Physiological stress responses in big gamefish after capture: Observations on plasma chemistry and blood factors

Physiological stress responses in big gamefish after capture: Observations on plasma chemistry and blood factors

03#-%29/86 Camp. Biochem. Physiel. Vol. 84A, No. 3, pp. 565-571, 1986 $3.00 + 0.00 F’ergamonJournals Ltd Printed in Great Britain PHYSIOLOGICAL S...

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03#-%29/86

Camp. Biochem. Physiel. Vol. 84A, No. 3, pp. 565-571, 1986

$3.00 + 0.00

F’ergamonJournals Ltd

Printed in Great Britain

PHYSIOLOGICAL STRESS RESPONSES IN BIG GAMEFISH AFTER CAPTURE: OBSERVATIONS ON PLASMA CHEMISTRY AND BLOOD FACTORS R. M. G. WELLS,* R. H. MC&TYRR,* A. K. MORGAN* and P. S. DAVIES *Department of Zoology, University of Auckland, Auckland, New Zealand. Telephone: (09) 737-999 tDepartment of Physiology and Anatomy, Massey University, Palmerston North, New Zealand. Telephone: (063) 69-099 (Received

19 November

1985)

Abstract-l. The plasma electrolytes, Na+, K+, Ca’+, Cl- and osmolarities had high values in capture-stressed big gamefish. 2. Blood metabolites measured after stress showed glucose and lactate elevations. 3. The activity of the plasma enzymes alkaline phosphatase, alanine aminotransf~a~, aspartate aminotransferase, creatine kinase and lactate dehydrogenase suggested tissue disruptions following severe capture stress. 4. Haematocrit values and methaernoglobin were high in capture-stressed gamefish. 5. The plasma chemistry of resting and capture-stressed snapper (Chrysophrys aurutu.s) was studied for

comparison. 6. Specific differences in plasma bi~hemistry

appeared to be the result of different strategies of fish

behavibur during capture.

INTRODUCTION It is now generally recognized that fish tend to react to stress from capture, severe exercise and handling with far more exaggerated disruptions to their physiology and biochemistry than those seen in higher vertebrates such as mammals. These disruptions to normal function are particularly marked in metabolic and osmotic regulation, and in respiratory control (Ali et al., 1980). Much of our knowledge about the stress responses in fish is based on studies of the rainbow trout which, in many laboratories, has now assumed the status of the University Rat. Marlin and tuna are very different fish in terms of their physiology and behaviour, and the value of recognising stress factors in these species might be relevant in appraising the success of tagging programmes, fish meat quality, and attempts to contain live specimens. Only 34% of the body weight of a typical teleost fish is accounted for by circulating blood, and a further 30% is composed of white muscle, in addition to a variable proportion of red muscle in large, active pelagic teleosts. It might therefore be expected that changes in muscle biochemistry will be refiected strongly by the composition of the blood. The aim of the present study was to see whether blood sampling from highly active marine fish could be used to provide useful indices of stress. Such indices might be found by analysis of plasma electrolytes, blood-borne metabolites and enzymes and haemato~ogical parameters. MATERIALS AND

