- Email: [email protected]
The role of vitamin C in calcium uptake by fish
Aquaculture, 19 (1980) 287-294 o Elsevier Scientific Publishing Company,
287 Amsterdam
- Printed in The Netherlands
THE ROLE OF VITAMIN C IN CALCIUM UPTAKE BY FISH
C.L. MAHAJAN
and N.K. AGRAWAL
Fish Biology Laboratory, 302004 (India) (Accepted
Department
of Zoology,
University
of Rajasthan,
Jaipur-
28 August 1979)
ABSTRACT Mahajan, C.L. and Agrawal, N.K., 1980. The role of vitamin C in calcium uptake by fish. Aquaculture, 19: 287-294, Calcium uptake in experimentally induced scorbutic snake heads, Channa ( = Ophicephalus) punctatus, fed vitamin C-deficient diet for 210 days is reported, using %a as tracer. When compared with a parallel control on completely synthetic diet, the results show decreased absorption and utilization of calcium by gill, skin, muscle and bone of scorbutic fii from surrounding water. The physiological significance of these results is discussed, and its practical utility in aquaculture is indicated.
INTRODUCTION
Studies on fish nutrition in the last two decades suggest that dietary deficiency of vitamin C results in poor growth, various skeletal deformities, such as lordosis and scoliosis, distorted hyperplastic gill cartilage and mottled sclerotic cartilages in the eye, as well as haemorrhages, loss of scales and delayed wound healing etc. (Kitamura et al., 1965; Poston, 1966; Kitamura, 1969; Halver et al., 1969, 1975; Ashley et al., 1975; Andrews and Murai, 1975; Lim and Love& 1978; Hilton et al., 1978). As a part of our extensive studies on the nutritional requirements of fish (Agrawal et al., 1978; John and Mahajan, 1979), most of the above mentioned changes have already been reported by us in Channa punctutus (Mahajan and Agrawal, 1979). Considering the importance of calcium as ground substance in bone, cartilage, wound healing, blood coagulation etc., investigations on the calcium uptake by various tissues of scorbutic fish using 45Ca as a tracer were made and are reported in this paper. MATERIAL
AND METHODS
Channa f = Ophicephalus) punctatus Bloch (average wt. 42.80 f 4.26 g, length 115.0 * 7.4 mm) of age 1 + years were collected from freshwater areas around Jaipur and, after a week’s acclimatization, were divided into the following groups of 80 each in laboratory aquaria:
288
Group I = control, fed complete synthetic diet (Table I) (at 3% body weight/ day). Group II = ascorbic acid-deficient, fed the same diet as Group I except that ascorbic acid was not added (at 3% body weight/day). The environmental conditions in holding aquaria were as follows: pH 7.8, temperature range 16-33”C, dissolved oxygen 6.5 mg/l, alkalinity 140 mg/l, dissolved calcium 58.76 + 0.28 mg/l, with traces of phosphates and nitrates. The light conditions varied with the season and daylight hours, the aquarium room being exposed to sky light. The routine was continued for 210 days, when increased mortality appeared in ascorbic acid-deficient group (Mahajan and Agrawal, 1979). At this point eight fish from group I and II each were randomly selected for calcium uptake studies. Fish from each group were kept separately in glass aquaria filled with 20 1 synthetic bicarbonate water (Table II). Water in the aquaria was constantly aerated through aerators in a closed system. After 24 h acclimatization, 50 mg/l calcium chloride and 50 pCi/l 45Ca (as CaCl,) were dissolved in each tank. The radioisotope was obtained from Bhabha Atomic Research Centre, Bombay, India. Immediately after adding the radioisotope, one millilitre water samples were taken in the aluminium planchet from each tank to determine the activity/ml of water. Four fish from each tank were sacrificed at 24 and 48 h. Gill, skin, muscle, bone, intestine and kidney tissue samples were carefully removed from identical sites from all the fish autopsied. A TABLE
I
Composition
of complete
Ingredient Vitamin-free casein Cellulose powder White dextrin Gelatin Corn oil Shark liver oil’ Vitamin mixture2 Mineral mixture3 Water
synthetic
diet (Modified
from Mahajan
and Yadave,
1974)
Weight (g/kg) 325 150 225 200 55 15 8 32 1200 ml
