Seasonal, size and comparative study of plasma proteins of four airbreathing freshwater fishes

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1 Prec. Indian Acad. Sci., Vol. 85 B, No. 6, 1977, pp Seasonal, size and comparative study of plasma proteins of four airbreathing freshwater fishes NEENA SIDDIQU1 Department of Zoology, Aligarh Muslim University, Aligarh MS received 30 October 1976 ABSTRACT Seasonal variations in total protein, alhumin, globulin, fibrinogen in the blood plasma of Clarias batrachus (L.) showed a well defined cycle. Feeding intensity and spawning were found to have marked influence on the plasma proteins. In summer, when fishes were fully ripe protein concentration was low. It appears that major part of the protein is diverted towards gonad formation. High values in the month of March and Aplil indicates higher metabolic activity of fishes in these months. Protein contents were also found to change with the inclease offish size. The increase is found to be associated with the changing dietary habit of the fish. Plasma proteins also vary widely from species to species. 1. ]NTRODUCTION INFORMATION concerning the chemical constituents of blood is useful for fishery biologists, to see whether a population of fish has properly adjusted in an environment or not. Water normally constitutes 90~o by weight of the plasma. The second principal constituent of plasma is represented by various proteins. Plasma protein is a mixture of albumin, globulin, and fibrinogen. Aside from fibrinogen which plays a role in blood coagulation, albumin and globulin maintain the water balance between the blood and tissue. 1 Plasma proteins serve as a source of nutrition for tissues of the body. It is generally recognised that the protein content of fish blood changes under varying conditions, such as season, stage of maturity, spawning, type of food consumed and feeding habits, z-4 Variations have also been found i n serum proteins as related to age, diet and water temperature3-6 Moore 7 found species differences in serum protein pattern. The present study reports seasonal changes in total protein, albumin, globulin and fibrinogen in plasma of C. batrachus. An attempt has also 384

2 PLASMA PROTEINS OF FRESHWATER FISHES 385 been made to correlate them with season, maturity stages, spawning, food and feeding habits. Variations according to age, and comparative pictures in four air-breathing fish species are also reported here. 2. MATERIALS AND METHODS For detailed investigations catfish, C. batrachus (L.) caught from a pond were immediately transported to the laboratory. They were in under tap water until sacrificed. Fishes measuring from 20.0 to 25-0 cm and weighing g were selected for seasonal studies. Monthly analyses were made over a period of 22 months. Data of overlapping months were pooled. The size range was kept constant throughout the seasonal studies in order to avoid differences in plasma proteins due to size differences. For size group studies fishes of different size ranges were collected and females were studied separately (table 2). A comparative study of the chemical constituents of the blood plasma of C. batrachus, Heteropneustes fossilis (Bloch), Channa punctatus (B[och) and Channa striatus (Bloch) was also made. These fishes were collected from the same pond and all the analyses were completed within one month to avoid seasonal variations. Each fish was bled by severing the caudal peduncle and collecting blood in small tubes containing double oxalate of ammonium and potassium at the concentration of 4 mg/2 ml of blood. For checking the blood coagulation double oxalate for catfishes and 3.2~ sodium citrate solution was used for murrels as an anticoagulant. To obtain plasma, blood was centrifuged at 3500 rpm as soon as possible. The modified micro-kjeldahl method was followed for estimation of plasma protein concentration. 3. RESULTS Seasonal and size variation were recorded in the values of plasma total protein and different, protein fractions, viz., albumin, globulin and fibrinogen (tables 1 and 2). These fractions also show variations between each species (table 2). TOTAL PROTEIN " The protein content varied from " 10 g~ to 6" g~ in males and from g~ to g~/o in females (table 1). The values of total protein in males were generally higher than in females. The lowest value ( g~) was recorded in July, while the maximum value ( g~o) was obtained in March. Total protein B3--June 77

3 386 NEENA SIDDIQUI Table 1. Seasonal variations in plasma proteins of C. batrachus, Months Sex Total Protein Albumin Globulin Fibrinogen January February March April May June July August gm/100 ml. M (10) "204-0"08 1" "604-0"20 F (10) 4"804-0"07 2"864-0"06 1"294-0" "02 C (10) 5"094-0"11 3"024-0"08 1"444-0" "02 M (12) 5"554-0" " " "01 F (10) 4" " "394-0" "02 C (25) 6"124-0"09 3"654-0"08 1"804-0"07 0"674-0"04 M (14) " "04 2"004-0" F (11) 5,984-0" "02 C (25) "08 1"804-0"07 0"67-4-0"04 M (12) 5"744-0" "08 1"734-0"09 0"614-0"02 F (18) 4"854-0' "06 1'464-0"09 0"664-0"01 C (30) 5"294-0"10 3"014-0"09 1"644-0"03 0'634-0"03 M (11) " ' "01 F (9) 4, ,18 1, ,604-0"02 C (20) " t-0.!, "04 M (10) "01 F (10) " "10 C (20) 3"784-0" '144-0" "03 M (11) ' " "01 F (10) " "02 C (21) 3"664-0" " " "04 M ( 8) 4"374-0" " " F (12) " " '01 C (20) 4"144-0"14 2"434-0" " "06 September M (15) " " ~0.03 F (17) 4.025:0" "014-0" C (20) " "254-0" "05 October November December M (10) " F (11) " "01 C (21) 4" "45-t-0" "13 0"564-0"04 M (14) "254-0" F (16) 3, " "03 C (30) "054-0" "02 M (9) 4, "03 F (15) 4"014-0" "604-0"02 C (24) " "04 M- Male Mean ± S.E. F--Female No. of fishes in parenthesis. C -- Combined, level of the plasma increased with the increasing length of the fish (table 2) and in all four size groups it ranged from 4-49 ± 0-17 g~o to g~ in males and from g~o to g~o in females. The concentration of protein was always higher in males than in the females.

