Metomidate anaesthesia in Atlantic salmon, Salmo salar, prevents plasma cortisol increase during stress

Aquaculture ELSEVIER

Aquaculture134(1995)155-168

Metomidate anaesthesia in Atlantic salmon, S&w salar, prevents plasma cortisol increase during stress Yngvar A. Olsen ‘*a**, Ingibjorg E. Einarsdottir b, Kjell J. Nilssen b aThe Norwegian College of Veterinary Medicine, Department of Morphology, Genetics and Aquatic Biology, P.O. Box 8146 Dep., N-0033 Oslo I, Norway b The University of Trondheim, BrattQra Research Station, Section of Aquaculture, N-7055 Dragvoll, Norway

Accepted 20 December 1994

Abstract Atlantic salmon parr (58 g) in fresh water at 5.O”C and adult salmon ( 1130 g) in sea water at 7.7”C were exposed to water containing different concentrations of metomidate in the range 1 to 10 mg l- r. Metomidate was efficacious in inducing anaesthesia (hypnosis), and efficacy increased with concentration over the interval tested. The anaesthetic was more potent in the adult salmon acclimated to sea water than in freshwater Parr. Metomidate at 3 mg l- ’ or higher completely prevented any plasma cortisol increase after a handling stressor when stressor and anaesthetic were applied concomitantly. The lack of a cortisol response seemed to he due to a blockage at the level of the interrenal cell, since exogenous ACTH injected intraperitoneally did not produce a cortisol increase in metomidate-anaesthetized fish but did in those anaesthetized with MS-222. Blood lactate levels and haematocrit increased in fish during metomidate anaesthesia. Keywords:

Anaesthetics; Metomidate; Salmo s&r; Cortisol; Stress

1. Introduction

When assaying the corticosteroid response to evaluate stress, a common procedure is to rapidly anaesthetize a group of fish before blood sampling. For certain types and concentrations of anaesthetics this procedure ensures that the hypothalamo-pituitary-interrenal axis (HP1 axis) is not activated due to the handling stress associated with sampling (Wedemeyer et al., 1990). Common anaesthetics for this purpose are tricaine methanesulphonate * Corresponding author. ’ Present address: Central Veterinary Laboratory, P.O. Box 8156 Dep., N-0033 Oslo 1, Norway. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SDI0044-8486(95)00008-9

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(MS-222), benzocaine, and phenoxyethanol. The mechanism preventing activation of the HP1 axis is not known. It is reasonable to assume, however, that the transmission of sensory information to the hypothalamus is blocked. There is evidence that sensory neural pathways in some cases may be only partially blocked. Etomidate and several other hypnotics have poor or ineffective analgesic properties in mammals (Reneman and Janssen, 1977; Hall and Clarke, 199 1) , indicating transmission of pain stimuli. Surgery and injection procedures will activate the HP1 axis even when the fish is under deep MS-222 anaesthesia (authors’ unpubl. obs.; Pickering et al., 1987). Anaesthetic agents that prevent the activation of the HP1 axis at proper concentrations may not be efficient at low concentrations. A low concentration may actually work as a stressor and give an exacerbated cortisol response (Strange and Schreck, 1978; Barton and Peter, 1982). A special case is demonstrated by ether anaesthesia in mammals which causes corticosteroid elevation, probably by a direct, humoral effect on the hypothalamus (Matsuda et al., 1964). Metomidate, dl- 1-( 1-phenylethyl) -5( metoxycarbonyl) imidazole hydrochloride, and the closely related compound etomidate, are rapid-acting, non-barbiturate hypnotics in several species. Metomidate is presently marketed as a fish anaesthetic. The anaesthetic has a short induction time in fish, although the recovery time is rather long (Gilderhus and Marking, 1987). A side-effect is muscle twitches, which can interfere with blood sampling. Etomidate is the better studied compound and was used until recently in human intensive care (Hall and Clarke, 1991). Preziosi and Vacca (1982) found a marked reduction in plasma level of corticosterone in rats after anaesthetic doses of etomidate. Their finding was later confirmed in studies on cortisol synthesis in humans and dogs (Fragen et al., 1984; Fraser et al., 1984). Etomidate affects the mitochondrial cytochrome P,,,-dependent enzymes that catalyse the synthesis of cortisol, especially 1lPhydroxylase and, at a higher concentration, the cholesterol side-chain cleavage enzyme (Vanden Bossche et al., 1984; Wagner et al., 1984). Whether metomidate also has this effect has not been rigorously established, but several imidazole compounds interfere with steroid synthesis (Feldman, 1986). The current experiments were designed to study the efficacy of metomidate as an anaesthetic in Atlantic salmon (Sdmo s&r) and whether metomidate was able to eliminate the normal plasma cortisol increase in this species during stress. Both freshwater parr (Experiment 1) and adult seawater-adapted salmon (Experiment 2) were studied. An inhibition of the cortisol response was demonstrated, and subsequently an attempt was made to locate the site of inhibition within the HP1 axis (Experiment 3). Haematocrit and blood lactate levels were measured in order to describe the haematologic and respiratory effects of metomidate anaesthesia.