annual Bay of Islands International Billflsh Tournament off the north-eastern coast of New Zealand (35”1O’S,175”lSE). Gamefish were caught using trolled lures or baits and their capture times and weights were noted from the official tournament records. The principal species sought were billfishes (Fam. Istiophoridae) and the following species were captured: striped marlin (Tetrapturus audax), blue marlin (Makuira nigricarzs) and the black marlin (Makuiru indict). In addition to these, two tuna species (Fam. Scombridae) were captured, skipjack (Katsuwon~ pelamis) and yellowtin tuna (Thunnus albacares), and two species of shark, the mako (Isurus oxyrhinchus) and the blue shark, Prionace gfaucu. Blood was collected into heparinized tubes from the ventral aorta soon after landing. Samples were then placed on ice and whole blood analyses were completed within 2-3 hr. Plasma was separated by ~nt~fugation at 12,000g for 3 min and stored frozen in solid CO? prior to analysis. It was clearly not possible to take blood samples from undisturbed gamefish in order to establish baseline values. With this in mind, eight specimens of an active teleost, the snapper (Chr~sophr~s uuru~a.~), were captured for comparison. Samples were taken in the same way as for capture-stressed specimens, and then the fish were anaesthetized in MS-222 and cannulated via the caudal vein using PE-50 polyethylene tubing. A recovery period of 48 hr was allowed before further samples were taken from unstressed fish and treated as above. Sodium, potassium, iron, calcium and inorganic phosphate were measured in a SMAC Analyzer (Technicon, USA) at a TELARC registered medical diagnostic laboratory. Chloride was measured using a Buchler-Cotlove automatic chloride titrator (Buchler Instruments, USA). Plasma osmolarity was measured using an Advanced Instruments (USA) 3WII osmometer calibrated with freezing-point standards.

METHODs

Metabolites

Fish capture and blood sampkrg

Whole blood adenosine t~phosphate

Blood was sampled from lish captured during the 13th 565

(ATP), lactate and

R. M. G.

566 Table

WELLS et

al.

I. Plasma electrolytes in capture-stressed big gamefish. The number of specimens is given in parentheses and the data are expressed as means + standard deviation where numbers are sufficient

Yellowfin tuna Skipjack tuna Striped marlin Blue marlin Black marlin Blue shark

(8) (I) (3) (1) (1) (2)

Sodium (mM) 233 * 55 217 253 + 25 308 288 309

Mako shark

(2)

276

Potassium (nW 20 f 2 15 8+5 16 9 7

9

Chloride (mM) 173 + 14 156 194+21 278 214 291

Calcium (nW 6.4 & 0.7 5.5 * 1.3 7.3 5.9 4.6

01M) 29k 10 II +I 5 12 17

254

4.8

7

glucose were measured enzymatically with the aid of a Pye-Unicam SP- 1750 U.V. spectrophotometer and Sigma test chemicals (combination kits 366 u.v., 826 U.V. and 15 U.V.respectively). The ATP kit is not specific for the various trinucleotides. Plasma concentrations of cholesterol, urea, uric acid, creatinine and bilirubin were measured by SMAC Analyzer. Enzymes and protein

Plasma enzyme activities and protein concentrations were measured by SMAC Analyzer. These included alkaline phosphatase (AP), alanine ammo transferase (ALT), aspartate amino transferase (AST), g-glutamyl transferase (GGT), lactate dehydrogenase (LDH) and creatine kinase (CK), in addition to albumin and total protein. Haematology

Whole blood haemoglobin (Hb) concentration was estimated from the cyanmethaemoglobin derivative (Dacie and Lewis, 1975) as recommended for fish blood (Cobum and Fischer, 1973). Haematocrit (Hct) values were obtained by centrifuging samples in 75 ~1 microhaematocrit tubes. Red cell counts were estimated using a Neubauer chamber. These data were used to calculate the derived parameters of mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemogtobin concentration (MCHC).

RESULTS

Values for plasma electrolytes in big gamefish are given in Table 1, metabolites in Table 2, enzymes in Table 3, and haematological parameters together with fish wet weights and capture times in Table 4. Table 5 is a compilation of data from eight capturestressed and cannulated resting snapper. Where significant differences are stated, the Mann-Whitney U-statistic was calculated and P < 0.05 accepted as significant. Plasma electrolytes (Table 1)

Plasma sodium, potassium, chloride, calcium, iron and inorganic phosphate concentrations were much higher in captured gamefish than in unstressed snapper and, in turn, values from stressed snapper were

Yellowfin tuna Skipjack tuna Striped marlin Blue marlin Black marlin Blue shark Make shark

(8) (1) (3) (1) (1) (2) (2)

