- Email: [email protected]
Primary and secondary stress responses in golden perch, Macquaria ambigua
Camp. Biochem. Physiol. Vol. 107A. No. I, pp. 49-56, 1994 Printed in Great Britain
0
0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
Primary and secondary stress responses in golden perch, Macquaria ambigua John F. Carragher
and Christine M. Rees
School of Aquatic Science and Natural Resources Management, Geelong, Victoria 3217 Australia
Deakin University,
Golden perch (Mucquuriu umbiguu), a species of Australian freshwater fish, were subjected to a number of simple stress procedures. Bloodsamples were taken and levels of commonly measured primary and secondary stress response parameters (cortisol, glucose and lactate) were determined. Anaesthesia and exertion of fish prior to bloodsampling affected resting levels of some of the parameters measured. Wild and aquarium-acclimated golden perch had low plasma cortisol levels ( < 2 ng/ml). Most fish appeared to adapt well to aquarium conditions, although occasional fish showed indications of being chronically stressed. Golden perch responded quickly to stress (< 5 min), with increased plasma levels of cortisol and lactate. In contrast, glucose levels did not increase until at least 10 min after the stress was initiated; by 30 min, however, the typical hyperglycaemic response was observed. Golden perch recover rapidly from acute stress (~2.5 hr). Golden perch seem to acclimate quickly to conditions of chronic stress. Key words: Golden perch; Macquaria ambigua; Stress response; Anaesthesia; Cortisol; Lactate; Glucose levels. Comp. Biochem. Physiol. 107A, 4%54, 1994.
Introduction Many biochemical and physiological pathways are involved in the response of fish to stress. For convenience they have been classified as secondary, or tertiary, depending primary, upon when they become evident and the mechanism involved (Mazeaud et al., 1977; Wedemeyer and McLeay, 1981). Primary responses include the release of catecholamines and corticosteroids (adrenaline and cortisol, respectively); these hormones affect tissues and thereby produce secondary effects (e.g. hyperglycaemia, hyperlactacemia and leucopenia; Mazeaud and Mazeaud, 1981; Pickering et al., 1982). Tertiary responses encompass the physiological effects of the secondary responses, and include decreased growth rate, reduced reproductive capacity and increased susceptibility to disease (Pickering and Pottinger, 1989; Pickering, 1990; Campbell et al., 1992). Most studies of stress in fish have considered only the
primary and secondary responses, with plasma cortisol, glucose and lactate levels being the parameters usually measured to describe the response. Generally, studies have examined the response of fish to stressors typical of aquacultural practice, e.g. confinement, netting, crowding, transportation, anaesthesia and poor water quality, in order to reduce the stress imposed by these procedures and, thus, improve production (Strange et al., 1977; Strange and Schreck, 1978; Barton and Peter, 1982; Carmichael et al., 1984; Barton et al., 1986; Barton and Schreck, 1987a; Robertson et al., 1988; Pickering, 1992). Particular aspects of the response can be useful for inter- and intraspecific comparisons of the ability of a fish to cope with stressors; examples of such characteristics include basal and maximum levels of the various parameters, rapidity of onset and duration of response. Most studies have been carried out on species important in aquaculture (especially salmonids). but with the recent worldwide trend away from the culture of introduced species, many other
J. F. Carragher, Animal Behaviour and Welfare Research Centre, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, New Zealand. Received 9 March 1993; accepted 14 April 1993. Correspondence to:
49
50
John F. Carragher and Christine M. Rees
species are undergoing trials to assess their suitability for aquaculture (Pillay, 1992). The golden perch (Macquaria ambigua; Percichthyidae) is one such candidate. The species is native to the large freshwater systems of central and south-eastern Australia, and wild populations support a modest fishery which is mostly recreational in nature (Battaglene and Prokop, 1987). Golden perch are currently propagated commercially, the fingerlings being sold for growout and fishout. The demand for golden perch as food and sportfish in Australia is considered likely to increase in the foreseeable future. In addition, fry have been exported to a number of other countries for assessment of aquaculture potential (O’Sullivan, 1991). Only one published study has examined any aspect of the golden perch stress response. Braley and Anderson (1992) investigated the effects of repeated bloodsampling on levels of blood metabolites. Blood glucose and amino acid levels were elevated in stressed fish during the 96-hr experimental period, whereas free fatty acid levels decreased. No change in blood lactate level was found. Thus, stress was shown to influence golden perch metabolism in a fashion that could affect growth. However, for two reasons it is difficult to compare the published golden perch stress response data with other species; firstly, plasma cortisol levels (the most commonly used indicator of stress) were not measured, and secondly, the stress procedure used was complex (Braley and Anderson, 1992). The present study was undertaken to further investigate the golden perch stress response. A number of experiments were performed in order to characterize different aspects of the golden perch stress response and assess the ability of this species to tolerate stressors.
Materials and Methods Aquarium experiments Eighteen golden perch (250-800 g) that had been caught from the wild at least 1 year before experiments began, were kept individually in 70 1 tanks supplied with a constant flow (approximately 1 l/min) recirculated freshwater. The water temperature was maintained at 20°C and a 12:12 hr 1ight:dark cycle was employed throughout. The fish were fed every other day with frozen prawns or pieces of ox liver. As the aquarium room was also used by other workers, the fish employed in this series of experiments were kept on the top row of the racking system (2.5 m above the ground), to reduce disturbance. The same fish were reused throughout the study and a minimum of 3 weeks was allowed for fish to recover between experiments.
Stressors Two types of stressor were employed in this study. The first was termed “netting and confinement” stress. This entailed the fish being netted from their home tank and placed in individual 75 1 plastic dustbins containing 7 1 of water. This filled the bin to 6cm, a depth which did not allow the fish to remain upright. Thereafter the fish were otherwise undisturbed until required for bloodsampling. The second type of stressor also utilized a low water level, but unlike previously, each fish remained in its home tank. The water level was lowered by adjusting the outlet pipe on the side of the tank such that 6 cm of water remained, a process that took approximately 10 min. The supply of fresh water to the tank was maintained throughout the stress period. The fish were not otherwise disturbed by the experimenter until bloodsampling. This was termed “shallow water” stress. Bloodsampling Bloodsamples were usually taken after fish had been anaesthetized with benzocaine (0.3 g/l; Sigma). In one experiment, however, the effect of anaesthesia on blood metabolite levels was assessed. Non-anaesthetized fish were rolled up in the capture net and wedged into a slit cut into a dense foam block which had been thoroughly wetted. In all cases blood (1.5 ml) was withdrawn from the caudal sinus, clotting was prevented by fluoride heparin and samples were kept on ice until centrifugation. Plasma was separated and frozen prior to assay. A number of experiments were performed during the present study, each designed to investigate a specific aspect of the golden perch stress response. The questions posed are presented as four experiments. Experiment 1. The eflect of anaesthesia on resting blood metabolite levels. All 18 aquarium acclimated fish were bloodsampled without being anaesthetized. It took less than 2 min from first disturbance to obtain the sample from each fish. The procedure was repeated several weeks later but this time each fish was anaesthetized before blood was taken. As a consequence, it took at least 4 min from first disturbance to sample each fish. Experiment 2. The time of onset of the stress response. Fish stressed by netting and confinement were bloodsampled at different times after first disturbance. Four separate experiments were performed. In one experiment fish were sampled at 0, 5 or 10 min; in another the sample times were 0, 10 and 30 min. Thirty minute samples were also taken in a further two experiments. Only data from the two former experiments will be presented here as it was found
Stress responses in
that results were consistent between replicate experiments. Experiment 3. Recovery from acute stress. Six fish were bloodsampled before any stress procedure began. The remaining 12 fish were subjected to shallow water stress. After 30 min of stress six fish were bloodsampled. The remaining six fish had their tanks refilled by readjusting the outlet pipes to their normal positions. Two and a half hr later these fish were bloodsampled (i.e. 3 hr after the stress was initiated). Experiment 4. Short term chronic stress. Beginning at 9 am, six golden perch were subjected to chronic shallow water stress. At noon a second group of six fish were subjected to the same chronic stress. At 3 pm the two groups of stressed fish and the six remaining unstressed animals were bloodsampled. Angling Eighteen golden perch (lo&350 g) were caught by rod and line from a backwater of the Murray river, near Mildura, Victoria. The fish were caught during daylight hours over 3 days in September 1992. The fish took less than 1 min to land whereupon they were immediately bloodsampled. Fluoride heparin-treated blood was kept on ice it could be separated and the plasma frozen.
