Cmnp. tlmhum. P/IJwJ/.. 1976, Vol 55A. pp. 329 to 336. Peryumm Pr~.~ss. Pnrirrd in Grmt Brliain

INFLUENCE OF ANOXIA ON THE ENERGY METABOLISM OF GOLDFISH CARASSIUS AURATUS (L.)

GIJ~DO VAN DEN THILLART, FANJA KESBEKE AND AREN VAN WAARDE

Zoological Laboratory, Department of Animal Physiology, State University of Leiden, Kaiserstraat 63, Leiden, The Netherlands

(Receirred 1 March 1976)

Abstract-l. Goldfish has a large anoxia tolerance. At 20°C 10 hr of complete anoxia can be survived without any problem. The concentration of some metabolites were determined in dependence of anoxia, both in whole fish and dorsal white muscle.

2. Lactate production does not cover the energy need. 3. Apart from an increase in lactate level we were able to demonstrate an increase in concentration

of alanine, creatine, total CO, and ADP. 4. Concentrations of ATP and creatine-phosphate decrease with anoxia while AMP, glutamate and

volatile fatty acids did not change significantly. 5. The “energy charge” in the white muscle falls during hypoxia from 1.0 till 0.8 and stabilizes

at that level during about 10 hr although the direct energy reserves of ATP and creatine-phosphate decrease.

INTRODUCTION

Energy metabolism of goldfish is mainly described by oxygen consumption: Fry & Hart (1948), Smit (1965), Blaika (1958), Beamish (1964), and carbon dioxide production: Kutty (1968). Although Blaika described the anoxia tolerance of crucian carp, a species from the same genus as goldfish, hardly any research was done on anoxia tolerance since. As described below, hypoxia effects on metabolism has had some atten- tion. Oxygen debt such as described by Brett (1972) for trout could not be demonstrated in goldfish (Smit et al., 1971), thus demonstrating that anaerobic glyco- lysis in goldfish is strongly depressed under physio- logical normal conditions. In carp (Cyprinus carpio) a closely related species, lactate accumulation was demonstrated by Wittenburger & Diaciuc (1965) after severe exercise under stress condition. Swimming goldfish without stress conditions do not build up an oxygen debt, as was indicated by Smit et al. (1971) in our laboratory. Nevertheless it was possible to demonstrate that resting goldfish could produce an oxygen debt, but only after a long period of anoxia. In those experiments, which will be published else- where it was found that the decrease of accumulated lactate was correlated with a decrease of oxygen debt. Heath & Pritchard (1965) have clearly demonstrated that there is not a one to one relationship between oxygen debt and lactate decrease as is generally assumed. They found, that the period of oxygen debt after hypoxia is always longer than the period of in- creased lactate concentration, indicating that there are other processes involved. Several intermediary metabolites have been shown to change in concen- tration, Heath & Pritchard (1965) described impor- tant changes of glycogen and glucose. Mazeaud (1969) showed a remarkable increase of glucose and a de- crease of fatty acids in carp-blood after anoxia. The most striking but poorly understood phenomenon in fish anaerobiosis is that the CO2 and NH, produc- tion is independent of OX availability (Kutty 1968,

1972). Hochachka et al. (1973) proposed several alter- native energy-producing anaerobic pathways with endproducts: propionate, succinate and alanine. These reduced products were found in the everte- brates Ascaris (Awapara & Campbell 1964) and Mytilus (de Zwaan 1971; Kluytmans et al., 1976), but proved to be quantitatively unimportant in fish. Although Blaika (1958) described a production of volatile fatty acids during anoxia in carp and trout, Burton & Spehar (1971) and Driedzic & Hochachka (1974) could not confirm his results. In all investiga- tions lactate seemed to be the most important end- product, although succinate and alanine increased sig- nificantly (Johnston 1975; Driedzic & Hochachka 1974).

