Dietary Calcium And Potassium On Growth Indices Biology Essay

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Two completely randomized experimental designs were conducted to investigate the effect of different levels of dietary Ca on some growth indices, body biochemical composition and some whole body elements in rainbow trout fingerlings in a culture system. Tow basic diets with 0.95% Ca and 0.72% K were prepared and with CaCO3 for experiment ? and K2CO3 for experiment ??, other dietary treatments were built. In initiation of each experiments, 25 pieces of rainbow trout fingerlings (12.18� 0.04 and 15.60� 0.05) introduced in each experimental units respectively, and were fed with dietary treatment ad libitum two times daily at 9:00 and 15:00 for a 8- week period. It was resulted that different levels of inorganic dietary-Ca in diets, had not significantly affected on growth factors (W1, WG, G%, SGR% day-1and TGC), but different levels of inorganic dietary-K in diet had significantly affected (p< 0.05) on these factors. FCR and survival rates did not show significant differences between the treatments in each experiment. In The first experiment, crud protein CP% and Ash% significantly increased and total lipid showed depletion with an increase in the dietary Ca (p< 0.05). Change in inorganic dietary-Ca had significantly affected on Ca, P, Mn, Zn, Cu and Fe of whole body contents (p< 0.05) and not affected the Mg and K of whole body. With increasing the inorganic dietary-K, diet with 0.9% total K, had significantly increased in CP% (p< 0.05), however not significant differences between the trails in Ash% and total lipid% were observed. The Ca, K, P, Mg, Zn, Fe and Cu of whole body were significantly changed (p< 0.05), and Mn had not significantly changed with increasing the inorganic dietary-K.

Results were obtained in these studies, showed that, changes in amount of inorganic Ca in diets at the range of 0.95- 1.61% could not significantly affect on growth indices but dietary-K at the range of 0.72- 1.3% affected the growth indices significantly (p< 0.05). Significant effects on biochemical composition and some whole body minerals of cultured rainbow trout fingerlings with changes the dietary Ca and K were observed.

Key words: Rainbow trout, dietary-Ca, dietary-K, growth indices, biochemical composition, whole body minerals.

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1. Introduction

Minerals are required for the normal life processes, and fish, need these inorganic elements. Fish may derive these minerals from the diet and also from ambient water [1-3]. All forms of aquatic animals require inorganic elements or minerals for their normal life processes [3]. Many essential elements are required in such small quantities that it is difficult to formulate diets and maintain an environment that is low in minerals to demonstrate a mineral deficiency [3]. Definition of the mineral requirements of fish is complicated by the facility with which they can take up certain ions from the water. Ca, Mg, Na, K, Fe, Zn, Cu and Se can all be obtained from the water. For marine fish, a full mineral package in the diet appears unnecessary and probably only P, Fe and Zn need to be supplied [4]. Calcium is one of the most abundant cations in the body of a fish and closely related to the development and maintenance of the skeletal system and participate in several physiological processes including the maintenance of acid�base equilibrium, osmoregulation, muscle contraction, bone mineralization, blood clot formation, nerve transmission, maintenance of cell membrane integrity, and activation of several important enzymes [3,5,6] and is readily derived from the water and occurs in adequate amounts in most diets consumed by fish [3]. Regulation of Ca influx and efflux occurs at the gills, fins, and oral epithelia. The endocrine control of Ca metabolism in fish is also regulated by hyper- and hypocalcemic hormones. Teleosts possess two hormones with hypocalcemic action: calcitonin, secreted by the ultimobranchial gland, and stanniocalcin (STC), secreted by the corpuscles of Stannius [7].

Very few studies have been conducted on the dietary Ca requirements of fish. Dietary Ca supplementation up to a certain level benefited the performance of blue tilapia reared in Ca-free water [8] but also fingerling scorpion fish reared in sea water [9]. A positive effect of dietary Ca supplementation was also found in American cichlid [10] and Atlantic salmon when dietary P level was inadequate [11] and in red sea bream at high dietary P levels [12]. However, excess dietary Ca has been reported to induce negative effects in other fish species [11, 13].

