Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium concentration

Abstract

This study assessed the responses of vitamin-D3 intraperitoneally injected to Rohu, Labeo rohita @ of 0 IU/kg bw (only solvent), 100 IU/kg bw and 500 IU/kg bw reared in 20 and 40 ppm of calcium (Ca) enriched water. The cellular changes in Corpuscles of Stannius (CS) gland, serum Ca, and inorganic phosphate (Pi) level were analysed up to the 60th day. Rohu administered with 100 IU/kg bw D3 and exposed to 40 ppm Ca-rich water exhibited notable hyperplasia of CS compared with their control groups. Notable changes with high serum Ca level (13.87±0.3 mg/dl) was detected on the 5th day in fish exposed to 40 ppm Ca-rich water, while related values attained (13.74±0.1 mg/dl) only after 7 days in 20 ppm Ca-rich water of 500 IU/kg bw vitamin D3 injection. Similarly, high serum Pi level (7.66±0.2 mg/dl) in 40 ppm Ca injected with D3 at 500 IU/kg bw. The results demonstrated that the Ca homeostasis of Labeo rohita is influenced by intra-peritoneal vitamin D3. Progressive studies should be conducted by increasing the dose of vitamin D3 to investigate optimum dose/supplement in feed for commercially important aquaculture teleost Labeo rohita for maximum and sustainable absorption of Ca from the variable water Calcium levels to maintain Ca²⁺ homeostasis.

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Original article
Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 adminis-
tration in teleost Labeo rohita (Hamilton, 1822) reared in water with two dif-
ferent levels of calcium concentration
U. Sivagurunathan, Prem Prakash Srivastava, Subodh Gupta
PII: S1319-562X(20)30333-8
DOI: https://doi.org/10.1016/j.sjbs.2020.07.033
Reference: SJBS 1819
To appear in: Saudi Journal of Biological Sciences
Received Date: 25 March 2020
Revised Date: 2 June 2020
Accepted Date: 28 July 2020
Please cite this article as: U. Sivagurunathan, P. Prakash Srivastava, S. Gupta, Responses of Corpuscles of
Stannius to intra-peritoneal vitamin-D3 administration in teleost Labeo rohita (Hamilton, 1822) reared in water
with two different levels of calcium concentration, Saudi Journal of Biological Sciences (2020), doi: https://
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COVER PAGE
Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost
Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium
concentration
U. Sivagurunathan1, Prem Prakash Srivastava1, *, Subodh Gupta1
1Fish Nutrition, Biochemistry and Physiology Division, ICAR - Central Institute of Fisheries
Education (Deemed University), Off Yari Road, Panch Marg, Versova, Mumbai– 400 061, India
*Corresponding author: E-mail: ppsrivastava@cife.edu.in (P.P. Srivastava)

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Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost
Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium
concentration
ABSTRACT
This study assessed the responses of vitamin-D3 intraperitoneally injected to Rohu, Labeo rohita
@ of 0 IU/kg bw (only solvent), 100 IU/kg bw and 500 IU/kg bw reared in 20 and 40 ppm of
calcium (Ca) enriched water. The cellular changes in Corpuscles of Stannius (CS) gland, serum
Ca, and inorganic phosphate (Pi) level were analysed up to the 60th day. Rohu administered with
100 IU/kg bw D3 and exposed to 40 ppm Ca-rich water exhibited notable hyperplasia of CS
compared with their control groups. Notable changes with high serum Ca level (13.87±0.3 mg/dl)
was detected on the 5th day in fish exposed to 40 ppm Ca-rich water, while related values attained
(13.74±0.1 mg/dl) only after 7 days in 20 ppm Ca-rich water of 500 IU/kg bw vitamin D3 injection.
Similarly, high serum Pi level (7.66±0.2 mg/dl) in 40 ppm Ca injected with D3 at 500 IU/kg bw.
The results demonstrated that the Ca homeostasis of Labeo rohita is influenced by intra-peritoneal
vitamin D3. Progressive studies should be conducted by increasing the dose of vitamin D3 to
investigate optimum dose/supplement in feed for commercially important aquaculture teleost
Labeo rohita for maximum and sustainable absorption of Ca from the variable water Calcium
levels to maintain Ca2+ homeostasis.
keywords: Labeo rohita, Vitamin D3, Ca enriched water, Corpuscles of Stannius, serum Ca and
inorganic phosphorus.