2. Materials and methods Experiment

1

This study was conducted at the Central Veterinary Laboratory, Oslo, Norway, in December 1991 using Atlantic salmon parr, Salmo salar L., (average weight 58 g, Akvaforsk strain) from Marenor A/S, Slemmestad, Norway. Precocious male parr were excluded. The fish were acclimated to dechlorinated city tap water for 10 weeks. The water had a pH of

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6.3 and a conductivity (25°C) of 2.9 mS m-l. The calcium content was 2.5 mg 1-l. The acclimation and test temperatures were 5.O”C. For the determination of efficacy, fish were taken directly from the holding tanks and transferred to a 40-l glass aquarium containing metomidate solution for the determination of anaesthesia stage duration, as described by Schoettger and Julin ( 1967). The following provides a brief description of the stages employed: stage 1, Sedation; stage 2, Partial loss of equilibrium; stage 3A and 3B, Total loss of equilibrium; stage 4, Loss of reflex activity; and stage 5, Medullary collapse. The times to reach each anaesthesia stage were determined in groups of six fish. Time was registered when three out of six individuals had entered that anaesthesia stage. Metomidate concentrations 1,2, 3, and 5 mg l- ’ were tested on different fish groups; 10 mg 1-l was not tested due to lack of fish. The observation period was 80 min. 180 fish were divided equally and at random among six black 53-l polyethylene tanks for the freshwater stress trial. The tanks were equipped with centre drains and were located inside separate small enclosures with a door in front, preventing visual contact with personnel. Above each tank a lamp supplied continuous incandescent light. The water was delivered at a flow-rate of 1.5 1 min - ’. The fish were acclimated to the experimental conditions for 6 weeks. They were not fed or disturbed in any other way for at least 24 h before sampling. Anaesthesia and sampling were performed in the following way: The door to the randomly selected tank (assigned to one metomidate concentration) was removed, the water supply to the tank was turned off and aeration began. Concentrated solutions of metomidate (MarinilTM Wildlife Labs., Inc., Fort Collins, CO, USA) in 100 ml water were added to make the final concentration in the tank water 0 (control), 1, 2, 3, 5, and 10 mg 1-l after mixing. Six fish were immediately dip-netted from the tank into a bucket containing a solution of 5 mg 1-i metomidate. Preliminary trials had shown that this concentration was sufficient to prevent an increase in plasma cortisol level. When the fish were immobilized, blood was sampled from the caudal vessels using Li-heparinized vacuum tubes (Venoject, Terumo Europe N.V., Leuven, Belgium). The blood samples were put on ice and not treated further until all six fish were sampled. This sample was termed 0 min. Sampling of five or six fish from the same tank was repeated at 10,20, 30, and 60 min by the same procedure. The sampling procedure was intentionally used as a stressor for the remaining fish in the tank.

Experiment

2

This study was performed at the Brattora Research Station, University of Trondheim, in January 1992. Adult seawater-adapted Atlantic salmon (Akvaforsk strain) of average weight of 1.13 kg (s.d. 0.38 kg) at the time of the experiment, were acclimated to square, 0.5-m3 fibreglass holding tanks containing 200 1 of sea water. Thirteen fish were placed in each of eight tanks and acclimated for 4 months. The water supply to the tanks was maintained at 5 1 min-‘. The 34 ppt salinity sea water was pumped from 70 m depth. The temperature during the acclimation period declined from 10.2 to 7.7”C. Fluorescent light was left on continuously. Extruded dry salmon feed (Felleskjapet A/S) was supplied by automatic feeders at a rate of 0.6% of body weight day-‘. Care was taken not to disturb the fish for at least 24 h before sampling.

