Abstract

Forty channel catfish (Ictalurus punctatus; Rafinesque) with a mean weight of 1.5 kg were individually identified, randomly assigned to four treatment groups and acclimated to an environment aimed at being "Stressfree". Tricaine methanesulphonate, metomidate and placebo treated controls were administered in aqueous solution while the flunixin meglumine treated group of fish was injected intraperitoneally. The fish were given the treatment at the start of the experiment and blood samples were taken over a 23 day period. During the first twenty-four hours both metomidate and tricaine methanpsulphonate (although not significantly different to each other during the first twenty-four hours) were significantly different (p ± 0.05) to flunixin and placebo treated controls. After the twenty-four hour period no significant differences were seen between any of the groups. The very low value of serum cortisol obtained for the metomidate treated group is discussed.

Introduction

On commercial fish farms, netting and close confinement of fish are frequently observed procedures which necessarily stress the animals. Factors affected by these procedures are serum glucose and serum cortisol (1,2). Routine collection of blood samples in this laboratory involves seining fish in ponds and holding them in keep nets prior to sampling. Since these procedures would obviously stress the fish, three chemical agents were investigated to observe their ability to modify the adaptive mechanisms of fish to stress. The chemicals tested were tricaine methanesulphonate (MS222, Fort-Dodge, Tows); flunixin meglumine (Banamine; Schering, Jew Jersey) and metomidate (Janssen; Beerse, Belgium). Acclimated fish were given a stressor (taken as the act of administering the chemical agent itself and blood samples were taken over a 23 day period.

Materials and Methods

Forty adult channel catfish were randomly selected from a commercial production pond. Spines from the dorsal and pectoral fins were removed to prevent aggressive behavior of fish causing traumatic damage to other fish in the group. Weights and fork lengths were recorded for each fish. All groups of fish had a mean weight (kg ± S.D.) of 1.543 1 0.32 and mean fork length (cm ± S.D.) of 53 ± 2.7. Each fish was individually identified using two methods. The first method utilized a Panjet dental inoculator (3), (Panjet, R Wright and Sons, Dundee, Scotland). This was filled with a filtered, supersaturated solution of Alcian Blue. Spots were made on specific sites of the ventral abdominal region of the fish; each site corresponding to a particular number. The second method of marking the fish utilized Silver Nitrate pencils; the number of the fish was drawn on the left flank between the dorsal fin and the lateral line. However, after the acclimation period (22 days) no marks made by the silver nitrate pencil could be identified and so recognition of each fish was dependent upon the panjet dye-marks. The fish were randomly assigned 3 to four swimming pools (volume 10,000 1) at a stocking density of 5 kg/m.³ The fish were then left for 22 days to acclimate to their surroundings. They were offered food twice a week until the start of the experiment and were not fed during the sampling period. During the 22 day acclimation period attempts were made to keep all vibrational and visual stimuli to a minimum. Total ammonia nitrogen (mg/l) and unionized ammonia (mg/1 ) were measured in all pools over the experimental period. Since there are no published reports on the use of flunixin in fish and only two reports (4,5) on the use of metomidate in fish, dose response studies for both these agents were undertaken in channel catfish fingerlings. Flunixin was found to be ineffective as a water treatment in fish weighing 50 g even up to 250-mg/1. Intra-peritoneal injection of flunixin at doses from 2-50 mg/kg body weight gave an LD 50 at 24 hours of 13.5 mg/kg body weight. A dose of 2.2 mg.kg was determined to be effective without signs of toxicity. A bioassay of fingerlings weighing approximately 10 g were effectively tranquilized at a dose of 5 mg/1 of metomidate (Francis-Floyd, Pars. Comm.). 60 mg/1 of MS222 was dissolved in 1 L of distilled water. The placebo treated control group was given 1 L of distilled water alone. The dosage of MS222 and metomidate were calculated to cause Stage 1, Plane I anesthesia (5). Metomidate was also dissolved in 1 L distilled water.

Treatments were given at time 0; sampling times were 10 min, 30 min, 1 hr, 3 hr, 8 hr, 12 hr, 18 hr, 1 dy, 3 dy, 9 dy, 15 dy and 23 dy. At time 0, the flunixin group of fish was gently seined to one end of the pool, individually identified, a blood sample taken and injected intraperitoneally with the weight related dose (2.2 mg/kg). The fish were seined to one end of each pool as gently as possible. Every attempt was made to minimize stress. Individual fish were lifted out of the pool, identified, held in a restraining device designed to keep the fish in a dark, quiet environment and 2 milliliters of blood collected from the caudal vein by 4 ml vacutainer tubes, using a 22 gauge x I inch needle. Serum was separated by centrifugation (10 minutes, 1500 x gravity) in a Bectman centrifuge (TJ-6R) and stored at -72°C until analyzed.