3
1. Introduction
Freshwater aquaculture in India is dominated by Indian Major Carps (Labeo rohita, Catla catla
and Cirrhinus mrigala), by contributing about 87% of the total freshwater fish production
(ICLARM, 2001). Among the IMC, Labeo rohita is being widely cultured throughout India and
accounts for a majority of the production, and have a great potential in terms of biodiversity as
well as consumer preferences.
Corpuscle of Stannius (CS) is endocrine tissue secretes hypocalcemic factor(s), Stanniocalcin
to prevent hypercalcemia by reducing branchial and whole body Ca uptake (Fontaine, 1964). Many
workers (Fenwick, 1976; Fenwick and Forster, 1972; Wendelaar Bonga and Greven, 1978; Pang
et al., 1975). Olivereau (1964) and Johnson (1972) and Gu et al (2015) reported that CS is more
active in seawater (rich in calcium) than in freshwater (poor in calcium) and inferred that calcium
content of the surrounding water has a direct impact on the CS activity and transcriptomic
responses of CS. Suryawanshi and Mahajan (1976) and Ahmad and Swarup (1979) have also
reported that the activity of CS increases in the fishes kept in calcium-rich freshwater. In past
years, a steady flow of papers has advanced evidence that Ca2+ influx in branchial, intestinal (and
likely in renal Ca2+ transporting cells) is controlled by stanniocalcin (STC) (Buttler, 1993; Flik et
al., 1993; Wendelaar Bonga and Pang, 1986a,b). STC is a hormone produced by the so-called CS
endocrine gland. In gill and intestine STC inhibits Ca2+ entry (Flik et al, 1993; Verbost et al., 1993)
and it thus, controls the permeability of Ca2+ of the apical membrane through a secondary
messenger (cAMP) dependent pathway most likely a Camp operated calcium channel (Verbost et
al., 1993). In the complex epithelium of the gills’ chloride cells, specialized ion transporting cells
mediated Ca2+ uptake (Flik et al., 1995; McCormic et al., 1993; Perry et al., 1992). Stanniocalcin
is a glycoprotein hormone important in the maintenance of Ca and Pi homeostasis in fish. Two
mammalian related stanniocalcin genes, STC1 and STC2, were found to be expressed in various

4
tissues of fish as paracrine regulators (Luo et al., 2005; Joshi, 2020). Although Stannniocalcin-1
(STC-1) was originally described in fish, it is now known to be present throughout the animal
kingdom in both vertebrates and invertebrates (Ishibashi and Ima, 2002; Yoshiko and Aubin,
2004; Tanega et al., 2004; Richards et al., 2012; Palma et al., 2019). The principal sources of
STC-1 in bony fish are endocrine glands known as the Corpuscles of Stannius (CS) which are
anatomically associated with the kidneys. STC-1 release is stimulated by a rise in serum levels of
ionic Ca above the physiological set point through the activation of Ca-sensing receptors (Richards
et al., 2012). The hormone then exerts regulatory effects on the epithelial transport of Ca and/or
phosphate across the gills, gut, and kidneys to restore normocalcemia as the inhibitory action of
STC reduces mucosa to serosa calcium transport, which means that the uptake in gills and intestine
and re-uptake from ultra-filtered plasma in the nephron is controlled (Flik and Verbost, 1996). The
transcriptomic responses of corpuscle of Stannius gland of Japanese eels (Anguilla japonica) to
Changes in Water Salinity are recently reported by Gu et al (2015). The effects of Euphorbia
royleana on the alteration of CS of Heteropneustes fossilis are demonstrated by Prasad et al.
(2017).
Administration of vitamin D3 and its derivative had been demonstrated in many fishes to make
it hypercalcemic and to know the changes in serum Ca and Pi level (Srivastav and Srivastav 1988;
Srivastav and Singh, 1992). Although vitamin D3 is abundantly present in fish liver, its role in Ca
homeostasis has been emphatically denied (Rao and Raghuramulu, 1995). It has been reported that
vitamin D3 and its metabolites induce hypercalcemia in fishes (Singh and Srivastav, 1996; Swarup
and Srivastav, 1982; Srivastav et al., 1985; Hayes et al., 1986; Srivastav and Singh, 1992; Srivastav
et al., 1998). However, there is not single literature on the effect of vitamin D3 on the physiological
response to Indian major Carps, the most important fishes for aquaculture in India and other
neighbouring Asian countries, and changes in serum Ca and Pi level. This study clearly explains