‘Used as a source of vitamin A (1500 III/g) and vitamin D (160 IU/g). ‘Thiamine hydrochloride, 40 mg; riboflavin, 160 mg; pyridoxine hydrochloride, 40 mg; choline chloride, 4.0 g; nicotinic acid, 600 mg; calcium pantothenate, 400 mg; inositol, 1.6 g; biotin 4 mg; folic acid, 12 mg; ascorbic acid, 800 mg; cyanocobalamine, 0.08 mg; manadione, 32 mg; alphatocopheryl acetate, 320 mg. ‘Aluminium chloride, 5 mg; zinc sulphate, 95 mg; cuprous chloride, 3 mg; manganese sulphate, 25 mg; potassium iodide, 5 mg; cobalt chloride, 30 mg; sodium chloride 1.395 g; magnesium sulphate 4.36 g; sodium biphosphate 2.774 g; potassium sulphate, 7.64 g; calcium biphosphate, 4.32 g; ferric citrate, 0.948 g; calcium lactate, 10.40 g.
289 TABLE II Composition of synthetic bicarbonate water (Phillips et al., 19SS) Mineral ions
Concentration
Ca’ + Na’+ K’+ Mga+ HCO,‘SiO,‘NO,‘PO,‘_ Trace*
50.0 40.7 3.2 5.0 100.0 5.0 1.0 5.0 0.035
(mg/litre)
*Trace minerals composed of 0.025 ppm each of Ala+, Ba*+, COa+, Cuz+, Fe’+, Mnl+, Nil+, Snl+, Zn’+, Bra-, F1-, I’-, CO,a-, Mo,O,,‘-.
sample of each tissue, 0.5-3 g, was weighed on a single pan electric balance, and digested in a minimum quantity (-0.5 ml) of concentrated nitric acid by heating in a boiling water bath for 3-5 min. The dissolved tissue was diluted to 4 ml with distilled water. Three samples of each tissue were prepared in this manner. One ml of each diluted sample was taken on an aluminium planchet and dried in an incubator (55-6O”C). Radiation activity in all the water and tissue samples was counted in a Nuclear-Chicago G.M. counter. The calculations made were on the general principle that the radioactivity of each tissue of the fish indicates the passage of calcium from the experimental water into that tissue during the time interval, and is a measure of the total calcium taken from this source and retained. One pCi of radioactivity represents the same amount of absorbed calcium in the particular experimental tank (Phillips et al., 1954). The results were calculated as follows: Counts/g tissue/min: Counts/ml tissue sample X dilution wt. of tissue (g)
(A)
Counts/CcCi of isotope/min: Counts/ml water sample (R)
Isotope (pCi)/ml water Amount of mineral ion labelled by/pCi isotope: Amount of mineral (pg)/ml water Amount of isotope (pCi)/ml water Uptake of mineral =$X
C (clg/g time)
WI
290 RESULTS
Analysis of the results presented in Table III and Fig. 1 shows that there was a statistically significant increase (P < 0.001) in calcium uptake in the first 24 h by the intestine of ascorbic acid deficient fish as compared to control. However, this trend slowed down considerably in the next 24 h so that the deficient group was found to have a decreased rate of uptake to such an extent that it was slightly lower (though statistically insignificant) than the control. Kidney tissue on the other hand, showed just the opposite trend; the calcium uptake in the first 24 h in the kidney of deficient group was far less compared with the control (- 4 pg/g compared with 16 pg/g in the control tissue), but in the next 24 h it increased to such an extent that it was significantly higher (P < 0.001) than the control (- 21 pg/g as compared with 13 pg/g in the control). In skin, gill and bone the uptake both at 24 and 48 h stages was less in the deficient group than in the control, even though the uptake values greatly increased between 24 and 48 h in both the control and deficient groups. However, there was considerable difference in the relative uptake values between the two groups at 48 h when compared to that at 24 h. Thus the difference in the rate of uptake remained almost constant in the skin, while in the gill and bone it was reduced. Despite these relative variations, the difference in calcium uptake by all these tissues between the control and deficient groups remained statistically highly significant. The decrease in calcium uptake in the muscle, however, was found to be statistically significant only at 48 h in the deficient group (P < 0.001) as compared with the control. At 24 h the calcium uptake in muscle, though less, was not found to be statistically significant.
