Anaerobic metabolism in goldfish (Carassius auratus) R. M. WALKER AND P. H. JOHANSEN Department of Biology, Queen's University, Kingston, Ont., Canada K 7 L 3N6

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Received January 17, 1977

WALKER,R. M . , and P. H. JOHANSEN. 1977. Anaerobic metabolism in goldfish (Carassius auralus). Can. J. Zool. 55: 1304-131 1. At 20°C goldfish survive anaerobic conditions for only a few hours while at 4'C survival is extended to several days. During the course of low-temperature anaerobiosis there was a rise in blood glucose and lactate, adecline in liver glycogen concentration, and an increase in liver water content, while liver size remained constant. The better cold anaerobic survival of winter and hypophysectomized goldfish compared with spring and sham-operated animals was correlated with greater glycogen stores in the livers of the former. It is concluded that liver glycogen is a necessary energy source during cold anaerobiosis, and it is suggested that the resulting hyperglycemia may represent a mechanism to increase glycolytic energy yield. Cold anaerobiosis also resulted in elevated liver glucose-6-phosphatase (EC 3.1.3.9) activity, suggesting an increase in glycogenolysis, but no change in glycogen phosphorylase (EC 2.4. I . 1) activity. While cold anaerobic survival is short term it is possible that liver glycogen may sustain goldfish for longer periods at low oxygen levels through a mixed aerobic-anaerobic metabolism. WALKER,R . M . , et P. H. JOHANSEN.1977. Anaerobic metabolism in goldfish (Carassius auralus). Can. J. Zool. 55: 1304-13 1 1. Des poissons rouges ne peuvent survivre que quelques heures dans des conditions anaerobiques a 20°C, alors qu'a 4OC, ils peuvent survivre plusieurs jours. L'anaerobie a basse temperature entraine une augmentation du glucose et du lactate sanguins, une reduction de la concentration de glycogene hepatique et une augmentation du contenu hydrique du foie; le volume du foie ne subit aucun changement. Des poissons rouges ont subi I'ablation de I'hypophyse; ces poissons survivent mieux a des conditions anaerobiques en hiver que ne le font des poissons qui n'ont subi qu'une operation simulee et qui sont places dans des conditions anaerobiques au printemps. La survie meilleure des poissons d'hiver est reliee a leurs reserves plus importantes de glycogene hepatique. On en conclut que le glycogene hepatique constitue une source d'energie necessaire durant I'anaerobie au froid et que I'hyperglycemie qui en resulte pourrait i t r e un mecanisme propre a augmenter la production d'energie glycolytique. L'anaerobie au froid produit egalement une augmentation de I'activite de la glucose-6-phosphatase (EC 3.1.3.9) dans le foie, ce qui indique peut-itre une augmentation de la glycogenolyse, mais reste sans effet sur I'activite de la glycogenephosphorylase (EC 2.4.1.1). La survie a des conditions anaerobiques au froid est de courte duree, mais il est possible que le glycogene hepatique puisse maintenir la survie des poissons rouges pour des periodes plus longues a des concentrations d'oxygene faibles par un metabolisme aerobique-anaerobique mixte. [Traduit par le journal]

Introduction Liver glycogen is a principal source of blood glucose, a readily available and usable energy source in all vertebrates. Through the action of glycogen phosphorylase (EC 2.4.1.1), glucose units as glucose-1-phosphate are cleaved from the glycogen complex. Glucose-1-phosphate is converted to glucose-6-phosphate which can enter directly the metabolic pathways of liver and muscle cells or in the case of the liver be released as free glucose into the blood by glucose-6-phosphatase (EC 3.1.3.9). While the ultimate function of liver glycogen is as an energy supply, the circumstances of

its use by fishes varies. Evidence suggests that it can be used during or after exercise (Black et al. 1966; Dean and Goodnight 1964; Miller et al. 1959); in starvation (Dave et al. 1975; Ince and Thorpe 1976): in osmoregulation as the potential source of elevated blood glucose (Umminger 1971); in a supercooled environment (Umminger 1970); and possibly under anoxic conditions that some fish are reportedly able to survive for extended periods (Blazka 1958; Mathur 1967; Coulter, 1967). Goldfish can survive at least short-term anoxia and are capable of anaerobic CO, production (Hochachka 1961; Kutty 1968; and van den