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A total of 18 fish were dip-netted out of two holding tanks in one operation, taking care not to disturb fish in neighbouring tanks. The fish were divided equally between three separate O.l-m3 circular polyethylene tanks containing 70 1 of aerated metomidate-inseawater solution, all three tanks containing the same metomidate concentration. One polyethylene tank was assigned for each sampling interval ( 10, 20, and 30 min). The 0 min sample fish were anaesthetized in a separate 0.1 -m3 tank containing a solution of 12 mg l- ’ metomidate. These 0 min samples were used to provide pre-treatment values for all concentrations tested on one day, owing to limitations on numbers of fish. Blood sampling started in the 0 min group as soon as the fish were immobilized. Each metomidate concentration was tested separately in the same manner. The tested metomidate concentrations in this experiment were 0 (control), 2, 4 and 8 mg l- ‘. Metomidate concentrations 0 and 2 mg 1-l were sampled on the first day, 4 and 8 mg l- ’ on the next. Two of the holding tanks sampled on day 1 were sampled again 25 h later (fish for the 4 mg l- ’ metomidate treatment regime). This time interval is normally sufficient for cortisol to return to resting level after an acute stress like dip-netting (Pickering et al., 1982). Blood was sampled from the caudal vessels using heparinized l-ml plastic syringes and transferred to centrifuge tubes. After centrifugation, plasma was stored at - 20°C until analysed for cortisol. Three tanks containing six fish each were tested to determine duration of anaesthesia stages as described in Experiment 1. The groups were exposed to 2,4, and 8 mg l- ’ metomidate, respectively, by pouring a concentrated solution of metomidate into the fish tank after the water supply had been turned off. Aeration was provided. The fish were observed for 10 min and time intervals were registered as described in Experiment 1. Experiment

3

This study was performed in July 1992 in the same laboratory as Experiment 1. Atlantic salmon (Akvaforsk strain) were purchased from Bandaksmolt A/S, Lkdal, Norway. The fish, which were smolts 2 months earlier but had not been in sea water, had an average weight of 63 g (s.d. 7.98 g). The fish tanks, the feeding regime and the water quality were identical to those in Experiment 1. The acclimation period to the water was 6 weeks, of which the last 3 weeks also were acclimation to the experimental conditions. The number of individuals in each tank was originally eight, but a few escaped during the acclimation period. One of the saline-injected fish in Tank 3 died during anaesthesia. A preliminary experiment showed that the cortisol response to stress could be completely eliminated 48 h after feeding with dry pellets (Ewos EST 93 Vextra) coated with dexamethasone (300 mg kg-‘; Sigma Chem. Co., St. Louis, MO, USA). The coating was done according to the procedure described by Pickering et al. ( 1987). Intraperitoneal injection of 0.1 ml saline (0.9% NaCl) gave no increase in cortisol level after 1 h in dexamethasone-treated fish. The same type of injection, but with 1 I.U. of porcine adrenocorticotropic hormone (ACTH; A6303, Sigma Chem. Co.) gave a plasma cortisol level in the range 560 to 980 nmol 1-i. Experiment 3 was run with four tanks. The water temperature was 8.6”C, but increased gradually to lO.O”C at blood sampling in Tanks 1 and 2 due to aerated, stagnant water. The fish were fasted for 2 days before initiation of dexamethasone feeding, and then fed ad libitum with coated pellets at 0 and 24 h. At 48 h, Tanks 1 to 4 were sampled in sequence. Tank 1 fish were anaesthetized by direct addition of bicarbonate-neutralized tricaine methanesulphonate (MS-222; Sigma Chem. Co.) solution to a final concentration of 70 mg l- ’