TABLE III Uptake of calcium by various tissues of fish fed complete synthetic diet (control) and ascorbic acid-deficient diets (-AA) for 210 days Tissue
Gastrointestinal tract Gill Muscle Bone Skin Kidney
Uptake in 24 h (mean i S.E.) (Is/g)
Uptake in 48 h (mean r S.E.) (rg/g)
Control
-AA
Control
-AA
7.851kO.435 59.262k3.318 3.989+0.160 68.421i2.433 51.929+0.897 16.65920.377
16.651+0.553* 32.073+1.016* 2.647iO.144 21.697*0.297* 31.027i2.410* 4.656t0.186’
8.905+0.271 139.209k4.268 4.457kO.091 75.027kO.945 103.219t1.353 13.181+0.297
7.671k0.393 91.880*7.630* 2.077+0.137* 61.456*1.748* 62.409+2.483* 21.228+0.137*
*P < 0.001 Asterisks in the -AA group indicate the significance of difference. No asterisk indicates the non-significant change.
291
Dr
[7
CONTROL
n
AA.
Dcf.
O-
T
0-
O-
0t
I aLh e
A
GILL
Fig. 1. Absorption
e
A
BONE
a
A
SKIN
of calcium by tiiues
G.I.TRACT
of ascorbic acid deficient
A
B
MUSCLE
KIDN
and control fish.
DISCUSSION
Calcium metabolism in fish is particularly interesting because of their aquatic environment. They are unusual, if not unique amongst vertebrates, in their ability to absorb calcium from surrounding water (Fleming, 1974). Unlike tetrapods, a definite calcium regulating system has not been demonstrated so far, and our knowledge about the presence of functional parathyroid gland and the exact role of calcitonin in the metabolism of this electrolyte is still limited (Copp et al., 1972). The results presented above show that in normal (control) fish, maximum calcium uptake in the intestine and kidney takes place in the fiit 24 h, while in gill, bone skin and muscle this occurs between 24 and 48 h. These findings are similar to those reported in earlier reports (Phillips et al., 1954; Mahajan and Yadave, 1975). In intestine and kidney, being the sites of absorption and excretion, the homeostasis in calcium uptake takes place within 24 h, while in the bone, skin and muscle, being the sites of storage and metabolism, absorption rate increases with time. The gill, which is the major site of calcium ingress to the fish’s body, distributes absorbed calcium rapidly to the different tissues via the blood and show steady uptake in first 24 h. However, after this period calcium deposition begins in this tissue (Phillips
292
et al., 1954) which may account for the increased uptake observed in this tissue during the present study in the 24-48 h interval. The present study clearly establishes that a chronic vitamin C deficiency results in decreased absorption and utilization of calcium, as shown by decreased calcium uptake by the gill, which is the major site of calcium absorption. There is also a similar trend in uptake by bones, muscles and skin which are the major sites of calcium utilization and storage. The decreased absorption of calcium by the gill may be due to distortion of gill filaments and gill supporting cartilages in scorbutic fish, as pointed out by Halver et al. (1975). The increased calcium uptake by intestine in ascorbic acid-deficient fish during first 24 h may be compensatory to the decreased uptake by gill. However, its lowering to the control level in the next 24 h is possibly due to quick distribution and/or excretion of the absorbed calcium. In the kidney, on the other hand, the uptake in first 24 h was less