Fish are able to consume their own structural pro- teins (Johnston & Goldspink 1973), it is also known that they have large quantities of free amino acids and other nitrogen containing metabolites like crea- tine and trimethylamine oxide (Shewan 195 I ). Although these substances are important in osmo- regulation, it is suggested that they must have other physiological functions. The position of these metabo- lites in energy metabolism has to be established.

To get a more accurate impression of the anaerobic energy metabolism we investigated the energy state, as reflected by the concentrations of ATP and crea- tine-phosphate, and some possible anaerobic endpro- ducts like lactate, alanine and volatile fatty acids.

MATERIALS AND METHODS

Coditioniny

Experiments were performed in summer 1975 with 3 yr old goldfish (+lOOg), acclimated at 20°C and 16 hr-light period. Oxygen saturation in the tanks was SCrlOO%. Minimal 24 hr before each experiment one goldfish was placed in a 61 respirometer (Fig. 1). Anoxia-experiments were carried out during the night in order to minimize activity. Experiments were initiated by switching off the feed-back control of the oxygen monitor. Thus the oxygen

329

330 Gums VAN DEN THILLART, FANJA KESBEKE AND AREN VAN WAARDE

RESP,ROMETER FOR ONE FIS”

I ~_____ 0, meter

-control -record

WB I _ Fig. 1. Water in respirometer (R) circulates by action of pump (Pl) and flows through an oxygen probe (E). The oxygen probe is temperature compensated and connected with an E.I.L. oxygen monitor type 9401. When the oxygen concentration fails below a desired level the magnetic valve (M) is activated. Saturated water flows in from container (0) till level is reached. Outflowing water is saturated by air diffusers and circulates via pump (P2) and container (0). Water is thermostated by a 40 I Tamson waterbath (WB) at 20 + 05°C and filtered through a carbonfilter (F).

saturation level decreased from 60 to OT/, within 1; hr. After a desired period of anoxia the experiment was terminated by anaesthetizing the fish with 100 ppm MS 222 (Tricaine- methanosulfonaat-Sandoz). Anaesthesia was always reached without disturbing the fish by injecting solubilized

MS 222 through the outflow hole. As a result of some preliminary experiments it was found necessary to follow this procedure to obtain reliable and reproducible results.

Both dorsal white muscles were excised as fast as poss- ible. After decapitation the dissection of the fish including following extractions were carried out at 2°C. While the remainder of the fish was ground in liquid NZ, one dorsal white muscle was extracted in 2 vols 67, HCIOl and the other in 9 vols 200 mM NaHCO, (pH 9.5).

The perchloric acid extract was neutralized with 30”~,, KOH to pH 7.5 within 15 min to protect the acid unstable phosphoryl groups, Control experiments with ATP and creatine-phosphate proved to be satisfactory (deviation <23<). Extracts were centrifugated 30 min at 20,000 9.

Determination qf intermediates

After grinding in liquid N, the remainder of the fish was extracted in 2 vols 6% HCIO+ Special precautions were taken to trap the liberated CO* in Ba(OH), solution (Fig. 2). The perchloric acid extract was neutralized with 30”; KOH and centrifugated 30min at 20,OOOg.

Neutralized (perchlorate) extracts were used directly to determine the concentration of the phosphorylated com- pounds ATP, ADP, AMP and creatine-phosphate. In the freeze-dried extracts were determined: creatine, alanine, glutamate and lactate. With the exception of lactate, according to Gercken (1960), all other compounds (ATP, ADP, AMP, alanine, glutamate, creatine-phosphate) were measured enzymatically according to the methods de- scribed in “Methoden der enzymatischen Analyse” (Berg- meyer 1971). One modification on the ADP-determination

was performed. The first ATP-producing reaction was done with pyruvate kinase and phosphoenol-pyruvate in- stead of with a combination of creatine-kinase and crea- tine-phosphate. This reaction had the following practical advantages: low cost, 100% yield, very fast, easily per- formed.