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after the water supply had been turned off. When the fish were immobilized (about 10 min) , four fish were injected intraperitoneally with ACTH and four with saline (same procedure as in preliminary experiment). The adipose fin was removed in the saline-injected fish for identification, and all fish were transferred back to the tank. The fish remained in the aerated, static anaesthetic solution until blood sampling, 1 h after injection (sampling as described in Experiment 1). Tank 2 fish were sampled identically to Tank 1, but in this case the anaesthetic was 5 mg l- ’ metomidate. Tank 3 fish were dip-netted from the tank into a bucket containing a neutralized and aerated MS-222 solution at double concentration ( 140 mg l- ‘). Tank 4 fish were similarly transferred to 10 mg 1-l metomidate. The fish in these two tanks were injected immediately after immobilization, and remained in the anaesthetic solution for a total of 10 min before they were transferred back to their tanks supplied with running fresh water. Blood sampling was performed 1 h after injection on re-anaesthetized fish from all tanks. Analytical procedure Haematocrit was determined using a Bayer Compur Ml 100 mini-centrifuge. For lactate analysis, 20-~1 sub-samples of blood were added to centrifuge tubes containing 200 ~1 of 0.3 M perchloric acid. After centrifugation, plasma and lactate supernatant were transferred to polyethylene storage tubes and kept at - 70°C until analysis. Plasmacortisol was analysed by radioimmunoassay according to the method described by Olsen et al. ( 1992). The cortisol antiserum (F3-3 14) was delivered by Endocrine Sciences, Tarzana, CA, USA. The sensitivity of the standard curve was 0.55 nmol l-i, and the within-assay and between-assay coefficients of variation were 4.0 and 14.6%, respectively. Blood lactate was analysed by the use of kit no. 139 084 (Boehringer Mannheim GmbH, Germany) and absorption was read at 340 nm in a Shimadzu UV-160A spectrophotometer. Statistical procedure The Shapiro-Wilk test was used to check for normality of data in Experiment 1 after all 0 min samples had been pooled. A normal distribution could not be rejected for haematocrit and lactate results; however, for cortisol the probability that the results were from a normal distribution was very small (P = 0.0001). Analysis of variance and t-test with Bonferroni correction was used for haematocrit and lactate. Non-parametric methods ( Kruskal-Wallis test and Wilcoxon Rank Sum test) were used in the analysis of cortisol results due to the non-normal distribution of this variable. The significance level was chosen as P = 0.05 in all tests. Analysis was performed by PC-SAS programs (SAS Institute Inc., Cary, USA).

3. Results Experiment 1 Table 1 shows the time in minutes required to reach the different anaesthesia stages. Within the metomidate concentration range tested, the time to reach the different anaesthesia stages was reduced with increasing metomidate concentration. Fish exposed to 1 mg l- ’ took 12 min to enter stage 3B (loss of equilibrium and tactile response), whereas fish exposed to 5 mg 1~ ’ took 2 min 10 s. Fish anaesthetized in 1 and 2 mg 1- ’ metomidate did

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Table 1 Time (min. s) for Atlantic salmon freshwater time of contact with metomidate solution Metomidate

cont. (mg 1-l)

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parr to reach anaesthesia

Anaesthesia

stages (Schoettger

and Julin, 1967) from

stage

1

2

1

2 00

400

2 3 5

1 00 040 0 29

201 1 12 0 50

3A

3B

4

8 00

12 00

-

331 152 1 30

544 340 2 10

39 00 8 13

Six fish were used for each concentration; time was registered when three fish had reached the proper stage. -: Stage not encountered during the observation period (80 mm). Temperature 5°C.

not reach stage 4 (loss of reflex activity) during the observation period of 80 min. Fish exposed to 3 mg 1-r reached stage 4 in 39 min. No fish entered stage 5 (medullary collapse, death) during the observation period, although ventilatory activity was almost terminated after 80 min in fish exposed to 5 mg I- ’ metomidate. Cortisol measurement on 0 min samples in Experiment 1 (Fig. 1) showed low plasma levels (median range 10 to 28 nmol l- ’ ) , probably representative of the resting condition. The control group showed a statistically significant increase (P = 0.003) in plasma cortisol level from a level of 19 nmol 1-r at 0 min to a maximum of 383 nmol 1-l at 30 min. The group receiving 1 mg 1-l metomidate also showed a significant increase in cortisol level (P = 0.0009)) but only reaching 175 nmoll- ’ in 20 min. This level remained nearly constant from 20 to 60 min. The fish exposed to the metomidate concentrations 2, 3,5, and 10 mg 1-r showed no significant increase in cortisol levels over the 60 min duration of the experiment. Results from 3, 5, and 10 mg 1-l metomidate groups were lumped together