than the control, but increased considerably in the next 24 h which may be due to the accumulation of calcium ions for excretion in the kidney because of decreased uptake by other tissues. The decrease in the total calcium absorption results in the mobilization of the calcium from the bones to maintain calcium homeostasis and results in the skeletal deformities reported earlier (Mahajan and Agrawal, 1979). Pang (1971) also reported withdraw1 of calcium from skeletal tissues in dark adapted vitamin C deficient juvenile Fundulus heteroclitus, where skeletal deformities preceeded by serum hypocalcemia, were observed. It is interesting to note that Siddiqui (1967) reported a seasonal correlation in ascorbic acid and calcium contents of various tissues of Channa ( = Ophicephalus) punctatus. The results of the present study and those of Pang (1971) and Siddiqui (1967) support the conclusion that calcium uptake by tissues is affected by vitamin C status of the fish. The influence of ascorbic acid deficiency on calcium uptake and collagen synthesis by impairing the hydroxylation of protocollagen proline and lysine (Barnes and Kodicek, 1972) explains the skeletal deformities reported by us in this fish and similar reports by others. CONCLUSION
These findings point to the desirability of ensuring an optimum vitamin C supply in the diet of intensively cultivated fish. This would enable the fish to utilize properly the available dissolved calcium in the water, thus resulting in healthy fish stocks, good growth and net productivity. For Channa punctutus, an air-breathing fish inhabiting swamps and ponds and presently being exploited for intensive cage culture, these conclusions may be kept in mind when formulating artificial diets.
293 ACKNOWLEDGEMENT
The study was supported by Indian Council of Agricultural Research, New Delhi. Thanks are due to the Head, Department of Zoology, University of Rajasthan, Jaipur for facilities and Prof. P.N. Srivastava for extending help in isotope studies.
REFERENCES Agrawal, N.K., Juneja, C.J. and Mahajan, C.L., 1978. Protective role of ascorbic acid in fishes exposed to organochlorine pollution. Toxicology, 11: 369-376. Andrews, J.W. and Murai, T., 1975. Studies on vitamin C requirements of channel catfish (Ictalururpunctatus) J. Nutr., 105: 557-561. Ashley, L.M., Halver, J.E. and Smith, R.R., 1975. Ascorbic acid deficiency in rainbow trout and coho salmon and effects on wound healing. In: W.E. Ribelin and G. Migaki (Editors), The Pathology of Fishes, University of Wisconsin Press, Medison, pp. 769785. Barnes, M.J. and Kodicek, E., 1972. Biological hydroxylation and ascorbic acid with special regard to collagen metabolism. In: R.S. Harris, P.L. Munson, E. Diczfaiusy and J. Glover (Editors), Vitamins and Hormones Vol. 30, Academic Press, New York and London, pp. 1-43. Copp, D.H., Byfield, P.G.H., Kerr, C.R., Newsome, F., Walker, V. and Watts, E.G., 1972. Calcitonin and ultimobranchial function in fishes and birds, In: R.V. Talmage and P.F. Munson (Editors), Calcium, Parathyroid hormone and Calcitonin. Proc. 4th Parathyroid Conference, Excerpta Medical, Amsterdam, pp. 12-20. Fleming, W.R., 1974. Electrolyte metabolism of teleosts, including calcified tissues. Chem. Zool. 8: 471-508. Halver, J.E., Ashley, L.M. and Smith, R.M., 