In previous studies we observed that the redlip mullet Liza haematocheila., tiger puffer Takifugu rubripes. and giant croaker Nibea japonica. could not absorb adequate Ca from seawater to fulfill their requirements [9, 14, 15]. In contrast, Ca absorption from seawater by red sea bream Pagrus major. and black sea bream Acanthopagrus requirements [16, 17]. In rainbow trout fry no effect of dietary Ca deficiency was noticed on growth performance and fish composition [18]. The Ca requirement of fish is affected by the water chemistry, the phosphorus level in the diet, and species differences [7, 19, 20] and between 0.3-0.65% [3] and approximate 1% [5] were determined. A low concentration of Ca (0.34% or less) is required in the diet of red sea bream, carp, eel, and catfish for optimum growth [12, 21-23]. Optimal dietary Ca:P ratios seem to vary between different species, but reported to be 0.2 in Atlantic salmon [11] and 1.3 in American cichlid [10]

Sodium, potassium, and chloride are the most abundant electrolytes in the body of living organisms. Sodium and chloride are the major cation and anion, respectively, of extracellular fluids of the body, whereas potassium and magnesium are the major intracellular cations. The osmotic pressure of the intracellular and extracellular fluids is rigidly controlled, largely through energy-dependent regulatory mechanisms that determine the rate of absorption of sodium ions and water by the epithelial membranes of the gill, gut, integument, and kidney [3].

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As with all animals, potassium is the most abundant intracellular ion in fish and plays many important physiological roles including the maintenance of cellular volume and membrane potentials and the generation of nerve impulses [24, 25]. In fish, potassium plays additional critical roles in osmo- and iono-regulation and acid/base balance [26, 27].

Plasma membranes contain an energy-dependent Na+ pump, which actively transfers Na+ from the intracellular to the extracellular environment. As Na+ exists, K+ enters, because the membrane is fully permeable to K+ and these ions are very similar in properties to the Na+ they are replacing [3, 19]. Potassium deficiency causes overall muscle weakness, resulting in intestinal distention, weakness of cardiac and respiratory muscles, and their ultimate failures [3, 19]. K supplementation was found necessary in purified diets for Chinook salmon. Juvenile Chinook salmon reared in freshwater required 0.8% K in their diet for maximum growth, and whole-body K saturation was reached at a K concentration between 0.6 and 1.2% of the diet [28]. Fish reared in seawater, where the K concentration is much higher than in freshwater, do not require K supplementation [16].

Effects of Ca on growth, biochemical composition and elements of whole body were studied in various researches [18, 29-32] but few studies focus on effects of K on these factors and had not exact data on requirements of rainbow trout for these tow minerals and requirements of rainbow trout may vary with the mineral content of the water [4, 18]. For these reasons, in the present study, we investigated the effect of different levels of inorganic dietary Ca and K on some growth indices, body biochemical composition and some whole body minerals in rainbow trout fingerlings in a culture system to access the new data in the widely range.

2. Materials and methods

2.1. Experimental fish and conditions

All rainbow trout (O. mykiss) fingerlings were provided by fish farm from Dohezar; Tonekabon (mazandaran- Iran) and transferred to experimental fish farm of university of Tehran (Karaj-Iran). After primary accumulation, 30 pieces of rainbow trout fingerling, introduced in each experimental units randomly, and fed with basic diets (5% biomass) for 10 days. Then, 25 pieces of fish (initial mean weight: 12.18� 0.04 gram for experiment ? and 15.60� 0.05 gram for experiment ??) introduced in to 12 fiberglass tanks containing 200 L water for each experiment respectively, and fed whit experimental diets for 8-week period. Water of each units were re- circulated with a water pump and filtered for collecting the fecal matters and other suspended solids. 20% of water were exchanged after tow days and aerated with one air pump. Temperatures were satiable at 16- 17 �C and light period was naturally regulated.