600

0

A

0

10

20

30

60

Time (min)

Fig. 1. Median plasma cortisol levels in Atlantic salmon parr in fresh water after concomitant stressor exposure and addition of metomidate to the tank water at time 0. The stressors were visual disturbance above water, water aeration, introduction of metomidate solution, and dip-netting of fish at 0, 10, 20, 30, and 60 min. Temperature 5°C. + signs indicate individual observations. N=5 or 6. n , 0 mg 1-l metomidate; 0, 1 mg I-‘; *, 2 mg I-‘; 0.3 mg 1-l; III,5 mg I-‘; 0,lO mg 1-l.

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0’

’

0

1

10

161

134 (1995) 155-168

I

20

I

/

30

Time (min) Fig. 2. Lactate levels in blood of Atlantic salmon parr (same blood samples as in cortisol analysis) during metomidate anaesthesia. Treatment, number of individuals, and symbols as described in legend of Fig. 1. * Significant difference from the control group at the same time (P < 0.05). Each point is the mean f s.e.m.

and this produced a median level of 13.8 nmol l- ’ and 95% confidence limits of the median with lower and upper bounds of 11 .O and 16.6 nmol l- ‘. The control and 1 mg 1- ’ metomidate treatment groups showed mean blood lactate levels of less than 1.25 mm01 1-l during the exposure period (Fig. 2; same blood samples as in Fig. 1). There was no significant increase in lactate levels in fish from either of these treatments. Fish exposed to metomidate concentrations of 2, 3, 5, and 10 mg 1-l showed significant increases in lactate levels during exposure (P = 0.019 for the 2 mg 1-l group and P=O.OOOl for the higher concentration groups). Statistical comparison between the control fish and the metomidate treatment groups at the different sampling times showed that the fish exposed to 5 and 10 mg 1-l were higher in lactate at 30 and 60 min, whereas the 3 mg l- ’ fish were higher only at 60 min. A significant increase in haematocrit (Fig. 3) occurred in fish exposed to all metomidate concentrations as well as the control (0 mg l- ‘, P = 0.001; 1 mg l- ‘, P = 0.020; P = 0.0001 55 50 45 40 35 30 25

-r t 0

I

10

/

20

30

Y

Time (min) Fig. 3. Haematocrit levels in Atlantic salmon parr during metomidate anaesthesia. Treatment, number of individuals, and symbols as in legend of Fig. 1. *Significantdifference from the. control group (P < 0.05). Each point is the mean f s.e.m.

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Table 2 Time (min. s) for adult Atlantic salmon in sea water to reach anaesthesia Metomidate

cont. (mg 1-l)

2 4 8 Time registered

Anaesthesia

stages during metomidate

anaesthesia

stage

1

2

3A

3B

4

044 0 24 0 12

1 54 046 021

2 34 1 10 0 50

5 30 1 30 1 20

644 150 140

and number of fish as in Table 1. Observation

period 10 min. Temperature

7.7”C.

for all higher concentrations). However, the increase in control fish and fish from metomidate concentration 1 mgl- ’ was less than the increase in fish from the other treatment groups. Multiple comparison procedures showed that haematocrit values in the four higher concentration groups were significantly different from the control at 60 min. Experiment 2 All tested metomidate concentrations were sufficient to produce stage 4 anaesthesia in the seawater-adapted fish in < 7 min (Table 2). The time required to enter stage 4 was similar in fish subjected to metomidate concentrations 4 and 8 mg 1-l ( 1 min 50 s and 1 min 40 s, respectively). Metomidate concentration 2 mg l- ’ showed a markedly prolonged time (6 min 44 s) for the fish to reach stage 4 compared with higher concentrations, although much shorter than the time required by the parr to reach this stage in Experiment 1. The fish were only observed for 10 min and stage 5 was not encountered during this time period. Plasma cortisol in seawater-adapted control fish increased significantly (P = 0.0009) to 243 nmol l- ’ at 30 min (Fig. 4). That in fish exposed to 2 mg l- ’ metomidate also changed significantly with time (P = 0.01) but did not rise higher than 50 nmol 1-l. Exposure of fish to 4 and 8 mg l- ’ metomidate prevented any stressor-induced effect through time on

