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Preservation of trout sperm in liquid or frozen state
Aquaculture, 14 (1978) 49-56 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
PRESERVATION
49
OF TROUT SPERM IN LIQUID OR FROZEN STATE
S. BUYUKHATIPOGLU
and W. HOLTZ
Znstitut fir Tienucht und Haustiergenetik der Uniuersita”t Giittingen, Albrecht-ThaerWeg 1, 3400 Giittingen (Federal Republic of Germany) (Received 20 October 1977; revised 16 December 1977)
ABSTRACT Bilyilkhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. A series of experiments to study the preservation of rainbow trout semen in the liquid and frozen state was carried out. For the preservation of liquid semen both undiluted and diluted semen was stored in total darkness at 20” or 4°C under a number of different gasses (air, 0,, 0, + N, (1 :l), N,, CO,, N, + H, + CO, (8 :l :l)). The best results were obtained when storing semen in an undiluted state at 4°C under 0, (hatching rate after 16 days was 80.6%, controls were 98.2%). Good results were also obtained under air (after 9 days 94.7% vs 94.9% for the controls). Anaerobic conditions, dilution or an increase in temperature all rendered less favourable results. For the purpose of cryopreservation a diluent was developed on the basis of the composition of rainbow trout seminal plasma. The semen was mixed with the diluent immediately before freezing at a ratio of 1 :l or 1:3. The pellet-technique (Nagase, 1964) was found to be more convenient and successful than freezing in straws. The pellets were thawed in a 1% NaHCO,-solution. The hatching results after storage periods of l-4 months were very variable (2.6--80.3%).
INTRODUCTION
The availability of suitable methods for successful preservation of trout semen might be extremely valuable both from an organisational and a breeding point of view (e.g. semen transport or crossing of strains with differing spawning seasons). One has to differentiate between the preservation of liquid semen for limited periods of time and cryopreservation for long-term semen storage. Brofeldt (1914) was one of the first to recognize that trout and salmon spermatozoa will survive longer if kept cool. Later experiments were conducted involving the storage of salmonid sperm at temperatures from 0 to 9°C (Scheuring, 1924; Smith and Quistorff, 1943; Barrett, 1951; Henderson and Dewar, 1959; Withler and Humphreys, 1967; Withler and Morley, 1968; Stein, 1975). Several investigations were aimed at increasing storage time by
50
adding electrolyte solutions or coelomic fluid from females (Dorier, 1949/ 1950,195l; Henderson and Dewar, 1959). In most cases “Cortland’‘-medium (Wolf, 1963) served as the basis for the development of diluents. Substances that were frequently added to diluents were lactose, fructose, lecithin, mannitol, glycin or egg yolk and, to prevent cell damage during freezing and thawing, ethylene glycol, propylene glycol, glycerol or dimethylsulfoxide (DMSO) (Truscott et al., 1968; Hoyle and Idler, 1968; Graybill and Horton, 1969; Ott and Horton, 1971a,b; Stein, 1975). A diluent developed on the basis of seminal plasma was first reported by Truscott and Idler (1969). To date no satisfactory techniques have been developed for either liquid preservation or cryopreservation of trout sperm. MATERIAL
AND METHODS
The milt that was used for the investigation was collected from 2-3 year old males which were kept under standardized conditions in fish tanks and were stripped at 1 or 2 week intervals. The milt was collected in pre-cooled test tubes and transported to the laboratory on water-ice. There its motility was examined within one hour by the technique described by Holtz et al. (1977). Only good quality semen was utilized for experimental purposes. Milt from different males was never mixed. Preservation of liquid semen
Samples of 1.5 ml of either undiluted semen or semen diluted at a ratio of 1 : 1 or 1 : 16 with a buffered isotonic diluent resembling the inorganic composition of seminal plasma (Holtz et al., 1976) were placed in open glass vials with a surface area of 3.14 cm2 and a height of 4.5 cm. Six of these vials were placed in a 1 1 Erlenmeyer-flask, the bottom of which was covered with moist cotton (Fig. 1). The flasks were stoppered and kept in darkness at either 20” or 4°C. Twice daily they were gassed with one of the following gasses: air, 02, N2, C02, N2-02 (1 : 1) or N2-H2-C02 (8 : 1 : 1). Each day the vials were swirled gently before a tiny drop of semen was taken from each. Motility was estimated as described by Holtz et al. (1977). In a follow-up trial undiluted milt kept at 4°C under air was added to a minimum of 500 freshly collected trout eggs after 2,4 or 9 days of storage. Milt kept at 4°C under pure O2 was added to a similar number of eggs after a storage period of 15 days. Cryopreservation
To a number of semen samples DMSO or glycerol was added at ratios of 1 : 0.1, 1 : 0.5,l : 1 or 1 : 3 before freezing them by the pellet-technique (Nagase, 1964). Most samples, however, were mixed at a ratio of 1 : 1 or
51
Fig. 1. Experimental
set-up for liquid preservation of rainbow trout semen.
