1 2 3 3 3 4

3

1 Division of Analytical Chemistry, Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, University of Inonu,

Malatya, Turkey; 2 Surgu Vocational School, University of Inonu, Malatya, Turkey; 3 Department of Animal Nutrition,

Faculty of Veterinary Science, Firat University, Elazig, Turkey; 4 Nutrition 21, Inc., Purchase, NY, USA

Arginine silicate inositol complex (ASI; arginine

49.47 g kg)1, silicon 8.2 g kg)1, inositol 25 g kg)1), a novel

composition that is a bioavailable source of silicon and

arginine, has potential benefits for vascular and bone health.

We have previously reported that bone mineral content in-

creased and the amount of Ca, P, Mg and Mn in the excreta

decreased in poultry with ASI supplementation. In the

present study, the effect of ASI supplementation at various

levels (0, 500, 1000 mg kg–1 ASI) on growth, feed intake,

feed conversion ratio (FCR) and concentrations of body

elements, operculum bone ash and activity of serum alkaline

phosphatase (ALP) in rainbow trout was evaluated. Ninety

0+ year-old rainbow trout with initial average weight of

50 ± 3 g were randomly assigned to three treatment

groups, three replicates of 10 fish each. The fish were fed

either a basal diet or the basal diet supplemented with either

500 or 1000 mg of ASI. Body weight gain (P = 0.25), feed

intake (P = 0.36) and feed efficiency (P = 0.42) were not

signifcantly influenced by the dietary ASI supplementation.

Per cent operculum bone ash (634 g kg)1 versus 558 g kg)1,

P = 0.001) and ALP activity (112 UL–1 versus 92 UL–1,

P = 0.001) linearly increased as dietary ASI supplementa-

tion increased. Increasing dietary ASI supplementation lin-

early increased serum and whole body Ca (P = 0.01), P

(P = 0.01), Mg (P = 0.05; P = 0.001) Mn (P = 0.05;

P = 0.01) and Zn (P = 0.01; P = 0.02) concentrations

respectively. In conclusion, ASI supplementation to the

basal diet significantly improved operculum bone ash and

whole body mineral content in rainbow trout and did not

impact feed consumption, weight gain or FCR.

KEY WORDSKEY WORDS: alkaline phosphatase, arginine silicate inositol,

bone, minerals, rainbow trout

Received 12 February 2007, accepted 7 August 2007

Correspondence: Kazim Sahin, Department of Animal Nutrition, Faculty of

Veterinary Science, Firat University, 23 1119, Elazig, Turkey. E-mail:

nsahinkm@yahoo.com

Arginine, one of 10 indispensable amino acids in fish feeds, is

involved both in the synthesis of substrates (polyamine and

LL-proline) implicated in collagen synthesis, and in the pro-

duction of growth hormone (GH), insulin-like growth factor-

I (IGF-I) and nitric oxide (Wilson 1985; Berge et al. 1997;

Chevalley et al. 1998; Colao et al. 1999; Buentello & Gatlin

2000) and utilized as a store of ATP and in organic phos-

phate in the form of phosphoarginine (Berge et al. 1997).

Arginine has also been reported to have an effect on immu-

nological functions in mammals (Park 1993; Berge et al.

1997). Several roles for silicon (Si) have been defined, largely

on the basis of rat and chicken studies (Carlisle 1972;

Schwarz & Milne 1972). Silicon, an abundant trace mineral

in nature is proving to be an essential ingredient for stronger

bones, better skin and more flexible joints (Carlisle 1972;

Schwarz & Milne 1972). It has been concluded that Si acts as

a regulating factor for the deposition of calcium and phos-

phorous in bone tissue (Carlisle 1970). Recent studies suggest

that dietary arginine and Si may play an important role in the

development, growth and modelling of long bones (Seaborn

& Nielsen 2002). On the other hand, there is a direct

relationship between Si and Ca in bone (Carlisle 1976).

Arginine silicate inositol complex (ASI; arginine 49.47%,

silicon 8.2%, inositol 25%; supplied by Nutrition 21 Inc,

Purchase, NY) is a novel composition that provides a bio-

available source of Si and arginine and has potential benefits

for bone health (Russell 2005). Several studies have reported

that no adverse effects are observed following administration

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2008 14; 257–262. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

doi: 10.1111/j.1365-2095.2007.00526.x

Aquaculture Nutrition

of 7 g arginine/three times daily for 1 week (Adams et al.

1995; Lerman et al. 1998), 12 g inositol day–1 for 4 weeks

(Levine 1997) or 45 mg silicon day–1 for 31 days (Van Dyck

et al. 1999) in humans. There are a few documents on the role

of ASI complex in bone metabolism of rats and quails

(Juturu et al. 2004; Komorowski et al. 2004; Onderci et al.