Volatile &try acids

Volatile fatty acids were extracted from 30 ml NaHCO, extract by the method of Mahadevan & Zieve (1969) modi- fied according to Kluytmans et al. (1976). After steam dis- tillation and freeze-drying the remainder was dissolved in 0.50 ml 25:< H3P04. On a column packed with 20% PEGA + 2% HjPOl placed in a Becker-Unigraph F 407 gaschromatograph with flame ionization detection, 1.0 ~1

of the H3P04 solution was injected. Quantification was achieved with the external standards: propionate, butyrate, acetate, valerianate and iso-butyrate in 25% H3P04.

RESULTS

Some details about the experimental fish are given in Table 1. Weight loss due to dissection was caused by moisture attached to plastic sheets. Total weight of control and anoxia group was almost the same with S.E. of about 10%.

The periods of anoxia were calculated by extrapola- tion (Fig. 3). Oxygen consumption remained constant between 60 and 20% saturation but decreased below 20% as a result of decreasing oxygen-gradient across the gill membranes. Thus the oxygen concentration will slow down asymptotically till zero. Therefore the onset of anoxia is hard to determine.

By extrapolating the oxygen consumption there is a point of time t, at which the oxygen saturation would have become zero. But due to the physiological restrictions, which lead to O2 transport problems, O2 consumption decreases (Hughes 1973). Point tI can easily be found in contrast with tl. We could state that at tr the aerobic energy production should have ended. The oxygen consumption calculated from the initial concentration decrease varied from 15 to 20 mg O,/lOOg/hr, which was equal to the mean day level but higher than night level (8-10 mg O,/lOOg/hr). Obviously fish were disturbed slightly during decrease of oxygen level, though at low levels the animals were almost inactive and rested at the bottom without

e IR FLOW

I 2 3 4 5

Fig. 2. Determination of CO2 in whole goldfish. After grinding a fish in liquid N, (-18O”C), the material was transferred to a 400ml Sorval omnimix stainless steel beaker. The beaker is modified as indicated with two air- flow connections. Flasks 1, 2 and 3 are filled with 0.010 M Ba(OH), solution covered with pentane. Flask 4 is filled with 200 ml 6% HClO., and flask 5 with 100 ml 30% KOH. While the Ba(OH)* solution was stirred with 3 magnetic stirrers, lOOm1 HC104 was added fast to the frozen fish. The mixer was turned to 10,000 rev/min and the remainder (1OOml) of the HC104 was added slowly (~lOml/min). When all HClO, was added, the airflow was continued with a higher speed (f20ml/min. One hr after the first addition of HC104 the air flow was stopped and the trapped CO2 determined by titrating the Ba(OH), solu- tions with O.lOM HCI, using thymol blue as indicator.

Influence of anoxia 331

Table 1.

Fish No.

controls l-h& r.7

7 8 9

10 I1 12 13 14 x*cr

0,’ / ” anoxia weight weight loss pXiOd

94.75 * 12.28 3.2 I: 1.9 .-_ -.

103.35 1.8 6 hr 30 min i14.15 3.3 8 hr 50 min 91.82 2.0 8 hr 40 min

t06.6I 1.4 12 hr 35 min 93.89 1.9 Ohr 15min 91.46 2.3 6 hr 10 min 90.89 I.9 6 hr 25 min

109.25 I.4 I.3 hr 30 min 100.18 i 9.26 2.0 + 0.60 7 hr 52 min

opercular movements. After 12 hr 35 min of anoxia fish No. 10 had lost equilibrium and fish No. 14 was dead after 13 hr 30min. All other fishes were able to swim even after 8 hr 50 min of anoxia.