1969. Ascorbic acid requirements of coho saimon and rainbow trout. Trans. Am. Fish. Sot., 98: 762-771. Halver, J.E., Smith, R.R., Tolbert, B.M. and Baker, E.M., 1975. Utilization of ascorbic acid in fii. Ann. N.Y. Acad. Sci., 258: 81-102. Hilton, J.W., Cho, C.Y. and Slinger, S.J., 1978. Effect of graded levels of supplemental ascorbic acid in practical diets fed to rainbow trout. J. Fish. Res. Bd. Can., 35: 431436. John, M.J. and Mahajan, C.L., 1979. The physiological response of fishes to a deficiency of cyanocobalamine and folic acid. J. Fish Biol., 14: 127-133. Kitamura, S., 1969. Summary on the hypovitaminosis of rainbow trout Salmo gairdneri. Fish Pathol. (Japan), 3: 73-92. Kitamura, S., Ohara, S., Suwa, T. and Nakagawa, K., 1965. Studies on vitamin requirements of rainbow trout, Salmo gairdneri I. On the ascorbic acid. Bull. Jap. Sot. Sci. Fish., 31: 818-826. Lim, C. and Lovell, R.T., 1978. Pathology of vitamin C deficiency syndrome in channel catfish (Zctaluruspunctatus). J. Nutr., 108: 1137-1141. Mahajan, C.L. and Agrawal, N.K., 1979. Vitamin C deficiency in Channo punctotus Bloch. J. Fish Biol., in press. Mahajan, C.L. and Yadave, J.S., 1974. Experiments on synthetic diet for carps - some preliminary observations and their relevance to aquaculture. J. Fish. Sot. Ind., 6: 178-184. Mahajan, CL. and Yadave, J.S., 1976. The effect of calcium level in water on the assimiiarion and distribution of calcium in Labeo rohifu. Proc. All India Symp. Comp. Animal Physiol. Suppl. Abs. pp. l-2.
294
Pang, P.K.T., 1971. The effect of complete darkness and vitamin C supplement on the killifish. Fundulus heterocfitus, adapted to sea water I. Calcium metabolism and gonadal maturation. J. Exp. Zool., 178: 15-21. Phillips, A.M. Jr., Lovelace, F.E., Podoliak, H.A., Brockway, D.R. and Balzer, G.C. Jr., 1954. The absorption of calcium by brook trout. N.Y. State Const. Dept., Fish Res. Bull., 18: 5-23. Phillips, A.M. Jr., Podoliak, H.A., Brockway, D.R. and Balzer, G.C. Jr., 1956. Effect of mineral content of aquaria water on the metabolic activity of brook trout. N.Y. State Const. Dept., Fish. Res. Bull., 20: 42-56. Poston, H.A., 1966. Effect of dietary L-ascorbic acid on immature brook trout. N.Y. State Const. Dept., Fish Res. Bull., 30: 46-51. Siddiqui, M.A., 1967. Seasonal variation in ascorbic acid content and calcium content of different tissues of Ophicephaluspunctatus Bloch. Ind. J. Exp. Biol., 5: 54-55.
287 Amsterdam
- Printed in The Netherlands
THE ROLE OF VITAMIN C IN CALCIUM UPTAKE BY FISH
C.L. MAHAJAN
and N.K. AGRAWAL
Fish Biology Laboratory, 302004 (India) (Accepted
Department
of Zoology,
University
of Rajasthan,
Jaipur-
28 August 1979)
ABSTRACT Mahajan, C.L. and Agrawal, N.K., 1980. The role of vitamin C in calcium uptake by fish. Aquaculture, 19: 287-294, Calcium uptake in experimentally induced scorbutic snake heads, Channa ( = Ophicephalus) punctatus, fed vitamin C-deficient diet for 210 days is reported, using %a as tracer. When compared with a parallel control on completely synthetic diet, the results show decreased absorption and utilization of calcium by gill, skin, muscle and bone of scorbutic fii from surrounding water. The physiological significance of these results is discussed, and its practical utility in aquaculture is indicated.