Time (min) Fig. 4. Median plasma cortisol levels in adult Atlantic salmon in sea and different seawater solutions of metomidate at time 0. The fish smaller aerated anaesthesia tanks containing the proper metomidate signs indicate individual observations. W, 0 mg 1-I metomidate; *,

water after concomitant exposure to stressor were dip-netted out of their holding tank to concentration. N = 6. Temperature 7.7’C. + 2 mg 1-I; X, 4 mg 1-I; +, 8 mg 1-l.

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z E

800 .

0

163

0

q

=51 600 - q ._ t: s 400 z P Q 200 -

-

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MS-222

MET0 Tank1 Tank2 1 h anaesthesia

0

a %

MS-222 MET0 Tank3 Tank4 10 min anaesthesia

Fig. 5. Individual plasma cortisol levels in dexamethasone-treated Atlantic salmon in fresh water 1 h after intrapzritoneal injection of porcine ACTH (0) or 0.9% NaCl(0). Fish in Tanks 1 and 2 (N= 8) stayed in the anacsthesia solution (70 mg I- ’ buffered MS-222 or 5 mg I- ’ metomidate) from injection to blood sampling. Fish in Tanks 3 and 4 (N= 5) were subjected to the anaesthesia solution for only 10 min, but at double concentration. Temperature was 8.6”C, increasing to 10°C in Tanks 1 and 2. MS-222, tricaine methanesulphonate. METO, metomidate.

plasma cortisol levels. There was a difference of 31 nmol lthe first and the second day.

’ between the 0 min groups of

Experiment 3 Dexamethasone-treated fish anaesthetized with MS-222 had very differentplasmacortisol levels depending on treatment (Fig. 5). Saline-injected fish showed a plasma cortisol level in the range 0 to 50 nmol l- ‘, whereas ACTH-injected fish produced a high cortisol response (from 458 to 864 nmol 1-l). The high cortisol response was almost completely abolished in ACTH-injected fish from Tank 2 (anaesthetized with metomidate) and their plasma cortisol level was significantly lower than the level in ACTH-injected fish from Tank 1 (P = 0.03). Their cortisol levels (range 14 to 39 nmol l- ‘), although higher than their saline-injected cohabitants, were of similar magnitude to the levels of the saline-injected fish in Tank 1. Tank 2 individuals had been in the metomidate solution for about 68 min and were in anaesthesia stage 4 when blood was sampled. The fish in Tank 4 were only exposed to the metomidate solution for 10 min. Although they were exposed to twice the metomidate concentration of Tank 2 fish, their cortisol response after ACTH injection was only partly suppressed (range 262 to 372 nmol 1-l). When tested against the ACTHinjected fish in Tank 3, the difference was not significant (P = 0.11).

4. Discussion In both the freshwater and the seawater experiments, the anaesthetic efficacy of metomidate increased with metomidate concentration within the range tested. The behaviour of