In Fig. 4 and 5 the influence of anoxia on some metabolites in dorsal white muscle is presented. The increase of the creatine concentration +7 ,U moles/g

hours without uxygen supply

Fig. 3. Decrease of oxygen-saturation in the respirometer. Explanation see text.

is probably correlated with a decrease of creatine- phosphate. The lactate level increases continuously from 6 to 14 p moles/g and is obviously much more important than the increase of alanine from 2 to 4.5 1~ moles/g. From the phosphorylated compounds (Fig. 5) AMP has the lowest level, almost zero. and ADP gives the most striking results. The rise of ADP from 0.2 till 3.6 is important in stabilizing the energy charge. The energy charge presents the amount of phosphorylat~d alanine nucleotides, which is given by Atkinson (1968) in the formula ATP + 4 ADPi ATP + ADP + AMP.

The energy charge is the most important regulation factor in energy metabolism. Therefore it is an impor- tant fact that the energy charge stabilizes during anoxia at a high level. The high energy compounds ATP and creatine-phosphate decrease during anoxia from 10 and 8 to 2 and I p moles/g respectively.

In Fig. 6 and 7 the anoxia dependency is presented for the same metabolites in whole goldfish. Though concentrations of metabolites in whole fish and in muscles are different the changes due to anoxia show

Od c 0 5 I

10 15 hours of anoxia

Fig. 4. Influence of anoxia on some metahohtes in dorsal white muscle. All metabolites are determined en~maticaiIy in neutralized perchloric acid extracts of goldfish white muscle. Fish were anaesthetized with 1OOppm MS 222 and killed by decapitation. The concentrations are given in pmoles/g wet wt. The first point indicated as c, is the mean of the control group (Table 2). o creatine (cone 2 x scale

value), &. lactate, q alanine, + glutamate.

332 Gum VAN DEN THILLART, FANJA KESBEKE AND AREN VAN WAARDE

hours of anoxia

Fig. 5. Influence of anoxia on phosphorylated compounds in dorsal white muscle. All metabolites are determined enzymatically in neutralized perchloric acid extracts of goldfish white muscle. Fish were anaesthetized with 100 ppm MS 222 and killed by decapitation. The concentrations are given in pmoIes/g wet wt. The first point indicated as c, is the mean of the control-group (Table 2). l energy charge (no dimension, 0,l x scale value), 0 ATP, n creatine-phosphate, 0 ADP, + AMP.

the same tendency. The creatine and alanine levels increase almost exclusively during hypoxia, while the lactate concentration increases continuously. The glu- tamate concentration however hardly changes. The concentration of the phosphorylated compounds ATP and creatine-phosphate tend to decrease slowly with anoxia, while ADP has a remarkable rise in concen- tration during hypoxia. As could be expected concen- trations of creatine-phosphate and the adenine nu- cleotides ATP and ADP are much higher in white muscle than in whole fish. However AMP concen- tration in muscle is found rather low and creatine- phosphate in whole fish rather high.

To compare anoxic with normoxic fish the means of observed values with standard errors are tabulated in Table 2. The probability of differences was tested according to Student’s t-test. Confidences >95”<, (P < 5”;;) are found in nearly all cases. Only AMP

and acetate levels proved not to change significantly. From Table 2 and Fig. 6 we can see that the sum of ATP. ADP and AMP decreases with anoxia indi- cating that adenine nucleotides disappear. Comparing muscle with whole fish, we deduce that in muscle. lactate and alanine levels increase 2 x , while in whole fish the levels increase 3 x as a result of anoxia. While concentrations are higher in muscle these sub- stances diffuse possibly to other parts of the body.

In the remaining fish there is a remarkable increase of total CO2 from 23 to 31 p moles/g (Table 2). In preliminary experiments we found however, in dorsal white muscle from normoxic goldfish < 10 p moles/g which is about 5”; of total CO*.

Volatile fatty acids from the polar phase of a CHCl,-MeOH-H,O extraction could be detected in minimal quantities in the white muscle. Acetate did not change, propionate decreased, other volatile

15 hours of anoxia

Fig. 6. Influence of anoxia on some metabolites in whole goldfish. All metabolites are determined enzymatically in neutralized perchloric acid extracts of whole fish. Goldfish were anaesthetized with 1OOppm MS 222, killed by decapitation and ground in liquid N,. 0 creatine, A lactate. q alaninc.