Lactate (nW 15+4 7 26 + 2 24 17 9 13

Cholesterol (mM) 9+2 7 5*1 2 5 I 2

Phosphate (nW 9+2 4 6+2 4 5 18

5

Osmolarity (mOsm) 498 + 21 461 542 + 38 616 607 1090

1063

higher than those from unstressed snapper. The exception was a low plasma chloride in a skipjack tuna, a specimen which also had the shortest capture time (3 min) and presumably the least capture stress. The electrolyte differences were matched by comparable differences in plasma osmolarity. The exceptionally high osmolarities in the sharks relate to their osmotic strategy whereby blood urea is normally maintained at high concentrations (see Table 2). Metabolites (Table 2)

Glucose was higher in stressed snapper than in fish permitted a period of rest and recovery (Table 5). Glucose values were highly variable among the gamefish, but were highest in the black marlin, which also had the longest fight time (408 min). It was further observed that glucose in striped marlin blood decreased rapidly after landing the fish. Blood lactate was high in all gamefish. Lactates were much lower in snapper, but showed the expected decrease during recovery. No consistent pattern of change was Seen in cholesterol, creatinine, bilirubin or uric acid for stressed versus rested snapper, or, with the exception of bilirubin, between snapper and gamefish. Red cell ATP levels were lower in stressed snapper, and even lower in capture-stressed gamefish. An elevated plasma urea was seen in stressed snapper and high levels of both urea and bilirubin occurred in teleost gamefish. Extremely high blood ureas in sharks are a normal feature of their plasma. Enzymes and protein (Table 3)

Total protein and albumin were higher in stressed than in rested snapper, and were also high in gamefish. Alkaline phosphatase (AP) was low and showed no significant difference in stressed versus rested snapper. Very high AP activities were seen in the big gamefish and these showed an obvious positive relationship with capture times (Table 4). Alanine amino transferase (ALT) was significantly higher in stressed than in rested snapper, but other enzymes were highly variable. ALT, AST, LDH and CK showed no consistent patterns in the gamefish.

Table 2. Blood metabolites in capture-stressed Glucose NM) 4.0 f 2.2 5.5 2.9 + 2.7 3.4 20.9 4.5 7.8

h0”

Urea (n-N 1.9f 2.9 1.5 0.7 * 0.2 0.7 1.7 294 322

big gametish Urate (nW 0.1 0.1 0.1 0.1 0.2 0.0 0.2

Creatinine (nW 0.02 0.03 0.03 0.01 0.05 0.04 0.05

Bilirubin (PM) 1.6 4.0 5.0 1.0 1.0 1.0 1.0

2.8 3.5 7.8 6.4 10.7 8.5 10.8

Stress factors in big gamefish Table 3. Plasma enzymes in capture-stressed AP Yellowlish tuna Skipjack tuna Striped marlin Blue marlin Black marlin Blue shark Mako shark

(8) (1) (3) (1) (1) (2) (2)

Table 4. Haematological

Yellowfish tuna Skipjack tuna Striped marlin Blue marlin Black marlin Blue shark Mako shark

G-glutamyl plasma. Haematology

(8) (1) (3) (1) (1) (2) (2)

transferase

lb + 29 2lp: 35 156 362 :9’

parameters,

big gamefish Albumin (g/l)

ALT

AST

LDH

CK

61 *II 142 65+29 99 2 21 3

390&211 203 56*25 178 45 44 534

1958 k2518 344 316 + 176 615 35 210 505

1132f294 1006 428 f I54 494 380 117 380

wet weights

Weight (kg)

Fight time (min)

27.3 f 4.6 5.1 76.7 * 10.8 325 179 104 85

13*4 3 5Ok 18 17 408 15 52

was not

567

detected

and average

in the

(Table 4)

Haemoglobin (Hb) was high in stressed snapper, and fell to lower levels during recovery. With the exception of the blue marlin, which had the shortest capture time among the billfish, Hb and Hct were both very high in teleostean gamefish. An appreciable fraction of the Hb in big gamefish was oxidized with metHb ranging from 13 to 57% of total Hb. MetHb was not detected (< 2%) in either stressed or rested snapper.