4 PLASMA PROTEINS OF FRESHWATER FISHES 387 Table 2. Plasma proteins in different size groups of C. batrachus. Size group Length (cm) Sex Total Albumin Globulin Fibrirogen protein protein gm/100 ml I 15" Male (13) "004, ± "03 Female (15) " ,0" "04 I! 20"0-24"0 Male (17) 4.894,0"16 3"294-0"09 1"144,0" Female (14) " "904-0" "01 III 25" Male (16) "21 3"514.0"14 1"304.0"10 0"514,0"02 Female (10)4-86:/: " "564.0"01 IV 30"0-34"0 Male (12) " "11 2"004-0" "05 Female (11) " "90+0"09 0"654.0"01 Mean 4. S.E. No. of fishes in parenthesis. Table 3. Total plasma protein and protein fractions of four air-breathing freshwater fishes. Species Length (cm) Total Albumin Globulin Fibrinogen protein C. batrachus (11) H. fossilis (10) C. punctatus (10) C. striatus (9) gm/100 ml 22"04-25"0 4"714-0" "02 20"04-24"0 5"114-0" ::k0"04 0"684-0" " "004-0"08 0"814-0, " " Mean ::1: S.E. No. of fishes in parenthesis. Total protein content was highest in It. fossilis ( g~o)and lowest in C. punctatus ( g~o). C. batrachus and C. striatus showed intermediate values (table 3). ALBUMIN : Seasonal trend in the plasma albumin content of C. batrachus is also apparent (table 1). The highest value (3.65 ~ 0.08 g~o) was recoided in March and the minimum (2" g~o) in June and July. During winter months the values remained comparatively high. Males contain more albumin than the females. It varied from 2.00 q- 0"10gYo to 3.57 :~ 0-10 g~ in females and fiom 2.30 ± 1.12 g~o to 3.84 ± 0.04g~ in males. After attaining the maximum value in March a gradual decrease in albumin

388 NEENA SIDDIQUI content was recorded till it reached its minimum level in June. Albumin increased with the size of fish and ranged from 3-00 ±0-13g,% to 3-87 ± 0.11 g~o in males and from 2.60 4-0.

5 388 NEENA SIDDIQUI content was recorded till it reached its minimum level in June. Albumin increased with the size of fish and ranged from 3-00 ±0-13g,% to 3-87 ± 0.11 g~o in males and from g~o to 3" g~o in females from first to fourth size groups (table 2). Maximum amount ( l0 gyo of albumin was recorded in 11. fossitis and the minmum in C. punetatus ( g~o). In C. batrachus and C. striatus the values were g~o and g~o respectively (table 3). GLOBULIN ; Globulin content of the plasma varied fiom g~/to "07g~o being highest in March ( g~) and lowest in July ( g~o). High values were also recorded during winter months. The values of plasma globulin were higher in males (table 1). The plasma globulin valied from g~o to g~ in males and from 0.10 ±0.08g~ to g~ in females. Globulin content of the plasma followed a similar pattern as reported for total and albumin proteins. However, the globulin levels were higher in males than in the females (table 2). 11. fossilis and C. batrachus contained g~o and g~ of globulin respectively, while gyo in C. striatus and g~o in C. punctatus were recorded (table 3). FIBRINOGEN : The fibfinogen values ranged from g~ to g~. The maximum value was recorded in March ( g~) and the minimum ( g~) in July (table 1). During winter months also the values were generally high, while low values were recoided during the summer months. More fibt'inogen was found in females than in the males. Fibrinogen values increased with the increasing size of fishes. Foi 1st, 2nd, 3rd and 4th length groups the fibrinogen values were 0.40 ~ 0-03 g~o, g~, g~o, ,g~ in males and g~, g~, g~, g~ in females respectively (table 1). Flbrinogen contents, like other proteins were higher in catfishes (C. batrachus and 11. Jbssilis) than in the murrels (C. punetatus and C. striatus) (table 3). 4. DISCUSSION High levels of plasma protein during winter months suggest that the fish continues its normal feeding at low temperatures. However, during the summer months the environmental conditions become quite adverse. The water of the seasonal ponds lies up, the water of perennial ponds shrinks

