ZOOLOGICAL SCIENCE 5: 893-895 (1988) © 1988 Zoological Society of Japan

[ C O M M U N I C A T I O N ]

Phosphocalcic Response to Vitamin D3 Treatment in Freshwater Snake, Matrix piscator

A J A I K. SRIVASTAV and LADLI R A N I

Department of Zoology, University of Gorakhpur, Gorakhpur-237 009, India

ABSTRACT—Vitamin D3 (20 IU/100 g bw) was admin­istered to the freshwater snake, Natrix piscator for 15 days. This treatment evokes hypercalcemia from day 3 to day 5. This response declines at day 10 and day 15. Vitamin D3 also induces hyperphosphatemia from day 5 till the end of the experiment (day 15).

INTRODUCTION

Little information is available on the role of Vitamin D and its metabolites in the lower vertebrates. There exist a few reports regarding the effects of vitamin D3 and its metabolites in teleosts [1-5] and amphibians [6-8]. However, in reptiles there is a single report [9] of vitamin D3-induced hypercalcemia. There seems to be no study concerning the effect of vitamin D3 on the serum inorganic phosphate concentration of rep­tiles. Hence, the present study was designed to investigate such an effect in freshwater snake, Natrix piscator.

MATERIALS AND METHODS

Adult specimens (both sexes) of N. piscator (bw 90-130 g) were collected locally and acclimatized to the laboratory conditions (ambient temperature and light ranged between 22-25°C and 10:20-10: 50 hr, respectively) for one week prior to use. An initial sampling of blood (from 6 specimens) was taken from the non-treated (normal) speci­mens before the start of the experiment. Then the

remaining animals were divided into two groups (A and B) of 30 specimens (20 males and 10 females) each.

Snakes from the both groups were given daily intraperitoneal injections of the following treat­ments for 15 days: Group A: 0.1 ml/100 g bw of vehicle (95% ethanol) and Group B: 20 I.U. of vitamin D3/100 g bw (dissolved in 95% ethanol). Vitamin D3 was provided by Prof. A. W. Norman (Univ. of Calif.). In all cases, the injection volume was 0.1 ml/100 g bw. The injections were per­formed at 10: 00 each day.

Six specimens (4 males and 2 females) from each group were anesthetized with ether 4 hr after the last injection on the 1st, 3rd, 5th, 10th and 15th day of the experiment. Blood samples were collected by caudal amputation. After clotting of the blood the sera were separated by centrifuga-tion (3500 rpm in a refrigerated centrifuge Janetzki K 24) and analysed for serum calcium and serum inorganic phosphate according to Trinder's [10] and Fiske and Subbarow's [11] methods, respec­tively.

The animals were not fed during experiment. Differences in the serum calcium and inorganic phosphate levels among different groups were analysed by Student's Mest.

RESULTS

In non-treated specimens the values of serum calcium and serum phosphate are 10.42 ± 0.28 and 4.38 ± 0.18 mg/100 ml, respectively.

Serum calcium level of N. piscator remains Accepted November 24, 1987 Received September 21, 1987

894 A. K. SRIVASTAV AND L. RANI

TABLE 1. Effect of vitamin D3 on serum calcium and phosphate in freshwater snake, Natrix piscator

Days

1

3

5

10

15

Serum calcium (mg/100 ml)

Group A

10.50 ± 0.18

10.63 ± 0.27

10.58 ± 0.26

10.45 ± 0.28

10.63 ± 0.16

Group B

10.95 ± 0.32

11.66 ± 0.293

12.38±0.29d

12.08±0.25c

11.12 ± 0.28

Serum phosphate (mg/100 ml)

Group A

4.31 ± 0.11

4.37 ± 0.13

4.27 ± 0.16

4.42 ± 0.12

4.32 ± 0.20

Group B

4.47 ± 0.20

4.86 ± 0.18

4.92 ± 0.12b

5.05 ± 0.14b

5.18 ± 0.18b

The values are mean ± S.E. of 6 determinations. a, b, c, and d, indicate significant responses: P<0.05, P<0.01, P<0.005 and P<0.001, respectively.

unaffected after day 1 following vitamin D3 treat­ment. After day 3, the value increases significant­ly. This increase continues up to day 5. Thereaft­er, a decline is noticed in the calcium level on day 10 and day 15 (Table 1 and Fig. 1). However, the

calcium value at day 10 is still significantly higher than the control level.

Serum inorganic phosphate levels of vitamin D3-treated specimens were not significantly devi­ated from the controls on day 3. From day 5 onwards, increased levels of phosphate are noticed (Table 1 and Fig. 1).

DISCUSSION

Our data demonstrate that vitamin D3 is effec­tive in inducing hypercalcemia and hyperphos­phatemia in TV. piscator. These results concur with earlier studies conducted with different species of vertebrates [1-9]. As the animals were not fed in the present study, the calcium needed for the hypercalcemic response to vitamin D3 possibly was derived from the demineralization of bones. The probability of getting calcium from water cannot be ruled out.

Parathyroid hormone has been reported to facilitate the transformation of the vitamin D3

metabolite 25-hydroxycholecalciferol to the active form 1, 25-dihydroxycholecalciferol [12, 13]. It has also been reported that increased calcium ions can inhibit this transformation by the direct effect on the parathyroid and calcitonin secretion rates [14]. The observed decrease in calcemic value in Natrix piscator on day 15 may be explained on the fact that the elevated blood calcium may have suppres­sed the secretion of parathyroid hormone and thus affecting the formation of 1, 25-dihydroxycholecalciferol. The decline of calcium

FIG. 1. Serum calcium and phosphate in freshwater snakes (N. Piscator) at various time intervals after vitamin D3 treatment. The values are mean ± S.E. a, b, c, and d indicate significant responses, P<0.05, <0.01, <0.005 and <0.001, respectively.

895 Phosphocalcic Response to Vitamin D 3

level on day 15 can also be attributed to the possible activation of the secretion of ultimobran-chial gland, which has been reported to contain a hypocalcemic factor - calcitonin [15, 16], in re­sponse to prolonged hypercalcemic challenge. Suzuki et al. [17] have reported that the small granular cells of ultimobranchial gland in sea snake, Laticauda semifasciata contain highly de­veloped Golgi complexes which are surrounded by immature granules in the process of formation. They have explained that the activity of the glandular cells is high because the snakes live in sea where the calcium load is high.

The hyperphosphatemic action of vitamin D3

suggests that nondietary phosphorus, possibly from the bone or soft tissues, can be mobilized.

Prior to the present report no data have been available concerning vitamin D3 effect on serum inorganic phosphate level from reptiles.

ACKNOWLEDGMENTS

This study was supported by a research grant F 23-334/84-SR II from University Grants Commission, India to A. K. Srivastav.

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