DISCUSSION

Physiological stress responses may be considered secondary alterations insofar as primary responses to stress are triggered through afferent sensory inputs resulting in principally endocrine outflow (Wedemeyer and McLeay, 1981; Haux et al., 1985). The physiological responses to severe exercise are activated by the release of catecholamines from the head kidney and it is these secondary responses which are described here. Release of excess catecholamines may produce such direct effects as cardiac lesions in highly stressed cold-blooded vertebrates (Carlsten et al., 1983), but more generalized disturbances occur to metabolism and other regulated processes. Comparison of our data with that from other sources presents several difficulties. Firstly, there are essential differences in osmotic strategy between freshwater and marine teleosts and with elasmobranchs. Secondly, the literature reports some factors as blood values, others as plasma or serum, with the latter occasionally confused. We have interpreted blood as inclusive of the formed elements, plasma as the heparinized acellular fraction and serum as the expressed fluid from clotted blood. Thirdly, because resting values from big gamefish are unobtainable, the basis for comparisons must be extrapolated from values for other smaller species at rest.

capture

Protein (g/l)

21 rt 3 24 17+3 7 IO 4 8

times for capture-stressed

62 + 6 58 bb* 14 19 29 8 30

big gamefish

(%

Hct (“/)

RBC (x 106/Pl)

MCHC (g/l)

MetHb (“/)

189k30 204 137+23 59 103 29 105

74*9 83 55 + 7 19 43 II 34

4.1 f 0.6 5.4 3.6 + 0.8 1.4 3.3 0.23 -

254 + 24 247 249 + I9 311 242 264 318

55 f 36 I3 24 f 6 31 50 51 39

Electrolytes

and water

Capture stress has profound effects on salt and water balance in sea-water fish resulting in elevated sodium, chloride levels and osmolarity (Eddy, 1981). The osmotic stresses imposed on severely exercised freshwater fish tend to differ from those of marine species, with the former tending to suffer water inflow, and the latter a water outflow (Love, 1980). Serum osmolarity increases following capture of marine teleosts (Umminger, 1970) and in sharks for up to 3 hr post-capture (Cliff and Thurman, 1984). The effects of capture stress in teleosts induces adrenaline release which, in turn, has a marked influence on branchial and erythrocyte ion fluxes (Eddy, 1981; De Vries and Ellory, 1981). It appears that in stressed elasmobranchs water shifts out of the vascular compartment in response to either raised intracellular

Table 5. Plasma chemistry and blood factors in snapper following capture stress and in cannulated snapper during recovery (N = 4) Rested Sodium (mM) Potassium (mM) Chloride (mM) Calcium (mM) Iron (JIM) Phosphate (mM) Osmolarity (mOsm) Glucose (mM) Lactate (mM) Cholesterol (mM) Urea (mM) Urate (mM) Creatinine (mM) Bilirubin @M) ATP (pM/gHh) AP (U/l) ALT (U/l) AST (U/l) LDH (U/l) CK (U/l) GGT (U/l) Albumin (g/l) Total protein (g/l) Hh (g/l) *Differences

significant

200.8 + 4.5 4.3 & 0.5 190+10 2.5 + 0.2 2.6 f 0.2 2.3 f 0.7 381 + 53 4.4 rt 0.4 0.2 f 0.07 2.4 + 0.4 0.7 + 0.39 0.03 + 0.01 0.01 * 0.00 0.5 + 0.6 26.5 k lb.5 20.0 f. 12.3 1.5 & 2.4 135.3 + 130.0 1553 + 1498 2467 + 2308 3.5 + 2.4 5.0 & 0.8 II&2 47.7 * 8.8 at P < 0.05.