INTRODUCTION
Studies on fish nutrition in the last two decades suggest that dietary deficiency of vitamin C results in poor growth, various skeletal deformities, such as lordosis and scoliosis, distorted hyperplastic gill cartilage and mottled sclerotic cartilages in the eye, as well as haemorrhages, loss of scales and delayed wound healing etc. (Kitamura et al., 1965; Poston, 1966; Kitamura, 1969; Halver et al., 1969, 1975; Ashley et al., 1975; Andrews and Murai, 1975; Lim and Love& 1978; Hilton et al., 1978). As a part of our extensive studies on the nutritional requirements of fish (Agrawal et al., 1978; John and Mahajan, 1979), most of the above mentioned changes have already been reported by us in Channa punctutus (Mahajan and Agrawal, 1979). Considering the importance of calcium as ground substance in bone, cartilage, wound healing, blood coagulation etc., investigations on the calcium uptake by various tissues of scorbutic fish using 45Ca as a tracer were made and are reported in this paper. MATERIAL
AND METHODS
Channa f = Ophicephalus) punctatus Bloch (average wt. 42.80 f 4.26 g, length 115.0 * 7.4 mm) of age 1 + years were collected from freshwater areas around Jaipur and, after a week’s acclimatization, were divided into the following groups of 80 each in laboratory aquaria:
288
Group I = control, fed complete synthetic diet (Table I) (at 3% body weight/ day). Group II = ascorbic acid-deficient, fed the same diet as Group I except that ascorbic acid was not added (at 3% body weight/day). The environmental conditions in holding aquaria were as follows: pH 7.8, temperature range 16-33”C, dissolved oxygen 6.5 mg/l, alkalinity 140 mg/l, dissolved calcium 58.76 + 0.28 mg/l, with traces of phosphates and nitrates. The light conditions varied with the season and daylight hours, the aquarium room being exposed to sky light. The routine was continued for 210 days, when increased mortality appeared in ascorbic acid-deficient group (Mahajan and Agrawal, 1979). At this point eight fish from group I and II each were randomly selected for calcium uptake studies. Fish from each group were kept separately in glass aquaria filled with 20 1 synthetic bicarbonate water (Table II). Water in the aquaria was constantly aerated through aerators in a closed system. After 24 h acclimatization, 50 mg/l calcium chloride and 50 pCi/l 45Ca (as CaCl,) were dissolved in each tank. The radioisotope was obtained from Bhabha Atomic Research Centre, Bombay, India. Immediately after adding the radioisotope, one millilitre water samples were taken in the aluminium planchet from each tank to determine the activity/ml of water. Four fish from each tank were sacrificed at 24 and 48 h. Gill, skin, muscle, bone, intestine and kidney tissue samples were carefully removed from identical sites from all the fish autopsied. A TABLE
I
Composition
of complete
Ingredient Vitamin-free casein Cellulose powder White dextrin Gelatin Corn oil Shark liver oil’ Vitamin mixture2 Mineral mixture3 Water
synthetic
diet (Modified
from Mahajan
and Yadave,
1974)
Weight (g/kg) 325 150 225 200 55 15 8 32 1200 ml
‘Used as a source of vitamin A (1500 III/g) and vitamin D (160 IU/g). ‘Thiamine hydrochloride, 40 mg; riboflavin, 160 mg; pyridoxine hydrochloride, 40 mg; choline chloride, 4.0 g; nicotinic acid, 600 mg; calcium pantothenate, 400 mg; inositol, 1.6 g; biotin 4 mg; folic acid, 12 mg; ascorbic acid, 800 mg; cyanocobalamine, 0.08 mg; manadione, 32 mg; alphatocopheryl acetate, 320 mg. ‘Aluminium chloride, 5 mg; zinc sulphate, 95 mg; cuprous chloride, 3 mg; manganese sulphate, 25 mg; potassium iodide, 5 mg; cobalt chloride, 30 mg; sodium chloride 1.395 g; magnesium sulphate 4.36 g; sodium biphosphate 2.774 g; potassium sulphate, 7.64 g; calcium biphosphate, 4.32 g; ferric citrate, 0.948 g; calcium lactate, 10.40 g.