+ glutamate.

Influence of anoxia 333

Fig. 7. Influence of anoxia on phosphorylated compounds in whole goldfish. All metabolites are deter- mined enzymatically in neutralized perchloric acid extracts of whole fish. Goldfish were anaesthetized with 100 ppm MS 222, killed by decapitation and ground in liquid N,. 0 ATP, A creatine-phosphate

(concentration 25 x scale value), 0 ADP, + AMP.

fatty acids were not found. Total lipid content of white muscle decreased significantly as obtained by evaporazing the apolar phase and vacuum drying above PzO,.

DISCUSSION

In energy metabolism the “energy charge” is a very important regulation factor. We have seen that this factor stabilizes in goldfish white muscle at 0.81 dur- ing anoxia. The concentrations of ATP and creatine- phosphate however decrease continuously. Therefore AMP or ADP would increase, but as shown in Table 2 the total concentration of ATP, ADP and AMP decreases during anoxia. Most likely inosine-mono- phosphate (IMP) is the substance into which all adenosine-compounds are converted. This IMP has long been known to accumulate in fish not only after death (Murray & Jones, 1957; Saito et al., 1959; Now- lan & Dyer, 1969) but also after severe exercise (Jones & Murray, 1960; Fraser et al., 1966). See Table 3.

The accumulation of IMP is possibly by action of the enzyme AMP-deaminase, which catalyses the reaction AMP + HzO+ IMP + NH,. This enzyme has been described in carp, mackerel and cod muscle (Dingle & Hines, 1967; Hidaka & Saito, 1960) and proved to be a highly active enzyme ( f 400 pmoles/ min/g). The physiological significance of IMP-accu- mulation however was described by Tomheim & Lowenstein (1972) in rat muscle. They found two antagonist enzymes AMP-deaminase and AMP-syn- thetase regulated by the energy charge. At high energy charge AMP was produced and at low energy charge AMP was converted into IMP. Of course NH, pro- duction have an important additional effect in neutra- lizing lactic acid. Fraser et al. (1966) described NH3 production in cod white muscle during severe exer- cise. They found IMP production almost equalizes the NH,-increase. Probably this could also explain the increase in NH,-production during anaerobiosis as described by Kutty (1972).

Table 2.

Compound nmoles/g

dorsal white muscle whole fish X+Cl Xi0 P* X&a x+0 control anoxia 0,

/0 control anoxia

ATP 10.22 * 1.37 4.79 + 2.58 <0.05 1.19 + 0.32 0.74 & 0.41 <2.5 ADP 0.21 & 0.24 2.57 k 1.08 <0.05 0.12 + 0.16 0.40 & 0.19 <I.0 AMP 0.01 * 0.01 0.07 * 0. I I > IO 0.05 * 0.09 0.11 + 0.18 >I0 creatine-phosphate 8.31 k 2.34 3.15 + 2.02 <0.05 2.55 k 0.92 1.27 k 1.18 <2.5 creatine 21.8 k 1.7 28.0 k 2.2 <0.05 9.48 + 2.33 11.78 f 1.21 < 2.5 alanine 2.08 k 0.79 4.10 + 0.71 <0.05 0.50 * 0.35 1.76 + 0.52 < 0.05 glutamate 0.71 * 0.21 1.14 + 0.34 1 .o 1.31 * 0.13 1.55 k 0.26 <5 lactate 5.82 k 1.42 12.14 k 4.06 <OS 2.45 k 0.46 7.30 f 1.60 <0.05 HCO;t 23.4 & 5.74 31.0 + 7.12 2.5 total lipid** 34.8 + 18.9 16.26 + 5.46 <2.5 acetate 0.48 + 0.51 0.31 * 0.21 > 10 propionate 0.48 + 0.54 0.08 i 0.08 <5 energy charge 0.99 & 0.01 0.82 _t 0.12 <0.5

* P: single tail probability, tested according to Student’s t-test. ** Total lipid in mg/g. t CO1 determined in remainder of fish.