Stressed 219.6 f 7.1’ 5.3 f 1.4 2OOk 17 2.8 + 0.5 3.7 + 1.2 4.0 f 2.2 492 + 143 13.7 + 5.8’ 0.810.1’ 2.4 +O.l 1.95 *0.51* 0.06 + 0.03 0.02 + 0.01 0.5 + 0.6 19.1 * 11.2 17.5 + 9.3 37.5f 1I .2* 301.8 + 205.4 2149 i 1833 4lOOFb50 1.3 +0.5 1.5 + 0.6. 23i l* 119.6 + 29.6’

568

R. M. 0. WELLS et al.

lactate or increased sodium influx into the blood (Piiper et al., 1972). Plasma sodium, chloride and potassium rise in severely exercise-stressed flounder (Wood et al., 1983) and similar observations are known for other fish (Haux et al., 1985), including elasmobranchs (Martini, 1974; Cliff and Thurman, 1984). Potassium levels seem to persist in these stressed fish, but in the absence of haemolysis the K+ is likely to be of intracellular (muscle) origin. Potassium e8Iuxes are stimulated by catecholamines (Boume and Cossins, 1982). Oikari and Soivio (1975) reported that K+ decreased in aged samples, especially in catecholamine-primed cells. Further evidence that the prolonged elevation in plasma K+ is due to intracellular acidosis was given by Turner et al. (1983). What are the effects of raised plasma potassium? Myocardial function is disrupted when K+ rises above 7 mM/l (Martini, 1974). Significantly, Fiinge (1976) found that the potassium levels were highest in the plasma of most active species (e.g. mackerel) and lowest in benthic inactive species (e.g. Lophius). Values of > 7 mM/l were obtained in all our captured big gamefish, but not in the snapper. The implication is that hyperkalaemia may be detrimental, or even a proximal cause of death. There is little information about calcium regulation in marine teleosts. The range of total plasma calcium was 4.6-7.3mM/l in gamefish, appreciably higher than average values of around 2.6mM/l in cod (Bjomsson and Deftos, 1985), or the 2.5-2.8mM/l found in either stressed or unstressed snapper. It has been suggested that elevated calcium and catecholamine levels protect the teleostean heart from acidosis which might result from stress (Farrell, 1984). In general, electrolyte levels determined in a range of marine species (FHnge, 1976) showed similar values to those obtained in our study, though those of the former study are probably from stressed fish. Rapid changes occur in the electrolyte composition of even mildly stressed fish (Rail0 et al., 1985), which suggests that capture stress has profound effects on salt and water balance in marine fishes. Metabolites The level of glucose in blood has been a useful indicator of stress (Love, 1970, 1980, Wedemeyer and McLeay, 1981; Swift, 1983). The rise of blood glucose in stressed snapper was consistent with the high values in game&h. Blood glucose in Pkureuectes rose to 5 mM/l following capture (Wardle, 1972). Similarly, in other teleosts, when physical activity exceeds what is normal for the species, glucose levels rise (Eddy, 1981; Swift, 1983). These findings are closely matched by glucose elevation in stressed elasmobranchs (Mazeaud ei al., 1977; Cliff and Thurman, 1984). The release of glucose appears to be promoted by rising circulated catecholamines (Patent, 1970; De Roos and De ROOS, 1978). Capture-stressed sharks which did not survive had very low blood glucose (Cliff and-Thurman, 1984) which is consistent with our data for gamefish. The glucose mobilization appears extremely rapidly, well before the lactate peak is manifested (Wardle, 1972). The appearance of lactate in the blood following

