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Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics
Aquaculture, 78 (1989) 229-236 Elsevier Science Publishers B.V., Amsterdam -
229 Printed in The Netherlands
Triploidy Induction in Recently Fertilized Atlantic Salmon Ova Using Anaesthetics R. JOHNSTONE’, R.M. KNOTTI, A.G. MACDONALD’ and M.V. WALSINGHAMI ‘DAFS Marine Laboratory, Victoria Road, Aberdeen AB9 8DB (Great Britain) ‘Department of Physiology, University of Aberdeen, AberdeenAB9 1AS (Great Britain) (Accepted 26 January 1989)
ABSTRACT Johnstone, R., Knott, R.M., Macdonald, A.G. and Walsingham, M.V., 1989. Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics. Aquaculture, 78: 229-236. The comparative effectiveness of five general anaesthetics as agents for inducing triploidy in recently fertilized Atlantic salmon ova is reported. Triploid rates and triploid yields following the exposures of eggs from a single female fish to nitrous oxide, and separately to Freon 22, were shown to be related to the partial pressure and duration of exposure. The maximum triploid yield (79.7% ) was observed following exposure to nitrous oxide at 11 atmospheres (atm) for the first 30 min after fertilization. Triploid yields following exposure to Freon (at 1 and 3 atm) were generally lower, the highest value (43.7%) occurring after exposure at ambient pressure for the first hour after fertilization. Triploid yields in these same eggs were low ( < 10%) when halothane and ethrane were used at partial pressures in the range 0.05-0.2 atm and cyclopropane at atmospheric pressure was ineffective. In a subsequent season the eggs from ten different individual female Atlantic salmon were shown to vary little in susceptibility following exposure to nitrous oxide treatment (O-30 min after fertilization, 11 atm) and experienced high yields of triploids (mean=80.4&6.6% SD).
INTRODUCTION
In a previous communication (Shelton et al., 1986) the first report of the use of an anaesthetic gas, nitrous oxide, to induce triploidy in recently fertilized rainbow trout, Salmo guirdneri, eggs was presented. The success of this experiment prompted us to confirm and extend our observations using another species, the Atlantic salmon, S. sahr. The results of two experimental series conducted in consecutive seasons are presented. In the first (1986)) batches of eggs from a single female farmed Atlantic salmon were exposed to that range of nitrous oxide treatments which had been used in the earlier trout study. We also attempted, using these same eggs, to test the hypothesis that triploid induction by nitrous oxide is part of a general phenomenon and that triploidy
0044-8486/89/$03.50
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230
can be brought about by an equi-narcotic dose of other general anaesthetics. In selecting a range of agents to test for this effect we included potent anaesthetics which, it was hoped, could be administered to eggs at partial pressures lower than the 11 atm required for nitrous oxide treatment and which might therefore be more conveniently adopted in commercial practice. In the subsequent season the eggs of ten salmon from the same farm were separately exposed to that nitrous oxide treatment which had proved most effective in the 1986 trial in order to investigate the extent of any hen-to-hen variability in susceptibility to treatment. Additionally, pools of eggs generated by mixing equal numbers of eggs from these ten hens were exposed to range of nitrous oxide treatments and handling protocols to further investigate the factors of importance in maximizing triploid yield during nitrous oxide exposure. MATERIALS AND METHODS
In 1986 the eggs of a single two-sea-winter (ZSW) farmed Atlantic salmon female were stripped out and maintained in an oxygen-rich atmosphere in a polythene bag on ice. Groups of eggs (ca 150) were removed in turn from this pool, fertilized with the mixed milt from three farmed Atlantic salmon males and washed with 10°C water. The eggs were then either taken as controls or exposed as a monolayer in g-cm-diameter glass dishes to the particular anaesthetic agent under study, using the partial immersion protocol reported previously (Shelton et al., 1986). In this protocol approximately 150 eggs were contained in the dishes together with 9 ml of incubation water, this being just sufficient to cover the eggs prior to their being placed in the treatment vessel. According to narcosis theory as applied to cells in general (see for example Macdonald and Wann, 1978), equipotent doses of the general anaesthetics used in this study were selected on the following basis. Eleven atmospheres nitrous oxide, the highly effective dose reported in Shelton et al. (1986)) is 7.3fold the dose which abolishes the righting reflex in 50% of mice (Miller et al., 1972). Similarly the lesser partial pressure previously used, 5.0 atm, is 3.3-fold the ED50 for the loss of righting reflex. On this basis 