2.2. Feed production and feeding regime

Tow basic diet with 0.95% total Ca (A1) for experiment ? and 0.72% total K (A2) for experiment ?? with choosing and chemical analyzing the integrates, were prepared (Table 1). Then with CaCO3 and K2CO3, respectively for each experiment, other dietary treatments (B1, C1 and D1) (1.21%, 1.41% and 1.61% � Ca) (Table 2) and (B2, C2 and D2) (0.9%, 1.1% and 1.3% - K) (Table 3) were built. The increases in Ca and K were made by weight replacement of a-cellulose with

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the mineral salts in the diets. Fish were fed with dietary treatment ad libitum two times daily at 9:00 and 15:00 for an 8- week period.

Table 1

Diet formulation (as % of wet matter)

Integrates [ %] experiment ? experiment ??

Fish meal 25 39

Meat meal 2 7

Fish oil 8 6

Wheat flour 12 15

Wheat bran 14 9

Soybean meal, solvent extracted 23 12

Cotton seed meal, solvent extracted 9.33 5.33

Brewers flour 1.66 4.66

Corn flour 2 8

Vitamin premix 1 1

a-cellulose CaCO3 a-cellulose K2CO3

A1 2 ---- A2 2 ---

B1 1.4 0.6 B2 1.69 0.31

C1 0.9 1.1 C2 1.34 0.66

D1 0.4 1.6 D2 0.99 1.01

100 100

Each liter of vitamin premix contain: vitamin A, 7000000 IU; vitamin D3, 70000 IU; vitamin E, 7000 IU;

Niacin, 7000 IU; Vitamin B1, 875 mg; Vitamin B2, 875 mg; Vitamin B6, 1.750 mg; Vitamin B12, 8.75 mg

2.3. Samplings and Chemical analyses

In first, five fish from each tank were collected and kept frozen at -20 �C for later whole body composition and mineral analyses. In the final sampling five fish from each tank were stored for whole body composition and mineral analyses. Individual fish weight measurements of the experimental population were taken at the start and from each tank on days 14, 28, and at the end of the experimental period. The survival of fish was calculated from daily mortality and from the final number of the surviving trout fingerling recorded in each tank.

Proximate composition of diets and body was determined according to the following procedures: dry matter after drying at 105 �C for 24 h, protein (N�6.25) by the Kjeldahl method after acid digestion(Kjeltec 1030 Autoanalyzer, Foss Tecator AB, Hogans, Sweden), ash by incineration at 550 �C for 16 h (AOAC 2000, ID 942.05) .Total lipid was determined by Soxhlet extraction (Soxtec System, HT, Foos Tecator 1043) (AOAC 2000, ID 920.39). Ca, K and the remaining minerals determined by atomic absorption spectrometry (ISO 6869-2000, Shimadzu AA-670) [33].

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Table 2

Proximate composition and mineral contents of experiment ? diets..

CP%* EE%** Ash% Ca% K% P% Mg% Mn (ppm) Zn (ppm) Cu (ppm) Fe(ppm)

A1 38.34 11.44 6.41 0.95 0.93 1.006 0.27 70.05 75.89 12.75 425.00

B1 38.34 11.44 6.41 1.21 0.93 1.006 0.27 70.05 75.89 12.75 425.00

C1 38.34 11.44 6.41 1.41 0.93 1.006 0.27 70.05 75.89 12.75 425.00

D1 38.34 11.44 6.41 1.61 0.93 1.006 0.27 70.05 75.89 12.75 425.00

CP*: Crud protein; EE**: Total lipid

Table 3

Proximate composition and mineral contents of experiment ?? diets.

CP%* EE%** Ash% Ca% K% P% Mg% Mn (ppm) Zn (ppm) Cu (ppm) Fe(ppm)

A2 37.93 10.81 6.65 1.22 0.72 1. 06 0.25 65.31 77.81 10.25 447.91

B2 37.93 10.81 6.65 1.22 0.90 1. 06 0.25 65.31 77.81 10.25 447.91

C2 37.93 10.81 6.65 1.22 1.10 1. 06 0.25 65.31 77.81 10.25 447.91

D2 37.93 10.81 6.65 1.22 1.30 1. 06 0.25 65.31 77.81 10.25 447.91

CP*: Crud protein; EE**: Total lipid

2.4. Calculations and statistical analyses

- Growth indices and feed conversion were determined according to the following formulas (W2=final bodyweight, W1=initial body weight):

- Growth percent: G%= (W2/W1)�100

- Specific growth rate: SGR (%) = (ln W2-ln W1)�100/feeding days.