5
the histological change and efficiency of CS and its hormone Stanniocalcin in freshwater fish
Labeo rohita. Thus the objective of the present study is to assess the response and behavior of Ca
and Pi regulating endocrine gland, Corpuscles of Stannius (CS) in Labeo rohita, most cultured fish
in aquaculture system when reared in two levels of Ca enriched water exposed to intra-peritoneal
doses of vitamin D3.
2.Materials and methods
2.1 Experimental fish
The experiment was conducted for 60 days at the wet laboratory of ICAR - Central Institute of
Fisheries Education (Deemed University), Mumbai. Healthy Labeo rohita (weight: 30±2g ;) fishes
were brought from Mahad fish farm, Mumbai in oxygen-filled polythene bags to CIFE, Mumbai.
The fish stock was acclimatized for one week in calcium-deficient water under an aerated condition
with a basal diet.
2.2 Experimental setup
The experimental setup consists of 18 uniform size plastic rectangular tanks (80 cm × 57 cm
× 42 cm, 150 L capacity) covered with perforated lids. Two hundred and sixteen fish were
randomly and equally distributed and stocked into experimental tanks with a 2×3 factorial design
in triplicates. The calcium level in water is enriched using Calcium chloride (CaCl2.2H2O) and the
experimental fish were stocked @12 fish/tank into 2 groups, namely group A- 20 ppm Ca, and
group B - 40 ppm Ca enriched water. The level of Ca in the experimental tank is measured using
EDTA titration method (APHA, 1998). Similarly, water quality parameters like pH, Dissolved
Oxygen, Ammonia, Nitrite, Nitrate, and Temperature were estimated periodically as per APHA
(1998) and maintained throughout the experiment.

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2.3 Intra-peritoneal injection
After stocking fish from both groups were injected intraperitoneally i.e., in the abdomen at a
45º angle between the pelvic fins and anal vent with vitamin D3 at different doses of, i) (0.0 IU/kg
bw only solvent, Arachis oil) ii) (100 IU/kg bw) and iii) (500 IU/kg bw). By considering 0.2ml
Vitamin D3 injection for 25g size fish, the 3Lakh IU Arachitriol (oil-based) was diluted with
Arachis oil. Out of 9 tanks from group A with 20 ppm Ca water, 3 tanks were given Vitamin D3
Intraperitoneal injection @ the dose of 0 IU/kg bw (control, only 0.2 ml solvent, Arachis oil), 3
tanks were given Vitamin D3 Intraperitoneal injection @ the dose of 100 IU/kg bw and 3 tanks
were given Vitamin D3 Intraperitoneal injection @ the dose of 500 IU/kg bw respectively. A,
similar setup is arranged for group B with 40 ppm Ca-rich water. The controls were given only
solvent (without vitamin D3 to give similar conditions in control and experimental fishes of both
the treatments A & B), thus, the administration of Vitamin D3 was not done to the control group.
The doses 100 IU/kg and 500 IU/kg is at par with many authors used in their studies (Swarup and
Srivastav, 1982; Swarup and Norman, 1996; Singh and Srivastav, 1996; Srivastav et al., 1998).
2.4 Fish Sampling and Serum Analysis
Sampling was done on Day 1, 2, 3, 5, 7, 9, 11, 13, 15, 30, and 60. In each sampling,
one fish from each replicate was sacrificed during the study period and taken for ionic activity,
and histological studies. Serum samples were taken by collecting blood in vials using a sterile
syringe and they are kept aside without any disturbance for separation of serum from a blood clot.
Then the vials were centrifuged for perfect separation of serum without any hemolysis to estimate
Ca and inorganic phosphate (Pi) level using Trinder (1960) and Fiske and Subbarow, (1925)
methods respectively.
2.5 Histological Studies