2006; Sahin et al. 2006). The aim of this study was to

investigate the effects of ASI complex supplementation on

growth, feed intake, feed conversion ratio (FCR) and oper-

culum bone ash, serum alkaline phosphatase (ALP) activity

and concentration of body elements of rainbow trout.

Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) with

an initial average weight of 50 g provided from Surgu

Vocational School Division of Inonu University, Malatya,

Turkey were used in the study. Fish were allowed to accli-

mate themselves to the experimental system for 2 weeks be-

fore starting the experiment. Uniform size fish were selected

from a large population, weighed and randomly distributed

in experimental tanks and the photo-period was adjusted to

12 h light and 12 h dark. The fish were kept in 1073 L

fibreglass tanks supplied with aerated spring water. The

water flow was 1 L min)1 kg)1 of fish biomass. A total of 90

rainbow trout were randomly divided into three groups each

group consisted of three tanks (three replicates) and each

tank contained 10 fish. Fish were fed either a basal diet

containing 450.0 g kg–1 crude protein and 16 MJ kg–1

metabolizable energy or the basal diet supplemented with

either 500 or 1000 mg kg–1 ASI. ASI complex (arginine

49.47%, silicon 8.2%, inositol 25%) was supplied by

Nutrition 21 Inc, Purchase, NY, USA. The basal diet was

formulated using NRC guidelines (NRC National Research

Council, USA 1993). Ingredients and nutrient composition of

the basal diet are shown in Tables 1 and 2. Diets were fed by

hands two times daily to apparent (near) satiation for

56 days. The water quality parameters were monitored

weekly during the experiment and the ranges were tempera-

ture 11 ± 0.3 �C, dissolved oxygen 8.5 ± 0.5 mg L)1

and pH 7.4 ± 0.7 (SE). No critical values were detected for

NO2 and NO3.

At the end of the study, 12 fish (four fish per replicate)

randomly chosen from each treatments were starved for 24 h

and blood was collected from the caudal vein into tubes. The

tubes containing blood samples were then centrifuged at

1860 · g for 10 min and serum was collected and stored at

)80 �C for later analysis of minerals. Twelve fish (four fish

per replicate) were killed for whole body mineral and bone

ash analysis. Left operculum was removed for bone ash

analyses. Operculum samples were dipped in boiling distilled

water for 30 s, and skin and connective tissues were removed.

Bones were dried for 24 h at 60 �C, and thereafter ashed at

550 �C for 16 h. Diet and body were digested according to

AOAC (1990). Serum and whole body concentrations of Ca,

Mg, Mn and Zn were determined using atomic absorption

Table 1 Ingredient and nutrient composition of the experimental

diets

Ingredients (g kg–1)

Fish meal 480.0

Corn gluten 100.0

Soybean meal 200.0

Wheat middling 139.0

Fish oil 60.0

Binder (Sodium alginate) 10.0

Vitamin mixture1 10.0

Mineral mixture2 1.00

Total 1000.0

1 Vitamin added to supply the following (per kg diet): vitamin A,

8000 IU; vitamin D3, 4500 IU; vitamin E, 300 IU; vitamin K3, 40 mg;

thiamine HCl, 50 mg; riboflavin, 70 mg; d-Ca pantothenate,

200 mg; biotin, 1.5 mg; folic acid, 20 mg; vitamin B12, 0.15 mg;

niacin, 300 mg; pyridoxine HCl, 20 mg; ascorbic acid, 300 mg; ino-

sitol, 400 mg; choline chloride, 2000 mg; butylated hydroxy tolu-

ene, 15 mg.2 As g kg of diet: calcium biphosphate 5.43; calcium lactate 5H2O

13.08; magnesium sulphate 5.51; potassium phosphate 9.59; so-

dium biphosphate 2H2 O 3.49; sodium chloride 5H2O 1.74; As mg

kg)1 of diet: manganous sulphate, 40; ferrous sulphate, 30; copper

sulphate, 5; zinc sulphate, 75; sodium selenite, 1; cobalt chloride,

2.5; sodium fluoride, mg.

Table 2 Nutrient composition of the experimental basal diet

Nutrients

Dry matter, g kg–1 920

Crude protein, g kg–1 450

Crude ash, g kg–1 75

Ether extraction, g kg–1 140

Metabolizable energy, MJ kg)1 16

Arginine, g kg–1 22

Ca, mg g–1 11.0

P, mg g–1 12.2

Mg, mg g–1 9.5

Mn, lg g–1 25.5

Zn, lg g–1 47.7

Si, lg g–1 150

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

� 2007 Blackwell Publishing Ltd Aquaculture Nutrition 14; 257–262

spectrophotometer (Perkin Elmer AAnalyst, 800, Norwalk,

CT, USA). Phosphorus was measured by a spectrophotom-

eter (Pearson 1976). Serum ALP activity was measured using

biochemical analyser (Olympus AU-660, Tokyo, Japan).

