The effect of dietary carbohydrate on the stress response in cod (Gadus morhua)

The effect of dietary carbohydrate on the stress response in cod (Gadus morhua)

319 Aquaculture, 95 ( 199 1) 3 19-328 Elsevier Science Publishers B.V., Amsterdam The effect of dietary carbohydrate on the stress response in cod (...

766KB Sizes 0 Downloads 4 Views

319

Aquaculture, 95 ( 199 1) 3 19-328 Elsevier Science Publishers B.V., Amsterdam

The effect of dietary carbohydrate on the stress response in cod (Gadus morhua) Gro-Ingunn Hemre, Georg Lambertsen and 0yvind Lie Directorate ofFisheries. Institute of Nutrition, Strandgaten 229, N-5024 Bergen. Norwa) (Accepted

10 October 1990)

ABSTRACT Hemre, G.-I., Lambertsen, G. and Lie, 0.. 1991. The effect of dietary carbohydrate response in cod (Gadus morhua). Aquaculture, 95: 319-328.

on the stress

150 cod (mean weight ~440 g) were divided into two groups and fed diets with or without carbohydrate for 60 days. No differences were found in total growth during the feeding period. Both fish groups were then stressed by handling and transported for 2 h. Five fish from each dietary group were collected for analysis at different time intervals up to 96 h. In terms of serum cortisol increase, the stress response was significant in both dietary groups and reached a maximum after half an hour. No response was found in blood hematocrit or in blood hemoglobin at any sampling time. The plasma glucose levels in resting fish reflected the dietary levels of carbohydrate. The plasma glucose response was also significantly more affected by stress in the carbohydrate-eating fish than in the fish on a carbohydrate-free diet. Blood lactate did not reflect the diet composition, while a significant increase was seen in both groups after 1.5 h of recovery. No change in muscle lactate was measured as a response to stress, but significantly higher muscle lactate levels were measured in the fish without dietary carbohydrate. Neither white muscle nor hepatic glycogen levels were affected by the diet, but a significant increase was seen in both organs in the fish on a carbohydrate feed after 3 h of recovery.

INTRODUCTION

Experiments with farmed cod have shown that this species has a low ability to utilize carbohydrates in the feed. From an apparent digestibility of 40-60% for starch, only 24% of absorbed glucose could be accounted for, either as blood glucose or liver and muscle glycogen, and the weight gain showed no protein-sparing effect from carbohydrate (Hemre et al., 1989). As known from other studies, both stress conditions and high levels of dietary carbohydrate affect the carbohydrate metabolism in fish (Srivastava and Sahai, 1987; Hemre et al., 1989). Among the primary stress responses is a pronounced elevation of plasma corticosteroids. As stated by Mazeaud and Mazeaud ( 198 1)) the osmotic stress response varies with species and type of stressor and may be explained by adrenergic activation affecting osmoregulation as a secondary response. This can be 0044-8486/91/$03.50

0 I99 I -

Elsevier Science Publishers

B.V.

320

G.-I. HEMRE ET AL.