334 Gumo VAN DEN THILLART, FANJA KESBEKE AND AREN VAN WAARDE

Table 3.

ATP [I moles/g

ADP AMP IMP energy charge

* rat muscle rested 3.03 0.41 0.12 working 1.83 3.10 0.04

t codling muscle rested 5.34 0.58 0.69 exhausted 0.26 0.43 0.57 postmortem 24 hr at 22’C 0.10 0.20 0.60

$. trout muscle rested 3.53 1.04 0.42 postmortem 24 hr at 0°C 0.17 0.35 0.14

$ seabass rested 11.73 1.80 0.84 postmortem 24 hr at 22°C 0.17 1.38 4.54

* goldfish rested 9.45 4.34 0.01 white muscle 15 hr anoxia 3.09 3.23 0.32

0.91 0.65

1.26 0.x5 5.86 0.38 4.30 0.22 3.36 0.81 6.09 0.63

0.88 __ 0.14

0.70 0.85 3.20 0.11

* Eddington er ul., 1973; t Murray et al., 1957; t Saito ef al., 1959; 5 Nagayama 1961; c Van den Thillart et NI., (unpublished).

Table 4.

Estimated oxygen stores in goldfish (1COg) mg Oz

swimbladdcr, 9?d Oz,* total gas 70/, 0.57 blood, 12.5 rnl~0~~~~ ml, SO:,;, saturated 0.47 myoglobin, 30% muscle weight, 1.5 ml O,/lOO g 0.60 tissue water, 60”,/, of total weight, 0.5 ml OJlOOg 0.39

2.03

*Oxygen content of goldfish swimbladder was deter- mined in preliminary experiments with Fry’s gasanalysator (9.2 + 3.94; 0,).

Lactate and alanine accumulate during anoxia indi- cating these are end products in anaerobic metabo- lism. Glutamate did not accumulate. In working mam~lian muscle glutamate is consumed and alanine is produced (Felig & Wahren, 1971). Because of the concentration differences of alanine and gluta- mate between muscle and whole fish it is highly prob- able that alanine flows out and glutamate flows into muscle tissue. In uitro experiments in rat muscle by Ozand & Tildon (1973) proved that alanine forma- tion was independent of glycolysis. Thus indicating anaerobic alanine formation did not follow normal glycolysis.

As mentioned in material and methods, anaesthesia was necessary to depress glycoIysis. Fraser et al. (1966) described the effect on creatine-phosphates ATP, lactate and glycogen in white cod muscle. ATP

and creatine-phosphate increases from 4 and 5 to 6 and 15 p moles/g respectively, while lactate decreases from 10 to 2 p moles/g. All these effects must be explained as a result from death struggle. The enor- mous stimulation of glycolysis during death struggle must therefore cause a lot of artefacts in measuring substrates. Thus in carp, “control” lactate levels were found between 9 and 16 p moles/g, much higher than our control level 5.8 f 1.4~ moles/g. As far as we know only Fraser et al. (1966) used anaesthetics to measure physiological relevant substrates.

By calculation we will estimate the energy con- sumption of goldfish over the mean anoxia period (7 hr 52 min). Because in the energy balance the whole fish must be examined, it is obvious that phosphory- lated compounds of other fish tissues are of minor importance, for their concentrations are about 1jlOth of the muscle value (Table 2). The units of energy will be expressed in ATP-equivaIents. During anaero- bic glycolysis glycogen stores are depleted first. If re- stricted to substrate level phosphorylation, and trans- amination, the catabolism of 1 glucose unit will be 2 lactate + 3 ATP or 2 alanine + 3 ATP. Aerobic conditions gave with the same substrate 37 ATP + 6 CO2 + 6 HzO. Because the aerobic ATP yield is only dependent of oxygen consumption we can calculate from the standard metabolic rate (SMR) the energy need of the fish. This SMR is at 20°C 3.0mg 02/100g/hr (Beamish & Moorherjii, 1964), which can give rise to 0.56 m-moles ATPjlOO g/k. Over a period of 7 hr 52 min anoxia, the minimal energy demand must be 4.4. m-motes ATP for a 100 g goldfish.