51
golden perch
did not differ between the two groups. In contrast, plasma lactate levels were 3-fold higher in anaesthetized fish (Fig. 1). To investigate whether aquarium-acclimated golden perch were representative of the species (being either chronically stressed or, conversely, too “relaxed”), bloodsamples were taken from wild golden perch caught by angling. The individual plasma cortisol levels in 10 of the 18 fish caught by angling were below the detection limit of the assay (0.4 ng/ml; for the purposes of statistical analysis they were each assigned a value of 0.2 ng/ml). The mean cortisol level of all of angled fish was 0.9 ng/ml, a value much lower than aquarium fish sampled by either of the other methods (Fig. 1). Plasma glucose and lactate levels in wild fish were significantly lower and higher, respectively, than in aquarium fish (Fig. 1). Experiment 2 Plasma cortisol levels rose quickly once golden perch were stressed. Five minutes after a netting and confinement stress was initiated the mean cortisol value had doubled to
Assays Plasma cortisol levels were determined by radioimmunoassay as described in Pankhurst et al. (1992). The interassay variation was 9% (n = 7), and the detection limit was 0.4 ng/ml. Plasma glucose and lactate were measured using calorimetric, enzymatic kits from Boehringer Mannheim and Sigma, respectively. Both manufacturers’ recommended protocols were modified to allow use of an ELISA plate reader. Statistics Data were analysed by one way analysis of variance and Tukey’s Honestly Significant Difference Test using the SYSTAT package. The means were considered to be significantly different if P < 0.05. The Figures show mean values + SEM.
Results Experiment 1 Aquarium-acclimated golden perch bloodsampled as quickly as possible after capture without anaesthesia had a mean plasma cortisol level of 4.1 ng/ml, anaesthetized fish had a mean value of 6.5 ng/ml (Fig. 1). Plasma glucose levels
1
2
3
SamplingMethod Fig. 1. The effect of sampling method on plasma cortisol. glucose and lactate levels in unstressed golden perch. The sampling methods are (I) nonanaesthetized aquarium-acclimated fish, (2) anaesthetized aquarium-acclimated fish, and (3) nonanaesthetized wild fish caught by angling. Mean f SEM (N = I8 fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
52
John F. Carragher and Christine M. Rees
-b
but decreasing to the initial value after 2.5 hr recovery (Fig. 3). Experiment 4 The mean blood cortisol level of golden perch was 57 ng/ml after 3 hr of chronic shallow water stress (Fig. 4). After a further 3 hr of stress the cortisol level had decreased to 28 ng/ml. Plasma glucose levels were significantly elevated after 3 and 6 hr of low water stress, the mean level at 6 hr was lower than at 3 hr, but the difference was not statistically significant. Plasma lactate levels did not differ significantly in this experiment, however the mean level at 3 hr was greater, and showed more variation, than at other sampling times (Fig. 4).
Discussion The present study examined various aspects of the golden perch stress response. The stressors used in the study were chosen for two reasons, first, they were simple, reproducible procedures, and second, they are relevant to aquacultural practice. These few simple experFig. 2. The effect of netting and confinement stress on plasma cortisol, glucose and lactate levels in golden perch. Fish were sampled at 0, 5, 10 or 30 min after the stress was initiated. Mean f SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
approximately 10 ng/ml and after a further 5 min it had reached 60 ng/ml (Fig. 2). The maximal mean cortisol level (240 ng/ml) was measured after 30 min of stress. Plasma glucose levels did not change significantly, although mean values decreased, in the first 10 min after the stress procedure was initiated. After 30 min of stress, however, stressed fish had significantly higher glucose levels than unstressed fish (Fig. 2). Plasma lactate levels were elevated after 5 min of netting and confinement stress, thereafter the levels continued to increase (Fig. 2). Experiment 3 Thirty minutes of shallow water stress increased the mean plasma cortisol level of golden perch to over, 90 ng/ml. In the group of fish in which the water level was subsequently normalized, the mean cortisol level 2.5 hr later was not significantly different to that found in unstressed fish (13 ng/ml c.f. 9.3 ng/ml; Fig. 3). Plasma glucose levels were not found to differ during this experiment. Plasma lactate levels followed a similar pattern to that of cortisol, being elevated after 30 min of stress
Fig. 3. Plasma cortisol. glucose and lactate levels in golden perch after 150 min recovery from 30 min of shallow water stress. Time is shown as min after the stress was initiated. Mean f SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
Stress responses in golden perch b -
0
3
6
w.wn Fig. 4. Plasma cortisol, glucose and lactate levels in golden perch after 3 and 6 hr of continuous shallow water stress. Mean + SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
iments have addressed several fundamental questions concerning the ability of golden perch to tolerate stressful conditions. These data may be useful in helping to decide whether this species is a suitable candidate for future intensive culture. It is difficult to obtain reliable resting values of stress-related parameters (e.g. plasma cortisol, glucose and lactate levels) in any species of animal. These parameters can be affected by pre-capture events (chasing and/or confinement), or the capture event itself (restraint and/or handling) (discussed in Pankhurst and Sharples, 1992). Golden perch are no exception. The lowest mean plasma cortisol level (the parameter most widely used as an indicator of stress) was found in fish caught from the wild by rod and line; which tends to suggest that these fish were least stressed. However, these fish had the lowest glucose and highest lactate levels of unstressed animals sampled by the three different methods. These differences suggest that either the diet and/or environment of wild fish affected basal plasma glucose and lactate levels (as has been reported, Driedzic and Hochachka, 1978), or alternatively, that the wild fish CBPA 10711-E
53
struggled, allbeit briefly, during capture and thus affected levels of these parameters. Aquarium-acclimated golden perch bloodsampled without anaesthesia had the highest plasma glucose and lowest plasma lactate levels of the three methods examined. We believe this reflects the fact that struggling, and hence anaerobic metabolism, was minimised by the immobilization technique used to obtain the bloodsample. In contrast, the mean cortisol level of this group was higher and more variable than expected. This happened because two of the fish had high ( > 10 ng/ml) cortisol levels, whereas most of the remainder had low levels ( < 2 ng/ml). These two fish also had high glucose and lactate levels, suggesting that they were chronically stressed before the experiment began (see below). Occasionally “unstressed” fish in other experiments were found to have similarly high levels of the three parameters (e.g. experiment 3). The reason why these fish were stressed is not known. The anaesthetized group of fish also included such an “outlier” (29 ng/ml); apart from that the range of individual cortisol values was from 1-13 ng/ml, with an overall mean of 6.5 ng/ml. The mean plasma glucose level was also similar to that in unanaesthetized aquarium fish. However, the plasma lactate level was significantly higher in anaesthetized fish. This finding is believed to reflect the extra time (2-3 min) taken to sample these fish and the physical exertions of the fish in the anaesthetic solution. Thus, aquarium-acclimated golden perch can be sampled quickly such that resting values of stress-reactive parameters are not affected. Anaesthesia, however, delays bloodsampling and, as a consequence, changes in plasma lactate levels can become apparent. In addition, it appears that occasional fish are chronically stressed by some aspect(s) of the holding conditions, and may confuse data gathered from experiments which are limited by fish numbers. The low levels of cortisol measured in wild and most tank-acclimated fish are in agreement with a number of studies which have suggested that unstressed fish generally have less than 5 ng cortisol/ml (Robertson et al., 1988; Pickering and Pottinger, 1989; Pankhurst et al., 1992). Golden perch respond quickly to stress. Measurable changes in plasma cortisol, glucose and lactate levels were detected only 5 min after initiation of the stress, however statistical analysis indicated these changes were significant only after 30 min. The conservative nature of the statistically significant differences indicated (e.g. the cortisol level after 10 min was IO-fold higher than that in unstressed animals, but statistically the two are not different) is due to the limited number of fish sampled at each timepoint.