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WALKER AN1D JOHANSEN

Thillart et al. 1976). Hochachka et al. (1973) presented a scheme for facultative anaerobic metabolism in invertebrates whereby anaerobic glycolysis is supplemented by the simultaneous metabolism of amino acids, which produces CO,, alanine, and succinate as end products. Whether such pathways could operate in potentially anaerobic fish such as goldfish or carp is not clear. While Johnston (1975) found that in carp exposed to low oxygen, alanine and succinate accumulated in red, but not white muscle, Driedzic and Hochachka (1975) found no amino acid mobilization during anaerobiosis in carp and no increase of the end products known to accumulate in invertebrates. Van den Thillart et al. (1976) concluded that only one third of goldfish energy needs were supplied during anoxia from stored 0, reserves, phosphorylated compounds, and glycolysis. Regardless of its mechanism, anaerobic metabolism requires a carbohydrate substrate. Therefore we investigated the effects of anoxia on aspects of carbohydrate metabolism in goldfish, especially since moderate starvation does not appreciably deplete the relatively large hepatic glycogen reserves (Stimpson 1965; Walker and Johansen 1977) and liver glycogen increases with cold acclimation (Umminger 1975).

Materials and Methods Holding Conditions and Operative Procedures Goldfish (4-9 g) were obtained from Hartz Mountain Pet Supplies, Rexdale, Ontario, Canada. They were held in 275-f tanks at 20°C for at least 1 month before experimentation and were fed once daily on Purina trout ration. To acclimate fish to low temperature, they were gradually brought down from 20°C to 4°C f 0.5"C over a 1-month period and then held at this temperature for at least 2 months. These fish were not fed except as stated in one experiment. Hypophysectomy and sham operations were performed as reported previously (Johansen 1967). The completeness bf hypophysectomy was judged by the colour-loss criterion (Johansen and ROY 1969) and by the absence of any p'ituitary remnants under moderate magnification at autopsy. The operated fish were held in a 0.2% NaCl solution to insure against osmoregulatory failure in the hypophysectomized fish. For the anaerobic studies pairs of fish were placed into 500-ml erlenmeyer flasks filled with aerated water of the desired temperature. The flasks were firmly stoppered with rubber bungs so as to exclude air bubbles. Control flasks were unstoppered and gently aerated. The flasks were then placed in a temperature-controlled water bath at 4'C f 0.5"C. The oxygen content of the water was measured with a model 54 oxygen meter

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and self-stirring electrode (Yellow Springs Instrument Company, Yellow Springs, Ohio, U.S.A.). The fish were killed by cervical section and tissues quickly excised for immediate biochemical analysis. A midventral incision exposed the liver which was then dissected free of the highly coiled intestine to which it adheres. White muscle was excised from the epaxial muscle in the area of the anterior portion of the dorsal fin. Blood was collected after 1 min of tricaine methanesulfonate (0.06%) anesthesia. The gills were immediately exposed and swabbed dry with absorbent cotton. Several filaments were slashed with a scalpel and blood was collected in a disposable micropipette. Blood samples were allowed to clot for 5 min and then centrifuged for 10 min. Biochemical Assays Muscle glycogen was determined by the method of Lo et al. (1970). Liver glycogen was determined by a modification of Lo's method as outlined previously (Walker and Johansen 1975). Liver lipid was determined by the method of Bligh and Dyer (1959). Protein was estimated by the biuret method (Layne 1957). Blood glucose was determined on 10-p1 samples of serum using the reagents of the Sigma Chemical Corporation, Saint Louis, Missouri, U.S.A., and a microadaptation of their protocol (bulletin No. 510). Blood lactate was determined by the method outlined in Sigma bulletin No. 826-UV using Sigma reagents. This latter test procedure was scaled down by a factor of two to adapt to the small blood volumes available for analysis. Enzyme assays for glucose-6-phosphatase and glycogen phosphorylase have been described (Walker and Johansen, 1977). Results are presented as the mean _+ standard error; sample sizes are in parentheses. Significance of the results was determined by two-way analysis of variance (2 x 2 ANOVA) or Student's t-test where applicable.