1.17 atm and 0.53 atm of Freon 22 (CHCLF,; chloro-difluoro-methane) would be equipotent with the N20 treatments and for convenience we used 3.0 and 1.0 atm. Similarly (Miller et al., 1972),a dose of cyclopropane (cC,H,) equipotent with 11 atm N,O would be 1.17atm and for convenience we used 1.0 atm. The ED50 (righting reflex) for halothane (2 bromo-2-chloro-l,l,l-trifluoroethane) is 0.017 atm (Smith, 1969) or 0.0077 atm (Miller et al., 1972) and, again for reasons of experimental convenience, the doses of this anaesthetic which were tested.were 0.2 atm by static exposure and 0.124 and 0.056 atm using diluted streams of gas. Ethrane (2-chloro-1,1,2-trifluoroethyl difluoromethyl ether), which is about half as potent as halothane, was administered by static exposure at 0.11
231
atm, that is at a dose equivalent to the lower dose of nitrous oxide previously described. Eggs were exposed to elevated pressures of nitrous oxide (Medical grade, BOC Ltd) and Freon 22 in a pressure vessel as previously described (Shelton et al., 1986) using commercially available cylinder supplies. After fertilization and washing (ca 1-2 min), the eggs were placed in the vessel which was then purged with gas (ca 1 min) before being raised to the working pressure. Ambient pressure treatments with Freon 22 and cyclopropane were separately performed in this vessel by streaming the gases through the vessel at a slow rate (ca 5 ml/min) throughout the treatment period. The lower halothane (M & B Ltd, Dagenham) partial pressures used in this study (0.124 and 0.056 atm) were administered to eggs using the air streaming method of Allison et al. (1970). Eggs, in partially immersed “monolayers” in petri dishes, were held in a glass desiccating flask; the plastic tubing used to duct the vapour to the chamber having been fully equilibrated with the particular partial pressure of halothane prior to treatment. A higher dose of halothane (0.2 atm) was administered by containing the monolayer of eggs in a glass desiccator containing static air, equilibrated with liquid halothane (magnetically stirred in an open dish) at 10°C. During treatment in this way the temperature of the incubation water containing the eggs rose from 10” C to 16’ C, presumably due to the heat of solution of the anaesthetic in water. Ethrane (Abbot Laboratories Ltd, Kent) was similarly administered by static exposure at a partial pressure of 0.11 atm. In 1987 batches of ca 150 eggs derived from ten individual farmed Atlantic salmon (2SW maturers) were exposed to NzO (11 atm, O-30 min after fertilization) in monolayers as before following the addition of 9 ml of incubation water. Control batches of eggs were taken from each hen and reared separately. Equal numbers of eggs were then taken from each hen to generate pools of 150 eggs. Four such pools were exposed to a 30-min nitrous oxide exposure beginning immediately after fertilization at either 5,7,9 or 11 atm. Five pools were exposed to a 30-min nitrous oxide exposure at 11 atm following the addition of differing amounts of incubation water (2,4,6,12 and 18 ml) in an attempt to investigate the effect of this variable on the kinetics of nitrous oxide uptake. Two pools were taken as controls. Following treatments the eggs were in both seasons flooded with water and maintained at ambient temperatures (l-6” C) in individual netting baskets until yolk sac absorption was nearly completed. Dead eggs and alevins were removed frequently and recorded. Following formalin fixation of the survivors, the ploidy status of individuals in the various treatment groups was estimated by comparison with control animals following the microdensitometric analysis of Feulgen-stained erythrocyte preparations (Johnstone and Lincoln, 1986). In those groups where fewer than 50 animals survived, all animals were taken for analysis. When more than this number survived, a random sample of 50
232
survivors was analysed. The triploid yield in each group was calculated as the product of the triploid rate and the numbers surviving to the point of sacrifice, expressed as a percentage of the number of eggs originally present. RESULTS
The results of those experiments performed in the 1986 season when groups of eggs from a single female salmon were exposed to differing anaesthetic treatments in the period immediately after fertilization are summarized in Table 1. The survival rate in control eggs taken during these experiments was 88.3%. Following treatment with nitrous oxide, triploid rates and survival rates varTABLE 1 The effect of different anaesthetics on ploidy status of recently fertilized Atlantic salmon eggs Anaesthetic
Treatment conditions (time of exposure: minutes after fertilization)
survival rate (%)
Triploid rate (%)
Triploid yield (% )
Nitrous oxide
5 atm 5 atm 11 atm 11 atm 11 atm 11 atm
79.9
28 50 78 98 100 25
22.4 33.0 54.7 79.7 61.8 12.4 25.5 43.7 20.4 11.9
(O-30) (O-60) (O-15) (O-30) (O-60) (30-60)
66.1 70.1 81.3 61.8 49.4