54
John F. Carragher and Christine M. Rees
Plasma cortisol levels increased throughout the 30 min confinement period, however, from the data it is not possible to indicate if the response peaks after 30 min, or at some later time. The maximal mean cortisol level found during this study (approximately 240 ng/ml) was similar to that found for other species of fish subjected to similar types and durations of stressors [several species (180-340 ng/ml), Davis and Parker, 1986; ted drum (250 ng/ml), Robertson et al., 1988; striped bass (> 300 ng/ ml), Davis and Parker, 1990; blue mao mao (250 ng/ml), Pankhurst et al., 19921, but higher than found in other species [brown trout (100-l 50 ng/ml) and rainbow trout (40-80 ng/ ml), Pickering and Pottinger, 1989; snapper (20 ng/ml), Pankhurst and Sharples, 19921. In the present study, plasma glucose levels decreased slightly, but not significantly, soon after the stress was initiated. This condition lasted for at least 10 min. The authors are not aware of any other published study in which this pattern has been observed, although few studies have looked at such short term changes. It is suggested that this initial decrease may be due to the increased utilization of circulating glucose as a substrate for metabolism and energy production. After 30 min of stress the more usual hyperglycaemic response was observed in golden perch, suggesting that glucose was being regenerated from lactate or liberated from body stores (glycogen, lipid or protein) to maintain the high metabolic readiness of the animal (Barton and Schreck, 1987b). In common with other studies the increase in plasma glucose level after 30 min of stress was not maximal. Plasma lactate levels were found to increase quickly and remain high during the 30 min of netting and confinement stress. Golden perch were surprisingly placid during the stress procedures, only exerting themselves occasionally and briefly. In contrast, rainbow trout confined in the same way are more agitated (Carragher, personal observation). Given this reduced response it is surprising that plasma lactate levels increased as they did; it suggests that golden perch have a low capacity for aerobic metabolism. The increase in lactate level in golden perch in these experiments was similar to those observed in some other studies (Zfold, Barton and Schreck, 1987a; 3-fold, Waring et al., 1992), but less than in others (dfold, Pickering et al., 1982). The severity of stressor does influence the magnitude of the response obtained (Driedzic and Hochachka, 1978). Thirty minutes of forced swimming, netting and confinement stress increased golden perch lactate levels approximately lo-fold (Carragher, unpublished results). Two and a half hours after a 30 min shallow water stress plasma cortisol levels had returned
to the level measured in control fish. Similar experiments on other species usually show longer recovery times. One hour of handling and confinement elevated plasma cortisol levels for at least 8 and 4 hr in brown and rainbow trout, respectively (Pickering and Pottinger, 1989). Plasma cortisol levels in Atlantic salmon and flounder confined underwater in a net for 9 min did not decrease to the level found in resting fish until at least 8 and 24 hr post-stress, respectively (Waring et al., 1992). Striped bass acclimated to 16 and 21 “C recovered (in terms of their plasma cortisol level) from a 12 min net confinement stress in less than 6 hr; at higher and lower acclimation temperatures the recovery was slower (Davis and Parker, 1990). Thus, golden perch appear to have the ability to recover quickly from acute stress. The 90 ng cortisol/ml measured after 30 min of shallow water stress in experiment 3 suggests that this stressor was less severe than the netting and confinement procedure used in experiment 2 which resulted in 240 ng/ml indicating that the magnitude of the golden perch stress response is related to the severity of the stressor. Similar graduated response to different severity stressors have been reported previously (Strange et al., 1977, 1978; Barton et al., 1980; Robertson et al., 1988; Pankhurst and Sharples, 1992). We may be able to take advantage of the fishes’ ability to discriminate between stressor severity to adapt and improve handling procedures. By assessing the physiological reaction of the fish to a stressor we avoid anthropomorphic judgements about perceived severity. Plasma glucose levels were not affected by 30 min of shallow water stress. No other published studies have documented such a response. The usual effect of acute stress is to cause an increase in plasma glucose level which continues for at least as long as elevated cortisol levels persist (e.g. Pickering et al., 1982; Barton et al., 1986; Davis and Parker, 1990; Waring et al., 1992). This was not the case in this experiment. Possibly the milder nature of this procedure, compared to the handling and confinement stress which did cause an increase, failed to affect glucose metabolism; or perhaps the 30 min bloodsample was taken when the glucose level was increasing after an initial slight decrease (as was found in experiment 1). Whatever the explanation, it is clear that 2.5 hr after a 30 min stress plasma glucose levels are the same as in unstressed fish. Thirty minutes of shallow water stress did, increase plasma lactate levels, however, suggesting that an appreciable amount of anaerobic metabolism was undertaken. Given this, it is even more surprising that plasma glucose levels did not change. This finding suggests that
Stress responses in golden perch
the lactate may have been converted back to glucose (gluconeogenesis) in the muscles where it was then used, and thus reduced the need for glucose to be mobilized from body stores. After 2.5 hr of recovery plasma lactate levels had returned to basal. Similar studies suggest that after acute stress plasma lactate levels return to prestress values soon after cortisol levels are normalized (Pickering et al., 1982; Barton et al., 1986). Thirty minutes of chronic shallow water stress produced a mean cortisol level of over 90 ng/ml (experiment 3; Fig. 3), after 3 hr it was 57 ng/ml (experiment 4; Fig. 4). The level decreased further to 28 ng/ml after 6 hr. This suggests that the fish were acclimating to the chronic stressor. It is not possible to extrapolate these data to predict when the cortisol level would return to that found in unstressed fish. Published studies in which similar procedures have been carried out suggest that this is a typical acclimation response. Robertson et al. (1988) found that cortisol levels in red drum returned to prestress values by the end of a 5.5 hr transportation stress. Barton and Peter (1982) found that rainbow trout showed signs of acclimating to transport stress 48 hr into the procedure. In contrast, rainbow trout kept in confined and crowded tanks took 6-10 days to acclimate; whilst brown trout took 3C40 days (Pickering and Pottinger, 1989). Plasma glucose levels were elevated compared to control fish after 3 and 6 hr of chronic stress. This is similar to the results of chinook salmon (Barton and Schreck, 1987a) and red drum (Robertson et al., 1988). This hyperglycaemia was probably maintained by the elevated plasma cortisol level continuing to stimulate glycogenolysis or gluconeogenesis. Plasma lactate levels did not differ significantly in this experiment, although according to the results of experiment 3, plasma lactate levels were elevated early in the stress period. The decrease which occurred during the stress may have been a consequence of reduced physical activity (and hence anaerobic metabolism), although fish were heard to be occasionally active throughout the 6 hr of stress. Alternatively, the rate of gluconeogenesis increased during the stress and reduced circulating lactate levels. Braley and Anderson (1992), in the only other published study dealing with the golden perch stress response, observed a maximal 4.5-fold increase in blood glucose, and the hyperglycaemic state persisted for at least 96 hr. In contrast, the maximal response obtained in the present study was only 2-fold and the level appeared to be decreasing after 6 hr of chronic stress. These differences are likely to reflect the complex nature of the stress applied in the
55
Braley and Anderson study (multiple handling, anaesthesia and bloodsampling), compared to the simple stressors used in the present study. Plasma lactate was the other parameter measured in both studies, this was not found to change after multiple bloodsampling (Braley and Anderson, 1992). They reported a mean lactate level of 1.9 mM, a value similar to that measured in anaesthetized unstressed fish in the present study. In the present study plasma lactate level was affected mainly by the method of bloodsampling (i.e. anaesthetized versus nonanaesthetized), and by short periods of stress (~3 hr; Fig. 4). Since the minimum sampling frequency in the Braley and Anderson (1992) study was 3 hr, it is likely that perturbations in lactate levels caused by handling and bloodsampling were completed before the subsequent sample was taken (unlike the compounded response of plasma glucose levels). Thus, golden perch can acclimate to aquarium conditions, they respond quickly to stressors with maximal cortisol levels similar to those reported for other species, they recover rapidly from acute stress and appear to acclimate quickly to chronic stress. These characteristics suggest that the golden perch is a species capable of tolerating stressors found in intensive aquaculture practice. Acknowledgements-The contributions of the following individuals and groups in the course of this study are gratefully acknowledged: Dr Ian MacDonald (Zoology Department, Melbourne University) for cortisol label, Dr Tom Watson (Deakin University) for cortisol standard. Dr Trevor Anderson (Deakin University) for comments on the manuscript, Hal Braley and Karen Drieberg for their angling skills, the Native Finfish group at Snobs Creek Research Station and Hatchery (Department of Conservation and Environment, Victoria) for access to golden perch, and The William Anglis (Victoria) Charitable Fund for financial support.