6 PLASMA PROTEINS OF FRESHWATER FISHES 389 considerably and becomes turbid. Thus C. batrachus like other air-breathing fishes either passes the summer at the bottom of the perennial ponds or bury themselves in the mud and aestivate in pits. In both conditions either very little food is consumed or almost no food is taken by the aestivating fish. Decreased values in summer months, therefore, were due to low metabolic activity. The increase in value from August to November appears to be related to more consumption of food, as the environmental conditions improve after the first onset of the monsoon. The quality of food consumed also appears to influence the protein content of blood plasma. High values of serum protein were found in these months when fish takes mainly protein food. The seasonal variations in plasma proteins appear to be not only due to exogenous factors such as possible shortage of food, feeding intensity and environmental changes, but also result from endogenous processes inherent to reproduction. Low values were obtained during the period when the fishes either contained maturing or ripe gonads. The body reserves might have been utilized in gonad building. The breeding season of this fish lasts from May to August, with peak in May and June. During this period a sharp decline in the concentration of total protein, albumin, globulin and fibrinogen was noticed, because the blood reserves are called upon to provide substrates for the gonad formation. The depletion of protein in the blood can be correlated with the increase of ovarian protein. Krishnamoorthy 8 reported an increase in the concentration of a number of amino acids in the ovary during gonad development and attributed it to increased protein metabolism in the ovary. Sreenivasan 9 reported an increase in the level of protein in the gonads, as the gonads advance towards maturation, and decrease in protein level in spent gonads. Obviously the plasma protein is mobilised during gonadal growth and accounts for its depletion in plasma during the breeding season and pre-spawning months. Daring post-spawning months the amount of total protein, albumin, globulin and fibrinogen increased indicating the recovery of the fish from the exhaustion of spawning. Shell l also found a decrease in plasma protein contents of small mouth bass from June to August and increased thereafter. The chemical composition of fish blood varied according to its size and age. Total protein and various protein fractions showed a relative increase along with the increasing length of fish. Qayyum and Qasim al have reported that the feeding habit of Ophiocephalus punctatus changes with age. C. batrachus also changes its feeding habits as it grows and consumes more proteinous food when adult. Food of juveniles is different from that of adults? ~ The fry take protozoanal, small crustaceans, algae, worms, rotifers, etc., while the adult fishes take insect B4--June 77

7 390 NEENA SIDDIQUI larvae, shrimps, worms, vegetable debris, etc. A gradual rise in total protein, albumin, globulin and fibrinogen was noted from first to fourth size group (table 2) whcih appears to be due to more consumption of proteinous food of the larger fishes. Plasma proteins also vary widely from species to species (table 3). Plasma protein was high in catfishes but was low in murrels. The differences in concentration of protein in various species appears to be affected by diet of the fish. Sulya 13 made electrophoretic study of 26 species of etasmobranches and teleostean fishes, and found a general increase in the amount of plasma proteins from lower to more specialized fishes. Naseem la reported low concentration of protein in carps and catfishes. ACKNOWLEDGEMENT Special thanks are extended to Dr. A. Q. Siddiqui for his valuable help and guidance and to Prof. S. M. Alam, Head, Department of Zoology, for providing the facilities. REFERENCES 1. Harrow, B., A Text Book of Biochemistry (4th ed.), W. B. Saunder's Co., Philadelphia and London (1946). 2. Booke, H., N.Y. Fish Game J (1964). 3. Siddiqui, M. A. and Siddiqui, M., Indian J. Exp. Biol (1965). 4. Elliot, J. W.,Fowler, L.G. and Burrows, R.E., Tech. Paper No. 8, Bur. Sport Fishery and Wildlife, pp (1966). 5. Koroleva, N. V., Dorkl. Akad. ]~auk. S.S.R (1963). 6. Phillips..A.M., etal., Cor land Hatch. Rep. 3i 51 (1963). 7. Moore, D. I-I., J. Biol. Chem (1965). 8. Krishnamoorthy, K. P., J. Zool. Soc. India (1957). 9. Sreenivasan, A., Sundera Raj., R. and Lakshmiragharam, S., Proc. Indian Sci. Congr. Calcutta 51st and 52nd Session Part Ill, p. 455 (1964). 10. Shell, E.W., U.S. Fish flrildlife Serv. Res. Rep. No (1961). 11. Qayyum, A. and Qasim, S.Z., J. Bombay l~'at. Hist. Soc (1964). 12. Mookerjee, H. K. and Mazumdar, S. R., Proc. Zool. Soc. Beng. 3 7 (1956). 13. Sulya, L. L., Box, B. E. and Gunter, G., Amer. J. Physiol (1961). 14. Naseem, S. M., Ph.D. Thesis, Aligarh Muslim Umversity, Aligarh (1968)

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