Chemical analysis of the basal diet was run using standard

procedures of AOAC (1990).

The data were initially analysed by analysis of variance

(ANOVAANOVA) using General Linear Models procedure of SAS

Institute (1999) for the effects of ASI. Differences in variables

were attained using Duncan�s post hoc test. Linear and

quadratic polynomial contrasts were used to evaluate the

effect of different levels of ASI. Statistical significance was

assumed at P < 0.05.

Table 3 summarizes the effect of feeding and levels of ASI on

performance of rainbow trout. Initial (50 g) and final (158 g)

body weights (P = 0.45) of fish were not different across the

dietary treatments. Body weight change (P = 0.25), feed

intake (P = 0.36) and FCR (P = 0.42) were not influenced

by the dietary ASI supplementation. As seen in Table 4,

increasing ASI levels linearly increased per cent operculum

bone ash (P = 0.001). Serum ALP activity (P = 0.001)

linearly increased as dietary ASI supplementation increased.

The effects of ASI supplementation on mineral concentra-

tions in the serum and whole body are shown in Table 5.

Increasing dietary ASI supplementation linearly increased

serum and whole body Ca (P = 0.01), P (P = 0.01), Mg

(P = 0.05 and P = 0.001), Mn (P = 0.05 and P = 0.01)

and Zn (P = 0.01 and P = 0.02) concentrations respec-

tively.

The present study was carried out to investigate the effect of

different levels of ASI supplementation in the diet on per-

formance, operculum bone ash, serum ALP activity in

rainbow trout. ASI supplementation to practical diets for

rainbow trout at any levels (0, 500 and 1000 mg kg–1 of

diet) did not show any significant effect on the growth of the

fish. This is in agreement with the studies on poultry

(Onderci et al.2006; Sahin et al. 2006) and rats (Russell

2005) that ASI supplementation had no significant influence

on the growth of the animals. Feed conversion in the

present study also did not differ among the treatments,

indicating that the tested ASI had no influence on the feed

intake and growth performance of the fish. Similar to our

results, it is reported that ASI complex treatment have no

significant effect on feed intake and feed efficiency in poultry

(Sahin et al. 2006).

Skeletal abnormalities may reduce the market value of

fishes as they affect the morphology, survival and health of

fishes (Koumoundouros et al. 1997; Paxton et al. 2006).

Handling stress, nutritional deficiencies including vitamins

and minerals, vaccination, sub-optimal environmental con-

ditions and artificial photoperiods may affect the minerali-

zation of vertebral bone, which is a slow process that takes

place secondly to the deposition of matrix proteins in smolt

production (Baeverfjord et al. 1998; Fjelldal et al. 2006;

Meunier 2006; Paxton et al. 2006). In the present study, the

bone ash level and serum ALP activity were significantly

higher with the inclusion of 1000 mg kg–1 ASI of diet

compared with that of 500 mg kg–1 ASI of diet, suggesting

the most effective amount of ASI being 1000 mg kg–1 ASI

of diet (Table 4). The results of the studies show that ASI

Table 3 Effects of supplemental ASI (mg kg–1) on performance of

rainbow trout (n = 30 per treatments; 10 per tank)

Item 0 500 1000

Body weight, g

Initial 51 ± 3.5 50 ± 4.3 50 ± 2.5

Final 158 ± 6.5 157 ± 8.2 159 ± 5.0

Body weight gain, g 107 ± 3.2 107 ± 7.7 109 ± 6.5

Feed intake, g 153 ± 2.4 154 ± 2.8 155 ± 5.5

Feed conversion ratio1 1.4 ± 0.2 1.4 ± 0.3 1.4 ± 0.1

Values are shown as mean ± SD.

ASI, arginine silicate inositol.1 g feed intake/g live weight gain.

Table 4 Effects of supplemental ASI

(mg kg–1) on bone ash (g kg–1) and ALP

activity (U L–1) of rainbow trout

(n = 12 per treatment; three per tank)

Item 0 500 1000 Linear Quadratic

Bone ash 553 ± 2.2c 617 ± 0.9b 634 ± 1.3a 0.001 NS

Serum ALP 92 ± 6.2c 105 ± 8.6b 112 ± 9.3a 0.001 NS

Values are shown as mean ± SD.

ASI, arginine silicate inositol; ALP, alkaline phosphatase; NS, not significant.a–c Means in line within main effect differ significantly (P < 0.05).

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� 2007 Blackwell Publishing Ltd Aquaculture Nutrition 14; 257–262

supplementation may decrease skeletal abnormalities (Sea-

born & Nielsen 2002; Onderci et al. 2006). In a similar

study involving poultry, Sahin et al. (2006) reported that

bone mineral density, serum osteocalcin and dehydroepi-

androsterone concentrations and ALP activity increased,

whereas tumour necrosis factor-a and C-reactive protein

concentrations decreased as dietary ASI supplementation

increased. Arginine could be involved in both conditions

osteoporosis and fractures or bone defects (Civitelli et al.