References Barton B. A. and Peter R. E. (1982) Plasma cortisol stress response in fingerling rainbow trout, Sulmo gairdneri Richardson, to various transport conditions, anaesthesia and cold shock. J. Fish Biol. 20, 39-51. Barton B. A. and Schreck C. B. (1987a) Influence of acclimation temperature on interrenal and carbohydrate stress responses in juvenile chinook salmon (Oncorhynchus
tshuwystcha).
Aquaculture
62, 299-310.
Barton B. A. and Schreck C. B. (1987b) Metabolic cost of acute physical stress in juvenile steelhead. Trans. Am. Fish. Sot. 116, 257-263.
Barton B. A., Peter R. E. and Paulencu C. R. (1980) Plasma cortisol levels of fingerling rainbow trout (Salmo guirdneri) at rest, and subjected to handling confinement, transport, and stocking. Can. J. Fish. Aquat. Sci. 37, 805-811.
Barton B. A., Schreck C. B. and Sigismondi L. A. (1986) Multiple acute disturbances evoke cumulative physiological stress responses in juvenile chinook salmon. Trans. Am. Fish. Sot. 115, 245-251.
56
John F. Carragher and Christine M. Rees
Battaglene S. and Prokop F. (1987) Golden perch. In A&cl A 3.2.2., Department of Agriculture, Sydney, New South Wales. Braley H. and Anderson T. A. (1992) Changes in blood metabolite concentrations in response to repeated capture, anaesthesia and blood sampling in the golden perch, Macquaria ambigua. Comp. Biochem. Physiol 103A, 445-450. Campbell P. M., Pottinger T. G. and Sumpter J. P. (1992) Stress reduces the quality of gametes produced by rainbow trout. Biol. Reprod. 47, 114&1150. Carmichael G. J., Tomasso J. R., Simco B. A. and Davis K. B. (1984) Characterization and alleviation of stress associated with hauling largemouth bass. Trans. Am. Fish. Sot. 113, 778-785.
Davis K. B. and Parker N. C. (1986) Plasma corticosteroid stress response of fourteen species of warmwater fish to transportation. Trans. Am. Fish. Sot. 115, 495499. Davis K. B. and Parker N. C. (1990) Physiological stress in striped bass: effect of acclimation temperature. Aquaculture 91, 349-358. Dried& W. R. and Hochachka P. W. (1978) Metabolism in fish during exercise. In FLsh Phvsiologv Vol. 7 (Edited bv Hoar W. S. and Randall D. J.), ~~-503-543. ‘Academic Press, New York. Mazeaud M. M. and Maxeaud F. (1981) Adrenergic responses to stress in fish. In Stress in Fish (Edited by Pickering A. D.), pp. 49-75. Academic Press, London. Mazeaud M. M., Mazeaud F. and Donaldson E. M. (1977) Primary and secondary effects of stress in fish: some new data with a general review. Trans. Am. Fish. Sot. 106, 201-212. O’Sullivan D. (1991) Status of Australian aquaculture in 1989190. AustAsia Aquaculture June, 2-13. Pankhurst N. W. and Sharples D. F. (1992) Effects of capture and confinement on plasma cortisol concentrations in the snapper, Pagrus aurafus. Aust. J. Mar. Freshwater Res. 43, 345-356. Pankhurst N. W., Wells R. M. G. and Carragher J. F. (1992) Effects of stress on plasma cortisol levels and blood viscosity in blue mao mao, Scorpis violaceus (Hutton),
a marine
teleost.
Comp.
Biochem.
Physiol.
lOlA,
335-339.
Pickering A. D. (1990) Stress and the suppression of somatic growth in teleost fish. In Progress in Comparative Endocrinology (Edited by Epple A., Scanes C. G. and Stetson M. H.), pp. 473479. Wiley-Liss, New York. Pickering A. D. (1992) Rainbow trout husbandry: management of the stress response. Aquacufture 100, 125-139. Pickering A. D. and Pottinger T. G. (1989) Stress responses and disease resistance in salmonid fish: effects of chronic elevation of plasma cortisol. Fish Physiof. Biochem. 7, 253-258. Pickering A. D., Pottinger T. G. and Christie P. (1982) Recovery of the brown trout, Salmo trutta L., from acute handling stress: a time course study. J. Fish Biol. 20, 229-244. Pillay T. V. R. (1992) Aquaculture and the Environment. Fishina News Books. Oxford. Roberts& L., ThornasP. and Arnold C. R. (1988) Plasma cortisol and secondary stress responses of cultured red drum (Sciaenops ocellatus) to several transportation procedures. Aquaculture 68, 115-130. Strange R. J. and Schreck C. B. (1978) Anaesthetic and handling stress on survival and cortisol concentration in yearling chinook salmon (Oncorhynchus tshawyfscha). J. Fish. Res. Board Can. 35, 345-349. Strange R. J., Schreck C. B. and Golden J. T. (1977) Corticoid stress responses to handling and temperature in salmonids. Trans. Am. Fish. Sot. 106, 213-217. Strange R. J., Schreck C. B. and Ewing R. D. (1978) Cortisol concentrations in confined juvenile chinook salmon (Oncorhynchus tshawystcha) Trans. Am. Fish. Sot. 107, 812-819. Waring C. P., Stagg R. M. and Poxton M. G. (1992) The effects of handling on flounder (PlatichthysJlesus L.) and Atlantic salmon (Salmo salar L.). J. Fish Biol. 41, 131-144. Wedemeyer G. A. and McLeay D. J. (1981) Methods for determining the tolerance of fishes to environmental stressors. In Stress in Fish (Edited by Pickering A. D.), pp. 247-275. Academic Press, London.