- Feed conversion ratio: FCR=feed consumed/biomass gain.

- Thermal growth coefficient: TGC= (W2 1/3-W1 1/3)�1000/S(t� feeding days). [34]

Percentage data were arc-sin transformed and data were checked for heterogeneity and normality prior to analysis and transformed, if necessary. Analytical data were subjected to one-way analysis of variance (ANOVA) using SPSS 18.0 for Windows and differences between means were tested using the Duncan test. Data are given as means� standard errors and effects were considered significant at a probability level of Pb 0.05.

3. Results

3.1. Growth indices

During the experiment ? and ?? period, fish doubled their initial body weight. With increased the inorganic dietary-C, The differences observed in final body weight, weight gain, growth percent (G %), SGR% and TGC% were not statistically significant (P>0.05) (Table 4). In experiment ??, with increasing the dietary-K, the weight gain, growth percent, SGR% and TGC% were significant difference (P<0.05) and increased significantly in treatment 3 (C2) (Table 4). Increasing the inorganic dietary-Ca and dietary-K (in rang of treatment) had no effect on survival rate (SR%) and feed conversion ratio (P>0.05)

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Table 4

Growth indices of rainbow trout fingerling fed graded dietary levels of inorganic Ca& K (means� std. error) *

Experiment ? Experiment ??

Diets A1 B1 C1 D1 A2 B2 C2 D2

Growth indices

Initial weigh 12.15� 0.02 **a* 12.16� 0.02 a 12.20� 0.04 a 12.17� 0.03 a 15.61� 0.01 a 15.57� 0.05 a 15.60� 0.06 a 15.62� 0.03 a

Final weight 27.30� 0.43 a 27.67� 0.17 a 27.13� 0.45 a 27.01� 0.73 a 34.09� 0.14 ab 33.89� 0.19 a 34.49� 0.17 b 33.92� 0.04 a

Weight gain 15.15� 0.42 a 15.51� 0.16 a 14.93� 0.47 a 14.84� 0.71 a 18.48� 0.14 ab 18.32� 0.21 a 18.89� 0.15 b 18.31� 0.72 a

SGR% 1.44� 0.03 a 1.47� 0/01 a 1.43� 0.03 a 1.42� 0.04 a 1.38� 0.01 ab 1.39� 0.01 a 1.42� 0.011 b 1.38� 0.01 a

G % 224.72� 3.39 a 227.52� 1.19 a 225.69� 3.41 a 221.86� 5.58 a 218.43� 0.94 ab 217.71� 1.53 a 221.13� 0.87 b 217.23� 0.66 a TGC % 0.77� 0.02 a 0.78� 0.01 a 0.78� 0.02 a 0.76� 0.03 a 0.75� 0.01 ab 0.74� 0.01 a 0.76� 0.01 b 0.74� 0.01 a

SR % 92.00� 4.00 a 98.69� 1.33 a 94.67� 1.33 a 91.33� 0.67 a 94.67� 1.95 a 98.69� 1.33 a 94.67� 1.33 a 93.33� 1.67 a

FCR 1.50� 0.02 a 1.49� 0.01 a 1.49� 0.01 a 1.49� 0.01 a 1.48� 0.01a 1.50� 0.01 a 1.49� 0.01 a 1.49� 0.01 a

Statistical differences within rows are shown with different superscript letters, (P<0.05)** .

3.2. Fish biochemical composition

Inorganic dietary-Ca, showed small variation, and was only significantly different on whole body protein content (CP %) between fish fed Diet D1 and fish fed with other diets at the end of experiment ? (p< 0.05) (Table 5). Whole body protein significantly increased at day 0 to day 56 in all treatments (p< 0.05) (Table 5). Significant increases in body ash content (Ash %) (p< 0.05) and significant reduction in total body lipid content (EE%) (p< 0.05) were found. No significant effect of inorganic dietary-Ca was noticed on dry matter (DM %) content of fish (p> 0.05) (Table 5). In experiment ??, inorganic dietary-K, significantly different on whole body protein content between fish fed Diet B2 and fish fed with other diets at the end of experiment (p< 0.05) (Table 6). Whole body protein significantly increased at day 0 to day 56 in all treatments (p< 0.05) (Table 6). Significant reduction in total body lipid content (p< 0.05) was found and fish fed with diet B2 (0.9% K) displayed lower whole-body lipid content compared to other dietary groups (at the end of the feeding trial) (Table 6).