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the fish was similar to the general description of stages for MS-222 anaesthesia (Schoettger and Julin, 1967). Gilderhus and Marking ( 1987) found that rainbow trout (Oncorhynchus mykiss) could be classified as “handleable” after 1.0 to 2.7 min in 5 mg 1-l metomidate ( 12”C, weight 73 g) . This term probably corresponds to stage 3B and, thus, their induction time compares well with our experiment. The higher temperature in the experiment of Gilderhus and Marking ( 1987) probably contributed to the higher efficacy in the rainbow trout. Temperature rise is known to increase the efficacy of several anaesthetics (Houston and Woods, 1976; Amend et al., 1982). Mattson and Ripple ( 1989) found that cod (Gadus morhua) anaesthetized with 5 mg 1-l metomidate at 9S”C reached stage 3B after 2 min (body weight 101 g) . This result also compares well with ours, in spite of the species difference. In the present experiment, the adult seawater-adapted salmon were anaesthetized more quickly than the salmon parr. A direct comparison of the changing efficacy of metomidate with size in our freshwater and seawater salmon is complicated by the temperature difference (2.7”C). In addition, different water media, different laboratories and genetic differences between fish strains are possible sources of variation. However, due to the large difference it is probably safe to conclude that induction of deep anaesthesia is faster in large salmon (weight approximately 1 kg) in sea water than in parr in fresh water when metomidate is administered through immersion. Fish body weight is known to influence anaesthetic efficacy. The efficacy normally increases with increasing fish size (McFarland, 1959; Houston and Corlett, 1976)) and this general rule also seems to hold for metomidate (Gilderhus and Marking, 1987). It should be remembered that the determination of the stage of anaesthesia, by the present criteria, is somewhat subjective and stage duration may thus vary between different observers. The second parts of Experiments 1 and 2 were aimed at determining the ability of metomidate to prevent a stressor-induced rise in plasma cortisol concentration. The initial plasma cortisol levels were low in all exposure groups of the freshwater parr. This indicated that the fish were not at a stage in their life history when resting levels are elevated, such as during smolting (Langhome and Simpson, 1986; Olsen et al., 1993). Plasma cortisol levels in Atlantic salmon are not well investigated. Plasma cortisol levels in Atlantic salmon increase to higher levels during stress than in rainbow trout (Oncorhynchus mykiss) exposed to the same stressor (Fevolden et al., 1991). True resting levels are difficult to determine due to the inevitable involvement of stressors associated with the tank environment and with blood sampling. Resting levels below 28 nmol 1-l ( = 10 ng ml-‘; Langhorne and Simpson, 1986) have been found in Atlantic salmon. In our opinion, resting plasma cortisol levels in Atlantic salmon are close to those found in brown trout, S. trutru ( < 14 nmol 1-l; Pickering and Pottinger, 1989). In the seawater-adapted salmon, for practical reasons it was not possible to have separate 0 min sub-groups for all metomidate concentrations. This was unfortunate since it could have led to biased results if the tanks were exposed to different background stressors. The difference in plasma cortisol level between 0 min groups on day 1 and day 2 indicated that this was the case. We believe that resting plasma cortisol levels in adult seawater-adapted salmon are not higher than in freshwater parr, and that the second day 0 min group were slightly stressed. The control groups (not exposed to metomidate) both in fresh water and sea water showed the expected rise in plasma cortisol level after handling. The cortisol level at 30 min was higher in the parr than in the adult seawater-

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adapted fish, but the levels can not be directly compared. This is due to the difference in type of stressors and factors like habituation, size and strain of fish which may have also influenced the cortisol level (Barton et al., 1987; Pickering and Pottinger, 1989; Fevolden et al., 1991). The 1 mg 1-l metomidate treatment group also showed a rise in cortisol level, albeit not as large as the control group. This indicates a threshold concentration for an effect of the anaesthetic on plasma cortisol levels. The normal response to a low (sedating) level of the anaesthetics MS-222 or quinaldine sulphate is a rise in plasma cortisol level (Strange and S&reck, 1978; Barton and Peter, 1982; Davis et al., 1982; Laidley and Leatherland, 1988; Robertson et al., 1988; Thomas and Robertson, 1991). However, this was not observed with metomidate (Thomas and Robertson, 1991; this study) or etomidate (Davis et al., 1982) and suggests a different mode of action. Metomidate immersion concentrations of 3 mg l- ’ or higher seem to prevent any plasma cortisol rise after moderate handling stress in Atlantic salmon at low temperatures. This raises the question of whether the absence of a cortisol increase in fish exposed to metomidate is due to a block of sensory transmission to the hypothalamus, or to some blockage within the HP1 axis. Thomas and Robertson ( 1991) found that injection of porcine ACTH produced high levels of plasma cortisol in red drum (Sciuenops ocelkztus) anaesthetized with MS-222 and quinaldine sulphate, but not in fish anaesthetized with metomidate. It is impossible to avoid some plasma cortisol increase after saline injection in MS-222-anaesthetized but otherwise untreated fish. In the present work dexamethasone treatment produced an almost complete block of endogenous cortisol secretion after intraperitoneal injection of saline. This was the case for both types of anaesthetic. By contrast, ACTH injection resulted in high levels of plasma cortisol in MS-222~anaesthetized fish, indicating that MS-222 is unable to inhibit the release of cortisol from the interrenal cells after ACTH stimulation. The effects of ACTH in metomidate-anaesthetized fish was different since there was no cortisol increase after ACTH injection in 1 h anaesthetized fish. It is reasonable to conclude that metomidate acts directly on the interrenal cells to prevent release of cortisol. The inhibitory effects of metomidate on cortisol release may be reversible and short-lasting. This could be deduced from the intermediate cortisol levels in ACTHtreated fish exposed to metomidate for 10 min, and then transferred to metomidate-free water before blood sampling. Work on etomidate effects in mammals has shown that the lack of cortisol secretion is due to an inhibition of the synthesis of mitochondrial P,,,-dependent enzymes, particularly 11Phydroxylase and the cholesterol side-chain cleavage enzyme (Vanden Bossche et al., 1984). In fish, the main pathway to synthesis of cortisol is identical to mammals. However, fish also have an alternative pathway via corticosterone (Kime, 1987). The possible end products from 1 lp-hydroxylase inhibition would therefore be 1 1-deoxycortisol or 1 ldeoxycorticosterone. No attempt was made to determine the concentration of these steroids in plasma. The cross-reactivities of our anti-cortisol antiserum to 1 I-deoxycortisol and 1 ldeoxycorticosterone were 4.5 and < 0.02%, respectively (information from the manufacturer); thus, these end products would not have substantially interfered with the cortisol assay. The lactate and haematocrit measurements in the freshwater pat-r were included to indicate possible respiratory and haematologic effects from metomidate anaesthesia. The increases in blood lactate levels in fish exposed to metomidate were not high compared to levels