1 atm streamed 1 atm streamed 3 atm 3 atm
(O-30) (O-60) (O-15) (O-30)
68.3 27.2 11.9
36 64 75 100
Cyclopropane
1 atm streamed 1 atm streamed
(O-15) (O-30)
86.2 83.3
0 0
Halothane
0.2 atm static 0.2 atm static 0.2 atm static 0.2 atm static
(O-10) (O-20) (10-20) (20-30)
56.3 42.0 73.3 75.2
8 22 0 8
0.124 atm streamed 0.124 atm streamed
(O-30) (O-60)
75.2 75.2
0 0
0 0
0.056 atm streamed 0.056 atm streamed
(O-30) (O-60)
90.8
0 0
0 0
0.11 atm static 0.11 atm static
(O-15) (O-30)
77.9
2 6
1.6 4.3
Freon 22
Ethrane
70.9
83.7
71.8
0 0
4.5 9.2 0 6
233
ied with the timing, duration and partial pressure of exposure. Treatments at 5 atm generated fewer triploids than those at 11 atm. A longer treatment (60 min) at the lower partial pressure induced significantly (PC 0.05) greater triploid rates than did a shorter treatment (30 min). Survival rates were decreased by the longer exposure but not significantly so. At 11 atm triploid rates increased with increasing exposure time in those treatments beginning immediately after fertilization but survival rates were less clearly related to the duration of treatment. Maximal triploid yields at 11 atm were generated by a 30-min treatment which began shortly after fertilization. Triploid rates and survival rates were poor in that treatment group in which exposure to nitrous oxide at 11 atm was delayed to encompass the period 30 to 60 min after fertilization. Triploids were also induced following exposure to Freon 22. As with nitrous oxide, Freon 22 was more effective in inducing triploidy at elevated partial pressure (3 atm) but this was associated with high mortality rates. Exposure to Freon at ambient pressure resulted in higher survival rates, the triploid rate in this instance being related to the duration of treatment. Cyclopropane when administered at ambient pressure had little effect on mortality rate and was without effect on ploidy status. Static exposure of eggs to the volatile agents halothane and ethrane resulted in only a low percentage of triploids. Halothane usage in particular was associated with increased mortalities especially in those exposures which began immediately after fertilization. Although survival rates following increased durations of exposure to diluted streams of halothane were higher, these treatments produced no triploids. Following exposure to nitrous oxide (O-30 min a.f., 11 atm), batches of eggs taken from different females in the 1987 season differed little in survival rate or in triploid rate and therefore in triploid yield (Table 2 ) . The mean survival rate relative to controls was 86.32 + 6.7% and the mean triploid rate and triploid yield were 93.2 2 3.2% and 80.4 -+6.6%, respectively. The relationship between the partial pressure of nitrous oxide during treatment and efficacy on pools of eggs generated from these same females are summarized in Table 3. Triploid rate increased and survival rate apparently decreased with increasing partial pressure of nitrous oxide exposure. The maximum triploid yield was observed following treatment at 9 atm. The consequence of an alteration in the amount of incubation water on the efficacy of a nitrous oxide treatment are also summarized in Table 3. At the normal rate of addition of incubation water adopted in these studies (9 ml water to ca 150 eggs in a 9 cm petri dish) the eggs were just covered by water at fertilization and came to be partially exposed as a result of water uptake during the water hardening process. At the highest rate of addition of water (18 ml) the eggs were wholly covered both before and after treatment. Following the addition of only 2 ml, little liquid water remained in the dish at the end
234 TABLE 2 The effect of nitrous oxide exposure (O-30 min a.f.; 11 atm) on ploidy status of different female farmed Atlantic salmon Fish no.
Control survival rate (%)
Survival rate (%)
Survival rate (%RC)a
Triploid rate (%)
Triploid yield (% RC)a
1 2 3 4 5 6 7 8 9 10 Mean k SD
92.9 97.3 96.8 95.1 96.0 87.6 88.9 95.1 92.1 91.3 93.31 k 3.3
71.2 88.2 83.0 82.4 85.3 84.8 71.6 75.9 76.0 86.9 80.53 f 6.3
76.6 90.6 85.7 86.6 88.9 96.8 80.5 79.8 82.5 95.2 86.32 f 6.7
96 94 90 94 86 96 94 96 92 94 93.2 k 3.2
73.5 85.2 77.1 81.4 76.5 92.9 75.7 76.6 75.9 89.5 80.4 f 6.6
“RC = relative to control. TABLE 3 The effect of different treatment protocols on the effectiveness of a nitrous oxide exposure (O-30 min a.f.) on ploidy status of Atlantic salmon egg pools* Treatment no. 1 2 3 4 Control 1
2 3 4 5 Control
Incubation water (ml)
Partial pressure (atm)
Survival (% )
Survival (% RC)a
Triploid rate (%)
Triploid yield (%)
9 9 9 9 9
5 7 9 11 0
68.7 60.7 62.3 50.4 75.3
91.2 80.6 82.7 66.9 100.0
28 IO 96 98 -
25.5 56.4 79.4 65.6 -
2 4 6 12 18 9
11 11 11 11 11 0
90.0 83.4 91.9 98.0 89.1 95.6
94.1 87.2 96.1 102.5 93.2 100.0
18 98 100 100 98 -
73.4 85.5 96.1 102.5 91.3 -
*Equal numbers of ova were taken from each of the ten individuals referred to in Table 2 to generate pools of 150 eggs. “RC = relative to control.