1 : 3 with a diluent developed on the basis of the chemical analyses of trout seminal plasma (Holtz et al., 1976; Biiyiikhatipoglu, 1977). The composition of the diluent is presented in Table I. Freezing took place either as pellets (Nagase, 1964) or in so called “Minitiib’‘-straws (Simmet, 1972) immediately after addition of the diluent. At the later stages of the experiment only pellets were made. For the purpose of thawing the samples were placed in test tubes at 0°C. After a few attempts without a thawing solution thawing took place in a 1% TABLE I Composition of the extender used for the cryopreservation Constituent NaCl KC.2 CaCl, MgEO, Tris (Hydroxymethylaminomethane) Citric acid Additives: Bovine serum albumen Promine-D Dimethylsulfoxide
of trout semen
Concentration 5.92
1.72 0.68 0.15 24.20 to pH 7.25 4.00 5.00-15.00 0.12
(mg/ml)
62
NaHC03solution (Stein, 1975). Motility and fertility of spermatozoa were tested after 1 to 4 months of storage. Only pellets were included in the fertility trials. By placing 100 pellets (each about 0.04 ml) into 20 ml thawing solution a sperm concentration of approximately 800 X lo6 (when diluting 1 : 1) or 400 X lo6 (when diluting 1 : 3) was obtained. This suspension was added to about 600 freshly collected eggs. About 600 eggs from each female were mixed with freshly collected trout milt to serve as controls. The techniques published by Hodgins and Ridgway (1964), Ott and Horton (1971b) and Stein (1975) were tested at the same time as the experiments recorded in this paper. RESULTS AND DISCUSSION
Preservation of liquid semen
Attempts to preserve trout milt at room temperature (20°C) turned out to be quite unsuccessful. Undiluted milt which, at a temperature of 4’C, kept its motility for 8.3 & 2.7 days had lost its ability to be activated within less than 2 days. Therefore these data are not presented. The data presented in Table II indicate that, contrary to the findings of Dorier (1951), dilution with an electrolyte solution had an unfavourable effect upon the survival of trout sperm. Contrary to our expectations partial or total reduction of O2 did not result in a prolongation of spermatozoa1 survival. Both under CO* and a combination of N?, Hz and COz (8 : 1 : 1) which is used for in vitro tissue culture under anaerobic conditions (Dowell, 1961), sperm had lost their ability of being activated within less than 24 hours. Under Nz undiluted semen maintained its ability of being activated for 3.6 * 1.3 days, under air or a mixture of O2 and Nz (1 : 1) for 8.3 + 2.7 or 9.0 f 0 days, respectively, and under pure oxygen for 12.4 + 1.8 days; i.e., the higher the oxygen content of the gas atmosphere the more favourable the storage conditions. These findings agree with observations by Scheuring (1924) and can serve as an explanation for failures experienced when storing milt in closed containers filled to the top (Smith and Quistorff, 1943; Barrett, 1951; Henderson and Dewar, 1959; Withler and Humphreys, 1967). The hatching rates during fertility trials with milt stored at 4°C are presented in Table III. Fertilizing capacity was maintained to a high degree by day 9 of storage under air. No tests.were conducted with milt stored for more than 9 days under these conditions. Semen samples kept under O2 at 4°C gave hatching rates of 80.6% when added to eggs after 15 days of storage with controls amounting to 98.2%. These results are considerably better than those reported in the literature (Brofeldt, 1914; Scheuring, 1924; Smith and Quistorff, 1943). Cryopreserva tion
Pellets turned out to be easier to handle than straws, particularly with
8.3 f 2.7 (16) 1.0 f 0 (6) 0 (6)
Air
Gas atmosphere
a Semen: diluent ratio. b Not tested.
Undiluted 1 :l 1 : 16
Dilution rate*
-
12.4~
0,
z.8 (14)
9.0 f 0 (2) -
0, + N, (1:l) 3.6 * 1.3 (10) 1.0 f 0 (6) 0 (6)
N*
0 (4) -
N,+H,+CO, (8:l:l)
0 (6) 0 (6) 0 (6)
CO,
-
Duration in days (Z * s) of spermatozoaI motility in trout milt stored at 4°C under different gas atmospheres at different dilution rates (The number of replicates is given in parentheses.)