Table 5

Whole body biochemical composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic Ca at the end of experiment ? (means� std. error)*

Treatment (diets) Day 0 A1 (day 56) B1 (day 56) C1 (day 56) D1(day 56)

biochemical composition

CP % 52.34� 1.54*a** 63.23� 0.99 b 63.68� 0.36 b 61.98� 0.69 b 67.95� 0.53 c

EE % 38.05� 0.15 d 20.45� 0.85 c 17.35� 0.75 b 19.40� 0.30 c 11.85� 0.25 a

Ash % 8.00� 0.20 a 11.00� 0.00 b 12.80� 0.20 c 13.10� 0.30 c 16.50� 0.25 d

DM % 99.50� 0.30 a 99.30� 0.30 a 99.75� 0.50 a 99.60� 0.20 a 99.65� 0.50 a

Statistical differences within rows are shown with different superscript letters, (P<0.05)** .

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Table 6

Whole body biochemical composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic k at the end of experiment ?? (means� std. error) *

Treatment (diets) Day 0 A2 (day 56) B2 (day 56) C2 (day 56) D2(day 56)

biochemical composition

CP % 52.34� 1.54*a** 67.93� 0.39 b 72.62� 2.16 c 63.71� 0.01 b 65.08� 0.04 b

EE % 38.05� 0.15 e 13.25� 0.25 b 8.40� 0.10 a 17.25� 0.65 d 14.95� 0.15 c

Ash % 8.00� 0.20 a 14.60� 0.60 b 15.90� 0.10 b 15.50� 0.90 b 14.90� 0.30 b

DM % 99.50� 0.30 a 99.70� 0.25 a 99.65� 0.30 a 99.60� 0.40 a 99.50� 0.50 a

Statistical differences within rows are shown with different superscript letters, (P<0.05)** .

3.3. Fish Whole body minerals

Change in inorganic dietary-Ca had significantly affected on Ca, P, Mn, Zn, Cu and Fe of whole body contents (p< 0.05) and not affected the Mg and K of whole body contents (Table 7) (Fig. 1, 2, 3 and 4).

Table 7

Whole body mineral composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic Ca at the end of experiment ? (means� std. error)*

Treatment (diets) Day 0 A1 (day 56) B1 (day 56) C1 (day 56) D1(day 56)

Minerals (%)

Calcium 2.06� 0.04*a** 2.48� 0.15 ab 2.46� 0.13 ab 2.52� 0.06 b 3.21� 0.31 c

Phosphorus 1.16� 0.01 a 1.84� 0.01 c 1.88� 0.01 d 1.82� 0.00 b 1.82� 0.00 b

Potassium 0.81� 0.01 a 0.80� 0.02 a 0.81� 0.01 a 0.81� 0.02 a 0.81� 0.01 a

Magnesium 0.16� 0.01 a 0.19� 0.01 c 0.19� 0.00 c 0.18� 0.00 b 0.20� 0.02 a

Statistical differences within rows are shown with different superscript letters, (P<0.05)** .

Figure 1. Whole body Zn composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ?. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

Figure 2. Whole body Mn composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ?. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

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The Ca, K, P, Mg, Zn, Fe and Cu of whole body were significantly changed (p< 0.05), and no significant effect of inorganic dietary-K were noticed between treatment on whole body Mn (Table 8) (Fig. 5, 6, 7 and 8).