of the treatment period, although the eggs were still obviously “moist”. Although survival rates were apparently unaffected by these differences, the triploid rate was lowered significantly (P
235 DISCUSSION
It has long been known that the potency of anaesthetic agents is closely correlated with their solubility in solvents such as olive oil or octanol (Miller et al., 1972). This implies the hypothesis that in order to exert their effects on biological systems these agents must first dissolve in or bind to molecular sites with a solubility parameter similar to that of the model solvents (Macdonald and Wann, 1978). The system under discussion here is that of the lipid-rich salmonid egg contained in incubation water. The more lipophilic and potent agents used in this study (the halogenated compounds) will be those which would be expected to equilibrate most slowly via the aqueous phase surrounding the eggs. In addition, highly hydrophobic anaesthetics may dissolve in irrelevant lipid “sinks”. The efficacy of these compounds as triploidization agents might therefore depend on kinetic factors as well as on their specific binding to sites whose unaltered function is necessary for the normal completion of those chromosomal movements typical of the meiotic division of recently fertilized salmonid eggs. The results of the experiments reported here show that in Atlantic salmon ova as in rainbow trout (Shelton et al., 1986), nitrous oxide is effective in generating high yields of triploids when given at an appropriate partial pressure and timing of exposure. The ability of other anaesthetic agents to manipulate ploidy status by interference with the second meiotic division of recently fertilized eggs has also been demonstrated. Nitrous oxide, the least hydrophobic and in mammalian systems the least potent of the agents used in this study, was increasingly effective in inducing triploid in salmon eggs when administered shortly after fertilization at partial pressures in excess of 5 atm. Cyclopropane, though ten times more potent than nitrous oxide in mammalian systems, is also ten times more hydrophobic (Miller et al., 1972). It was ineffective in this study when used at ambient pressure presumably because of its greater degree of hydrophobicity. It can be postulated therefore that in order for it to have an effect, cyclopropane would need to be delivered at elevated partial pressure, but perhaps not at pressures as high as those necessary for effective treatment with nitrous oxide. Freon 22 is very similar in anaesthetic potency and hydrophobicity to cyclopropane (Miller et al., 1972 ) and was more effective, which suggests that there may be specific binding differences between these two compounds. Though even more effective at elevated partial pressure, Freon was extremely toxic when used in this way. The volatile liquid agents halothane and ethrane are highly lipophilic and relatively ineffective in inducing triploidy, perhaps primarily because of their slow rate of equilibration. Halothane was relatively toxic in those exposures which began immediately after fertilization. The temperature rise observed in the static halothane exposures would have affected the halothane solubility and kinetics and this factor may therefore have contributed to its toxicity.
Mean triploid yields were satisfactorily high (80.4 + 6.6 SD% relative to controls) when eggs from ten individual female fish were exposed in 1987 to the preferred nitrous oxide treatment indicated in the 1986 experiments. The effectiveness of nitrous oxide was tested further in this season by treating pools of eggs in which these same individuals were equally represented. The results of these treatments demonstrated nitrous oxide to be effective over a relatively wide range of partial pressures and incubation protocols. Somewhat to our surprise, triploid rate was diminished by a paucity of incubation water and not by an excess. A delay in the initiation of nitrous oxide exposure to 30 min after fertilization resulted in higher mortalities and a lowered triploid rate (25%, Table 1) due presumably to the increasing degeneration of the polar body and its exclusion from the fusion nucleus. These results, together with the lack of any marked interindividual difference in susceptibility amongst salmon and the similarity of response observed in salmon to that seen in trout, suggest the nitrous oxide method has wide tolerance. In comparative trials with heat, pressure and nitrous oxide under experimental conditions we find nitrous oxide exposure gives higher yields of Atlantic salmon triploids than does our preferred heat shock for this species, but lower yields than our preferred pressure shock treatment (Johnstone, unpublished results, 1988). In summary, therefore, these results confirm the suggestion made in an earlier paper (Shelton et al., 1986) that certain anaesthetics may be useful in ploidy manipulation. They also suggest that the choice of agents for further study should take pharmokinetic factors into consideration as well as the biological systems which it is intended to disturb. REFERENCES Allison, A.C., Hulands, C.H., Nunn, J.F., Kitching, J.A. and Macdonald, A.C., 1970. The effect of inhalational anesthetics on the microtubular system in Actinosphaerium nucleofilum. J. Cell Sci., 7: 483-499. Johnstone, R. and Lincoln, R.F., 1986. Ploidy estimation using erythrocytes from formalin-fixed salmonid fry. Aquaculture, 55: 145-148. Macdonald, A.G. and Wann, K.T., 1978. Physiological Aspects of Anaesthetics and Inert Gases. Academic Press, London, 308 pp. Miller, K.W., Paton, W.D.M., Smith, E.B. and Smith, R.A., 1972. Physicochemical approaches to the mode of action of general anaesthetics. Anesthesiology, 36: 339-351. Shelton, C.J., Macdonald, A.G. and Johnstone, R., 1986. Induction of triploidy in rainbow trout using nitrous oxide. Aquaculture, 58: 155-159. Smith, E.B., 1969. The role of exotic gases in the study of narcosis. In: P.B. Bennett and D.H. Elliott (Editors), Physiology and Medicine of Diving and Compressed Airwork. Bailliere, Tinda11and Cassell, London, pp. 183-192.