severe stress takes some time to peak (Wood et al., 1977), in fact several hours in elasmobranchs swum to exhaustion (Piiper et al., 1982) and the actual values attained may reach 15 mM/l or more in elasmobranch and teleostean gamefish (Table 2, Wells and Davie, 1985) as well as in less active fish (Jensen et al., 1983). Whether the lactate load is a potentially lethal flood or whether it is released into the circulation because the fish is dying has received some comment. Wood et al. (1983) suggested that it is the intracellular acidosis which is lethal, not the lactate anion per se, and that the greatest mortalities occur some hours after activity. These ideas remain to be tested in big gamefish, though it is interesting to observe that the black marlin had low lactate levels even after a capture time of several hours. Cholesterol and other lipids appear to vary considerably with season and reproductive status of fish (Deb et al., 1983), and are higher in active, fastswimming teleosts (Fiinge, 1976). Bourke (1983) found that serum cholesterol was higher in stressed skipjack tuna, but we found little evidence for this in snapper, or particularly high values in gamefish, including the single skipjack tuna. Bilirubin is normally excreted at a constant rate by liver cells, and severe liver dysfunction might be indicated by altered values of the metabolite. High levels were found in gamefish plasma. Similarly, raised serum creatinine is indicative of decreased glomerular filtration rate. Creatinine levels were similar in the plasma of rested and stressed snapper and did not differ substantially in the gamefish, providing no clear evidence for renal failure.. Raised urate indicates purine catabolism, chiefly from liberated nucleoprotein. There were no high levels in our study, although the levels was in fact doubled in stressed snapper. Nucleoside triphosphates (NTP), including ATP, are implicated in the preservation of dorsal aortic oxygen content following strenuous exercise, by favouring the dissociation of bound oxygen from haemoglobin (Nikinmaa et al., 1984). The time course for rising ATP was slow in a freshwater teleost (Jensen er al., 1983), but occurred rapidly in a marine teleost (Ling and Wells, 1986a). These changes were apparently related to the preservation of oxygen uptake in the gills during stress, when erythrocyte ATP levels were decreased, thereby increasing blood-oxygen affinity. No significant changes to NTP were seen in severely stressed skipjack tuna (Bourke, 1983) or in our snapper. In more severely stressed gamefish, possible alterations in ATP could not be assessed because organic phosphate levels are highly species-specific, and resting values could not be obtained in big gamefish. Enzymes and protein Among the range of proteins found in vertebrate plasma, the albumins account for most of it and play an important role in maintaining colloid osmotic pressure. Albumin values were clearly higher in gamefish, though lying within the range of plasma values obtained from other teleosts which were most likely under some stress (Poluhowich and Parks, 1970) and from elasmobranchs (Urist and van de Putte, 1967). The snapper data showed a stress-