TABLE II
54
TABLE III Hatching rate in percent of eggs fertilized with undiluted semen from rainbow trout stored at 4°C under air or 0, (> 500 eggs/sample) Category of semen
Stored semen Control (fresh) semen
Under air
Under 0,
2 days
4 days
9 days
15 days
90.2 98.6
95.0 99.0
94.7 94.9
80.6 98.2
respect to the processes of thawing and addition to eggs. Therefore straws were abandoned and all results reported in the following refer to pelleted semen. Samples that were frozen after addition of only DMSO or glycerol showed very little motility after thawing and were not tested for fertility. The results obtained when freezing semen with a diluent containing different concentrations of Promine-D are summarized in Table IV. Since the dilution rate (1 : 1 or 1 : 3, respectively) did not seem to matter data were pooled. Promine-D, a soybean product by “Central Soya”, has been shown to be able to replace egg yolk in bull semen extenders (Coulter and Foote, 1975). It was decided to incorporate it into our extender as a source of protein as it is easy to obtain and convenient to store and to handle. The data presented in Table IV are of preliminary character as the variabilty was extremely high, not allowing for the drawing of final conclusions as far as practical applicability of the technique is concerned. The results compare favourably with those reported by Bratanov and Dikov (1960), Graybill and Horton (1969) and Ott and Horton (1971 a,b). Better results were reported only by Stein (1975). Attempts to fertilize eggs with trout semen frozen by the techniques of Hodgins and Ridgway (1964) and Ott and Horton (1971b) TABLE IV The proportion of hatched eggs after fertilization with semen frozen after addition of a diiuent with different levels of Promine-D (> 600 eggs/sample) Concentration (mgM)
0 5.00 7.50 10.00 15.00
of Promine-D
No. of replicates
2 3 4 3 1
Proportion of eggs hatched (% of controls)
x
x min
x max
26.2 37.8 51.0 32.2 2.6
6.1 17.2 14.8 10.3 -
46.3 59.7 80.3 73.6 -
55
were not successful. The technique of Stein (1975) gave us an average hatching rate of 30.9%. The results presented in this paper give some indication of the possibilities of storing trout semen either in a liquid or frozen state. It appears obvious though that further investigation is needed to eventually render techniques suitable for routine application under practical hatchery conditions. ACKNOWLEDGEMENTS
The authors would like to express their gratitude to Prof. Langholz and Prof. Smidt for encouraging and supporting this project, to Mrs Ebeling and Miss Pith for valuable technical assistance and to Dr Heydom and Mr Ritter from the “Tiertiztliches Institut” as well as the trout breeders Dr Miiller and Mr Lehmann for their help. This project was supported by a grant of the Deutsche Forschungsgemeinschaft. REFERENCES Barrett, I., 1951. Fertility of Salmonid eggs and sperm after storage. J. Fish. Res. Board Can., 8: 125-133. Bratanov, K. and Dikov, V., 1960. Uber gewisse Eigenarten des Spermas bei Fischen. Landw. Zentralblatt, 7 (3): 1301 (1962). Brofeldt, P., 1914. Uber Transport von Fischrogen und Milch ohne Wasser in Glastijpfen. Cited in Scheuring, 1924. Biiyilkhatipoglu, S., 1977. Nutzung der Spermareserven bei Regenbogenforellen. Diss. GSttingen, 120 pp. Coulter, G.H. and Foote, R.H., 1975. Lipid deficient extender for bovine spermatozoa: its development and use in measuring freezing-induced lipid. J. Dairy Sci., 58: 82-87. Dorier, A., 1949/1950. Action du liquide coelomique sur les spermatozoides de Truite arc-enciel. Trav. Lab. Hydrobiol. Pisc. Univ. Grenoble, 40: 69-73. Dorier, A., 1951. Conservation de la vitalit et du pouvoir fgcondant des spermatozoides de Truite arc-en-ciel. Trav. Lab. Hydrobiol. Pisc. Univ. Grenoble, 41: 75-85. Dowell, V.R., 1961. Methods for obtaining anaerobiasis. In: Laboratory Methods in Anaerobic Bacteriology. NCDC Laboratory Manual. Atlanta, National Communicable Disease Center, pp. 29-30. Graybill, J.R. and Horton, H.F., 1969. Limited fertilization of steelhead trout eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 26: 1400-1404. Henderson, N.E. and Dewar, J.E., 1959. Short-term storage of brook trout milt. Prog. Fish Cult., 21(4): 169-171. Hodgins, H.O. and Ridgway, G.J., 1964. Recovery of viable salmon spermatozoa after fast-freezing. Prog. Fish Cult., 26: 95. Holtz, W., Bilyilkhatipoglu, S., Stoss, J., Oldigs, B. and Langholz, H.J., 1976. Preservation of trout spermatozoa for varying periods. FAO Technical Conference on Aquaculture, Kyoto, Japan, 26.5.1976-2.6.1976, pp. l-3. Holtz, W., Stoss, J. and Biiyilkhatipoglu, S., 1977. Beobachtungen zur Aktivierbarkeit von Forellenspermatozoen mit Fruchtwasser, Bachwasser und destilliertem Wasser. Zuchthygiene, 12: 82-88. Hoyle, R.J. and Idler, D.R., 1968. Preliminary results in the fertilization of eggs with frozen sperm of Atlantic salmon (Sulmo s&r). J. Fish. Res. Board Can., 25: 1295-1297.
