermatozoa. J. Cell. Comp. Physiol., 62: 243-249.
275 -
Printed
in The Netherlands
The Role of Sample Depth in Storing Chilled Rainbow Trout (SaZmo guirdneri) Semen under Oxygen J. STOSS, L. GERIES
and W. HOLTZ
Znstitut fiir Tierzucht und Haustiergenetik der Uniuersitiit GSttingen, Albrecht-Thaer- Weg 1, D-3400 Cattingen (Federal Republic of Germany) (Accepted
5 December
1986)
ABSTRACT Stoss, J., Geries, L. and Holtz, W., 1987. The role of sample depth in storing chilled rainbow trout (Salmo gairdneri) semen under oxygen. Aquaculture, 61: 275-279. In order to investigate the role of oxygen exposure when storing rainbow trout semen at 0’ C, three experiments were conducted. In experiment 1, semen was placed in a test tube of 14.5 mm diameter which had a vertical slit covered with latex tubing to allow repeated sampling at different levels. After 3.5 days of storage, the motility rating at and as low as 5 mm below the surface was approximately 30%. At depths of 7.5 mm or lower it was 6.5% and less, and at 17.5 mm motion had ceased. After 7 days of storage motility was between 11.8 and 12.8% in the upper 5 mm, 3.0% at 7.5 mm and 0% at 10 mm andlower. In experiment 2, storage vials of different diameters were compared with each other. In narrow vials (31.0 mm sample depth) motility ceased rapidly. In very shallow vials (2.5 mm sample depth) motility was maintained at a reasonable level until day 26. Best results were obtained with an intermediate sample depth of 6.5 mm; motility rates of more than 20% were maintained until day 31. In a third experiment it was established that evaporation is not a problem in storing rainbow trout semen as long as the gas atmosphere is moisture-saturated. In conclusion, when storing chilled semen in open vials under oxygen, sample depth should be 5-6 mm and agitation of samples should be reduced to a minimum.
INTRODUCTION
The availability of a suitable method for preserving rainbow trout semen for a period of several days could be of considerable value. In our laboratory we have attempted to devise such a technique. Biiyiikhatipoglu and Holtz (1978) found that rainbow trout semen is stored best undiluted at low temperature under aerobic conditions. Subsequent trials by Stoss et al. (1978) revealed that a temperature just above freezing is most suitable, that addition of antibiotics
0044-8486/87/$03.50
0 1987 Elsevier Science Publishers
B.V.
276
controls bacterial growth and that agitation of stored semen should be avoided. Taking these points into consideration, Stoss and Holtz (1983) obtained satisfactory fertilization results with semen stored up to 34 days. In those experiments, fertilization trials were conducted with excessive amounts of semen, and this possibly concealed the fact that only part of the sperm cells had maintained their ability to fertilize. The present experiment was conducted with the intention of further improving the technique and, in an empirical way, studying the role of oxygen in this context. MATERIAL AND METHODS
The semen used in this experiment was stripped from 3- and 4-year-old anesthetized males in the middle of the spawning season. From each stripping 4ml aliquots were kept for 2-4 h in open test tubes on crushed ice. During that period motility estimates were conducted according to the method described by Holtz et al. (1977). The criteria recorded were (1) percent motile sperm, (2) intensity of motility and (3) duration of mass motility. In order to obtain sperm-free seminal plasma, pooled semen from eight strippings with more than 70% motile sperm was spun down at 600 g for 15 min. The plasma was used to dissolve penicillin and streptomycin at concentrations of 20 000 IU and 20 000 ,ug per ml, respectively. Of this liquid 25 ~1 was added to each ml of stored semen. In experiment 1, polyethylene test tubes with internal diameter 14.5 mm and having a l-mm slit running down the side were inserted into tight-fitting latex tubing. By piercing the tubing over the slit with a 25-g hypodermic needle attached to a l-ml tuberculin syringe it was possible to sample the content of the test tube at different levels without stirring it. Each of three such test tubes was filled with semen pooled from eight strippings. The tubes were placed into a 10-l desiccator kept in the dark at 0 + 0.5 ‘C and gassed daily with moisturesaturated oxygen as described by Stoss and Holtz (1983). After 3.5 and 7.0 days of storage, motility estimates were conducted on samples of 0.01-0.02 ml that were taken at 2.5-mm intervals from the surface. Each sampling was replicated five times for each of the three tubes. Experiment 2 was conducted with pooled semen from ten males, selected, pooled and treated as described above. Aliquots of 2 ml were pipetted into glass cylinders, five each with internal diameters 32, 20 and 9 mm. The cylinders were kept at 0 ? 0.5”C in a desiccator under oxygen as described above. At 4-6day intervals motility was estimated in a drop of semen adhering to a dissecting needle after it was dipped to the bottom of the cylinder. The experiment was repeated for the 32 and 20 mm cylinders. This time motility estimation was started on day 16 and continued at 5-day intervals. In order to find out whether evaporation plays a significant role during the
277 TABLE 1 Motility of sperm obtained from different levels below the surface of semen stored in test tubes after 3.5 and 7.0 days: each mean represents 15 estimates (3 tubes, 5 replications/tube)