7
The Corpuscle of Stannius along with the adjoining portion of the kidney was removed from
the fish of each treatment group and they were fixed in aqueous Bouin’s fluid. The fixed samples
were processed by standard techniques following a series of alcohol treatment, cleared in xylene,
and embedded in paraffin wax. Thin (5 μm) sections were prepared with a microtome and stained
with Haematoxylin/Eosin (HE).
2.6 Statistical analysis
The results of each experiment are expressed as mean  standard deviation and analysed by
multivariate analysis of variance (ANOVA) using statistical package SPSS version 16 to test the
significance of the difference between the control and experimental groups. A probability level of
0.05 was used to find out the significance in all cases.
3. Results
Change in serum Ca and inorganic phosphate (Pi)level and cellular changes of Corpuscle of
Stannius in two different Ca enriched groups
Control group (A and B)
The serum Ca and Pi level was observed on days; 0, 1st, 2nd, 3rd, 5th, 7th, 9th, 11th, 15th, 30th, and
60th day. And there was no rise in serum Ca and Pi levels from day 1 to day 60 (Table-1, Fig. 1-4).
The corpuscles of Stannius also possess oval or rounded nuclei without any cellular changes (Fig.
5 and 6).
Group A (20 ppm Ca) – Experimental group, AL and AH
Calcium analysis
In low dose (100 IU/kg bw) there is an increase in Ca level and reached the peak at 5th day up
to 11.45±0.4 mg/dl and again started decreasing till 60th day (Table 1). At the same time for high
dose (500 IU/kg bw) the uptake of Ca is higher than low dose and reached maximum on 7th day at
a range of 13.74±0 mg/dl. and again it reduced till 60th day (Fig. 1).

8
Inorganic Phosphate analysis
In low dose (100 IU/kg bw) there is an increase in Pi level and reaching the peak at 5th day up
to 6.23±0.5 mmol/l and again started decreasing till 60th day (Table 2). Similarly for high dose
(500 IU/kg bw) the uptake of Pi was higher than the low dose and reached maximum on 7th day at
a range of 7.04±0.2 mmol/l and again it reduced till 60th day (Fig. 3).
Histological analysis
Cellular structures are shown in Figs. 7 and 8 on day- 5 showing the nuclear volume of cells
records an increase and they become partially de-granulated as is evident by their weak staining
response. There was an increased dilatation of sinusoids (Fig.-8) and these changes get
exaggerated. The results of cellular activities are in correspondence with the serum levels of Ca
and inorganic phosphate and demonstrating that there was the hypocalcemic response of CS gland.
Group B (40 ppm Ca) - Experimental group, BL and BH
Calcium analysis
In low dose (100 IU/kg bw) there was an increase in Ca level and reaching peak at 5th day
up to 12.48±0.5 mg/dl and started decreasing till 60th day (Table 1, Fig. 2). At the same time for
high dose (500 IU/kg bw) the uptake of Ca was higher than the low dose and reached maximum
on 5th day at a range of 13.87±0.3 mg/dl and it reduced till 60th day (Fig. 2).
Inorganic Phosphate analysis
In low dose (100 IU/kg bw) there was an increase in Pi level and reaching peak at 5th day up
to 6.89±0.2 mmol/l and started decreasing till 60th day (Table 2, Fig. 4). At the same time for high