Results After placing two fish in a flask of 4°C aerated water ( 0 2 , 12.5-13.2 ppm) and sealing it to achieve anaerobiosis, the oxygen content fell to less than 1.0 ppm within 12 h and ranged from 0.35 to 0.40 ppm after 24 h. No further decline occurred for the duration of the experiment. Time zero was taken from the point of placement of the fish into sealed flasks. Therefore, while results are expressed as days under anaerobic conditions, it took about the first half day for the fish to consume the usable oxygen. Upon being placed into flasks the fish swam about vigorously, but gradually their activity slowed. Once anaerobiosis was achieved the fish became very quiet, with occasional respiratory movements or body twitches. Disturbing the flask induced minor locomotor activity. Upon completion of the experiment when the

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CAN. .I.ZOOL. VOL. 55, 1977

various biochemical tests were to be made the fish were judged to be alive if they moved when handled and the heart was still beating. Experiments carried out in midwinter suggest that the 'winter' fish were better able to survive anaerobically than those in experiments performed 3 months later in the spring (Fig. 1). The 'winter' fish had a greater liver glycogen content as manifested by their larger liver somatic index (LSI, 5.76 1 0.48) and higher glycogen concentrations (246 f 20 mg/g) compared with 'spring' fish (4.49 0.20 and 191 k 8 mg/g). The reason for this seasonal difference between groups held under similar conditions is not known. In anaerobic experiments carried out at 20°C on 20°C-acclimated goldfish, it was found that the oxygen content of the water in the sealed flasks was reduced from 6.5 to 0.3 ppm in 3.5 h and that the liver glycogen concentration fell slightly (Table 1). Under these conditions all fish died after 4 to 5 h. Table 2 presents a comparison of various aspects of metabolism in 2 0 " ~aerobic, 4°Cacclimated aerobic, and 4°C-acclimated goldfish held under anaerobic conditions for 5 days. Under aerobic conditions cold acclimation results in a significant iIIcrease in LSI, liver glycogen concentration, blood glucose, and blood lactate, but a significant decline in liver water, lipid, and protein concentrations when compared with warm-acclimated fish. There was no change in muscle glycogen concentration. After 5 days under cold anaerobic conditions there was a significant decline in liver and muscle glycogen concentrations and significant increases in liver water, blood glucose, and blood lactate concentrations when compared with cold-acclimated aerobic fish. Figure 2 more fully documents the changes in blood glucose, liver glycogen, and liver water levels as a function of time under anaerobic conditions. The steady decline in liver glycogen concentration was concomitant with the rise in blood glucose, which levels off after 4 or 5 days, and the continuous rise in liver water content. Hypophysectomy of goldfish offers a mechanism by which the liver glycogen content can be manipulated (Walker and Johansen 1975) so as to further study its importance in cold anaerobic survival. Before the operation cold-acclimated fish were fed half the ration of 20°C-acclimated fish for 6 weeks. The LSI

+

100

80

60

.

: 2

3

40

20

o

I

2 DAYS

4 UNDER

6

ANAEROBIC

8

10

CONDITIONS

7;

at l i ~ ~ ~ $ ~ ~ ~v ? '~ ! ' ~ ~ ~~ ~ ~~ ~ ~ ~ n ~ ~ a figure based on at least 12 fish except the 7- and 9day points for spring and winter fish respectively which are based o n 6 fish.

and liver glycogen levels of these cold fed fish at the time of operation were 11.34 f 0.74 ( N = 6) and 234 8 mg/g (N = 6) respectively. After operation the fish were starved for 24 weeks before anaerobic studies were carried out. A Chi-square analysis of the combined data of 3- and 4-day anaerobic fish shows that hypophysectomized fish were better able to survive these conditions than shamoperated controls (Table 3, P < 0.005). Hypophysectomized goldfish had greater LSI's and liver glycogen concentrations, and hence much greater liver glycogen stores (as expressed by milligrams per gram fish) than sham-operated fish (Table 4). Table 4 also shows that the operated fish like the unoperated ones (Table 2), responded to anaerobic conditions with elevated blood glucose and reduced liver glycogen concentrations. The glucose-6-phosphatase activities found after cold aerobic and 5-day anaerobic conditions are summarized in Table 5. Though