Serum cortisol concentrations were determined using a radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, California) according to published methodology (6). Statistical analysis for the experiment utilized the Rammage IT (Brigham Young University) program for a repeated measures study.

Results

During the 45 day observation period, water quality parameters did not vary excessively. Water temperature ranged from 22°C to 27°C, PH from 8.4 to 9.2, total ammonia nitrogen values from 0.4 to 2.2 mg/l and unionized ammonia from 0.087 to 0.583 mg/L. Observations were made on all groups of fish with respect to handling, tranquilization and other external factors.

The control fish remained active at all sampling times. During the first 24 hours of sampling, repeated seining of the fish was accompanied by increasingly severe struggling. As a consequence greater restraint became necessary. By day 3 of the experiment, epidermal pericaudal sloughing was observed in all fish due to firm handling of the caudal peduncle. This effect was most marked in the control group, but also occurred with treated fish.

Tranquilization was observed in the MS222 treated fish between 10 mins and 18 hr after administration. The effect was most marked at 60 min. The fish were easy to restrain but struggled when blood was taken. There was no muscle fasciculation. In the metomidate treated group tranquilization occurred 10 min to 24 hr after administration. These fish showed no reaction to handling for up to 24 hr; there was muscle fasciculation from 10 min to day 3, with a maximum effect at 3 hr. The flunixin treated fish showed no evidence of tranquilization and no muscle fasciculation. They reacted to restraint at each time interval but showed no response to caudal vein puncture.

Mortalities over the whole sampling period were as follows: 2/9 in the MS222 treated group; 3/8 in the flunixin treated group; 0/10 in the mitome treated group and 2/9 in the placebo treated control group.

Mean serum cortisol concentrations (ug/dl) are given In Figure 1. During the first 24 hours of sampling both MS222 and metomidate are significantly lower (p < 0.05) than flunixin and controls. Flunixin and control fish demonstrated no significant differences from each other. Also, metomidate and MS222 demonstrated no significant difference between each other. After 24 hours and for the duration of the experiment all 4 groups were not significantly different from each other.

In the metomidate treated group, mean serum cortisol concentrations decreased from a mean of 1.7 ug/dl to 0.91 ug/dl by 30 minutes and remained very low throughout the experiment.

Extreme traumatic damage (epidermal pericaudal sloughing) may have been a factor in the death of five out of ten control fish and factors other than overdosage of metomidate may also be incriminated in the high mortality rate seen in this group. Although six out of ten fish died In the metomidate treated group, one died when it was inadvertently left in the seine net between sampling times. In retrospect the 5 mg/l dose was probably too high. Our metomidate treated group showed extremely low cortisol values which were significantly different from the MS222 group. However, we still may not say that these values represent normal base-line values for channel catfish. This result could be a function of a true suppression of cortisol production. Alternatively, metomidate may exert some effect in vivo on cortisol such that detection of the hormone of RIA is not possible. It has been shown (11) that etomidate (the ethyl analog of metomidate) selectively suppresses the production of cortisol through inhibition of 11-β hydroxylation of cholesterol. Bels (Pers. Comm.) has stated that metomidate in chickens does not suppress corticosteroid production. Since these animals have a very weak 11-β hydroxylase system (corticosterone, not cortisol, being the major metabolite) this would putatively corroborate the effect of metomidate on the 11-β hydroxylase system.

Flunixin meglumine is a potent, non-narcotic, non-steroidal analgesic with anti-inflammatory and anti-pyretic activity in several species of domestic animals, in the horse flunixin acts as an anti-prostaglandin (via inhibition of the cyclooxygenase system) but possesses no tranquilizing effect. Its use in fish has not been previously reported.

The flunixin treated group of fish had to be handled in order to administer the drug intraperitoneally. Although there was no tranquilization, analgesia appeared to be present since the fish did not respond to the insertion of the needle for caudal venipuncture. However, serum cortisol values were not significantly different from controls. It is tempting to speculate that fish do not need to feel pain or even discomfort to be physiologically stressed. Both the flunixin and the control group appeared to be equally cognizant of all events taking place around them and their physiological stress response was activated accordingly.

This experiment was designed to demonstrate the efficacy of MS222, metomidate and flunixin in prevention of stress. It appears that analgesia alone is insufficient to prevent activation of the stress response. Tranquilization, even without analgesia, is much more beneficial. MS222 was shown to be effective in preventing stress at the dose given as measured by a reduction of cortisol. Metomidate is worthy of further investigation as a potentially useful agent to prevent a response to stress in channel catfish.

Figure

References

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