Table 8

Whole body mineral composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic K at the end of experiment ?? (means� std. error) *

Treatment (diets) Day 0 A2 (day 56) B2 (day 56) C2 (day 56) D2(day 56)

Minerals (%)

Calcium 2.06� 0.04*a** 3.60� 0.13 b 3.50� 0.01 b 3.59� 0.18 b 4.21� 0.01 c

Phosphorus 1.16� 0.01 a 1.79� 0.00 c 1.84� 0.00 e 1.82� 0.02 d 1.80� 0.00 b

Potassium 0.81� 0.01 a 0.83� 0.00 a 0.90� 0.02 b 0.95� 0.02 bc 0.97� 0.01 c

Magnesium 0.16� 0.01 a 0.20� 0.01 c 0.22� 0.02 d 0.19� 0.00 b 0.21� 0.02 c

Statistical differences within rows are shown with different superscript letters, (P<0.05)** .

Figure 5. Whole body Zn composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ??. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

Figure 3. Whole body Cu composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ?. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

Figure 4. Whole body Fe composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ?. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

Figure 6. Whole body Mn composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ??. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

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4. Discussion

4.1. Growth and biochemical composition

In this study we found that, increasing the inorganic dietary-Ca (between 0.95- 1.61%) had not significantly affected on some growth indices. The diet B1 with 1.21% total Ca showed the best growth indices and trail 4 (D1) with 1.61% Ca had lowest in this indices. Very studies showed that, dietary Ca is not required for some fish species held in freshwater such as common carp, chum salmon, rainbow trout, channel catfish and guppy (Poecilia reticulata.) [21, 22, 35-37]. In Atlantic salmon reared in freshwater and rainbow trout fry, the dietary Ca, had no significant effects on growth and final body weight [11, 18]. In Atlantic cod (Gadus morhua), WG, final body weight, SGR% and TGC% were not affected by dietary-Ca between 0.4 � 1.2 % in diet [32]. This is in contrast with the generally accepted view that fish do not require a Ca supplement, because they can easily absorb Ca from the surrounding water [38-41]. Takagi et al. (1989) Observed that both water and dietary Ca was necessary for normal calcification of regenerating scales in tilapia. Some marine fish such as redlip mullet, giant croaker and tiger puffer also showed a necessity for dietary Ca supplementation when fed purified or semi-puri-fied diets [9, 15, 42]. In contrast, Sakamoto and Yone (1976) reported that a dietary Ca supplement was dispensable for red sea bream fed a purified diet. A dietary Ca supplement was reported not to be essential in tilapia (Tilapia mossambica.) when they are reared in artificial seawater [43]. Therefore, the Ca requirement in marine fishes appears to be species-specific [9]. In many studies, the Ca and P ratio (Ca:P) in diets was important and in this research, the Ca:P ratio were changed with increased the Ca of the diets (0.95, 1.2, 1.4 and 1.6). In juvenile Grouper (Epinephelus coioides), increasing the Ca supplementation in diet caused to decreased in growth factors [31]. Growth of rainbow trout fry was not affected by dietary P or Ca level in agreement with several other studies that have shown that the requirement for P is lower for growth than for maximum P deposition [44-46]. Channel catfish and tilapia reared in calcium-free water required

Figure 7. Whole body Cu composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ??. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

Figure 8. Whole body Fe composition of rainbow trout fingerling in first and fish fed graded dietary levels of inorganic-Ca at the end of experiment ??. Means not sharing a common superscript letter are significantly different (P<0.05) according to one-way ANOVA followed by a Duncan�s test.

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0.45% and 0.7% Ca in the diet, respectively. The uptake of Ca from seawater was not sufficient to meet the Ca requirement of red sea bream, and it required 0.34% Ca in the diet [12, 15, 16, 47. 48] found a dietary Ca supplement was necessary for redlip mullet, Japanese flounder and scorpion fish, but not for black sea bream[9].

Increasing the inorganic dietary-k (between 0.72- 1.3%) had significantly affected on some growth indices (WG, final body weight, SGR% and TGC %). The diet C2 with 1.1% total K showed the best growth indices and trail 2 (B2) with 0.9 % and then the trail 4 (D2) with 1.3% K had lowest in this indices. Results we found in this research showed that, the dietary-K had significantly affected on rainbow trout fingerlings growth. K supplementation was found necessary in purified diets for juvenile Chinook salmon reared in freshwater (required 0.8% K in their diet for maximum growth) [28]. Growth reduction in P. japonicus when K in the diet was increased from 0.9% to 1.8% K [49] and similar to P. monodon [50]. The lack of a necessary mix of essential ions, including potassium (K+) has been demonstrated to limit growth and survival of shrimp [51, 52]. Increase in Litopenaeus vannamei PL survival and mean individual weight concurrent with an increase in the amount of K+ [19]. Results we found in this research showed that, the dietary-Ca and K requirements for fish growth were species-specific and dependent to water chemistry, ecosystem and interaction between minerals especially Ca and P.