0
0300-9629/94 $6.00 + 0.00 1993 Pergamon Press Ltd
Primary and secondary stress responses in golden perch, Macquaria ambigua John F. Carragher
and Christine M. Rees
School of Aquatic Science and Natural Resources Management, Geelong, Victoria 3217 Australia
Deakin University,
Golden perch (Mucquuriu umbiguu), a species of Australian freshwater fish, were subjected to a number of simple stress procedures. Bloodsamples were taken and levels of commonly measured primary and secondary stress response parameters (cortisol, glucose and lactate) were determined. Anaesthesia and exertion of fish prior to bloodsampling affected resting levels of some of the parameters measured. Wild and aquarium-acclimated golden perch had low plasma cortisol levels ( < 2 ng/ml). Most fish appeared to adapt well to aquarium conditions, although occasional fish showed indications of being chronically stressed. Golden perch responded quickly to stress (< 5 min), with increased plasma levels of cortisol and lactate. In contrast, glucose levels did not increase until at least 10 min after the stress was initiated; by 30 min, however, the typical hyperglycaemic response was observed. Golden perch recover rapidly from acute stress (~2.5 hr). Golden perch seem to acclimate quickly to conditions of chronic stress. Key words: Golden perch; Macquaria ambigua; Stress response; Anaesthesia; Cortisol; Lactate; Glucose levels. Comp. Biochem. Physiol. 107A, 4%54, 1994.
Introduction Many biochemical and physiological pathways are involved in the response of fish to stress. For convenience they have been classified as secondary, or tertiary, depending primary, upon when they become evident and the mechanism involved (Mazeaud et al., 1977; Wedemeyer and McLeay, 1981). Primary responses include the release of catecholamines and corticosteroids (adrenaline and cortisol, respectively); these hormones affect tissues and thereby produce secondary effects (e.g. hyperglycaemia, hyperlactacemia and leucopenia; Mazeaud and Mazeaud, 1981; Pickering et al., 1982). Tertiary responses encompass the physiological effects of the secondary responses, and include decreased growth rate, reduced reproductive capacity and increased susceptibility to disease (Pickering and Pottinger, 1989; Pickering, 1990; Campbell et al., 1992). Most studies of stress in fish have considered only the
primary and secondary responses, with plasma cortisol, glucose and lactate levels being the parameters usually measured to describe the response. Generally, studies have examined the response of fish to stressors typical of aquacultural practice, e.g. confinement, netting, crowding, transportation, anaesthesia and poor water quality, in order to reduce the stress imposed by these procedures and, thus, improve production (Strange et al., 1977; Strange and Schreck, 1978; Barton and Peter, 1982; Carmichael et al., 1984; Barton et al., 1986; Barton and Schreck, 1987a; Robertson et al., 1988; Pickering, 1992). Particular aspects of the response can be useful for inter- and intraspecific comparisons of the ability of a fish to cope with stressors; examples of such characteristics include basal and maximum levels of the various parameters, rapidity of onset and duration of response. Most studies have been carried out on species important in aquaculture (especially salmonids). but with the recent worldwide trend away from the culture of introduced species, many other
J. F. Carragher, Animal Behaviour and Welfare Research Centre, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, New Zealand. Received 9 March 1993; accepted 14 April 1993. Correspondence to:
49
50
John F. Carragher and Christine M. Rees
species are undergoing trials to assess their suitability for aquaculture (Pillay, 1992). The golden perch (Macquaria ambigua; Percichthyidae) is one such candidate. The species is native to the large freshwater systems of central and south-eastern Australia, and wild populations support a modest fishery which is mostly recreational in nature (Battaglene and Prokop, 1987). Golden perch are currently propagated commercially, the fingerlings being sold for growout and fishout. The demand for golden perch as food and sportfish in Australia is considered likely to increase in the foreseeable future. In addition, fry have been exported to a number of other countries for assessment of aquaculture potential (O’Sullivan, 1991). Only one published study has examined any aspect of the golden perch stress response. Braley and Anderson (1992) investigated the effects of repeated bloodsampling on levels of blood metabolites. Blood glucose and amino acid levels were elevated in stressed fish during the 96-hr experimental period, whereas free fatty acid levels decreased. No change in blood lactate level was found. Thus, stress was shown to influence golden perch metabolism in a fashion that could affect growth. However, for two reasons it is difficult to compare the published golden perch stress response data with other species; firstly, plasma cortisol levels (the most commonly used indicator of stress) were not measured, and secondly, the stress procedure used was complex (Braley and Anderson, 1992). The present study was undertaken to further investigate the golden perch stress response. A number of experiments were performed in order to characterize different aspects of the golden perch stress response and assess the ability of this species to tolerate stressors.
Materials and Methods Aquarium experiments Eighteen golden perch (250-800 g) that had been caught from the wild at least 1 year before experiments began, were kept individually in 70 1 tanks supplied with a constant flow (approximately 1 l/min) recirculated freshwater. The water temperature was maintained at 20°C and a 12:12 hr 1ight:dark cycle was employed throughout. The fish were fed every other day with frozen prawns or pieces of ox liver. As the aquarium room was also used by other workers, the fish employed in this series of experiments were kept on the top row of the racking system (2.5 m above the ground), to reduce disturbance. The same fish were reused throughout the study and a minimum of 3 weeks was allowed for fish to recover between experiments.
Stressors Two types of stressor were employed in this study. The first was termed “netting and confinement” stress. This entailed the fish being netted from their home tank and placed in individual 75 1 plastic dustbins containing 7 1 of water. This filled the bin to 6cm, a depth which did not allow the fish to remain upright. Thereafter the fish were otherwise undisturbed until required for bloodsampling. The second type of stressor also utilized a low water level, but unlike previously, each fish remained in its home tank. The water level was lowered by adjusting the outlet pipe on the side of the tank such that 6 cm of water remained, a process that took approximately 10 min. The supply of fresh water to the tank was maintained throughout the stress period. The fish were not otherwise disturbed by the experimenter until bloodsampling. This was termed “shallow water” stress. Bloodsampling Bloodsamples were usually taken after fish had been anaesthetized with benzocaine (0.3 g/l; Sigma). In one experiment, however, the effect of anaesthesia on blood metabolite levels was assessed. Non-anaesthetized fish were rolled up in the capture net and wedged into a slit cut into a dense foam block which had been thoroughly wetted. In all cases blood (1.5 ml) was withdrawn from the caudal sinus, clotting was prevented by fluoride heparin and samples were kept on ice until centrifugation. Plasma was separated and frozen prior to assay. A number of experiments were performed during the present study, each designed to investigate a specific aspect of the golden perch stress response. The questions posed are presented as four experiments. Experiment 1. The eflect of anaesthesia on resting blood metabolite levels. All 18 aquarium acclimated fish were bloodsampled without being anaesthetized. It took less than 2 min from first disturbance to obtain the sample from each fish. The procedure was repeated several weeks later but this time each fish was anaesthetized before blood was taken. As a consequence, it took at least 4 min from first disturbance to sample each fish. Experiment 2. The time of onset of the stress response. Fish stressed by netting and confinement were bloodsampled at different times after first disturbance. Four separate experiments were performed. In one experiment fish were sampled at 0, 5 or 10 min; in another the sample times were 0, 10 and 30 min. Thirty minute samples were also taken in a further two experiments. Only data from the two former experiments will be presented here as it was found
Stress responses in
that results were consistent between replicate experiments. Experiment 3. Recovery from acute stress. Six fish were bloodsampled before any stress procedure began. The remaining 12 fish were subjected to shallow water stress. After 30 min of stress six fish were bloodsampled. The remaining six fish had their tanks refilled by readjusting the outlet pipes to their normal positions. Two and a half hr later these fish were bloodsampled (i.e. 3 hr after the stress was initiated). Experiment 4. Short term chronic stress. Beginning at 9 am, six golden perch were subjected to chronic shallow water stress. At noon a second group of six fish were subjected to the same chronic stress. At 3 pm the two groups of stressed fish and the six remaining unstressed animals were bloodsampled. Angling Eighteen golden perch (lo&350 g) were caught by rod and line from a backwater of the Murray river, near Mildura, Victoria. The fish were caught during daylight hours over 3 days in September 1992. The fish took less than 1 min to land whereupon they were immediately bloodsampled. Fluoride heparin-treated blood was kept on ice it could be separated and the plasma frozen.