+

~

WALKER AND JOHANSEN

TABLE1. Effects of hypoxic conditions on goldfish at 20°C. Fish were held in sealed or aerobic flasks for 3.5 h. Results are presented as mean k SEM; sample sizes are given in parentheses

LSIa

Liver glycogen mg/g of liver

4.71k0.29 (6) 4.87k0.42 (6) 4.88k0.63 (6)

162+7 (6) 155f9 (6) 112f 14b (6)

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Fish group Controls (holding tanks) Controls (aerobic flasks) Anaerobic (sealed flasks) -

--

-

-

.(Grams of liver + grams of fish) x 100. bsignificantly diRerent from aerobic controls (P < 0.05).

TABLE2. The effects of temperature and 5 days of anaerobic conditions on goldfish variables. Experiments were performed during winter. Results are presented as mean k SEM; sample sizes are given in parentheses Variable LSI" Liver water content, % Liver glycogen, mg/g of liver Liver protein, mg/g of liver Liver lipid, mg/g of liver Muscle glycogen, mg/g of muscle Blood glucose, mg/100 ml Blood lactate, mg/ 100 ml

20°C (aerobic)

4°C (aerobic)

4°C (anaerobic)

3.47+0.25b (6) 70.28f 0.32b (10) 174k 15b (6) 104.1k3.0b (10) 26.41k1.85b (10) 4.74k0.41 (5) 23.8k1.7b (6) 30.7f-2.gb (6)

5.76k0.48 (6) 68.31k0.54 (10) 246 k 20 (6) 67.5k7.5 (6) 15.51k5.80 (6) 4.91.kO.69 (5) 38.3k3.1 (6) 42.0k2.4 (6)

5.49k0.55 (6) 81.49+1 .03b (6) 123k 24b (6) 60.2k3.1 (6) 15.83k4.20 (6) 1.67k0.13b (4) 254 k 7b (8) 193k 15b (6)

O(Grams of liver + grams of fish) x 100. bSignificantlydifferent from 4'C aerobic fish (P < 0.02, or better).

TABLE3. Survival of hypophysectomized and sham-operated goldfish after 3 and 4 days under anaerobic conditions. All fish were fed for 6 weeks before operation and starved for 24 weeks post operation. Results are presented as mean + SEM; sample sizes are given in parentheses No. of fish -

Experiment

Time days

1

3

2

4

Combined"

3 and 4

Total

Operation

Dead

Alive

Total

Hypophysectomized Sham operated Hypophysectomized Sham operated Hypophysectomized Sham operated

0 3 1

8 5 9 3 17 8 25

8 8 10 10 18 18 36

7

I 10 11

'Chi-square analysis of the combined experiments reveals that hypophysectomized fish survive significantly longer than sham-operated animals (P c 0.005).

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CAN. J. ZOOL. VOL. 55. 1977

TABLE 4. The effect of 3 days of anaerobic conditions on hypophysectomized and shamoperated goldfish. After operation all fish were kept at 4°C for 24 weeks without food. Anaerobic fish were the survivors of experiment 1, Table 3. Results are presented as mean SEM; sample sizes are given in parentheses

+

Variable

Condition

Blood glucose," mg/100 ml

Sham operated

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Hypophysectomized Liver Somatic index,*

Sham operated Hypophysectomized

Liver glycogen,' mg/g of liver

Sham operated Hypophysectomized

Calculated liver glycogen, mg/g of fish

Sham operated Hypophysectomized

Aerobic

Anaerobic

53.5k1.8 (4) 61.5k6.5 (4) 2.93k0.21 (4) 8.23k0.79 (4) 126+ 3 (4) 230k 11 (4) 3.7 18.9

159+ 20 (4) 207 27 (4) 2.87k0.24 (4) 8.2720.66 (4) 58k7 (4) 196+10 (4) 1.7 16.2

+

'Two-way ANOVA reveals a significant difference between aerobic and anaerobic fish groups (P

Anaerobic metabolism in goldfish (Carassius auratus).

Anaerobic metabolism in goldfish (Carassius auratus) R. M. WALKER AND P. H. JOHANSEN Department of Biology, Queen's University, Kingston, Ont., Canada...
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