56
Nagase, H., 1964. Deep freezing bull semen in concentrated pellet form. Proc. Int. Congr. Reprod. Anim. Insem. Artif., Trento, 3, p. 503 (abstract). Ott, A.G. and Horton, H.F., 1971a. Fertilization of chinook and coho salmon eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 28: 745-748. Ott, A.G. and Horton, H.F., 1971b. Fertilization of steelhead trout (S&no gairdneri) eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 28: 1915-1918. Scheuring, L., 1924. Biologische und physiologische Untersuchungen an Forellensperma. Arch. Hydrobiol., Suppl., 4: 181-318. Simmet, L., 1972. Ein vollautomatisches Verfahren zur Konfektionierung von Bullensperma in KunststoffrBhrchen nach der Landshuter Methode. 6. Int. Kongr. Fortpfl. Bes., Miinchen, 2, pp. 1357-1362. Smith, R.T. and Quistorff, E., 1943. Experiments with the spermatozoa of the steelhead trout, Salmo gairdnerii, and the chinook salmon, Oncorhyncus tschawytscha. Copeia 3: 164-167. Stein, H., 1975. Spezielle Untersuchungen am Fischsperma unter besonderer Berilcksichtigung der Spermakonservierung. Dim. T.U. Milnchen, 26 pp. Truscott, B. and Idler, D.R., 1969. An improved extender for freezing Atlantic salmon spermatozoa. J. Fish. Res. Board Can., 26: 3254-3258. Truscott, B., Idler, D.R., Hoyle, R.J. and Freeman, H.C., 1968. Sub-zero preservation of Atlantic salmon sperm. J. Fish. Res. Board Can., 25: 363-372. Withler, F.C. and Humphreys, R.M., 1967. Duration of fertility of ova and sperm of sockeye (Oncorhynchus nerka) and pink (0. gorbuscha) salmon. J. Fish. Res. Board Can., 24: 1573-1578. Withler, F.C. and Morley. R.B., 1968. Effects of chilled storage on viability of stored ova and sperm of sockeye and pink salmon. J. Fish. Res. Board Can., 25: 2695-2699. Wolf, K., 1963. Physiological salines for fresh-water teleosts. Prog. Fish Cult., 25: 135-140. KURZFASSUNG Bilyilkhatipoglu, S. und Holtz, W., 1978. Flllssig- und Gefrierkonservierung von Regenbogenforellensperma. Aquaculture, 13: 49-56. Mit dem Ziel, Forellensperma in flilssigem Zustand fiir einige Tage oder durch die Gefrierung filr einen 1Pngeren Zeitraum aufzubewahren, wurden Versuch zur Kurz- und Langzeitkonservierung durchgefiihrt : Fiir die Kurzzeitkonservierung wurde verdfinntes und unverdiinntes Sperma unter verschiedenen feuchtigkeitsgesgttigten Gasmilieus (Luft, O,, N, + 0, (l:l), N,, CO,, N, + H, + CO, (8:l:l) bei valliger Dunkelkeit entweder bei Raumtemperatur oder bei + 4°C aufbewahrt. Die besten Ergebnisse wurden erzielt bei Aufbewahrung von unverdtinntem Sperma bei 4°C unter Sauerstoff (nach 15 Tagen noch Schliipfrate von 80.6% gegeniiber 98.2% bei den Kontrollen), doch such unter Luft wurden gute Resultate erzielt (nach 9 Tagen eine Schliipfrate von 94.7% gegenilber 94.9% bei den Kontrollen). Anaerobe Verhliltnisse, Verdiinnung oder erhBhte Temperatur wirkten sich auf die Lebensfiihigkeit der Spermatozoen in jedem Falle nachtellig aus. Fiir die Gefrierkonservierung wurde eine Verdilnnerlijsung entaprechend der Zusammensetzung von Samenplasma der Regenbogenforelle entwickelt. Der Verdiinner wurde dem Sperma unmittelbar vor dem Einfrieren im Verhatnis 1~1 bzw. 1:3 zugesetzt. Besser als das Pailletten-Verfahren bewiihrte sich die Pelletierung nach Nagase (1964) mit anschliessender Aufbewahrung in fliissigem Stick&off. Zum Auftauen erwies sich eine l%ige NaHCO,L&ung als gllnstig. Die Ergebnisse der Befruchtungsversuche mit fiir ZeitrZiume von l-4 Monaten gelagertem Sperma erwiesen sich als stark variabel und bewegten sich in Relation zu Kontrollen mit Frlschsperma zwischen 2.680.3% geschllipfter Brut.