51
golden perch
did not differ between the two groups. In contrast, plasma lactate levels were 3-fold higher in anaesthetized fish (Fig. 1). To investigate whether aquarium-acclimated golden perch were representative of the species (being either chronically stressed or, conversely, too “relaxed”), bloodsamples were taken from wild golden perch caught by angling. The individual plasma cortisol levels in 10 of the 18 fish caught by angling were below the detection limit of the assay (0.4 ng/ml; for the purposes of statistical analysis they were each assigned a value of 0.2 ng/ml). The mean cortisol level of all of angled fish was 0.9 ng/ml, a value much lower than aquarium fish sampled by either of the other methods (Fig. 1). Plasma glucose and lactate levels in wild fish were significantly lower and higher, respectively, than in aquarium fish (Fig. 1). Experiment 2 Plasma cortisol levels rose quickly once golden perch were stressed. Five minutes after a netting and confinement stress was initiated the mean cortisol value had doubled to
Assays Plasma cortisol levels were determined by radioimmunoassay as described in Pankhurst et al. (1992). The interassay variation was 9% (n = 7), and the detection limit was 0.4 ng/ml. Plasma glucose and lactate were measured using calorimetric, enzymatic kits from Boehringer Mannheim and Sigma, respectively. Both manufacturers’ recommended protocols were modified to allow use of an ELISA plate reader. Statistics Data were analysed by one way analysis of variance and Tukey’s Honestly Significant Difference Test using the SYSTAT package. The means were considered to be significantly different if P < 0.05. The Figures show mean values + SEM.
Results Experiment 1 Aquarium-acclimated golden perch bloodsampled as quickly as possible after capture without anaesthesia had a mean plasma cortisol level of 4.1 ng/ml, anaesthetized fish had a mean value of 6.5 ng/ml (Fig. 1). Plasma glucose levels
1
2
3
SamplingMethod Fig. 1. The effect of sampling method on plasma cortisol. glucose and lactate levels in unstressed golden perch. The sampling methods are (I) nonanaesthetized aquarium-acclimated fish, (2) anaesthetized aquarium-acclimated fish, and (3) nonanaesthetized wild fish caught by angling. Mean f SEM (N = I8 fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
52
John F. Carragher and Christine M. Rees
-b
but decreasing to the initial value after 2.5 hr recovery (Fig. 3). Experiment 4 The mean blood cortisol level of golden perch was 57 ng/ml after 3 hr of chronic shallow water stress (Fig. 4). After a further 3 hr of stress the cortisol level had decreased to 28 ng/ml. Plasma glucose levels were significantly elevated after 3 and 6 hr of low water stress, the mean level at 6 hr was lower than at 3 hr, but the difference was not statistically significant. Plasma lactate levels did not differ significantly in this experiment, however the mean level at 3 hr was greater, and showed more variation, than at other sampling times (Fig. 4).
Discussion The present study examined various aspects of the golden perch stress response. The stressors used in the study were chosen for two reasons, first, they were simple, reproducible procedures, and second, they are relevant to aquacultural practice. These few simple experFig. 2. The effect of netting and confinement stress on plasma cortisol, glucose and lactate levels in golden perch. Fish were sampled at 0, 5, 10 or 30 min after the stress was initiated. Mean f SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
approximately 10 ng/ml and after a further 5 min it had reached 60 ng/ml (Fig. 2). The maximal mean cortisol level (240 ng/ml) was measured after 30 min of stress. Plasma glucose levels did not change significantly, although mean values decreased, in the first 10 min after the stress procedure was initiated. After 30 min of stress, however, stressed fish had significantly higher glucose levels than unstressed fish (Fig. 2). Plasma lactate levels were elevated after 5 min of netting and confinement stress, thereafter the levels continued to increase (Fig. 2). Experiment 3 Thirty minutes of shallow water stress increased the mean plasma cortisol level of golden perch to over, 90 ng/ml. In the group of fish in which the water level was subsequently normalized, the mean cortisol level 2.5 hr later was not significantly different to that found in unstressed fish (13 ng/ml c.f. 9.3 ng/ml; Fig. 3). Plasma glucose levels were not found to differ during this experiment. Plasma lactate levels followed a similar pattern to that of cortisol, being elevated after 30 min of stress
Fig. 3. Plasma cortisol. glucose and lactate levels in golden perch after 150 min recovery from 30 min of shallow water stress. Time is shown as min after the stress was initiated. Mean f SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
Stress responses in golden perch b -
0
3
6
w.wn Fig. 4. Plasma cortisol, glucose and lactate levels in golden perch after 3 and 6 hr of continuous shallow water stress. Mean + SEM (N = six fish in each case). Columns with different letters are significantly different from each other (P < 0.05).
iments have addressed several fundamental questions concerning the ability of golden perch to tolerate stressful conditions. These data may be useful in helping to decide whether this species is a suitable candidate for future intensive culture. It is difficult to obtain reliable resting values of stress-related parameters (e.g. plasma cortisol, glucose and lactate levels) in any species of animal. These parameters can be affected by pre-capture events (chasing and/or confinement), or the capture event itself (restraint and/or handling) (discussed in Pankhurst and Sharples, 1992). Golden perch are no exception. The lowest mean plasma cortisol level (the parameter most widely used as an indicator of stress) was found in fish caught from the wild by rod and line; which tends to suggest that these fish were least stressed. However, these fish had the lowest glucose and highest lactate levels of unstressed animals sampled by the three different methods. These differences suggest that either the diet and/or environment of wild fish affected basal plasma glucose and lactate levels (as has been reported, Driedzic and Hochachka, 1978), or alternatively, that the wild fish CBPA 10711-E
53
struggled, allbeit briefly, during capture and thus affected levels of these parameters. Aquarium-acclimated golden perch bloodsampled without anaesthesia had the highest plasma glucose and lowest plasma lactate levels of the three methods examined. We believe this reflects the fact that struggling, and hence anaerobic metabolism, was minimised by the immobilization technique used to obtain the bloodsample. In contrast, the mean cortisol level of this group was higher and more variable than expected. This happened because two of the fish had high ( > 10 ng/ml) cortisol levels, whereas most of the remainder had low levels ( < 2 ng/ml). These two fish also had high glucose and lactate levels, suggesting that they were chronically stressed before the experiment began (see below). Occasionally “unstressed” fish in other experiments were found to have similarly high levels of the three parameters (e.g. experiment 3). The reason why these fish were stressed is not known. The anaesthetized group of fish also included such an “outlier” (29 ng/ml); apart from that the range of individual cortisol values was from 1-13 ng/ml, with an overall mean of 6.5 ng/ml. The mean plasma glucose level was also similar to that in unanaesthetized aquarium fish. However, the plasma lactate level was significantly higher in anaesthetized fish. This finding is believed to reflect the extra time (2-3 min) taken to sample these fish and the physical exertions of the fish in the anaesthetic solution. Thus, aquarium-acclimated golden perch can be sampled quickly such that resting values of stress-reactive parameters are not affected. Anaesthesia, however, delays bloodsampling and, as a consequence, changes in plasma lactate levels can become apparent. In addition, it appears that occasional fish are chronically stressed by some aspect(s) of the holding conditions, and may confuse data gathered from experiments which are limited by fish numbers. The low levels of cortisol measured in wild and most tank-acclimated fish are in agreement with a number of studies which have suggested that unstressed fish generally have less than 5 ng cortisol/ml (Robertson et al., 1988; Pickering and Pottinger, 1989; Pankhurst et al., 1992). Golden perch respond quickly to stress. Measurable changes in plasma cortisol, glucose and lactate levels were detected only 5 min after initiation of the stress, however statistical analysis indicated these changes were significant only after 30 min. The conservative nature of the statistically significant differences indicated (e.g. the cortisol level after 10 min was IO-fold higher than that in unstressed animals, but statistically the two are not different) is due to the limited number of fish sampled at each timepoint.