229 Printed in The Netherlands
Triploidy Induction in Recently Fertilized Atlantic Salmon Ova Using Anaesthetics R. JOHNSTONE’, R.M. KNOTTI, A.G. MACDONALD’ and M.V. WALSINGHAMI ‘DAFS Marine Laboratory, Victoria Road, Aberdeen AB9 8DB (Great Britain) ‘Department of Physiology, University of Aberdeen, AberdeenAB9 1AS (Great Britain) (Accepted 26 January 1989)
ABSTRACT Johnstone, R., Knott, R.M., Macdonald, A.G. and Walsingham, M.V., 1989. Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics. Aquaculture, 78: 229-236. The comparative effectiveness of five general anaesthetics as agents for inducing triploidy in recently fertilized Atlantic salmon ova is reported. Triploid rates and triploid yields following the exposures of eggs from a single female fish to nitrous oxide, and separately to Freon 22, were shown to be related to the partial pressure and duration of exposure. The maximum triploid yield (79.7% ) was observed following exposure to nitrous oxide at 11 atmospheres (atm) for the first 30 min after fertilization. Triploid yields following exposure to Freon (at 1 and 3 atm) were generally lower, the highest value (43.7%) occurring after exposure at ambient pressure for the first hour after fertilization. Triploid yields in these same eggs were low ( < 10%) when halothane and ethrane were used at partial pressures in the range 0.05-0.2 atm and cyclopropane at atmospheric pressure was ineffective. In a subsequent season the eggs from ten different individual female Atlantic salmon were shown to vary little in susceptibility following exposure to nitrous oxide treatment (O-30 min after fertilization, 11 atm) and experienced high yields of triploids (mean=80.4&6.6% SD).
INTRODUCTION
In a previous communication (Shelton et al., 1986) the first report of the use of an anaesthetic gas, nitrous oxide, to induce triploidy in recently fertilized rainbow trout, Salmo guirdneri, eggs was presented. The success of this experiment prompted us to confirm and extend our observations using another species, the Atlantic salmon, S. sahr. The results of two experimental series conducted in consecutive seasons are presented. In the first (1986)) batches of eggs from a single female farmed Atlantic salmon were exposed to that range of nitrous oxide treatments which had been used in the earlier trout study. We also attempted, using these same eggs, to test the hypothesis that triploid induction by nitrous oxide is part of a general phenomenon and that triploidy
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers B.V.
230
can be brought about by an equi-narcotic dose of other general anaesthetics. In selecting a range of agents to test for this effect we included potent anaesthetics which, it was hoped, could be administered to eggs at partial pressures lower than the 11 atm required for nitrous oxide treatment and which might therefore be more conveniently adopted in commercial practice. In the subsequent season the eggs of ten salmon from the same farm were separately exposed to that nitrous oxide treatment which had proved most effective in the 1986 trial in order to investigate the extent of any hen-to-hen variability in susceptibility to treatment. Additionally, pools of eggs generated by mixing equal numbers of eggs from these ten hens were exposed to range of nitrous oxide treatments and handling protocols to further investigate the factors of importance in maximizing triploid yield during nitrous oxide exposure. MATERIALS AND METHODS
In 1986 the eggs of a single two-sea-winter (ZSW) farmed Atlantic salmon female were stripped out and maintained in an oxygen-rich atmosphere in a polythene bag on ice. Groups of eggs (ca 150) were removed in turn from this pool, fertilized with the mixed milt from three farmed Atlantic salmon males and washed with 10°C water. The eggs were then either taken as controls or exposed as a monolayer in g-cm-diameter glass dishes to the particular anaesthetic agent under study, using the partial immersion protocol reported previously (Shelton et al., 1986). In this protocol approximately 150 eggs were contained in the dishes together with 9 ml of incubation water, this being just sufficient to cover the eggs prior to their being placed in the treatment vessel. According to narcosis theory as applied to cells in general (see for example Macdonald and Wann, 1978), equipotent doses of the general anaesthetics used in this study were selected on the following basis. Eleven atmospheres nitrous oxide, the highly effective dose reported in Shelton et al. (1986)) is 7.3fold the dose which abolishes the righting reflex in 50% of mice (Miller et al., 1972). Similarly the lesser partial pressure previously used, 5.0 atm, is 3.3-fold the ED50 for the loss of righting reflex. On this basis 1.17 atm and 0.53 atm of Freon 22 (CHCLF,; chloro-difluoro-methane) would be equipotent with the N20 treatments and for convenience we used 3.0 and 1.0 atm. Similarly (Miller et al., 1972),a dose of cyclopropane (cC,H,) equipotent with 11 atm N,O would be 1.17atm and for convenience we used 1.0 atm. The ED50 (righting reflex) for halothane (2 bromo-2-chloro-l,l,l-trifluoroethane) is 0.017 atm (Smith, 1969) or 0.0077 atm (Miller et al., 1972) and, again for reasons of experimental convenience, the doses of this anaesthetic which were tested.were 0.2 atm by static exposure and 0.124 and 0.056 atm using diluted streams of gas. Ethrane (2-chloro-1,1,2-trifluoroethyl difluoromethyl ether), which is about half as potent as halothane, was administered by static exposure at 0.11