289 TABLE II Composition of synthetic bicarbonate water (Phillips et al., 19SS) Mineral ions
Concentration
Ca’ + Na’+ K’+ Mga+ HCO,‘SiO,‘NO,‘PO,‘_ Trace*
50.0 40.7 3.2 5.0 100.0 5.0 1.0 5.0 0.035
(mg/litre)
*Trace minerals composed of 0.025 ppm each of Ala+, Ba*+, COa+, Cuz+, Fe’+, Mnl+, Nil+, Snl+, Zn’+, Bra-, F1-, I’-, CO,a-, Mo,O,,‘-.
sample of each tissue, 0.5-3 g, was weighed on a single pan electric balance, and digested in a minimum quantity (-0.5 ml) of concentrated nitric acid by heating in a boiling water bath for 3-5 min. The dissolved tissue was diluted to 4 ml with distilled water. Three samples of each tissue were prepared in this manner. One ml of each diluted sample was taken on an aluminium planchet and dried in an incubator (55-6O”C). Radiation activity in all the water and tissue samples was counted in a Nuclear-Chicago G.M. counter. The calculations made were on the general principle that the radioactivity of each tissue of the fish indicates the passage of calcium from the experimental water into that tissue during the time interval, and is a measure of the total calcium taken from this source and retained. One pCi of radioactivity represents the same amount of absorbed calcium in the particular experimental tank (Phillips et al., 1954). The results were calculated as follows: Counts/g tissue/min: Counts/ml tissue sample X dilution wt. of tissue (g)
(A)
Counts/CcCi of isotope/min: Counts/ml water sample (R)
Isotope (pCi)/ml water Amount of mineral ion labelled by/pCi isotope: Amount of mineral (pg)/ml water Amount of isotope (pCi)/ml water Uptake of mineral =$X
C (clg/g time)
WI
290 RESULTS
Analysis of the results presented in Table III and Fig. 1 shows that there was a statistically significant increase (P < 0.001) in calcium uptake in the first 24 h by the intestine of ascorbic acid deficient fish as compared to control. However, this trend slowed down considerably in the next 24 h so that the deficient group was found to have a decreased rate of uptake to such an extent that it was slightly lower (though statistically insignificant) than the control. Kidney tissue on the other hand, showed just the opposite trend; the calcium uptake in the first 24 h in the kidney of deficient group was far less compared with the control (- 4 pg/g compared with 16 pg/g in the control tissue), but in the next 24 h it increased to such an extent that it was significantly higher (P < 0.001) than the control (- 21 pg/g as compared with 13 pg/g in the control). In skin, gill and bone the uptake both at 24 and 48 h stages was less in the deficient group than in the control, even though the uptake values greatly increased between 24 and 48 h in both the control and deficient groups. However, there was considerable difference in the relative uptake values between the two groups at 48 h when compared to that at 24 h. Thus the difference in the rate of uptake remained almost constant in the skin, while in the gill and bone it was reduced. Despite these relative variations, the difference in calcium uptake by all these tissues between the control and deficient groups remained statistically highly significant. The decrease in calcium uptake in the muscle, however, was found to be statistically significant only at 48 h in the deficient group (P < 0.001) as compared with the control. At 24 h the calcium uptake in muscle, though less, was not found to be statistically significant.
TABLE III Uptake of calcium by various tissues of fish fed complete synthetic diet (control) and ascorbic acid-deficient diets (-AA) for 210 days Tissue
Gastrointestinal tract Gill Muscle Bone Skin Kidney
Uptake in 24 h (mean i S.E.) (Is/g)
Uptake in 48 h (mean r S.E.) (rg/g)
Control
-AA
Control
-AA
7.851kO.435 59.262k3.318 3.989+0.160 68.421i2.433 51.929+0.897 16.65920.377
16.651+0.553* 32.073+1.016* 2.647iO.144 21.697*0.297* 31.027i2.410* 4.656t0.186’
8.905+0.271 139.209k4.268 4.457kO.091 75.027kO.945 103.219t1.353 13.181+0.297
7.671k0.393 91.880*7.630* 2.077+0.137* 61.456*1.748* 62.409+2.483* 21.228+0.137*
*P < 0.001 Asterisks in the -AA group indicate the significance of difference. No asterisk indicates the non-significant change.
291
Dr
[7
CONTROL
n
AA.
Dcf.