54
John F. Carragher and Christine M. Rees
Plasma cortisol levels increased throughout the 30 min confinement period, however, from the data it is not possible to indicate if the response peaks after 30 min, or at some later time. The maximal mean cortisol level found during this study (approximately 240 ng/ml) was similar to that found for other species of fish subjected to similar types and durations of stressors [several species (180-340 ng/ml), Davis and Parker, 1986; ted drum (250 ng/ml), Robertson et al., 1988; striped bass (> 300 ng/ ml), Davis and Parker, 1990; blue mao mao (250 ng/ml), Pankhurst et al., 19921, but higher than found in other species [brown trout (100-l 50 ng/ml) and rainbow trout (40-80 ng/ ml), Pickering and Pottinger, 1989; snapper (20 ng/ml), Pankhurst and Sharples, 19921. In the present study, plasma glucose levels decreased slightly, but not significantly, soon after the stress was initiated. This condition lasted for at least 10 min. The authors are not aware of any other published study in which this pattern has been observed, although few studies have looked at such short term changes. It is suggested that this initial decrease may be due to the increased utilization of circulating glucose as a substrate for metabolism and energy production. After 30 min of stress the more usual hyperglycaemic response was observed in golden perch, suggesting that glucose was being regenerated from lactate or liberated from body stores (glycogen, lipid or protein) to maintain the high metabolic readiness of the animal (Barton and Schreck, 1987b). In common with other studies the increase in plasma glucose level after 30 min of stress was not maximal. Plasma lactate levels were found to increase quickly and remain high during the 30 min of netting and confinement stress. Golden perch were surprisingly placid during the stress procedures, only exerting themselves occasionally and briefly. In contrast, rainbow trout confined in the same way are more agitated (Carragher, personal observation). Given this reduced response it is surprising that plasma lactate levels increased as they did; it suggests that golden perch have a low capacity for aerobic metabolism. The increase in lactate level in golden perch in these experiments was similar to those observed in some other studies (Zfold, Barton and Schreck, 1987a; 3-fold, Waring et al., 1992), but less than in others (dfold, Pickering et al., 1982). The severity of stressor does influence the magnitude of the response obtained (Driedzic and Hochachka, 1978). Thirty minutes of forced swimming, netting and confinement stress increased golden perch lactate levels approximately lo-fold (Carragher, unpublished results). Two and a half hours after a 30 min shallow water stress plasma cortisol levels had returned
to the level measured in control fish. Similar experiments on other species usually show longer recovery times. One hour of handling and confinement elevated plasma cortisol levels for at least 8 and 4 hr in brown and rainbow trout, respectively (Pickering and Pottinger, 1989). Plasma cortisol levels in Atlantic salmon and flounder confined underwater in a net for 9 min did not decrease to the level found in resting fish until at least 8 and 24 hr post-stress, respectively (Waring et al., 1992). Striped bass acclimated to 16 and 21 “C recovered (in terms of their plasma cortisol level) from a 12 min net confinement stress in less than 6 hr; at higher and lower acclimation temperatures the recovery was slower (Davis and Parker, 1990). Thus, golden perch appear to have the ability to recover quickly from acute stress. The 90 ng cortisol/ml measured after 30 min of shallow water stress in experiment 3 suggests that this stressor was less severe than the netting and confinement procedure used in experiment 2 which resulted in 240 ng/ml indicating that the magnitude of the golden perch stress response is related to the severity of the stressor. Similar graduated response to different severity stressors have been reported previously (Strange et al., 1977, 1978; Barton et al., 1980; Robertson et al., 1988; Pankhurst and Sharples, 1992). We may be able to take advantage of the fishes’ ability to discriminate between stressor severity to adapt and improve handling procedures. By assessing the physiological reaction of the fish to a stressor we avoid anthropomorphic judgements about perceived severity. Plasma glucose levels were not affected by 30 min of shallow water stress. No other published studies have documented such a response. The usual effect of acute stress is to cause an increase in plasma glucose level which continues for at least as long as elevated cortisol levels persist (e.g. Pickering et al., 1982; Barton et al., 1986; Davis and Parker, 1990; Waring et al., 1992). This was not the case in this experiment. Possibly the milder nature of this procedure, compared to the handling and confinement stress which did cause an increase, failed to affect glucose metabolism; or perhaps the 30 min bloodsample was taken when the glucose level was increasing after an initial slight decrease (as was found in experiment 1). Whatever the explanation, it is clear that 2.5 hr after a 30 min stress plasma glucose levels are the same as in unstressed fish. Thirty minutes of shallow water stress did, increase plasma lactate levels, however, suggesting that an appreciable amount of anaerobic metabolism was undertaken. Given this, it is even more surprising that plasma glucose levels did not change. This finding suggests that
Stress responses in golden perch
the lactate may have been converted back to glucose (gluconeogenesis) in the muscles where it was then used, and thus reduced the need for glucose to be mobilized from body stores. After 2.5 hr of recovery plasma lactate levels had returned to basal. Similar studies suggest that after acute stress plasma lactate levels return to prestress values soon after cortisol levels are normalized (Pickering et al., 1982; Barton et al., 1986). Thirty minutes of chronic shallow water stress produced a mean cortisol level of over 90 ng/ml (experiment 3; Fig. 3), after 3 hr it was 57 ng/ml (experiment 4; Fig. 4). The level decreased further to 28 ng/ml after 6 hr. This suggests that the fish were acclimating to the chronic stressor. It is not possible to extrapolate these data to predict when the cortisol level would return to that found in unstressed fish. Published studies in which similar procedures have been carried out suggest that this is a typical acclimation response. Robertson et al. (1988) found that cortisol levels in red drum returned to prestress values by the end of a 5.5 hr transportation stress. Barton and Peter (1982) found that rainbow trout showed signs of acclimating to transport stress 48 hr into the procedure. In contrast, rainbow trout kept in confined and crowded tanks took 6-10 days to acclimate; whilst brown trout took 3C40 days (Pickering and Pottinger, 1989). Plasma glucose levels were elevated compared to control fish after 3 and 6 hr of chronic stress. This is similar to the results of chinook salmon (Barton and Schreck, 1987a) and red drum (Robertson et al., 1988). This hyperglycaemia was probably maintained by the elevated plasma cortisol level continuing to stimulate glycogenolysis or gluconeogenesis. Plasma lactate levels did not differ significantly in this experiment, although according to the results of experiment 3, plasma lactate levels were elevated early in the stress period. The decrease which occurred during the stress may have been a consequence of reduced physical activity (and hence anaerobic metabolism), although fish were heard to be occasionally active throughout the 6 hr of stress. Alternatively, the rate of gluconeogenesis increased during the stress and reduced circulating lactate levels. Braley and Anderson (1992), in the only other published study dealing with the golden perch stress response, observed a maximal 4.5-fold increase in blood glucose, and the hyperglycaemic state persisted for at least 96 hr. In contrast, the maximal response obtained in the present study was only 2-fold and the level appeared to be decreasing after 6 hr of chronic stress. These differences are likely to reflect the complex nature of the stress applied in the
55
Braley and Anderson study (multiple handling, anaesthesia and bloodsampling), compared to the simple stressors used in the present study. Plasma lactate was the other parameter measured in both studies, this was not found to change after multiple bloodsampling (Braley and Anderson, 1992). They reported a mean lactate level of 1.9 mM, a value similar to that measured in anaesthetized unstressed fish in the present study. In the present study plasma lactate level was affected mainly by the method of bloodsampling (i.e. anaesthetized versus nonanaesthetized), and by short periods of stress (~3 hr; Fig. 4). Since the minimum sampling frequency in the Braley and Anderson (1992) study was 3 hr, it is likely that perturbations in lactate levels caused by handling and bloodsampling were completed before the subsequent sample was taken (unlike the compounded response of plasma glucose levels). Thus, golden perch can acclimate to aquarium conditions, they respond quickly to stressors with maximal cortisol levels similar to those reported for other species, they recover rapidly from acute stress and appear to acclimate quickly to chronic stress. These characteristics suggest that the golden perch is a species capable of tolerating stressors found in intensive aquaculture practice. Acknowledgements-The contributions of the following individuals and groups in the course of this study are gratefully acknowledged: Dr Ian MacDonald (Zoology Department, Melbourne University) for cortisol label, Dr Tom Watson (Deakin University) for cortisol standard. Dr Trevor Anderson (Deakin University) for comments on the manuscript, Hal Braley and Karen Drieberg for their angling skills, the Native Finfish group at Snobs Creek Research Station and Hatchery (Department of Conservation and Environment, Victoria) for access to golden perch, and The William Anglis (Victoria) Charitable Fund for financial support.