231
atm, that is at a dose equivalent to the lower dose of nitrous oxide previously described. Eggs were exposed to elevated pressures of nitrous oxide (Medical grade, BOC Ltd) and Freon 22 in a pressure vessel as previously described (Shelton et al., 1986) using commercially available cylinder supplies. After fertilization and washing (ca 1-2 min), the eggs were placed in the vessel which was then purged with gas (ca 1 min) before being raised to the working pressure. Ambient pressure treatments with Freon 22 and cyclopropane were separately performed in this vessel by streaming the gases through the vessel at a slow rate (ca 5 ml/min) throughout the treatment period. The lower halothane (M & B Ltd, Dagenham) partial pressures used in this study (0.124 and 0.056 atm) were administered to eggs using the air streaming method of Allison et al. (1970). Eggs, in partially immersed “monolayers” in petri dishes, were held in a glass desiccating flask; the plastic tubing used to duct the vapour to the chamber having been fully equilibrated with the particular partial pressure of halothane prior to treatment. A higher dose of halothane (0.2 atm) was administered by containing the monolayer of eggs in a glass desiccator containing static air, equilibrated with liquid halothane (magnetically stirred in an open dish) at 10°C. During treatment in this way the temperature of the incubation water containing the eggs rose from 10” C to 16’ C, presumably due to the heat of solution of the anaesthetic in water. Ethrane (Abbot Laboratories Ltd, Kent) was similarly administered by static exposure at a partial pressure of 0.11 atm. In 1987 batches of ca 150 eggs derived from ten individual farmed Atlantic salmon (2SW maturers) were exposed to NzO (11 atm, O-30 min after fertilization) in monolayers as before following the addition of 9 ml of incubation water. Control batches of eggs were taken from each hen and reared separately. Equal numbers of eggs were then taken from each hen to generate pools of 150 eggs. Four such pools were exposed to a 30-min nitrous oxide exposure beginning immediately after fertilization at either 5,7,9 or 11 atm. Five pools were exposed to a 30-min nitrous oxide exposure at 11 atm following the addition of differing amounts of incubation water (2,4,6,12 and 18 ml) in an attempt to investigate the effect of this variable on the kinetics of nitrous oxide uptake. Two pools were taken as controls. Following treatments the eggs were in both seasons flooded with water and maintained at ambient temperatures (l-6” C) in individual netting baskets until yolk sac absorption was nearly completed. Dead eggs and alevins were removed frequently and recorded. Following formalin fixation of the survivors, the ploidy status of individuals in the various treatment groups was estimated by comparison with control animals following the microdensitometric analysis of Feulgen-stained erythrocyte preparations (Johnstone and Lincoln, 1986). In those groups where fewer than 50 animals survived, all animals were taken for analysis. When more than this number survived, a random sample of 50
232
survivors was analysed. The triploid yield in each group was calculated as the product of the triploid rate and the numbers surviving to the point of sacrifice, expressed as a percentage of the number of eggs originally present. RESULTS
The results of those experiments performed in the 1986 season when groups of eggs from a single female salmon were exposed to differing anaesthetic treatments in the period immediately after fertilization are summarized in Table 1. The survival rate in control eggs taken during these experiments was 88.3%. Following treatment with nitrous oxide, triploid rates and survival rates varTABLE 1 The effect of different anaesthetics on ploidy status of recently fertilized Atlantic salmon eggs Anaesthetic
Treatment conditions (time of exposure: minutes after fertilization)
survival rate (%)
Triploid rate (%)
Triploid yield (% )
Nitrous oxide
5 atm 5 atm 11 atm 11 atm 11 atm 11 atm
79.9
28 50 78 98 100 25
22.4 33.0 54.7 79.7 61.8 12.4 25.5 43.7 20.4 11.9
(O-30) (O-60) (O-15) (O-30) (O-60) (30-60)
66.1 70.1 81.3 61.8 49.4
1 atm streamed 1 atm streamed 3 atm 3 atm
(O-30) (O-60) (O-15) (O-30)
68.3 27.2 11.9