measured as changes in blood hematocrit (Hct) and hemoglobin (Hb) values (Fletcher, 1975; Casillas and Smith, 1977; Wells et al., 1984; and Srivastava and Sahai, 1987). The most pronounced effects of cortisol administration are elevated blood glucose (Casillas and Smith, 1977; Mazeaud and Mazeaud, 198 1; Leach and Taylor, 1982; Tam et al., 1987) and elevated blood lactate (Piiper and Baumgarter-i, 1969), which are normally found as cortisol declines in plasma. In addition, the blood glucose level is affected by the dietary carbohydrate content (Hemre et al., 1989). The formation of muscle lactate is the main source of the pH decrease in slaughtered fish and influences fillet quality. Flight situations are known to promote an increase in muscle lactate as described for coalfish (Gadus virens L. ) by Johnston and Goldspink ( 1973 ) , for plaice (Pleuronectes platessa ) by Wardle ( 198 1) and for Atlantic salmon (Salmo salar) by Biirjeson and Hoglund ( 1975 ). The source of muscle lactate may be catabolized amino acids and/or glycogen (Driedzic and Hochachka, 1976). However, no correlation was found between the diet composition and the amount of white muscle glycogen in an earlier experiment with cod (Hemre et al., 1989). Contradictory results have been reported concerning the use of liver glycogen as an energy reserve during stress. Increased blood glucose and liver glycogen were found when exposing Notopterus notopterus to air (Narasimhan and Sundararaj, 197 1). Paxton et al. ( 1984) found elevated hepatic glycogen levels at recovery, as the plasma cortisol declined in goldfish (Carassius auratus) and eel (Anguilla japonica), while in cyprinodonts (Poecilias latipinna and Fundulus heteroclitus) and in cyprinids, glucocorticoids appeared to promote mobilization of hepatic glycogen. Studies have shown that the dietary content of carbohydrate is reflected in blood glucose levels and in deposition of glycogen in the liver in cod (Hemre et al., 1989, 1990)) and that the carbohydrate metabolism in fish is highly altered as a secondary stress response (Mazeaud et al., 1977). The purpose of this experiment was to test whether the diet fed prior to stress exerted any effect on the stress reaction in cod. The experimental approach included measurements of plasma cortisol, blood hematocrit and hemoglobin, plasma glucose and blood lactate levels, as well as muscle lactate and glycogen in liver and muscle, prior to and at time intervals after handling. MATERIALS

AND METHODS

Experimental design 150 cod, with an initial weight of 440 5 138 g (meant s.d. ), were caught in Masfjorden, Western Norway, and distributed into two 1.5-m2 tanks at the Aquaculture Station Matre. The fish were fed ad libitum once a day for 9 weeks prior to handling stress. Water temperature (8 "C) and salinity (20 g/

EFFECT OF DIETARY CARBOHYDRATE

ON THE STRESS RESPONSE IN COD

321

1) remained fairly constant throughout the feeding period. Five fish from each tank were collected for analysis before the remaining fish were caught by a landing net and transferred to two separate transport tanks ( 1 m3) with oxygenated water. The fish were then transported by car for two hours, after which groups of five fish from each regime were distributed in individual tanks to avoid any further disturbance. Five fish from each group were anaesthetised and removed for analysis after 0.5, 1.5, 3, 6, 12,24, 36 and 96 h. Diet composition Group A was given a diet without carbohydrate and group B a diet containing 25% carbohydrate on a dry-weight basis. The dietary lipid content ranged from I3 to 15% (dry weight) and protein was substituted by carbohydrate. The feed composition differed in carbohydrate and protein content similar to what is found in “natural” cod prey and “commercial” marine feed. Extruded wheat was used as the carbohydrate source and capelin oil as the lipid source. A mixture of saithe fillet and squid mantle was used as the protein source. See Table 1 for the exact diet composition. Sample collection Five fish from each group were collected simultaneously. From each fish, blood was collected from the vena caudalis by a medical syringe. The blood TABLE 1 Dietary composition respectively

Protein source= Capelin oil Extruded wheat Binder (guar-gum) Vitamin mixtureb Mineral mixtureC Analyses Dry matter Protein Fat Carbohydrate

(g/kg). Group A and B fish were given a feed without and with carbohydrates,

Group A

Group B

961 27

781 37 170 10 1 1

10 1 1

190 155 29

300 142 39 75

“Saithe tillet mixed with 10% squid mantle (dry matter I9.8%, protein 16.9% and fat 0.9%). bComposition (g/kg vitamin mix): thiamine-HC12.5; riboflavin 5.0; pyridoxine 5.0; Ca-pantothenate IO.@,niacin 37.5; folic acid I .25; ascorbic acid 100; vitamin A and D as Rovimix AD,, Type 5001 100 1.O; alpha-tocopherol acetate as Rovimix E-50 Adsorbate 15.0. ‘Commercial standard mineral mixture used for poultry and swine, (g/kg): phosphorus 60; calcium 240; sodium 60; magnesium 10; iron 2; manganese 2; zinc 2.5; copper 0.4; iodine 0.075; selenium 0.008.

G-1. HEMRE ET AL.