PRESERVATION
49
OF TROUT SPERM IN LIQUID OR FROZEN STATE
S. BUYUKHATIPOGLU
and W. HOLTZ
Znstitut fir Tienucht und Haustiergenetik der Uniuersita”t Giittingen, Albrecht-ThaerWeg 1, 3400 Giittingen (Federal Republic of Germany) (Received 20 October 1977; revised 16 December 1977)
ABSTRACT Bilyilkhatipoglu, S. and Holtz, W., 1978. Preservation of trout sperm in liquid or frozen state. Aquaculture, 14: 49-56. A series of experiments to study the preservation of rainbow trout semen in the liquid and frozen state was carried out. For the preservation of liquid semen both undiluted and diluted semen was stored in total darkness at 20” or 4°C under a number of different gasses (air, 0,, 0, + N, (1 :l), N,, CO,, N, + H, + CO, (8 :l :l)). The best results were obtained when storing semen in an undiluted state at 4°C under 0, (hatching rate after 16 days was 80.6%, controls were 98.2%). Good results were also obtained under air (after 9 days 94.7% vs 94.9% for the controls). Anaerobic conditions, dilution or an increase in temperature all rendered less favourable results. For the purpose of cryopreservation a diluent was developed on the basis of the composition of rainbow trout seminal plasma. The semen was mixed with the diluent immediately before freezing at a ratio of 1 :l or 1:3. The pellet-technique (Nagase, 1964) was found to be more convenient and successful than freezing in straws. The pellets were thawed in a 1% NaHCO,-solution. The hatching results after storage periods of l-4 months were very variable (2.6--80.3%).
INTRODUCTION
The availability of suitable methods for successful preservation of trout semen might be extremely valuable both from an organisational and a breeding point of view (e.g. semen transport or crossing of strains with differing spawning seasons). One has to differentiate between the preservation of liquid semen for limited periods of time and cryopreservation for long-term semen storage. Brofeldt (1914) was one of the first to recognize that trout and salmon spermatozoa will survive longer if kept cool. Later experiments were conducted involving the storage of salmonid sperm at temperatures from 0 to 9°C (Scheuring, 1924; Smith and Quistorff, 1943; Barrett, 1951; Henderson and Dewar, 1959; Withler and Humphreys, 1967; Withler and Morley, 1968; Stein, 1975). Several investigations were aimed at increasing storage time by
50
adding electrolyte solutions or coelomic fluid from females (Dorier, 1949/ 1950,195l; Henderson and Dewar, 1959). In most cases “Cortland’‘-medium (Wolf, 1963) served as the basis for the development of diluents. Substances that were frequently added to diluents were lactose, fructose, lecithin, mannitol, glycin or egg yolk and, to prevent cell damage during freezing and thawing, ethylene glycol, propylene glycol, glycerol or dimethylsulfoxide (DMSO) (Truscott et al., 1968; Hoyle and Idler, 1968; Graybill and Horton, 1969; Ott and Horton, 1971a,b; Stein, 1975). A diluent developed on the basis of seminal plasma was first reported by Truscott and Idler (1969). To date no satisfactory techniques have been developed for either liquid preservation or cryopreservation of trout sperm. MATERIAL
AND METHODS
The milt that was used for the investigation was collected from 2-3 year old males which were kept under standardized conditions in fish tanks and were stripped at 1 or 2 week intervals. The milt was collected in pre-cooled test tubes and transported to the laboratory on water-ice. There its motility was examined within one hour by the technique described by Holtz et al. (1977). Only good quality semen was utilized for experimental purposes. Milt from different males was never mixed. Preservation of liquid semen
Samples of 1.5 ml of either undiluted semen or semen diluted at a ratio of 1 : 1 or 1 : 16 with a buffered isotonic diluent resembling the inorganic composition of seminal plasma (Holtz et al., 1976) were placed in open glass vials with a surface area of 3.14 cm2 and a height of 4.5 cm. Six of these vials were placed in a 1 1 Erlenmeyer-flask, the bottom of which was covered with moist cotton (Fig. 1). The flasks were stoppered and kept in darkness at either 20” or 4°C. Twice daily they were gassed with one of the following gasses: air, 02, N2, C02, N2-02 (1 : 1) or N2-H2-C02 (8 : 1 : 1). Each day the vials were swirled gently before a tiny drop of semen was taken from each. Motility was estimated as described by Holtz et al. (1977). In a follow-up trial undiluted milt kept at 4°C under air was added to a minimum of 500 freshly collected trout eggs after 2,4 or 9 days of storage. Milt kept at 4°C under pure O2 was added to a similar number of eggs after a storage period of 15 days. Cryopreservation
To a number of semen samples DMSO or glycerol was added at ratios of 1 : 0.1, 1 : 0.5,l : 1 or 1 : 3 before freezing them by the pellet-technique (Nagase, 1964). Most samples, however, were mixed at a ratio of 1 : 1 or
51
Fig. 1. Experimental
set-up for liquid preservation of rainbow trout semen.