O-
T
0-
O-
0t
I aLh e
A
GILL
Fig. 1. Absorption
e
A
BONE
a
A
SKIN
of calcium by tiiues
G.I.TRACT
of ascorbic acid deficient
A
B
MUSCLE
KIDN
and control fish.
DISCUSSION
Calcium metabolism in fish is particularly interesting because of their aquatic environment. They are unusual, if not unique amongst vertebrates, in their ability to absorb calcium from surrounding water (Fleming, 1974). Unlike tetrapods, a definite calcium regulating system has not been demonstrated so far, and our knowledge about the presence of functional parathyroid gland and the exact role of calcitonin in the metabolism of this electrolyte is still limited (Copp et al., 1972). The results presented above show that in normal (control) fish, maximum calcium uptake in the intestine and kidney takes place in the fiit 24 h, while in gill, bone skin and muscle this occurs between 24 and 48 h. These findings are similar to those reported in earlier reports (Phillips et al., 1954; Mahajan and Yadave, 1975). In intestine and kidney, being the sites of absorption and excretion, the homeostasis in calcium uptake takes place within 24 h, while in the bone, skin and muscle, being the sites of storage and metabolism, absorption rate increases with time. The gill, which is the major site of calcium ingress to the fish’s body, distributes absorbed calcium rapidly to the different tissues via the blood and show steady uptake in first 24 h. However, after this period calcium deposition begins in this tissue (Phillips
292
et al., 1954) which may account for the increased uptake observed in this tissue during the present study in the 24-48 h interval. The present study clearly establishes that a chronic vitamin C deficiency results in decreased absorption and utilization of calcium, as shown by decreased calcium uptake by the gill, which is the major site of calcium absorption. There is also a similar trend in uptake by bones, muscles and skin which are the major sites of calcium utilization and storage. The decreased absorption of calcium by the gill may be due to distortion of gill filaments and gill supporting cartilages in scorbutic fish, as pointed out by Halver et al. (1975). The increased calcium uptake by intestine in ascorbic acid-deficient fish during first 24 h may be compensatory to the decreased uptake by gill. However, its lowering to the control level in the next 24 h is possibly due to quick distribution and/or excretion of the absorbed calcium. In the kidney, on the other hand, the uptake in first 24 h was less than the control, but increased considerably in the next 24 h which may be due to the accumulation of calcium ions for excretion in the kidney because of decreased uptake by other tissues. The decrease in the total calcium absorption results in the mobilization of the calcium from the bones to maintain calcium homeostasis and results in the skeletal deformities reported earlier (Mahajan and Agrawal, 1979). Pang (1971) also reported withdraw1 of calcium from skeletal tissues in dark adapted vitamin C deficient juvenile Fundulus heteroclitus, where skeletal deformities preceeded by serum hypocalcemia, were observed. It is interesting to note that Siddiqui (1967) reported a seasonal correlation in ascorbic acid and calcium contents of various tissues of Channa ( = Ophicephalus) punctatus. The results of the present study and those of Pang (1971) and Siddiqui (1967) support the conclusion that calcium uptake by tissues is affected by vitamin C status of the fish. The influence of ascorbic acid deficiency on calcium uptake and collagen synthesis by impairing the hydroxylation of protocollagen proline and lysine (Barnes and Kodicek, 1972) explains the skeletal deformities reported by us in this fish and similar reports by others. CONCLUSION
These findings point to the desirability of ensuring an optimum vitamin C supply in the diet of intensively cultivated fish. This would enable the fish to utilize properly the available dissolved calcium in the water, thus resulting in healthy fish stocks, good growth and net productivity. For Channa punctutus, an air-breathing fish inhabiting swamps and ponds and presently being exploited for intensive cage culture, these conclusions may be kept in mind when formulating artificial diets.
293 ACKNOWLEDGEMENT
The study was supported by Indian Council of Agricultural Research, New Delhi. Thanks are due to the Head, Department of Zoology, University of Rajasthan, Jaipur for facilities and Prof. P.N. Srivastava for extending help in isotope studies.
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