322

sample was immediately divided into two samples. One sample was diluted with 8% (w/v) perchloric acid to stabilize lactate (see Procedure No. 826UV, Sigma Chemical Co. ) and one sample was heparinized for analysis of hematocrit (Hct ) and hemoglobin (Hb) before centrifugation (5000 r.p.m. ). After centrifugation, plasma was frozen at - 20’ C until analysed for cortisol and glucose. The fish were then weighed and liver and fillet were dissected and immediately frozen on dry ice. The samples were stored at - 20’ C until analysed for lactate and glycogen. Chemical analysis Plasma cortisol was measured using a radioimmunoassay kit (Dada, Baxter Travenol Diagnostics, MA). This kit has been validated for fish plasma as described by Stefanson et al. ( 1989). Hct and Hb were determined on heparinized blood, Hct within one hour of sampling. Vitrex Pari microhematocrit tubes were used for Hct determinations as described by Sandnes et al. ( 1988 ) and Hb was determined spectrophotometrically (540 nm) using the cyanmethemoglobin method (Kit 525-A, Sigma Chemical Co. ). Blood lactate was determined at 340 nm using a quantitative enzymatic method (Procedure No. 826~UV, Sigma Chemical Co. ) . Plasma glucose, liver and muscle glycogen were determined as described by Hemre et al. ( 1989). Muscle lactate was analysed using a HPLC-ion-exclusion method according to Haller and Lackner ( 1987). The eluant used was sulfuric acid (0.017 N) and the column temperature was kept at 50°C. Protein (Nx 6.25 ) was analysed using a method described by Crooke and Simpson ( 197 1) and fat was measured gravimetrically using ethyl acetate extraction. Statistics The data were tested using one-way ANOVA analysis of variance, linear regression analysis and Friedman Two-Way Analysis by Ranks (“Statgraphits” (A plus*Ware’“Product, STSC) ). RESULTS

AND DISCUSSION

The fish behaved normally during the feeding period and no diseases were registered. The feed intake was good, and the fish almost tripled their weight during the 9 weeks of feeding (Table 2). No differences were found in weight gain between the two feeding groups nor in hepatosomatic index. The weight gain and the hepatosomatic index approximated what have been found for well-conditioned wild cod (Pedersen and Jobling, 1989)) indicating good experimental conditions. As also found in earlier experiments (Hemre et al., 1989), the feed conversion factor increased as the carbohydrate content in the diet increased. The fish behaved calmly and seemed relatively undisturbed after two hours

EFFECT OF DIETARY CARBOHYDRATE ON THE STRESS RESPONSE IN COD

323

TABLE 2 Growth and hepatosomatic index of cod fed diets without (group A) and with (group B) carbohydrates for 9 weeks prior to handling stress

Initial weight (g) sd. (n=75) Final weight (g) s.d. (n=75) Feed consumption (g/fish) Weight gain (g) Weight gain (%) Feed conversion (g dry feed/g weight gain) Hepatosomatic index” sd. “(Liver weight/final

weight)

x

Group A

Group B

443.0 138.1 1299.7 195.4 2480.0 856.7 193.4 0.6

435.6 104.8 1241.2 188.2 1745.0 805.6 184.9 0.7

4.4 1.8

5.1 1.7

100.