9
dose (500 IU/kg bw) the uptake of Pi was higher than the low dose and reached maximum on 7th
day at a range of 7.66±0.2 mmol/l and it reduced to normal condition as in case of control till 60th
day (Fig. 4).
Histological analysis
The cellular structures are shown in Fig. 9 and 10 on day-7 showing the nuclear volume of
cells records an increase and they become partially de-granulated as is evident by their weak
staining response. Also, there is an increased dilatation of sinusoids (Fig.10) and these changes get
further exaggerated and complete exhaustion of the gland was recorded.
4. Discussion
Serum Ca concentration
Serum Ca concentration level in Labeo rohita is calculated and it is found to be higher on the
5th day of fish, which is reared in group A with a high dose (500 IU/kg bw) of vitamin D3. At the
same time, it is found to be higher on the 7th day of fish which is reared in group B with high dose
(500 IU/kg bw) of vitamin D3. Srivastav et al (1997a), who also observed vitamin D metabolites
affect the serum Ca level in freshwater catfish Heteropneustes fossilis, in which there is an increase
in serum Ca level at the day of 3 and 5, which were injected with intraperitoneal Vitamin D3 and
Srivastav et al. (1997b) also done a similar experiment in freshwater mud eel Amphipnous cuchia
and reported that there is an increase in serum Ca level at day 10 when it is reared in Ca-rich
environment and injected with 100 ng of vitamin D3 for 100g bw/ day. Bansal et al. (1979) showed
increased serum Ca levels in Labeo rohita due to chronic chlordane exposure. This result shows
similarity to the experiment done by Srivastava et al. (2012) in which the fish Notopterus
notopterus treated with three level of vitamin D3 dosage like 100, 500, 1000 IU/kg bw. In this,

10
there is a peak levels of serum Ca is found on day 5 on the three levels. But the amount or level of
Ca intake varies with the dosage. The dosage with 1000 IU/kg bw shown higher absorption of Ca
from the environment which is followed by 500 and 100 IU. A number of authors studied
hypercalcemic effects of vitamin D3 metabolites (Singh and Srivastav, 1996; Swarup and
Srivastav, 1982; Srivastav et al., 1985; Hayes et al., 1986; Srivastav and Singh, 1992; Srivastav et
al., 1998), and showed that hypercalcemia depends on exposure time as well as on the type and
concentration of the vitamin D3 metabolite used (Swarup et al., 1983; Srivastav et al., 1993).
Responses to vitamin D treatments vary not only within but also among species. For example,
injecting 1,25(OH)2D3 in emerald rock cod (Pagothenia bernacchii) reduced free plasma Ca but
left total plasma Ca levels unchanged, suggesting an increased fractional binding of Ca to plasma
proteins (Fenwick et al., 1984).
In contrast, Sundell et al. (1993) repeatedly injected 1,25(OH)2D3 in the Atlantic cod and
observed an increase of free Ca while total Ca levels remained unchanged. In male Mozambique
tilapia (O. mossambicus) IP injections of 1,25(OH)2D3 increased total plasma Ca without altering
the free Ca levels (Srivastav et al., 1998). Vitamin D3 injected male catfish (Clarias batrachus)
acclimated to low Ca water increased their serum total Ca levels as compared with control fish.
However, this increase doubled in vitamin D3 injected catfish from water supplemented with extra
Ca when compared to controls from the same water, although the duration of this increase was
shorter than the one in fish from low Ca water (Swarup and Srivastav, 1982). A similar observation
was performed in freshwater mud eel, Amphipnous cuchia (Srivastav, 1983). Magnitude and
duration of the increase of plasma Ca in response to vitamin D3 are dependent on the Ca
concentration in the water (Srivastav et al., 1997). If Ca is not sufficiently available from the water
or the diet, then fish can supplement plasma Ca from internal sources. Intra-peritoneal injections
of unfed common carp with physiological doses of either vitamin D3 or 1,25(OH)2D3 resulted in