Energy yield by used phosphorylated compounds: ATP 0.120 - 0.074 = 0.046 m-moles ATP

4 ADP 0.006 - 0.020 = -0.014 m-moles ATP Cr-P 0.255 - 0.127 = 0.128 m-moles ATP

0.160 0.160 m-moles ATP Energy yield by increase

of anaerobic endoproducts: lactate 3/2 (0.730 - 0.245) = 0.728 m-moles ATP

alanine 312 (0.176 - 0.050) = 0.189 m-moles ATP 0.917 0.917 m-moles ATP

Total 1.08 m-moles ATP

Influence of anoxia 335

Thus <$ of the needed energy can be covered by observed metabolic changes. Did the fish have other possible anaerobic endproducts? From the anaerobic endproducts mentioned in the introduction we did not measure the ammonia and succinate production. As already mentioned the ammonia production seems to be a secondary product. Succinate however could play an important role. Johnston (1975) reported in carp red muscle an increase from I to 6pmoles/g at very low ~0~. there was no increase in white muscle. As lactate levels for both red and white muscle were much higher than the succinate levels, succinate is less important than lactate as anaerobic endproduct. However we will have to take into consideration possible oxygen stores in fish. Oxygen occurs, dis- solved in tissue water, bound by myoglobin and hemoglobin and as a gas in the swim bladder. To

estimate oxygen stores we used figures from Black (1940), Schmidt-Nielsen (1975). (See Table 4).

AWAPARA J. & CAMPBELL J. (1964) Utilization of 14C02 for formation of some amino-acids in three invertebrates. Camp. Biochem. Biophys. 11, 231-235.

BEAMISH F. (1964) Respiration of fishes with special emphasis on standard oxygen consumption. III Influence of bxygen. Can. J. 2001..42, 355-366..

BEAMISH F. & MOOKHERJII P. (1964) Resuiration of fishes . with special emphasis on standard oxygen consumption. I Influence of weight and temperature on respiration of goldfish, Carassius auratus L. Can. J. Zoo/. 42, 161-177.

BERGMEYER H. (1971) Editor. Methoden der onzymatischen Analysr. Verlag Chemie. Weinheim.

BILINSKI E. (1974) Biochemical aspects of fish swimming. In Biochemical and Biophysicul Prrspectices in Marine Biology. (Edited by MALINS-SARGENT), Vol. 1. pp. 239-288. Academic Press, New York.

BLACK E. C. (1940) The transport of oxygen by the blood of freshwater fish. Biol. Bull. 79. 215-229.

BLA~KA P. (1958) The anaerobic metabolism of fish. Phy- siol. Zool. 31, 117~128.

Thus the total oxygen store in fish tissue can give 2.03/32 x 6 = 0.37 m-moles ATP equivalent. Although this is a considerable quantity, it is obvious that the total amount of energy produced is less than the SMR. Calculating from anaerobic endproducts and oxygen stores were found 1.45/4.40.100~/, = 32?/, of the needed energy.

BRETT J. (1972) The metabolic demand for oxygen in fish, particularly salmonids and a comparison with other ver- tebrates. Resp. physiol. 14, 151-170.

BURTON D. & SPEHAR A. (1971) A reevaluation of the anaerobic end-products of freshwater fish exposed to en- vironmental hypoxia. Comp. Biochem. Ph~siol. 40A, 945-954.