1992). On the other hand, arginine acts as a precursor for

the synthesis of polyamine and LL-proline that act as sub-

strates for collagen and it is involved in the synthesis of GH

and IGF and in nitric oxide production (Chevalley et al.

1998; Colao et al. 1999). Silicon has been implicated as an

important component in bone formation and Si deprivation

results in abnormal skeletal development in chicks and rats

(Carlisle 1972; Schwarz & Milne 1972).

In the present study, serum and whole body Ca, P, Mg,

Mn and Zn concentrations increased with increasing sup-

plemental dietary ASI. In fish fed diets with no ASI, Ca and

P concentrations were 1312 and 2570 lg g–1 respectively.

The supplementation of diet with the ASI increased the

concentration of Ca and P to 1563 and 2815 lg g-1 respec-

tively. Our result is in agreement with the results of previous

studies on arginine and Si in chicks and rats (Carlisle 1972;

Schwarz & Milne 1972; Fiore et al. 2000; Clementi et al.

2001). Carlisle (1986) and Visser & Hoekman (1994) indi-

cated that dietary Si and arginine influence mineral meta-

bolism and bone mineralization of broiler chicks. Sahin

et al. (2006) reported that the amount of Ca, P, Mg and Mn

in the excreta decreased, while concentrations of these

minerals in tibia ash increased in quail by ASI supplemen-

tation. Similar to our results, Calomme & Vanden Berghe

(1997) reported that supplementation Si significantly in-

creased serum Ca in calves. A relationship between Si, Mg

and fluorine (F) in the formation of bone in the chick has

also been reported (Carlisle 1981). The mineral data is

higher than that reported by other authors (Wheaton &

Lawson 1985; Lall 1995). The concentrations of the major

mineral elements Ca and P in the trout body found in the

present study are in accordance with the finding of Vielma

et al. (1999a). On the other hand, Gokoglu et al. (2004)

reported lower values (632 mg kg–1). Similar to our results,

Wheaton & Lawson (1985) found that the P contents ranged

from 1520 to 2600 mg kg–1 in trout. Similar Mn values were

reported by other authors (Wheaton & Lawson 1985; Lall

1995). Wheaton & Lawson (1985) have reported that Zn

content of trout ranged from 1.4 to 26 mg kg-1. In contrast

to our findings, high body mineral content was observed by

Vielma et al. (1999b) and Roy & Lall (2006) and Shearer

(1984).

The results of the present study indicate that supplemental

ASI in the diet of rainbow trout increased serum and whole

body mineral contents, operculum bone ash, and did not

impact growth and feed consumption of rainbow trout. The

results also showed that the most effective amount of ASI

to get the effects observed in this study was 1000 mg kg–1 of

diet.

The authors thank the Surgu Vocational School Division

of Inonu University, Malatya-Turkey, for providing the

experimental facility, Cagatay Feed Company for provid-

ing diet and Nutrition 21, NY, USA for providing the

ASI.

Table 5 Effects of supplemental ASI

(mg kg)1) on serum and whole body Ca,

P, Mg, Mn and Zn, concentrations (wet

wt basis) of rainbow trout (n = 12 per

treatment; three per tank)

Item 0 500 1000 Linear Quadratic

Serum (lg mL–1)

Ca 2.44 ± 0.11c 2.65 ± 0.02b 2.78 ± 0.10a 0.01 NS

P 0.62 ± 0.05b 0.74 ± 0.06b 0.77 ± 0.03a 0.01 NS

Mg 0.69 ± 0.01b 0.73 + 0.05ab 0.78 ± 0.03a 0.05 NS

Mn 0.07 ± 001b 0.10 ± 0.05ab 0.14 ± 0.005a 0.05 NS

Zn 16.3 ± 0.95c 17.2 ± 0.82b 18.3 ± 0.63a 0.01 NS

Whole body (lg g–1)

Ca 1312 ± 98c 1480 ± 120b 1563 ± 110a 0.01 NS

P 2570 ± 150b 2750 ± 80a 2815 ± 98a 0.01 NS

Mg 190 ± 15b 225 ± 12a 236 ± 11a 0.001 NS

Mn 0.88 ± 0.09b 1.10 ± 0.05ab 1.25 ± 0.09a 0.01 NS

Zn 15.3 ± 1.2c 16.5 ± 0.98b 18.3 ± 1.2a 0.02 NS

Values are shown as mean ± SD.

ASI, arginine silicate inositol; NS, not significant.a–c Means in line within main effect differ significantly (P < 0.05).

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