of handling and transport. However, the rapid increase (PC 0.0 1) in plasma cortisol confirmed a stressed condition in both fish groups (Fig. 1). Maximum cortisol levels were found after half an hour of recovery, with no significant differences between the two dietary groups. The narrow cortisol peak is a typical response for a short time stressor, while a long-term stressor results in a cortisol increase which remains elevated for a longer time, as described for rainbow trout (Oncorhynchus mykiss) by Flos et al. ( 1988). Hematocrit ranged from 21 to 27% and hemoglobin from 3.5 to 4.4 g 100 ml- ‘, independent of diet and handling stress. The hematological values are in accordance with earlier reported values for cod (Lie et al., 1990). The relatively low and stable hematological values indicate that this species has a high tolerance to handling stress. One of the effects described as a secondary stress response is disturbances of osmoregulatory mechanisms (Fletcher, 1975; Casillas and Smith, 1977; Mazeaud and Mazeaud, 198 1; Wells et al., 1984 and Srivastava and Sahai, 1987), which can be measured as changes in hematological values. A significant (P< 0.01) diet response was seen in plasma glucose between the two dietary groups with group B values about a third higher than group A (Table 3), in accordance with earlier studies in cod (Hemre et al., 1989). Plasma glucose rose sharply in both groups upon handling and maximum levels were measured after l-3 h. In group B given carbohydrate, the blood glucose level rose to more than double the resting value, and remained high during the rest of the recovery period. This indicates a stress-related change in carbohydrate metabolism depending on the feed composition in this trial. Carnivorous fish react diabetic-like after feeding high levels of carbohydrates (Bergot, 1979; Hemre et al., 1990). The delayed regulation of blood glucose

324

G.-l. HEMRE ET AL.

cortisol ng/ml 16

01

End

10

20

of handling

30

40

50

60

70 hours

80 after

90

100

handling

Fig. 1. Plasma cortisol levels (ng/ml) prior to (from 0) and after (indicated with an arrow on the x-axis) handling stress in cod fed diets without (whole line) or with (broken line) carbohydrates.

in group B fish together with the high cortisol level upon stress may affect the health of the fish by lowering the resistance to infectious diseases. Wiik et al. ( 1989) found a significant increase in susceptibility to Aeromonas salmonicida after dietary cortisol administration in Atlantic salmon (Salmo sular). The blood lactate levels were not influenced by the dietary composition. These results are not in accordance with an earlier experiment showing a blood lactate concentration of 1.9 mmol/l when feeding a diet without carbohydrate and 2.7 mmol/l when feeding a carbohydrate-containing diet (Hemre et al., 1990). However, a small but significant (P~0.05) increase in blood lactate was found in both groups after 1.5 h of recovery in the present study. Both groups regained resting values 3 h after stress (Table 3 ) . The blood lactate concentration may vary with the ambient temperature as described by Wendt ( 1964) in Salvelinus fontinalis, and with the degree of stress, as described by Wells ,et al. ( 1984) in Antarctic cod (Dissostichus mawoni). A

EFFECT OF DIETARY CARBOHYDRATE

ON THE STRESS RESPONSE IN COD

325

TABLE 3 Plasma ghxose (mmol/l) and blood lactate (mmol/l) (s.e.m. n= 5) in cod fed a diet without (group A) and with (group B) carbohydrates for 9 weeks prior to handling stress. Blood was removed before handling stress and after 0.5, 1.5, 3,6, i2,24, 36 and 96 h Blood lactate

Plasma glucose

Before handling (s.e.m.)* 0.5 h after (s.e.m.) 1.5 h after (s.e.m.) 3 h after (s.e.m. ) 6 h after (s.e.m.) 12 h after (s.e.m.) 24 h after (s.e.m. ) 36 h after (s.e.m. ) 96 h after (s.e.m.)

Group A

Group B

Group A

Group B

3.3 4.9 4.9 4.3 4.2 4.8 4.4 3.8 3.9

4.4 6.7 10.3 9.8 8.0 7.3 9.4 6.8 7.1

0.3 (o.l)e 0.7 (0.2)’ 1.1 (0.4)’ 0.4 (0.2)e nm** 0.2 (0.1) nm nm nm

0.3 0.4 0.6 0.4

(0.1)” (0.2 )b (0.4)b (0.4)b (0.9)b (0.7)b (0.2)b (0.3)= (0.5)a

(O.S)b (0.4)’ (2.3)d (2.8)d (l.l)d (1.5)Cd (1.7)d (1.0)’ (1.6)’

(0.1)’ (0.2)e ( 0.2)e’

(0.1)’

nm

0.4 (0.1 ) nm nm nm

*s.e.m. =standard error mean. **nm = not measured. “b”d”fDifferent letters indicate significant difference between groups and/or sampling times (P-C 0.05).