11
hypercalcemia and hyperphosphatemia (Swarup et al., 1991), which suggests that the minerals
must have been derived from internal sources. Similarly, daily injections with vitamin D3 or 1,
25(OH)2D3 in fed American eel (A. rostrata), increased plasma Ca and phosphorus, while this
effect was absent in unfed eels (Fenwick et al., 1984). The plasma Ca levels in the fish are actually
not controlled and/ or regulated by the endocrine system only; other hormones like stanniocalcin
(Pierson et al., 2004), parathyroid hormone and related protein (Guerreiro et al., 2007; Abbink and
Flik, 2007), and the prolactin (Flik et al., 1984, 1989; Seale et al., 2006) are involved in the control
mechanism as well.
Serum inorganic phosphate (Pi) concentration
In this experiment serum phosphorus concentration level in Labeo rohita is calculated and it
is found to be higher on the 7th day of fish which is reared in group A with a high dose (500 IU/kg
bw) of vitamin D3. At the same time, it is found to be higher on the 7th day of fish which is reared
in group B with a high dose (500IU/kg bw) of vitamin D3. Srivastav et al (1997a) who also
observed vitamin D metabolites affect the serum phosphorus level in freshwater catfish
(Heteropneustes fossilis), in which there is an increase in serum phosphorus level from day 3 to
10, and showing a hyperphophatemic reaction which was injected with intraperitoneal Vitamin D3.
Srivastav et al. (1997b) done a similar experiment in freshwater mud eel Amphipnous cuchia and
reported that there is an increase in serum phosphorus level at 5th, when it is reared in Ca-rich
environment and injected with 100 ng of vitamin D3 for 100g bw/day.
In contrast to Ca, fish must obtain phosphate via the diet as water phosphate levels are normally
very low, and direct uptake of phosphate from the water is likely insignificant in fishes. Little
information on the involvement of vitamin D3 in phosphate metabolism in fish exists. Responses
to vitamin D3 metabolites on plasma phosphate vary between species. Daily intraperitoneal

12
injection with vitamin D3 or 1, 25(OH)2D3 increase plasma phosphate in catfish (C. batrachus)
(Swarup et al., 1984), American eel (Fenwick et al., 1984) and C. carpio (Swarup et al., 1991), but
not in Mozambique tilapia (Rao and Raghuramulu, 1999). In unfed American eel (Fenwick et al.,
1984) and freshwater mud eel, A. cuchia (Srivastav, 1983) plasma phosphate increased after intra-
peritoneal vitamin D3 injection. Apparently, in addition to phosphate reabsorption in the kidney
(Fenwick and Vermette, 1989), it can be mobilized by vitamin D3 metabolites from a non-dietary
source, presumably bone or soft tissues (Lopez et al., 1977).
Histology of Corpuscles of Stannius (CS)
In the present study, Labeo rohita injected with vitamin D3 and kept in two different calcium
environment exhibits degranulation of the CS cells by increase in the volume of the cells and
sinusoidal dilatation. Earlier workers have considered hypertrophy of CS cells as an indication of
the activity of CS in response to hypercalcemia (Olivereau and Olivereau, 1978; Srivastav et al.,
1985; Srivastav and Srivastav, 1988). The volume and density of CS cells increase as a result of
external Ca concentration (Urasa and Wendelaar Bonga, 1987). Suryawanshi and Mahajan (1976)
and Ahmad and Swarup (1979) have also reported that the activity of CS increases in the fishes
kept in calcium-rich freshwater. In Labeo rohita hypercalcemia results in degranulation of AF-
positive cells. Aida et al. (1980) have suggested that the secretory activity of cells of CS may
be directly affected by plasma ion levels, especially Ca ions. The present study supports this
suggestion and also it agrees with the observations of Wendelaar Bonga et al (1980). According
to Wendelaar Bonga et al (1980) the type-1 cells of CS are more active in the fish adapted to
diluted or full-strength seawater than in freshwater specimens. For this response they have stated
that the high activity of this cell in seawater is apparently due to the high Ca concentration of
seawater. The degranulation of the cells of CS of Labeo rohita can be attributed to the increased