36 64 75 100
Cyclopropane
1 atm streamed 1 atm streamed
(O-15) (O-30)
86.2 83.3
0 0
Halothane
0.2 atm static 0.2 atm static 0.2 atm static 0.2 atm static
(O-10) (O-20) (10-20) (20-30)
56.3 42.0 73.3 75.2
8 22 0 8
0.124 atm streamed 0.124 atm streamed
(O-30) (O-60)
75.2 75.2
0 0
0 0
0.056 atm streamed 0.056 atm streamed
(O-30) (O-60)
90.8
0 0
0 0
0.11 atm static 0.11 atm static
(O-15) (O-30)
77.9
2 6
1.6 4.3
Freon 22
Ethrane
70.9
83.7
71.8
0 0
4.5 9.2 0 6
233
ied with the timing, duration and partial pressure of exposure. Treatments at 5 atm generated fewer triploids than those at 11 atm. A longer treatment (60 min) at the lower partial pressure induced significantly (PC 0.05) greater triploid rates than did a shorter treatment (30 min). Survival rates were decreased by the longer exposure but not significantly so. At 11 atm triploid rates increased with increasing exposure time in those treatments beginning immediately after fertilization but survival rates were less clearly related to the duration of treatment. Maximal triploid yields at 11 atm were generated by a 30-min treatment which began shortly after fertilization. Triploid rates and survival rates were poor in that treatment group in which exposure to nitrous oxide at 11 atm was delayed to encompass the period 30 to 60 min after fertilization. Triploids were also induced following exposure to Freon 22. As with nitrous oxide, Freon 22 was more effective in inducing triploidy at elevated partial pressure (3 atm) but this was associated with high mortality rates. Exposure to Freon at ambient pressure resulted in higher survival rates, the triploid rate in this instance being related to the duration of treatment. Cyclopropane when administered at ambient pressure had little effect on mortality rate and was without effect on ploidy status. Static exposure of eggs to the volatile agents halothane and ethrane resulted in only a low percentage of triploids. Halothane usage in particular was associated with increased mortalities especially in those exposures which began immediately after fertilization. Although survival rates following increased durations of exposure to diluted streams of halothane were higher, these treatments produced no triploids. Following exposure to nitrous oxide (O-30 min a.f., 11 atm), batches of eggs taken from different females in the 1987 season differed little in survival rate or in triploid rate and therefore in triploid yield (Table 2 ) . The mean survival rate relative to controls was 86.32 + 6.7% and the mean triploid rate and triploid yield were 93.2 2 3.2% and 80.4 -+6.6%, respectively. The relationship between the partial pressure of nitrous oxide during treatment and efficacy on pools of eggs generated from these same females are summarized in Table 3. Triploid rate increased and survival rate apparently decreased with increasing partial pressure of nitrous oxide exposure. The maximum triploid yield was observed following treatment at 9 atm. The consequence of an alteration in the amount of incubation water on the efficacy of a nitrous oxide treatment are also summarized in Table 3. At the normal rate of addition of incubation water adopted in these studies (9 ml water to ca 150 eggs in a 9 cm petri dish) the eggs were just covered by water at fertilization and came to be partially exposed as a result of water uptake during the water hardening process. At the highest rate of addition of water (18 ml) the eggs were wholly covered both before and after treatment. Following the addition of only 2 ml, little liquid water remained in the dish at the end
234 TABLE 2 The effect of nitrous oxide exposure (O-30 min a.f.; 11 atm) on ploidy status of different female farmed Atlantic salmon Fish no.
Control survival rate (%)
Survival rate (%)
Survival rate (%RC)a
Triploid rate (%)
Triploid yield (% RC)a
1 2 3 4 5 6 7 8 9 10 Mean k SD
92.9 97.3 96.8 95.1 96.0 87.6 88.9 95.1 92.1 91.3 93.31 k 3.3
71.2 88.2 83.0 82.4 85.3 84.8 71.6 75.9 76.0 86.9 80.53 f 6.3
76.6 90.6 85.7 86.6 88.9 96.8 80.5 79.8 82.5 95.2 86.32 f 6.7
96 94 90 94 86 96 94 96 92 94 93.2 k 3.2
73.5 85.2 77.1 81.4 76.5 92.9 75.7 76.6 75.9 89.5 80.4 f 6.6
“RC = relative to control. TABLE 3 The effect of different treatment protocols on the effectiveness of a nitrous oxide exposure (O-30 min a.f.) on ploidy status of Atlantic salmon egg pools* Treatment no. 1 2 3 4 Control 1
2 3 4 5 Control
Incubation water (ml)
Partial pressure (atm)
Survival (% )
Survival (% RC)a
Triploid rate (%)
Triploid yield (%)
9 9 9 9 9
5 7 9 11 0
68.7 60.7 62.3 50.4 75.3
91.2 80.6 82.7 66.9 100.0
28 IO 96 98 -
25.5 56.4 79.4 65.6 -
2 4 6 12 18 9
11 11 11 11 11 0
90.0 83.4 91.9 98.0 89.1 95.6
94.1 87.2 96.1 102.5 93.2 100.0
18 98 100 100 98 -
73.4 85.5 96.1 102.5 91.3 -
*Equal numbers of ova were taken from each of the ten individuals referred to in Table 2 to generate pools of 150 eggs. “RC = relative to control.