relatively low water temperature (S’C) and the narrow cortisol peak indicating a mild stress condition in the present experiment may explain the low blood lactate levels (Wendt, 1964; Hemre et al., 1990). In comparison with results found in Antarctic cod, which responded mildly after anaesthetic cannulation, showing no secondary increase in blood lactate (Wells et al., 1984)) the stress response in the present experiment was sufficient to give a substantial increase in plasma glucose as well as blood lactate, but not severe enough to disturb osmoregulatory mechanisms as judged from the hematological analyses. It is known from both electromyographical records (Bone, 1966) and biochemical measurements (Black et al., 1966) that the white muscle is involved in providing the energy required for short bursts of high-speed swimming as in flight situations. In the present experiment, muscle lactate was not affected by stress (Table 4), whereas significantly (P< 0.05) higher muscle lactate levels were found in group A compared to group B over all sampling times. A very quick lactate production has been measured during bursts of high speed swimming (Black et al., 196 I), which may indicate that the sampling procedure (capturing fish with a landing net) would affect both the muscle lactate values already detected in the fish taken out before handling and the transport stress in the present study. The constant muscle lactate levels found during the recovery period are not in accordance with results found in coalfish (Johnston and Goldspink, 1973 ), plaice (Wardle, 198 1) and Atlantic salmon (Borjeson and Hoglund, 1975 ). Glycogen stores in white muscle were low and equal in both dietary groups,

G.-I. HEMRE ET AL.

326 TABLE 4

Liver and muscle glycogen and muscle lactate in fish fed a diet without carbohydrate with carbohydrate (group B) prior to and after handling stress

Before handling s.e.m. 0.5 h s.e.m. 3h s.e.m. 12h s.e.m. 96 h s.e.m.

(group A) and

Liver glycogen (%)

Muscle glycogen (O/o)

Muscle lactate (%)

Group A

Group B

Group A

Group B

Group A

Group B

2.1” 0.6 2.4” 0.6 1.2” 0.4 1.3” 0.4 1.5” 0.2

3.0” 0.6 2.6” 0.4 5.6 0.1 4.3b 0.3 4.0b 1.2

0.3’ 0.0 0.2’ 0.0 0.3’ 0.1 0.3’ 0.0 0.3’ 0.1

0.2’ 0.0 0.2’ 0.0 0.4d 0.0 0.4d 0.0 0.4d 0.1

0.5"

0.3’ 0.1 0.2” 0.0 0.3” 0.1 0.3’ 0.0 0.4’ 0.1

0.0 0.4” 0.0 0.5” 0.1 0.4’ 0.0 0.5’ 0.1

(s.e.m. values ~0.04 are given as 0.0 in the table.) “b”d’Different letters indicate significant difference between groups and/or sampling times (P-C 0.05).

and no decreases were found at any sampling time. However, a significant (PC 0.05 ) increase in muscle glycogen was measured in group B after 3 h of recovery, and was maintained at this level during the rest of the recovery period. Driedzic and Hochachka ( 1976) found an activation of phosphofiuctokinase and pyruvate kinase simultaneously with an increase in the NH: content in white muscle of common carp (Cyprinus carpio) during activity. This indicates the use of free amino acids as well as glycogen as energy sources in white muscle. In a preliminary study with cod, we found exhausted muscle glycogen stores after 6 h of transport (Hemre, unpublished). In the present experiment, muscle lactate was significantly higher (PC 0.05) in group A than in group B, the source of this may be catabolized amino acids and/or glycogen (Driedzic and Hochachka, 1976). The increase in muscle lactate together with the stable glycogen values after 2 h of handling may point to a use of free amino acids as a “first” energy reserve and a use of glycogen as a “second” energy reserve in white muscle of cod. The increase in muscle glycogen only in the carbohydrate feeding fish may also indicate that the high plasma glucose levels in this group favour white muscle glycogen synthesis. For group A, however, our results are in accordance with Chan and Woo ( 1978) showing that, while injections of cortisol increased the plasma glucose level, no transport of glucose to the muscle could be detected. The difference in liver glycogen concentration found between the two groups in response to diet was not significant. After 3 h of recovery a significant (PC 0.05 ) increase was measured only in group B fish, and this level was maintained during the rest of the recovery period. The results from group B are in accordance with studies in goldfish and eel as reported by Paxton et al.