References Barton B. A. and Peter R. E. (1982) Plasma cortisol stress response in fingerling rainbow trout, Sulmo gairdneri Richardson, to various transport conditions, anaesthesia and cold shock. J. Fish Biol. 20, 39-51. Barton B. A. and Schreck C. B. (1987a) Influence of acclimation temperature on interrenal and carbohydrate stress responses in juvenile chinook salmon (Oncorhynchus
tshuwystcha).
Aquaculture
62, 299-310.
Barton B. A. and Schreck C. B. (1987b) Metabolic cost of acute physical stress in juvenile steelhead. Trans. Am. Fish. Sot. 116, 257-263.
Barton B. A., Peter R. E. and Paulencu C. R. (1980) Plasma cortisol levels of fingerling rainbow trout (Salmo guirdneri) at rest, and subjected to handling confinement, transport, and stocking. Can. J. Fish. Aquat. Sci. 37, 805-811.
Barton B. A., Schreck C. B. and Sigismondi L. A. (1986) Multiple acute disturbances evoke cumulative physiological stress responses in juvenile chinook salmon. Trans. Am. Fish. Sot. 115, 245-251.
56
John F. Carragher and Christine M. Rees
Battaglene S. and Prokop F. (1987) Golden perch. In A&cl A 3.2.2., Department of Agriculture, Sydney, New South Wales. Braley H. and Anderson T. A. (1992) Changes in blood metabolite concentrations in response to repeated capture, anaesthesia and blood sampling in the golden perch, Macquaria ambigua. Comp. Biochem. Physiol 103A, 445-450. Campbell P. M., Pottinger T. G. and Sumpter J. P. (1992) Stress reduces the quality of gametes produced by rainbow trout. Biol. Reprod. 47, 114&1150. Carmichael G. J., Tomasso J. R., Simco B. A. and Davis K. B. (1984) Characterization and alleviation of stress associated with hauling largemouth bass. Trans. Am. Fish. Sot. 113, 778-785.
Davis K. B. and Parker N. C. (1986) Plasma corticosteroid stress response of fourteen species of warmwater fish to transportation. Trans. Am. Fish. Sot. 115, 495499. Davis K. B. and Parker N. C. (1990) Physiological stress in striped bass: effect of acclimation temperature. Aquaculture 91, 349-358. Dried& W. R. and Hochachka P. W. (1978) Metabolism in fish during exercise. In FLsh Phvsiologv Vol. 7 (Edited bv Hoar W. S. and Randall D. J.), ~~-503-543. ‘Academic Press, New York. Mazeaud M. M. and Maxeaud F. (1981) Adrenergic responses to stress in fish. In Stress in Fish (Edited by Pickering A. D.), pp. 49-75. Academic Press, London. Mazeaud M. M., Mazeaud F. and Donaldson E. M. (1977) Primary and secondary effects of stress in fish: some new data with a general review. Trans. Am. Fish. Sot. 106, 201-212. O’Sullivan D. (1991) Status of Australian aquaculture in 1989190. AustAsia Aquaculture June, 2-13. Pankhurst N. W. and Sharples D. F. (1992) Effects of capture and confinement on plasma cortisol concentrations in the snapper, Pagrus aurafus. Aust. J. Mar. Freshwater Res. 43, 345-356. Pankhurst N. W., Wells R. M. G. and Carragher J. F. (1992) Effects of stress on plasma cortisol levels and blood viscosity in blue mao mao, Scorpis violaceus (Hutton),
a marine
teleost.
Comp.
Biochem.
Physiol.
lOlA,
335-339.
Pickering A. D. (1990) Stress and the suppression of somatic growth in teleost fish. In Progress in Comparative Endocrinology (Edited by Epple A., Scanes C. G. and Stetson M. H.), pp. 473479. Wiley-Liss, New York. Pickering A. D. (1992) Rainbow trout husbandry: management of the stress response. Aquacufture 100, 125-139. Pickering A. D. and Pottinger T. G. (1989) Stress responses and disease resistance in salmonid fish: effects of chronic elevation of plasma cortisol. Fish Physiof. Biochem. 7, 253-258. Pickering A. D., Pottinger T. G. and Christie P. (1982) Recovery of the brown trout, Salmo trutta L., from acute handling stress: a time course study. J. Fish Biol. 20, 229-244. Pillay T. V. R. (1992) Aquaculture and the Environment. Fishina News Books. Oxford. Roberts& L., ThornasP. and Arnold C. R. (1988) Plasma cortisol and secondary stress responses of cultured red drum (Sciaenops ocellatus) to several transportation procedures. Aquaculture 68, 115-130. Strange R. J. and Schreck C. B. (1978) Anaesthetic and handling stress on survival and cortisol concentration in yearling chinook salmon (Oncorhynchus tshawyfscha). J. Fish. Res. Board Can. 35, 345-349. Strange R. J., Schreck C. B. and Golden J. T. (1977) Corticoid stress responses to handling and temperature in salmonids. Trans. Am. Fish. Sot. 106, 213-217. Strange R. J., Schreck C. B. and Ewing R. D. (1978) Cortisol concentrations in confined juvenile chinook salmon (Oncorhynchus tshawystcha) Trans. Am. Fish. Sot. 107, 812-819. Waring C. P., Stagg R. M. and Poxton M. G. (1992) The effects of handling on flounder (PlatichthysJlesus L.) and Atlantic salmon (Salmo salar L.). J. Fish Biol. 41, 131-144. Wedemeyer G. A. and McLeay D. J. (1981) Methods for determining the tolerance of fishes to environmental stressors. In Stress in Fish (Edited by Pickering A. D.), pp. 247-275. Academic Press, London.