of the treatment period, although the eggs were still obviously “moist”. Although survival rates were apparently unaffected by these differences, the triploid rate was lowered significantly (P
235 DISCUSSION
It has long been known that the potency of anaesthetic agents is closely correlated with their solubility in solvents such as olive oil or octanol (Miller et al., 1972). This implies the hypothesis that in order to exert their effects on biological systems these agents must first dissolve in or bind to molecular sites with a solubility parameter similar to that of the model solvents (Macdonald and Wann, 1978). The system under discussion here is that of the lipid-rich salmonid egg contained in incubation water. The more lipophilic and potent agents used in this study (the halogenated compounds) will be those which would be expected to equilibrate most slowly via the aqueous phase surrounding the eggs. In addition, highly hydrophobic anaesthetics may dissolve in irrelevant lipid “sinks”. The efficacy of these compounds as triploidization agents might therefore depend on kinetic factors as well as on their specific binding to sites whose unaltered function is necessary for the normal completion of those chromosomal movements typical of the meiotic division of recently fertilized salmonid eggs. The results of the experiments reported here show that in Atlantic salmon ova as in rainbow trout (Shelton et al., 1986), nitrous oxide is effective in generating high yields of triploids when given at an appropriate partial pressure and timing of exposure. The ability of other anaesthetic agents to manipulate ploidy status by interference with the second meiotic division of recently fertilized eggs has also been demonstrated. Nitrous oxide, the least hydrophobic and in mammalian systems the least potent of the agents used in this study, was increasingly effective in inducing triploid in salmon eggs when administered shortly after fertilization at partial pressures in excess of 5 atm. Cyclopropane, though ten times more potent than nitrous oxide in mammalian systems, is also ten times more hydrophobic (Miller et al., 1972). It was ineffective in this study when used at ambient pressure presumably because of its greater degree of hydrophobicity. It can be postulated therefore that in order for it to have an effect, cyclopropane would need to be delivered at elevated partial pressure, but perhaps not at pressures as high as those necessary for effective treatment with nitrous oxide. Freon 22 is very similar in anaesthetic potency and hydrophobicity to cyclopropane (Miller et al., 1972 ) and was more effective, which suggests that there may be specific binding differences between these two compounds. Though even more effective at elevated partial pressure, Freon was extremely toxic when used in this way. The volatile liquid agents halothane and ethrane are highly lipophilic and relatively ineffective in inducing triploidy, perhaps primarily because of their slow rate of equilibration. Halothane was relatively toxic in those exposures which began immediately after fertilization. The temperature rise observed in the static halothane exposures would have affected the halothane solubility and kinetics and this factor may therefore have contributed to its toxicity.
Mean triploid yields were satisfactorily high (80.4 + 6.6 SD% relative to controls) when eggs from ten individual female fish were exposed in 1987 to the preferred nitrous oxide treatment indicated in the 1986 experiments. The effectiveness of nitrous oxide was tested further in this season by treating pools of eggs in which these same individuals were equally represented. The results of these treatments demonstrated nitrous oxide to be effective over a relatively wide range of partial pressures and incubation protocols. Somewhat to our surprise, triploid rate was diminished by a paucity of incubation water and not by an excess. A delay in the initiation of nitrous oxide exposure to 30 min after fertilization resulted in higher mortalities and a lowered triploid rate (25%, Table 1) due presumably to the increasing degeneration of the polar body and its exclusion from the fusion nucleus. These results, together with the lack of any marked interindividual difference in susceptibility amongst salmon and the similarity of response observed in salmon to that seen in trout, suggest the nitrous oxide method has wide tolerance. In comparative trials with heat, pressure and nitrous oxide under experimental conditions we find nitrous oxide exposure gives higher yields of Atlantic salmon triploids than does our preferred heat shock for this species, but lower yields than our preferred pressure shock treatment (Johnstone, unpublished results, 1988). In summary, therefore, these results confirm the suggestion made in an earlier paper (Shelton et al., 1986) that certain anaesthetics may be useful in ploidy manipulation. They also suggest that the choice of agents for further study should take pharmokinetic factors into consideration as well as the biological systems which it is intended to disturb. REFERENCES Allison, A.C., Hulands, C.H., Nunn, J.F., Kitching, J.A. and Macdonald, A.C., 1970. The effect of inhalational anesthetics on the microtubular system in Actinosphaerium nucleofilum. J. Cell Sci., 7: 483-499. Johnstone, R. and Lincoln, R.F., 1986. Ploidy estimation using erythrocytes from formalin-fixed salmonid fry. Aquaculture, 55: 145-148. Macdonald, A.G. and Wann, K.T., 1978. Physiological Aspects of Anaesthetics and Inert Gases. Academic Press, London, 308 pp. Miller, K.W., Paton, W.D.M., Smith, E.B. and Smith, R.A., 1972. Physicochemical approaches to the mode of action of general anaesthetics. Anesthesiology, 36: 339-351. Shelton, C.J., Macdonald, A.G. and Johnstone, R., 1986. Induction of triploidy in rainbow trout using nitrous oxide. Aquaculture, 58: 155-159. Smith, E.B., 1969. The role of exotic gases in the study of narcosis. In: P.B. Bennett and D.H. Elliott (Editors), Physiology and Medicine of Diving and Compressed Airwork. Bailliere, Tinda11and Cassell, London, pp. 183-192.