Stress factdrs in big pmcfish related increase in plasma albumin and total protein. An increase in plasma protein was also noted for fish following exhausting exercise, which indicated haemoconcentration (Wood et al., 1983), although lowered colloid osmotic pressure was noted in marlin with long capture times (Hargens, 1984). The total protein fraction includes, in addition to albumin, a remarkable range of enzymes which serve no obvious function in the plasma. They are presumably intracellular in origin and derive from either leakage from cells or from degraded cells. Several pathological conditions are accompanied by marked changes in the levels of certain plasma enzymes and have proved useful in a number of higher vertebrates where specific organ damage may be ascertained. A raised activity of serum alkaline phosphatase (AP) is generally associated with liver dysfunction and has been reported in fish which appear stressed (Urist and van de Putte, 1967; Cvancara and Conte, 1970). No significant changes in AP activity were observed in severely stressed skipjack tuna (Bourke, 1983), or in our snapper (Table 5). By contrast, high plasma activity suggested that tissue damage had occurred in severely stressed big gamefish. AP might therefore be a useful plasma index of extreme stress. Creatine kinase becomes elevated in the serum of capture-stressed sharks (Rasmussen and Rasmussen, 1967; Cliff and Thurman, 1984). The rise is indicative of increased muscle permeability or loss of integrity of skeletal or cardiac muscle. Increased values were noted in stressed snapper, but values for severely stressed gamefish were not especially high. LDH plasma values may also indicate damage to contractile tissue. An exceptionally high catalytic rate by LDH is characteristic of the fast twitch muscles of tuna (Mommsen, 1984), and thus repeated and exhausting attempts to escape capture may have resulted in the high levels recorded for big gamefish. Alanine aminotransferase (ALT) has a high activity in both fish gills and in cardiac tissue (Mommsen, 1984), where it is involved in oxidative pathways. Thus the presence of ALT in stressed snapper and in gamefish plasma may indicate disruption to both gill and cardiac tissues. Aspartate aminotransferase (AST) is also involved in oxidative reactions with the liver containing a rich source of the enzyme in marine fish (Casillas et al., 1982) and it apparently shows a stress-related increase in piasma activity. It is worth noting that the range of serum aminotransferase activities in fish, and the conditions for its optimal assay are remarkably close to those for mammalian systems (Casillas et al., 1982), which points to the potential value of serum enzyme determinations in assessing stress and organ-specific damage in fish. In general, the serum enzyme activities of rested trout (cJ Hille, 1982) broadly conform to the resting snapper values obtained in this study, and encompass the lowest values of stressed gamefish. There appear to be some grounds for suggesting that extreme capture stress is manifested by high absolute values of enzyme activity. The marlin data show a consistent pattern of response in plasma enzymes. The black with the longest capture time (6-7 hr) had the lowest measured ALT, but the highest AP; it also had lower CK and LDH than the other marlin. By contrast, the blue with the shortest capture time (17 min) had the

569

highest ALT, lowest AP and higher CK and LDH. The striped marlins, with a mean capture time of 50min, are intermediate. These physiological responses may be the result of different strategies of fish behaviour when captured. Haematblogy The concentration of blood cell constituents in response to stress is well known and may result either from loss of plasma or from the release of cells from the spleen (Turner et al., 1983). Among highly stressed fish, survivors show a brief haemoconcentration (Wood et al., 1983). Haematocrit (Hct) measurements, which are often used as a measure of blood oxygen carrying capacity, may increase in response to stress at a different rate than that of the haemoglobin that the erythrocytes contain. These observations of stress-induced swelling are known for several fish and appear to be mediated by circulating catecholamines (Soivio and Nyholm, 1973; DeVries and Ellory, 1981; Nikinmaa and Heustis, 1984; Railo et al., 1985; Ling and Wells, 1986b). It is to be expected that active, pelagic species will have higher haematocrits than those in less active, demersal fish. Big gamefish going about their daily business will undoubtedly have a relatively high Hct, but the extraordinarily high values noted in Table 4 must present a great resistance to perfusion and thus possibly compromise exchange in the gill epithelium. Values of ~60% have been reported for two species of tuna (Alexander et al., 1980), but whether these values approach routine values is unknown. Despite the high Hb concentrations in stressed gamefish, a large proportion of it was oxidized to metHb (Table 4) and if the situation existed in viva then an appreciable fraction of the Hb is unavailable for oxygen transport. It was once thought that reptiles possessed high concentrations of metHb in vivo but it was subsequently shown that the oxidized derivative was not characteristic of healthy, unstressed animals (Gruca and Grigg, 1980). The reductase system responsible for maintaining haem proteins in the reduced ferrous state is particularly active in yellowfin tuna, which have unstable, rapidly autoxidizing haem proteins when compared with those of other vertebrates (Levy et al., 1985). Low blood pH in stressed gamefish (see Wells and Davie, 1985) is a condition which would be expected to enhance metHb formation. CONCLUSIONS

The black marlin had high glucose and, according to Cliff and Thurman (1984), would probably have a better survival chance if we could extrapolate. The black clearly worked aerobically against the angler in order to survive for 408 min. It appeared to have retained control of haematocrit and osmolarity. A long capture time may thus be associated with maintenance of blood and aerobic tissues. The knell of the black may have been sounded by liver failure as evidenced by the high AP value, or through renal failure (raised urea and creatin). The blue marlin, on the other hand, showed aerobic tissue failure (high ALT, CK and LDH) and had a lower blood Hb and Hct. This fish sustained an apparently anaerobic fight