1 : 3 with a diluent developed on the basis of the chemical analyses of trout seminal plasma (Holtz et al., 1976; Biiyiikhatipoglu, 1977). The composition of the diluent is presented in Table I. Freezing took place either as pellets (Nagase, 1964) or in so called “Minitiib’‘-straws (Simmet, 1972) immediately after addition of the diluent. At the later stages of the experiment only pellets were made. For the purpose of thawing the samples were placed in test tubes at 0°C. After a few attempts without a thawing solution thawing took place in a 1% TABLE I Composition of the extender used for the cryopreservation Constituent NaCl KC.2 CaCl, MgEO, Tris (Hydroxymethylaminomethane) Citric acid Additives: Bovine serum albumen Promine-D Dimethylsulfoxide
of trout semen
Concentration 5.92
1.72 0.68 0.15 24.20 to pH 7.25 4.00 5.00-15.00 0.12
(mg/ml)
62
NaHC03solution (Stein, 1975). Motility and fertility of spermatozoa were tested after 1 to 4 months of storage. Only pellets were included in the fertility trials. By placing 100 pellets (each about 0.04 ml) into 20 ml thawing solution a sperm concentration of approximately 800 X lo6 (when diluting 1 : 1) or 400 X lo6 (when diluting 1 : 3) was obtained. This suspension was added to about 600 freshly collected eggs. About 600 eggs from each female were mixed with freshly collected trout milt to serve as controls. The techniques published by Hodgins and Ridgway (1964), Ott and Horton (1971b) and Stein (1975) were tested at the same time as the experiments recorded in this paper. RESULTS AND DISCUSSION
Preservation of liquid semen
Attempts to preserve trout milt at room temperature (20°C) turned out to be quite unsuccessful. Undiluted milt which, at a temperature of 4’C, kept its motility for 8.3 & 2.7 days had lost its ability to be activated within less than 2 days. Therefore these data are not presented. The data presented in Table II indicate that, contrary to the findings of Dorier (1951), dilution with an electrolyte solution had an unfavourable effect upon the survival of trout sperm. Contrary to our expectations partial or total reduction of O2 did not result in a prolongation of spermatozoa1 survival. Both under CO* and a combination of N?, Hz and COz (8 : 1 : 1) which is used for in vitro tissue culture under anaerobic conditions (Dowell, 1961), sperm had lost their ability of being activated within less than 24 hours. Under Nz undiluted semen maintained its ability of being activated for 3.6 * 1.3 days, under air or a mixture of O2 and Nz (1 : 1) for 8.3 + 2.7 or 9.0 f 0 days, respectively, and under pure oxygen for 12.4 + 1.8 days; i.e., the higher the oxygen content of the gas atmosphere the more favourable the storage conditions. These findings agree with observations by Scheuring (1924) and can serve as an explanation for failures experienced when storing milt in closed containers filled to the top (Smith and Quistorff, 1943; Barrett, 1951; Henderson and Dewar, 1959; Withler and Humphreys, 1967). The hatching rates during fertility trials with milt stored at 4°C are presented in Table III. Fertilizing capacity was maintained to a high degree by day 9 of storage under air. No tests.were conducted with milt stored for more than 9 days under these conditions. Semen samples kept under O2 at 4°C gave hatching rates of 80.6% when added to eggs after 15 days of storage with controls amounting to 98.2%. These results are considerably better than those reported in the literature (Brofeldt, 1914; Scheuring, 1924; Smith and Quistorff, 1943). Cryopreserva tion
Pellets turned out to be easier to handle than straws, particularly with
8.3 f 2.7 (16) 1.0 f 0 (6) 0 (6)
Air
Gas atmosphere
a Semen: diluent ratio. b Not tested.
Undiluted 1 :l 1 : 16
Dilution rate*
-
12.4~
0,
z.8 (14)
9.0 f 0 (2) -
0, + N, (1:l) 3.6 * 1.3 (10) 1.0 f 0 (6) 0 (6)
N*
0 (4) -
N,+H,+CO, (8:l:l)
0 (6) 0 (6) 0 (6)
CO,
-
Duration in days (Z * s) of spermatozoaI motility in trout milt stored at 4°C under different gas atmospheres at different dilution rates (The number of replicates is given in parentheses.)
