Phosphocalcemic responses of vitamin D 3 administered intact or hypophysectomized Heteropneustes fossilis maintained either in artificial freshwater or calcium deficient freshwater

The serum calcium level of vehicle injected fish (group-A) exhibits no alteration throughout the experiment. Vitamin D3 administration to fish Heteropneustes fossilis exhibited hypercalcemia from day 3 to day 7 (group-B). However hypophysectomized and vehicle injected fish showed hypocalcemia from day 3 to day 7 (group-C). While hypophysectomized and vitamin D3 injected fish exhibited hypercalcemia on day 3 till the end of experiment (group -D). The serum phosphate of group A fishes is unaltered throughout experiment. The phosphate level of vitamin D3 treated fish (group-B) exhibits hyperphosphatemia from day 3 to day 7. In group C fishes exhibited a significant decrease in phosphate level throughout the experiment. While the group D fishes showed hyperphosphatemia from day 3 to day 7. In group E fishes the serum calcium level remains unaffected up to day 3 the level decreases on day 5 to day 7. In group F fishes showed hypercalcemia from day 3 to day 7. In group H fishes a progressive decrease in serum calcium level is noticed on day 3 to day 7. The group H fishes shows increase in the serum calcium level from day 3 to day 7. The serum phosphate level of group E fishes showed a decrease from day 3 to day 5. The phosphate level increases from day 3 to day 7 in group F fishes. The phosphate level exhibited a decrease from day 3 to day 7 (group G). The phosphate level increases from day 5 to day 7 of group H fishes.

regulates the metabolism and absorption of minerals and determines serum calcium and phosphorus levels. In vertebrates, the vitamin D endocrine system is a major regulator of calcium and phosphate homeostasis (Norman et al., 2002). Liver and fat stores large quantity of vitamin D in fishes. That is why fish are an important dietary source of vitamin D. Vitamin D is not synthesized by fish and are fully dependent on dietary source (Lock et al., 2010). Fish absorb vitamin D directly from their diet (Dusso et al., 2011). Uptill 1970s it was considered that the fish accumulate vitamin D but not metabolize vitamin D. But now it is clear that the fish have a vitamin D endocrine system with similar function like mammals (Lock et al., 2010). 1, 25 (OH)2 D3 (calcitriol), an active metabolite of vitamin D3 exerts its hypercalcemic effects through high affinity vitamin D3 receptor (Lock et al., 2010). Calcitriol plays a role in fish calcium metabolism by stimulation of intestinal calcium absorption and is a key factor in bone formation (Haga et al., 2004); the effects of calcitriol can be considered hypercalcaemic in mammals and fish alike.