Results we found in this research showed that, increasing the inorganic dietary-Ca and K, causing to increase the crud protein and decrease the total lipid (Table 5 and 6) of whole body composition. No dietary impact on fish protein content or protein utilization was found in haddock, similar to that found in Atlantic salmon [29], but different from that found in haddock, where P inadequacy caused a decrease in the whole body protein content [30]. It is well known that dietary Ca can lower the net absorption of dietary fat by its precipitating in the digestive tract resulting in the increased fat excretion in faeces [53]. The whole body lipid level of cod increased when the dietary P level was inadequate. The same effect has also been observed in haddock [30], Atlantic salmon [29], rainbow trout [45, 46] and other farmed fish species [10, 54-56]. The mechanism behind this effect remains to be elucidated. Assumptions were made that this is caused by the accumulation of fatty acids due to impaired �-oxidation [54] or oxidative phosphorylation due to P deficiency, leading to the inhibition of the TCA cycle and accumulation of acetyl- CoA and an increase in fatty acid synthesis [46]. Interestingly, Ca supplement under the two P levels decreased whole body and muscle lipid, and liver protein, which suggests Ca supplement has a similar effect with P supplement on decreasing lipid accumulation, and a relationship between Ca and protein metabolism.

The role of dietary Ca in lipid and protein metabolism remains to be elucidated [31]. Increasing the inorganic dietary-Ca (between 0.95 - 1.61%) caused significantly increased the whole body ash and no significant effect of inorganic dietary-K (between 0.72 - 1.3%) and Ca were noticed between treatment on whole body ash and dry matter contents. The whole body ash content in cod steadily increased with the increased dietary P and Ca supplementation. An increased body ash with increased dietary P has previously been reported in haddock [30] and in several other farmed fish species [10, 21, 36, 55, 57]. Different from that found in Atlantic salmon [11], the dietary Ca supplementation did not have a significant effect on body ash levels in cod. In other fish species increased levels of dietary Ca has produced a positive effect on vertebrae ash levels when dietary P levels were sufficient [58, 59].

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4.2. Whole body mineral content

Whole body minerals content showed small variation. Significant increases in whole body Ca, P and Cu (P<0.05) and a significant reduction in whole body Mg, Zn and Fe content (P<0.05) were found in fish whit increased the dietary-Ca (between 0.95 - 1.61%) in trails (Table 5 and Fig. 1, 3 and 4). No significant effect of dietary-Ca was noticed on Mn and K of whole body content (Table 5 and Fig. 2).Increasing the inorganic dietary-K (between 0.72 - 1.3%) showed Significant increases in whole body Ca and K (P<0.05) (Table 6) and a significant reduction in whole body Cu and Zn content (P<0.05) (Fig. 5 and 7). Significant increases in body P and Mg in trail 1 and 2 and then a significant reduction in trail 3 and 4 (P<0.05) (Table 6). Significant increases in whole body Fe in trail 4 (Fig. 8) and no significant effect of dietary-Ca was noticed on Mn of whole body content (Fig. 6)

There are several reports showing that changes in the levels of Ca affects the availability of other minerals both in terrestrial animals [60] and in fish [61] probably due to competitive inhibition of these cations during intestinal absorption [30]. When Ca is oversupplied, P absorption by the intestine may be hindered by its combination with Ca to form biologically unavailable calcium phosphates [62]. Dietary Ca also affected the growth but not vertebral Ca and P contents of channel blue tilapia and catfish when reared in Ca-free water [8, 23]. However, O�Connell and Gatlin (1994) observed in blue tilapia that a dietary Ca supplement increased the bone Ca content when they were reared in low-Ca water. In addition to the potential dietary essentiality, dietary Ca may interact with other essential dietary minerals such as P, magnesium (Mg) and zinc (Zn) [11, 13, 63, 64]. For instance, Nakamura (1982) reported excess dietary Ca inhibited P absorption in common carp. Increasing levels of dietary P have caused an increase in P, Ca, Mg and K levels in rainbow trout [45], a decrease in Zn and/or Mn levels in rainbow trout [63, 65] as well as a decrease in Atlantic salmon vertebrae Mg levels [11].