TABLE II
54
TABLE III Hatching rate in percent of eggs fertilized with undiluted semen from rainbow trout stored at 4°C under air or 0, (> 500 eggs/sample) Category of semen
Stored semen Control (fresh) semen
Under air
Under 0,
2 days
4 days
9 days
15 days
90.2 98.6
95.0 99.0
94.7 94.9
80.6 98.2
respect to the processes of thawing and addition to eggs. Therefore straws were abandoned and all results reported in the following refer to pelleted semen. Samples that were frozen after addition of only DMSO or glycerol showed very little motility after thawing and were not tested for fertility. The results obtained when freezing semen with a diluent containing different concentrations of Promine-D are summarized in Table IV. Since the dilution rate (1 : 1 or 1 : 3, respectively) did not seem to matter data were pooled. Promine-D, a soybean product by “Central Soya”, has been shown to be able to replace egg yolk in bull semen extenders (Coulter and Foote, 1975). It was decided to incorporate it into our extender as a source of protein as it is easy to obtain and convenient to store and to handle. The data presented in Table IV are of preliminary character as the variabilty was extremely high, not allowing for the drawing of final conclusions as far as practical applicability of the technique is concerned. The results compare favourably with those reported by Bratanov and Dikov (1960), Graybill and Horton (1969) and Ott and Horton (1971 a,b). Better results were reported only by Stein (1975). Attempts to fertilize eggs with trout semen frozen by the techniques of Hodgins and Ridgway (1964) and Ott and Horton (1971b) TABLE IV The proportion of hatched eggs after fertilization with semen frozen after addition of a diiuent with different levels of Promine-D (> 600 eggs/sample) Concentration (mgM)
0 5.00 7.50 10.00 15.00
of Promine-D
No. of replicates
2 3 4 3 1
Proportion of eggs hatched (% of controls)
x
x min
x max
26.2 37.8 51.0 32.2 2.6
6.1 17.2 14.8 10.3 -
46.3 59.7 80.3 73.6 -
55
were not successful. The technique of Stein (1975) gave us an average hatching rate of 30.9%. The results presented in this paper give some indication of the possibilities of storing trout semen either in a liquid or frozen state. It appears obvious though that further investigation is needed to eventually render techniques suitable for routine application under practical hatchery conditions. ACKNOWLEDGEMENTS
The authors would like to express their gratitude to Prof. Langholz and Prof. Smidt for encouraging and supporting this project, to Mrs Ebeling and Miss Pith for valuable technical assistance and to Dr Heydom and Mr Ritter from the “Tiertiztliches Institut” as well as the trout breeders Dr Miiller and Mr Lehmann for their help. This project was supported by a grant of the Deutsche Forschungsgemeinschaft. REFERENCES Barrett, I., 1951. Fertility of Salmonid eggs and sperm after storage. J. Fish. Res. Board Can., 8: 125-133. Bratanov, K. and Dikov, V., 1960. Uber gewisse Eigenarten des Spermas bei Fischen. Landw. Zentralblatt, 7 (3): 1301 (1962). Brofeldt, P., 1914. Uber Transport von Fischrogen und Milch ohne Wasser in Glastijpfen. Cited in Scheuring, 1924. Biiyilkhatipoglu, S., 1977. Nutzung der Spermareserven bei Regenbogenforellen. Diss. GSttingen, 120 pp. Coulter, G.H. and Foote, R.H., 1975. Lipid deficient extender for bovine spermatozoa: its development and use in measuring freezing-induced lipid. J. Dairy Sci., 58: 82-87. Dorier, A., 1949/1950. Action du liquide coelomique sur les spermatozoides de Truite arc-enciel. Trav. Lab. Hydrobiol. Pisc. Univ. Grenoble, 40: 69-73. Dorier, A., 1951. Conservation de la vitalit et du pouvoir fgcondant des spermatozoides de Truite arc-en-ciel. Trav. Lab. Hydrobiol. Pisc. Univ. Grenoble, 41: 75-85. Dowell, V.R., 1961. Methods for obtaining anaerobiasis. In: Laboratory Methods in Anaerobic Bacteriology. NCDC Laboratory Manual. Atlanta, National Communicable Disease Center, pp. 29-30. Graybill, J.R. and Horton, H.F., 1969. Limited fertilization of steelhead trout eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 26: 1400-1404. Henderson, N.E. and Dewar, J.E., 1959. Short-term storage of brook trout milt. Prog. Fish Cult., 21(4): 169-171. Hodgins, H.O. and Ridgway, G.J., 1964. Recovery of viable salmon spermatozoa after fast-freezing. Prog. Fish Cult., 26: 95. Holtz, W., Bilyilkhatipoglu, S., Stoss, J., Oldigs, B. and Langholz, H.J., 1976. Preservation of trout spermatozoa for varying periods. FAO Technical Conference on Aquaculture, Kyoto, Japan, 26.5.1976-2.6.1976, pp. l-3. Holtz, W., Stoss, J. and Biiyilkhatipoglu, S., 1977. Beobachtungen zur Aktivierbarkeit von Forellenspermatozoen mit Fruchtwasser, Bachwasser und destilliertem Wasser. Zuchthygiene, 12: 82-88. Hoyle, R.J. and Idler, D.R., 1968. Preliminary results in the fertilization of eggs with frozen sperm of Atlantic salmon (Sulmo s&r). J. Fish. Res. Board Can., 25: 1295-1297.