13
release of hypocalcemic factor (Stanniocalcin) from CS to encounter the elevated level of Ca
caused by vitamin D3 treatment. The sinusoidal dilatations in response to hypercalcemia in
Labeo rohita is similar to the observations on Clarias batrachus (Srivastav et al., 1985;
Srivastav and Srivastav, 1988).The degeneration among few corpuscular cells observed in
Labeo rohita in response to prolonged hypercalcemia is in agreement with the results obtained
by Hiroi (1970) on Oncorhynchus sp, Srivastav et al (1985) and Srivastav and Srivastav (1988)
on Clarias batrachus and histological and ultrastructure studies of CS in African catfish, Clarias
gariepinus (Karkit et al., 2019). Advanced evidence that Ca2+ influx in branchial, intestinal (and
likely in renal Ca2+ transporting cells) is controlled by stanniocalcin (STC) (Buttler, 1993; Flik et
al., 1993; Wendelaar Bonga and Pang, 1986a,b).
. The degeneration is due to the exhaustion of corpuscular cells. In the CS of Labeo rohita
kept in low Ca freshwater there is an increased storage of granules. This can be attributed to the
observed decrease in the serum Ca and inorganic phosphate levels. Hypoactive cells have been
noticed in the fish expose to low-Ca seawater (Wendelaar Bonga et al., 1980). Storage of secretory
granules within calcitonin cells (which secrete a hypocalcemic factor in mammals) in response to
hypocalcemia has also been reported earlier by Gittes et al., (1968) and Srivastav and Swarup
(1982). In mammals, it has been suggested by Hirsch and Munson (1969) that the heavy
accumulation of secretory granules in calcitonin cells during hypocalcemia results due to little or
no calcitonin secretion and continuance of its biosynthesis. In the present study, the same principle
seems to be involved. In a freshwater medium, there is no change in the cells of CS after vehicle-
injected fish. This may be due to the non-involvement of this cell type in Ca homeostasis as
according to Wendelaar Bonga et al (1976, and 1980). In Ca-rich medium the cells of CS of fish
and Ca/Pi levels after vehicle or vitamin D3-treatment exhibit a decrease in the volume. This
conforms with the reports of Wendelaar Bonga et al (1976; 1980) and Meats et al (1978) who

14
have reported indications for a reduction of secretory activity of CS from fish transferred
from freshwater to seawater. Buttler, (1993), Flik et al. (1993), and Wendelaar Bonga and Pang
(1986a,b) had reported Ca2+ influx in branchial, intestinal, renal Ca2+ transporting cells is
controlled by stanniocalcin in marine fishes.
5. Conclusion
The hypercalcemic and hyperphophatemic responses were more elevated in a higher doses of
vitamin D3 when fishes are reared in both the concentrations of calcium levels. Further, it is
observed that the normocalcemic and normophosphatemic responses were faster in a lower dose
of D3 in both rearing conditions. It is of interest to note in the present study that CS cells exhibit
an increased nuclear volume in Ca-rich when compared to those observed in low Ca
freshwater. This increased activity of CS cells in Ca-rich freshwater may be attributed to the
possible increase in the serum Ca and inorganic phosphate levels. Thus, this study helps in
understanding the Ca and Pi regulation of freshwater fish Indian major carp, Labeo rohita by the
endocrinal gland Corpuscles of Stannius in different calcemic environments of water for
aquaculture. A future study is needed to establish the mechanism of action and regulation of
calcium homeostasis in important culturable food fish species reared in different water/soil
conditions with special reference to saline and/or sodic soil water areas that are rising day by day
in many countries including India.
Acknowledgments
Authors are thankful to the Director and Vice-Chancellor, ICAR–Central Institute of Fisheries
Education, Mumbai; for providing all the facilities required for the present work. The funds

15
provided by Indian Council of Agricultural Research, New Delhi, for the research work are also
acknowledged.
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