Some studies in other fish species [13, 66, 67] the dietary Ca supplementation without additional P did not result in a significant decrease in vertebrae P or Ca content, but it did induce a significant decrease in the levels of vertebrae Mg and Mn. A negative effect of high dietary Ca supplementation on vertebrae Cu, Fe, Zn and Mn, has been observed in scorpion fish [9]. An antagonistic relation in fish between Ca and P, Mg and Zn utilization has been reported [11, 63] and although fish are able to take up Mg from the water. Indicating dietary Ca supplement was not necessary for Mg deposition whether P was supplemented or not and excess Ca had a negative on Mg and Zn deposition. High concentration of dietary Ca interferes with the absorption and retention of certain trace elements and Mg [3]. An inhibited effect of dietary Ca supplement on Mg deposition in scales and vertebrae has been reported in Atlantic salmon [11]. It has been reported in scorpion fish that a supplementation of 2.5% Ca as tricalcium phosphate decreased Zn content of vertebrae from 0.162 to 0.076 mg/g [48] and 4% tricalcium phosphate supplementation in the diet of rainbow trout decreased bone Zn from about 0.18 to 0.11 mg/g [68]. Rafiee and saad (2005) found that the red tilapia could assimilate 11.46% Fe, 13.43% Zn, 6.81% Mn, 3.55% Cu, 26.81 Ca %, 20.29% Mg, 32.53% N, 7.16% K and 15.98% P content of the feed supply during a culture period [69]

Fontagne' et al., (2009) showed that dietary Ca deficiency did not affect whole-body composition. Requirement of rainbow trout fry can be met in large part by absorption of Ca through gills and skin in freshwater [3], especially when the Ca content of water is relatively high (83 mg/L) as in the present study [18]. Fish fed diets without P supplements showed reduced

12

mineral (Ca, P and Mg) deposition in scales, vertebrae and opercula. When P was not supplemented, Ca supplement of 6 g kg-1 had no significant effect on mineral (Ca, P, Mg and Zn) deposition in scales and vertebrae. When diets were supplemented with 6 g kg-1 P, Ca supplement of 6 g kg-1 had no significant effect on mineral (Ca, P and Mg) content in scales, vertebrae and opercula, but excess Ca supplement (up to 12 g kg-1) had a negative effect on scale mineral (Ca, P and Mg) deposition and vertebrae Zn deposition. Ca supplement of 6 g kg-1 (Ca /P=1) might be optimum when diet was supplemented with 6 g kg-1 P.

In marine fishes the Ca content of whole body and vertebrae were not affected by the lack of dietary Ca supplement. In other words, Ca absorption from seawater was sufficient for maintaining normal tissue Ca but not for normal growth. We obtained similar results in our pervious studies with redlip mullet, giant croaker and tiger puffer [9, 15, 42].

They were not found the data about the effects of inorganic dietary-K on Whole body minerals content of rainbow trout.

5. Conclusions

The present experiment suggests that inorganic dietary-Ca in tested range had no significant effect on growth, and significant effect on whole body minerals and biochemical composition, which is in agreement with the generally accepted view that most fish can absorb Ca from the aquatic environment or from feed ingredients to meet their requirements. Inorganic dietary-K had significant effect on growth, whole body minerals and biochemical composition. The role of K in diet on reduction of Zn and Cu of whole body content was important. Suggested that the optimums of inorganic dietary Ca and K for rainbow trout fingerlings growth are 1.21 % Ca and 1.1 % K.

Acknowledgements

This study was supported by the department of fisheries of University of Tehran- karaj- Iran.

The authors would like to thank A.R. Naisi for the technical assistance and care of fish and A. Nazarzade for technical support.

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