EFFECT OF DIETARY CARBOHYDRATE ON THE STRESS RESPONSE IN COD

327

( 1984) showing elevated hepatic glycogen levels during recovery, as the cortisol concentration declined in plasma. The variation in hepatic glycogen during this experiment in general follows the same pattern as white muscle glycogen, and does not indicate the use of hepatic glycogen as an energy reserve during and after stress, but indicates an induced glycogen synthesis in group B fish, related to the higher blood glucose level. It may be noted that Chan and Woo ( 1978) suggested that the carbon skeleton used for the synthesis of blood glucose and liver glycogen in eel (Anguilla jupoizica) came from the breakdown of peripheral tissue such as muscle, releasing fat and amino acids for gluconeogenesis in the liver. The difference in stress response between the two dietary groups shows that the physiological reactions with respect to blood glucose as well as lactate and muscle and liver glycogen were affected by the diet fed prior to stress. Transport of fish frequently occurs in commercial farming, often followed by increased mortality. This trial may give reason to believe that a change in diet in advance of handling and transport could reduce some of the losses. Further studies are needed to clarify whether the combined effects of dietary carbohydrate and stress affect susceptibility to infectious diseases and general health in fish. It may be added that the results do indicate that the carbohydrate content of the feed does not affect pH values in the fillet after slaughter in commercial cod farming.

REFERENCES Bergot, F., 1979. Effects of dietary carbohydrates and oftheir mode ofdistribution on glycaemia in rainbow trout (Salmo gairdneri Richardson). Comp. Biochem. Physiol. A, 64: 543-547. Black, EC., Robertson, A.C. and Parker, R.R., 196 I. Some aspects of carbohydrate metabolism in fish. In: A.W. Martin (Editor), Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals. University of Washington Press, Seattle, WA, pp. 89-122. Black, E.C., Bosomworth, N.J. and Dochery, G.E., 1966. Combined effects of starvation and severe exercises on glycogen metabolism of rainbow trout, Salmo gairdneri. J. Fish. Res. BoardCan., 23: 1461-1463. Bone, Q., 1966. On the function of the two types of myotomal muscle fibre in elasmobranch fish. J. Mar. Biol. Assoc. UK, 46: 321-349. Borjeson, H. and Hoglund, L.B., 1975. Muscle and blood lactate in juvenile Safmo salar exposed to high pCOZ. Rep. Inst. Freshwater Res. Drottningholm, 54: 5-7. Casillas, E. and Smith, L.S., 1977. Effect of stress on blood coagulation and hematology in rainbow trout (Salmo gairdneri). J. Fish Biol., 10: 48 l-49 1. Chan, D.K.O. and Woo, N.Y.S., 1978. Effect of cortisol on the metabolism of the eel, Anguilla japonica. Gen. Comp. Endocrinol., 35: 169-178. Crooke, W.M. and Simpson, W.E., 197 1. Determination of ammonium in Kjeldahl digests of crops by an automated procedure. J. Sci. Food Agric., 22: 9- 10. Driedzic, W.R. and Hochachka, P.W., 1976. Control of energy metabolism in fish white muscle. Am. J. Physiol., 230 (3): 579-582. Fletcher, G.L., 1975, The effects of capture, stress and storage of whole blood on the red blood

328

G.-I. HEMRE ET AL.