BJECTIVES:
The objective of the present study is to investigate the Phosphocalcemic responses of vitamin D3 administered intact or hypophysectomized Heteropneustes fossilis maintained either in artificial freshwater or calcium deficient freshwater.
ATERIALS AND METHODS: Area of study: Adult specimens of Heteropneustes fossilis (bw 27-36g) were procured locally and acclimatized to the laboratory conditions for one week (temp 22-26⁰C) An initial sampling of blood was collected (from 10 fish) before the start of the experiment (zero hour). Then they were divided into following groups and given following treatments: Group A: This group received daily intraperitoneal injection of vehicle (0.1 mL of 96% ethanol /100g bw) and kept in artificial freshwater media. Group B: Fish from this group were given a daily intraperitoneal injection of vitamin D3 (100 ng/100g bw) and kept in artificial freshwater. Group C: In this group hypophysectomized (HYPX) fish was given daily intraperitoneal injection of vehicle (0.1 mL of 96% ethanol /100g of bw) and maintained in artificial freshwater. Group D: In this group the HYPX fish were given daily intraperitoneal injection of vitamin D3 (100 ng/100g bw) and kept in artificial freshwater. Group E: This group received daily intraperitoneal injection of vehicle (0.1 mL of 96% ethanol/100g bw) and kept in calciumdeficient freshwater. Group F: Fish from this group were given daily intraperitoneal injection of Vitamin D3 (100ng/100g bw) and kept in calciumdeficient freshwater. Group G: In this group HYPX fish were given daily inpraperitoneal injection of vehicle (0.1 mL of 96% ethanol/100g bw) and maintained in calcium-deficient freshwater.
Group H: In this group HYPX fish were given daily intraperitoneal injection of vitamin D3 (100ng/100g bw) and kept in calcium-deficient freshwater. Vitamin D3 was dissolved in 96% ethanol. From each of above mentioned groups 10 fishes were anaesthetized with MS222 and blood samples were taken after 1, 3, 5 and 7 days following initiation of the treatments. Statistical analysis: All data were presented as the mean ± S.E of six specimens and Student's t test was used to determine statistical significance. In all cases the experimental group was compared to its specific time control group. ESULTS: Serum calcium: The serum calcium level of vehicle injected fish (group A) exhibit no alteration throughout the experiment (figure 1). After day 1 following vitamin D3 treatment (Group B) the serum calcium level remain unchanged. An increase in the serum calcium level has been recorded on day 3 which progresses till day 7 (figure 1). Figure 1: Changes in the serum calcium level of intact hypophysectomized Heteropneustes fossilis kept in artificial freshwater and treated with vehicle or Vitamin D. Each value represents mean ±S.E of six specimens. Asterisk indicates significant differences (P˂0.05) as compared with vehicleinjected specimen In hypophysectomized and vehicle injected fish (group C) serum calcium level significantly decreases from day 3 to day 7 ( figure 1). An increase in the serum calcium level of hypophysectomized and vitamin D3 treated fish (group D) has been recorded on day 3 which persists till the end of the experiment (day 7) (figure 1). Serum phosphate: Serum phosphate levels of fish injected with vehicle (group A) have not shown any change throughout the experiment (figure 2). The serum phosphate level of fish treated with vitamin D3 (group B) remains unaltered on day1. The serum phosphate level fish of group B exhibits progressive increase from day 3 to 7 (figure 2). Hypophysectomized fish injected with vehicle (Group C) exhibit significant decrease in serum phosphate level from day 1 to day 7 (figure 2). In hypophysectomized fish treated with vitamin D3 (group D) A significant increase has been recorded in serum phosphate level from day 3 to day 7 (as compared to group C) (figure 2). iscussion: In the present study vitamin D3 treatment provoked elevation in the serum calcium and phosphate levels of the fish kept in artificial freshwater. This study derives support from the reports of other investigators who have also noticed increased total calcium levels in fish treated with vitamin D3/ vitamin D3 metabolites (Lock et al., 2010). Fenwick et al. (1984) injected Pagothenia bernacchii with 1, 25(OH)2D3 and reported reduced free plasma calcium, however, total plasma calcium remained unchanged. However, Sundell et al. (1993) treated Atlantic cod with 1, 25(OH)2D3 and recorded an increase in free calcium while total calcium levels were not affected. Increased total plasma calcium without any change in free calcium levels have been observed by Srivastav and Flik (1998) after injecting male Mozambique tilapia with 1,25(OH)2D3. In contrast, other investigators have reported that vitamin D3 fails to affect blood calcium level in sharks, rays and cyclostomes and lungfish. Srivastav et al. (1997) has suggested that the magnitude and duration of the increase of blood calcium in response to vitamin D3/1,25(OH)2D3 treatment are dependent on the availability of calcium content in ambient water. Also, fish can supplement plasma calcium from internal sources if calcium is not sufficiently available from external sources (food/water). Physiological doses of vitamin D3/1,25(OH)2D3 administered to unfed common carp provoked hypercalcemia and hyperphosphatemia which indicates that these electrolytes R D must have been mobilized from internal sources. In contrast, vitamin D3/1,25(OH)2D3 treatment to fed American eel (Anguilla rostrata) caused elevation in calcium and phosphate levels whereas in unfed eels this effect was absent (Fenwick et al., 1984). MacIntyre et al. (1976) observed hyperphosphatemia in eels treated with vitamin D3 metabolite (1,25(OH)2D3) but no change in calcium levels. These authors have suggested that this metabolite mediates phosphate homeostasis in marine fish which live in the environment rich in calcium but poor in phosphorus. In calcium-deficient freshwater administration of vitamin D3 to H. fossilis provoked hypercalcemia and hyperphosphatemia. The hypocalcemic response after vitamin D3 treatment to the fish kept in calcium-deficient freshwater cannot be attributed to calcium absorption through intestine as the fish were not fed and surrounding medium lack calcium. In vehicle-injected fish H. fossilis kept in calcium-deficient freshwater the serum calcium and phosphate levels showed a decrease. This derives support from the studies of other investigators who have also reported hypocalcemia and/or hypophosphatemia in fish kept in calcium-free environment. Wendelaar Bonga et al. (1984) noticed a significant hypocalcemia in tilapia after 5 days of its transference to lowambient calcium. This can be due to the increased efflux of these ions through gills. Flik et al. (1986) reported that calciumfree environment would allow diffusion of intracellular Ca ++ out of the fish. The observed hypocalcemia in vehicle-injected fish kept in calcium-deficient medium derives support from report of Wendelaar Bonga and Van der Meij (1981) who have reported increased integumental water permeability in low Ca ++ environment. Fenwick (1981) suggested that increased water uptake in low-calcium media may increase urine production which leads to calcium loss from the fish.
In the present study significant decrease in serum calcium and phosphate levels have been noticed in HYPX H. fossilis. Earlier, hypocalcemia has been reported after removal of pituitary. Prolactin in fish acts on the gills to stimulate the enzyme high affinity Ca ++ ATPase (Flik et al., 1986) which is considered to be the driving force for transmembrane calcium transport. In the foregoing study the observed hypocalcemia in hypophysectomized H. fossilis suggest the involvement of the pituitary in the uptake of calcium from the ambient water. This derives support from the studies of Flik et al. (1994) who have also reported that exogenous prolactin stimulates calcium uptake from the environment.
onclusion: It has been concluded from the present study that vitamin D plays a major role in the regulation of serum calcium and phosphate levels in fish Heteropneustes fossilis.
ONFLICT OF INTEREST: Authors have no conflict of interest.