510

R. M. G. WELLS et al.

and in doing so compromised the blood, which led to the failure of oxygen-dependent tissues. The striped marlin, as shown by their capture times, showed intermediate enzyme levels, [email protected] a combination of aerobic and anaerobic responses. Comparing our tuna data with those of Bourke (1983), whose capture times were in the order of seconds (pole and barbless hooks), then blood and aerobic tissues were certainly compromised. Perhaps the viscosity of blood with Hct of the order of 80% simply does not permit circulation. In this case the skeletal enzymes would not appear in blood to be sampled from the skipjack, given a circulation time of - 1 min, but, as expected, appear in the plasma of the yellowfln tuna with slightly longer capture times. Of the plasma electrolytes, potassium appeared to be a potentially useful indicator of handling and capture stress in fish, because its elevated concentration persists after other electrolytes decline. Extremely high concentrations may ‘cause cardiac dysfunction and be a proximal cause of mortality in extreme stress. Plasma enzymes promise to be a useful indicator of extreme stress and may give specific information about organ dysfunction. Useful indicators might be alkaline phosphatase, aminotransferases and possibly lactate dehydrogenase. Specific differences in the plasma enzymes may be the result of different strategies of fish behaviour during capture. Haematological indices, particularly Hct and the derived parameter MCHC, are useful when the haematology of the species is known in the absence of stress but, in other cases, the interspecific variation is too high to provide a useful index. Acknowledgements-We

are grateful to the Auckland University Research Committee for funds through project grant Nos. 141-Z-85 and 141-Z-168. P.S.D. gratefully acknowledges support from the NZ Big Game Fishing Council, and we thank the Bay of Islands Swordfish Club for their cooperation. The Pacific Gamefish Research Foundation is acknowledged for logistic support. N. Ling and A. Jerrett kindly assisted with capture and maintenance of snapper.

Casillas E., Sundquist J. and Ames W. E. (1982) Optimization of assay conditions for, and the selected distribution of, alanine aminotransferase and aspartate aminotransferaae of English sole, Paruphrys vetulus CS-arCt.J. F&It Blol. 21, I97-204. Cliff G. and Thurman G. D. (1984) Pathological and physiological effects of stress during capture and transport in the juvenile dusky shark, Carcharhimcs obscurus. Comp. Biocbem. Physiol. ISA, 167-173. Coburn C. B. and Fischer B. A. (1973) Red blood cell haematoloav of fishes: a critique of tecbni~ues and a compilation of published data. J. mar. Sci. i, 37-58. Cvancara V. A. and Conte F. P. (1970) Gill alkaline phosphatase activity during salt water’ adaptation of sockeye salmon (Onchorhynchus nerku) Walbaum. Int. J. Biochem. 1, 597-604. Dacie J. V. and Lewis S. M. (1975) Practical Iiaematology. Churchill Livingstone, London. Deb S., Mukherjee D. and Bhattacharya S. (1983) Interrelationship between plasma and ovarian choksterol in a tekost fish. Experientia 39, 427428. De Roos R. and De Roos C. (1978) Elevation of plasma glucose levels by catecholamines in elasmobranch ftsh. Gen. camp. EndocrinoI. 34,447--4X2. DeVries A. L. and Emory J. C. (1981) The effect of stress on ion transport in fish erythrocytes. J. Physiol. 324, 5 1. Eddy F. B. (1981) Effects of stress on osmotic and ionic regulation in fish. In Stress and Fish (Edited by Pickering A. D.), Ch. 4, IJD. 77-102. Academic Press, London. FHnge R. (1976).T% internal environment of marine fish. In Comparative Physiology: Water, Ions and Fluid Mechanics

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