As mentioned in the introduction the carbon diox- ide production seemed to be independent of oxygen availability. This phenomenon was explained by Bilinsky (1974) as a buffer of the HCO; reserve in the fish. Because, as a result of lactic acid production, HCO; will take up H+ forming H2C03 which is excreted as CO,. However, in Table 2 the HCO; reserve in fish is presented as being increased during anoxia instead of decreased. This observation indi- cates the important role COZ production must play in the energy productiqn, because CO1 production is always catalysed by dehydrogenases. NADH or NADPH formed in this way must be oxidized by elec- tron-acceptors, for the amount of adenine-nicotine- amidenucleotides is limited. At this moment however, we do not known a possible electron acceptor.

DINGLE J. & HINES J. (1967) Extraction and some proper- ties of AMP-aminohydrolase from prerigor and postri- gor muscle of cod. J. Fish. Res. Bd. Can. 24, 1717-1730.

DRIEDZIC W. & HOCHACHKA P. (1974) The unanswered question of high anaerobic capabilities of carp white muscle. Can. J. Zoo/. 53, 706-712.

EDDINGTON D., WARD G. & SAVILLE W. (1973) Energy metabolism of working muscle: concentration profiles of selected metabolites. Am. J. Physiol. 224, 137551380.

FELIG P. & WAHREN J. (1971) Aminoacid metabolism in man. J. clin. invest. 50, 2703-2714.

FRASER D., DYER W., WEINSTEIN H., DINCLE J. & HINES J. (1966) Glycolytic metabotites and their distribution at death in the white and red muscle of cod following various degrees of antemortem muscular activity. Can. J. Biochem. 44, 1015-1033.

In our calculation about the energy need of goldfish during a period of anoxia, we assumed the energy need to be equal to the SMR. This SMR however must be considered as an energy consumption level without any activity (Beamish & Mookherjii, 1964). Oxygen consumption levels of resting goldfish were found to be 2.5 x higher than the SMR. Because we found in these experiments fish able to swim even after a long period of anoxia, it is probable that the energy need was much higher than the SMR.

Therefore we can conclude from our experiments, that energy produced during anoxia from O2 reserves, phosphorylated compounds and glycolysis, can only cover l/3 of the energy need.

Acknowledyements-We are grateful to Dr. Jaques Kluytmans for his help with the analysis of volatile fatty acids, performed at the Laboratory of Chemical Animal Physiology (Utrecht).

~‘RY F. & HART J. (1948) The relation of temperature to oxygen consumption in goldfish. Biol. Bull. 94, 66-67.

GERCKEN G. (1960) Die quantitativen enzymatischen Dehydrierung von L- Laktaat fiir die Mikroanalyse. Z. Phys. Chem. 320, 180-187.

HEAP A. & PRITCHARD A. (1965) Effects of severe hypoxia on carbohydrate energy stores and metabolism in two species of freshwater fish. Physiol. Zool. 38, 325-334.

HIDAKA T. & SAITO K. (1960) Studies on the biochemical change in fish muscle. X. On the AMP-deaminase acti- vity of some fractions prepared from fish muscle. Bull. Jap. Sot. Fish. 26, 1001~1005.

HOCHACHKA P., FIELDS J. & MUSTAFA T. (1973) Animal life without oxygen: basic biochemical mechanisms. Am. zoo/. 13, 543-555.

HUGHES G. M. (1973) Respiratory responses to hypoxia in fish. Am. Zool. 13. 475-489.

We should also like to thank Prof. Dr. Albert Addink for his help in the preparation of the manuscript.

JOHNSTON J. (1975) Anaerobic metabolism in the carp, Car- assius auratus L. Comp. Biochem. Physiol. 51B, 235-241.

JOHNSTON J. & GOLDSPINK G. (1973) A study of the swimming performance of crucian carp, Carassius caras- sius (L.), in relation to the effects of exercise and recovery on the biochemical changes in the myotomal muscles and liver. J. Fish. Biol. 5, 249-260.

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