plasma cells, plasma proteins, glucose and electrolytes of the winter flounder (Pseudopleuronectes americanus). Can. J. Zoo]., 53: 197-206. Flos, R., Reig, L., Torres, P. and Tort, L., 1988. Primary and secondary stress responses to grading and hauling in rainbow trout (Salmo gairdneri). Aquaculture, 7 1: 99-106. Haller, T. and Lackner, R., 1987, Screening for organic acids in fish tissues with special reference to the distribution of taurine in Rutilus rutilus L. Fish Physiol. Biochem., 3: 145-l 49. Hemre, G.I., Lie, 0., Lied, E. and Lambertsen, G., 1989. Starch as an energy source in feed for cod (Gadus morhua): digestibility and retention. Aquaculture, 80: 261-270. Hemre, G.I.. Lie, 0., Lambertsen, G. and Sundby, A., 1990. Dietary carbohydrate utilization in cod (Gadus morhua). Hormonal response of insulin, glucagon and glucagon-like-peptide to diet and starvation. Comp. Biochem. Physiol., 97: 41-44. Johnston, LA. and Goldspink, G., 1973. A study of glycogen and lactate in the myotomal muscles and liver of the coallish (Gadus virens L. ) during sustained swimming. J. Mar. Biol. Assoc. U.K., 53: 17-26. Leach, G.J. and Taylor, M.H., 1982. The effects of cortisol treatment on carbohydrate and protein metabolism in Fundulus heteroclitus. Gen. Comp. Endocrinol., 48: 76-83. Lie, 0, Lied, E. and Lambertsen, G., 1990. Hematological values in cod (Gadus morhua). Fiskeridir. Skr., Ser. Errtaring (Directorate of Fisheries, Bergen, Norway), 3: 1 l-l 7. Mazeaud, M.M. and Mazeaud, F., 198 1. Adrenergic responses to stress in fish. In: A.D. Pickering (Editor), Stress and Fish. Academic Press, London, pp. 49-75. 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 ( 3 ): 20 l-2 12. Narasimhan, P.V. and Sundararaj, B.I., 197 1. Effects of stress on carbohydrate metabolism in the teleost Notopterus notopterus (Pallas). J. Fish Biol., 3: 441-451. Paxton, R., Gist, D.H. and Umminger, B., 1984. Serum cortisol levels in thermally-acclimated goldfish (Carassius auratus) and killifish (Fundulus heteroclitus): implications in control of hepatic glycogen metabolism. Comp. Biochem. Physiol., 78B(4): 8 13-8 16. Pedersen, T. and Jobling, M., 1989. Growth rates of large, sexually mature cod, Gadus morhua, in relation to condition and temperature during an annual cycle. Aquaculture, 8 1: 16 1- 168. Piiper. J. and Baumgarten, D., 1969. Blood lactate and acid-base balance in the elasmobranch Scyliorhinus stellaris after exhaustion activity. Pubbl. Stn. Zool. Napoli, 37: 84-94. Sandnes, K., Lie, 0. and Waagbo, R., 1988. Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon (Salmo salar). J. Fish Biol., 32: 129- 136. Srivastava, A.K. and Sahai, I., 1987. Effects of loading density on carbohydrate metabolism and hematology in Indian freshwater catfish, Heteropneustes fossilis. Aquaculture, 66: 275-286. Stefanson, S.O., Nrevdal, G. and Hansen, T., 1989. The influence of three unchanging photoperiods on growth and Parr-smolt transformation in Atlantic salmon (Salmo salar). J. Fish Biol., 35 (2): 237-247. Tam, W.H., Birkett, R.M., Payson, P.D., Whitney, D.K. and Yu, C.K.-C., 1987. Modification of carbohydrate metabolism and liver vitellogenic function in brook trout (Salvelinus fontinalis) by exposure to low pH. Can. J. Fish. Aquat. Sci., 44: 630-635. Wardle, C.S., 1981. Physiological stress in captive fish. In: A.D. Hawkins (Editor), Aquarium Systems. Academic Press, London, pp. 403-4 13. Wells, R.M.G., Tetens. V. and Devries, A.L., 1984. Recovery from stress following capture and anaesthesia of antarctic fish: hematology and blood chemistry. J. Fish Biol., 25: 567-576. Wendt, C., 1964. Liver and muscle glycogen and blood lactate in hatchery-reared Salmo salar L. following exercise in winter and summer. Rep. Inst. Freshwater Res. Drottningholm, 46: 167-184. Wiik, R., Andersen, K., Uglenes, I. and Egidius, E., 1989. Cortisol-induced increase in susceptibility of Atlantic salmon, Salmo salar, to Vibrio salmonicida, together with effects on the blood cell pattern. Aquaculture, 83: 20 l-2 15.