(mm)
Motility score (o/o) f SD
Intensity of motility (Oto5) x SD
(s) f
SD
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5
28.0 31.0 31.0 6.5 2.5 2.0 2.6 0.0
9.9 10.7 6.8 5.1 3.0 2.8 2.6 0.0
3.8 3.7 3.5 3.0 2.8 3.0 4.2 3.5
1.3 0.9 0.9 1.0 0.9 1.1 0.4 0.7
16.2 15.0 16.0 14.7 16.1 14.3 15.4 0.0
3.6 1.9 1.7 1.6 0.9 1.5 1.8 0.0
11.0 11.0 12.8 3.0 0.0
7.5 7.0 2.8 2.2 0.0
4.3 4.1 4.1 3.6 3.6
0.6 0.9 0.5 0.6 0.5
15.6 15.8 13.3 14.7 0.0
1.9 2.5 1.0 1.8 0.0
Storage time
Sampling depth
(days ) 3.5
7.0
0
2.5 5.0 7.5 10.0
Mass motility
storage period, experiment 3 was conducted. Pooled fresh semen from seven males was distributed to 20 vials of 22 mm internal diameter as 2-ml aliquots. Ten vials were placed into each of two desiccators, one of which was gassed with dry oxygen, the other with moisture-saturated oxygen. Gassing took place twice daily, otherwise conditions were as mentioned above. Vials were weighed at 4-day intervals until day 24. RESULTS
As shown in Table 1, motility in the 36-mm semen column decreased from the surface toward the bottom of the tube. Beyond 5 mm from the surface, motility test ratings were extremely low, reaching zero at 17.5 mm within 3.5 days and at 10 mm within 7 days. As long as cells were activated upon dilution, intensity and duration of motility were essentially unaffected (Table 1) . The concentration of spermatozoa near the surface of the semen column decreased with time, presumably as a result of sedimentation. The results of the first part of experiment 2 indicated that motility in the narrow vials (sample depth 31 mm, surface 64 mm2) had ceased by day 4 and that frequent sampling was disadvantageous. When the experiment was repeated with the other two types of vials (sample depth/surface area being 6.5 mm/314 mm2 and 2.5 mm/804 mm’, respectively) which were left untouched for 16 days, the motility rating at first sampling had dropped from 80% to 67%
in the 2.5mm vials and to 37% in the 6.5mm vials. Thereafter the decline in motility continued gradually, the zero point being reached by day 31 in the 2.5 mm vials and by day 41 in the 6.5-mm vials. As observed in experiment 1, intensity and duration of motility were maintained at a constant level as long as cells remained motile. Experiment 3 indicated that with semen that was gassed with dry oxygen twice daily there was a gradual weight loss, amounting to 1.25% by day 24. When using moisturized oxygen, no weight loss was recorded. DISCUSSION
The results of experiments 1 and 2 indicate that oxygen supply to sperm cells plays a decisive role in storing rainbow trout semen. In experiment 1 most cells sampled at more than 5 mm from the interphase between semen and oxygen had perished within 3.5 days. This observation supports the opinion of Terner and Korsh (1963) and Mounib (1967)) who suggest that metabolism of rainbow trout spermatozoa is primarily aerobic. The short survival time of sperm in very shallow semen samples (2.5 mm) with large surface area (804 mm’), on the other hand, suggests that in the presence of an abundance of oxygen the limited supply of reducible substances (Holtz et al., 1979) is exhausted rather rapidly, particularly since rainbow trout semen has a very high sperm density (5 to 20 x log/ml). It appears that in deep semen samples with small surface area, the perishing cells at the bottom of the container exert a detrimental effect on those located close to the surface, because in these, too, motility ceased within a rather short period. Better survival rates in samples that were left undisturbed until day 16 in comparison to those sampled throughout the storage period confirm earlier findings by Stoss et al. (1977) who showed that agitation of samples was detrimental to sperm survival. The result of experiment 3 suggests that, under the storage conditions applied in this experiment, there is essentially no evaporation loss. It may be concluded from this investigation that, when rainbow trout semen is stored at 0” C in a moisture-saturated oxygen atmosphere, it should be kept in uncapped vials filled to a level of 5-6 mm and not be stirred until it is used to fertilize eggs.
REFERENCES Biiyiikhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. Holtz, W., Stoss, J. and Biiyiikhatipoglu, S., 1977. Beobachtungen zur Aktivierbarkeit von Forellensperma mit Fruchtwasser, Bachwasser und destilliertem Wasser. Zuchthygiene, 12: 82-88.
219 Holtz, W., Btiyukhatipoglu, S., Stoss, J., Oldings, B. and Langholz, H.-J., 1979. Preservation of trout spermatozoa for varying periods. In: T.V.R. Pillay and W.A. Dill (Editors), Advances in Aquaculture. Fishing News Books, Famham, Great Britain, pp. 141-142. Mounib, M.S., 1967. Metabolism of pyruvate, acetate and glyoxylate by fish sperm. Comp. Biochem. Physiol., 20: 987-992. Stoss, J. and Holtz, W., 1983. Successful storage of chilled rainbow trout (Salmo guirdneri) spermatozoa for up to 34 days. Aquaculture, 31: 269-274. Stoss, J., Biiyiikhatipoglu, S. and Holtz, W., 1977. Short-term and cryopreservation of trout sperm. Symposium on Reproductive Physiology of Fish, 19-20 September 1977, Paimpont, France. Stoss, J., Btiyiikhatipoglu, S. and Holtz, W., 1978. Short-term and cryopreservation of rainbow trout (Salmoguirdneri Richardson) sperm. Ann. Biol. Anim. Biochim. Biophys., 18: 1077-1082. Terner, C. and Korsh, G., 1963. The oxidative metabolism of pyruvate, acetate and glucose in isolated fish spermatozoa. J. Cell. Comp. Physiol., 62: 243-249.