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measured after heavy physical exercise (above 20 mmol l- ‘; Wendt and Saunders, 1973). The present lactate increase can not be ascribed to increased physical activity since the activity dropped with increasing metomidate concentration. The low levels of plasma lactate observed in our experiment may not be directly indicative of the severity of respiratory distress, as there is probably a low correlation between plasma lactate and intracellular acidosis in fish (Wood et al., 1983). Increases in plasma lactate levels have been observed by several authors during MS-222 anaesthesia (Houston et al., 1971; Soivio et al., 1977) although not consistently (Black and Connor, 1964; Wells et al., 1984). Lactate increases in blood occur when insufficient oxygen is available for aerobic cell metabolism. This could be due to reduced ventilation and circulation, common side-effects of several anaesthetics (Randall et al., 1965; Houston et al., 1971; Iwama et al., 1989). Etomidate decreases the heart rate and the blood pressure in rainbow trout (Fredricks et al., 1993). Increases in haematocrit have been observed during MS-222 (Houston et al., 1971; Reinitz and Rix, 1977; Soivio et al., 1977; Lowe-Jinde and Niimi, 1983) and benzocaine (Soivio et al., 1977) anaesthesia; this is consistent with our findings. The haematocrit increase was possibly due to a swelling of the red blood cells (Nikinmaa, 1986). The stimulus for haematocrit increase could be low oxygen tensions, increased plasma catecholamine concentrations, or both. In summary, metomidate is a potent immersion anaesthetic in Atlantic salmon at low water temperatures. It induces rapid anaesthesia in salmon parr in fresh water, and is even more efficacious for large salmon in sea water. Metomidate is able to prevent cortisol increase after a handling stressor at concentrations of 3 mg 1-l or higher (temperature 57.7”C). The metomidate-induced inhibition of cortisol secretion is probably located at the level of the interrenal cell. Metomidate anaesthesia results in increased blood lactate concentration and haematocrit. One promising application for this anaesthetic in fish would be to induce a”chemica1 interrenalectomy” where metomidate could be used as a tool to separate catecholamine from cortisol effects. The possibilities regarding transportation of sedated fish without plasma cortisol increase should be considered. Metomidate is well suited for stress studies since the blockage of cortisol secretion is rapid and efficient.

Acknowledgements This work was supported by a scholarship from The Norwegian Agricultural Research Council (presently The Research Council of Norway) to Y.A.O., and by an operating grant from The Royal Norwegian Council for Scientific and Industrial Research to I.E.E.

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