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Nagase, H., 1964. Deep freezing bull semen in concentrated pellet form. Proc. Int. Congr. Reprod. Anim. Insem. Artif., Trento, 3, p. 503 (abstract). Ott, A.G. and Horton, H.F., 1971a. Fertilization of chinook and coho salmon eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 28: 745-748. Ott, A.G. and Horton, H.F., 1971b. Fertilization of steelhead trout (S&no gairdneri) eggs with cryo-preserved sperm. J. Fish. Res. Board Can., 28: 1915-1918. Scheuring, L., 1924. Biologische und physiologische Untersuchungen an Forellensperma. Arch. Hydrobiol., Suppl., 4: 181-318. Simmet, L., 1972. Ein vollautomatisches Verfahren zur Konfektionierung von Bullensperma in KunststoffrBhrchen nach der Landshuter Methode. 6. Int. Kongr. Fortpfl. Bes., Miinchen, 2, pp. 1357-1362. Smith, R.T. and Quistorff, E., 1943. Experiments with the spermatozoa of the steelhead trout, Salmo gairdnerii, and the chinook salmon, Oncorhyncus tschawytscha. Copeia 3: 164-167. Stein, H., 1975. Spezielle Untersuchungen am Fischsperma unter besonderer Berilcksichtigung der Spermakonservierung. Dim. T.U. Milnchen, 26 pp. Truscott, B. and Idler, D.R., 1969. An improved extender for freezing Atlantic salmon spermatozoa. J. Fish. Res. Board Can., 26: 3254-3258. Truscott, B., Idler, D.R., Hoyle, R.J. and Freeman, H.C., 1968. Sub-zero preservation of Atlantic salmon sperm. J. Fish. Res. Board Can., 25: 363-372. Withler, F.C. and Humphreys, R.M., 1967. Duration of fertility of ova and sperm of sockeye (Oncorhynchus nerka) and pink (0. gorbuscha) salmon. J. Fish. Res. Board Can., 24: 1573-1578. Withler, F.C. and Morley. R.B., 1968. Effects of chilled storage on viability of stored ova and sperm of sockeye and pink salmon. J. Fish. Res. Board Can., 25: 2695-2699. Wolf, K., 1963. Physiological salines for fresh-water teleosts. Prog. Fish Cult., 25: 135-140. KURZFASSUNG Bilyilkhatipoglu, S. und Holtz, W., 1978. Flllssig- und Gefrierkonservierung von Regenbogenforellensperma. Aquaculture, 13: 49-56. Mit dem Ziel, Forellensperma in flilssigem Zustand fiir einige Tage oder durch die Gefrierung filr einen 1Pngeren Zeitraum aufzubewahren, wurden Versuch zur Kurz- und Langzeitkonservierung durchgefiihrt : Fiir die Kurzzeitkonservierung wurde verdfinntes und unverdiinntes Sperma unter verschiedenen feuchtigkeitsgesgttigten Gasmilieus (Luft, O,, N, + 0, (l:l), N,, CO,, N, + H, + CO, (8:l:l) bei valliger Dunkelkeit entweder bei Raumtemperatur oder bei + 4°C aufbewahrt. Die besten Ergebnisse wurden erzielt bei Aufbewahrung von unverdtinntem Sperma bei 4°C unter Sauerstoff (nach 15 Tagen noch Schliipfrate von 80.6% gegeniiber 98.2% bei den Kontrollen), doch such unter Luft wurden gute Resultate erzielt (nach 9 Tagen eine Schliipfrate von 94.7% gegenilber 94.9% bei den Kontrollen). Anaerobe Verhliltnisse, Verdiinnung oder erhBhte Temperatur wirkten sich auf die Lebensfiihigkeit der Spermatozoen in jedem Falle nachtellig aus. Fiir die Gefrierkonservierung wurde eine Verdilnnerlijsung entaprechend der Zusammensetzung von Samenplasma der Regenbogenforelle entwickelt. Der Verdiinner wurde dem Sperma unmittelbar vor dem Einfrieren im Verhatnis 1~1 bzw. 1:3 zugesetzt. Besser als das Pailletten-Verfahren bewiihrte sich die Pelletierung nach Nagase (1964) mit anschliessender Aufbewahrung in fliissigem Stick&off. Zum Auftauen erwies sich eine l%ige NaHCO,L&ung als gllnstig. Die Ergebnisse der Befruchtungsversuche mit fiir ZeitrZiume von l-4 Monaten gelagertem Sperma erwiesen sich als stark variabel und bewegten sich in Relation zu Kontrollen mit Frlschsperma zwischen 2.680.3% geschllipfter Brut.