The effects of dietary inulin on growth ... - Wiley Online Library

Aquaculture Nutrition doi: 10.1111/anu.12155

2015 21; 242–247

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Young Researchers and Elite Club, Lahijan Branch, Islamic Azad University, Lahijan, Iran; Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran; 3 Department of Fisheries, Lahijan Branch, Islamic Azad University (IAU), Lahijan, Iran; 4 Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, Tromsø, Norway

This study was conducted to investigate the effects of dietary inulin on growth performance, diet utilization, survival rate, carcass composition and digestive enzymes activities (amylase, lipase and protease) of carp (Cyprinus carpio) fry (0.55 0.02 g). After acclimation, fish were allocated into 9 tanks (40 fish per tank) and triplicate fish groups were fed, control diet (0 g) or diets containing 5 g and 10 g inulin kg 1 for 7 weeks. No significant effect on growth performance and diet utilization of fish fed inulin compared with the control group was observed. However, supplementation of inulin significantly increased survival rate and carcass lipid content, while carcass protein content significantly decreased. Dietary inulin had no significant effects on digestive lipase, protease and amylase activities. KEY WORDS:

Cyprinus carpio, digestive enzymes, growth performances, inulin, prebiotic, survival

Received 18 August 2013; accepted 14 November 2013 Correspondence: Seyed Hossein Hoseinifar, Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. E-mail: [email protected]

Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of and/or activity of health-promoting bacteria in the intestinal tract (Gibson & Roberfroid 1995; Gibson 2004). In aquaculture, prebiotics have received increased attention since the first study was published by Hanley et al. (1995)

and stimulated growth performances, feed utilization, positive effects on gut microbiota, gut morphology and immune system, and disease resistance have been reported (Merrifield et al. 2010; Ringø et al. 2010, 2014). Inulin is one of the most studied prebiotic and consists predominantly of polydisperse b-(2?1)-linked fructan and is naturally present in a number of common foods such as garlic, onion, artichoke and asparagus (Van Loo et al. 1999; Mahious & Ollevier 2005; Roberfroid 2007). Despite some negative results (Olsen et al. 2001; Akrami et al. 2009; Reza et al. 2009), several studies have reported positive effects of inulin as growth promoter (Mahious et al. 2006; Burr et al. 2010; Ibrahem et al. 2010; Ortiz et al. 2013). Although numerous studies have been conducted on administration of prebiotics in aquaculture, no information is available on the effects of prebiotics on growth performance, carcass composition and digestive enzymes activities in early life stages of common carp (Cyprinus carpio) (Ringø et al. 2010, 2014). For numerous aquatic species, the commercial production of larvae and fry is a bottleneck. Thus, the aim of this study was to determine the effect of inulin on growth parameters, survival, carcass composition and digestive enzymes activities common carp fry.

Inulin used in this study was kindly provided by Orafti (Raffinerie Tirlemontoise, Tienen, Belgium). According to the manufacturer, composition of the product was 982 g kg 1 dry matter and 18 g kg 1 crude ash.

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ª 2014 John wiley & Sons Ltd

In addition to the basal diet, two experimental diets were prepared with different levels of inulin; 5 and 10 g kg 1 (Table 1). The ingredients were blended thoroughly in a mixer and pelleted using a meat grinder equipped with a 1.5-mm die. The pelleted diets were air-dried and stored in plastic bags at 4 °C until further use.

Three hundred and sixty common carp fry from the Dr. Keyvan Marine Science and Technology Research Center (Gilan province, Iran) were adapted to experimental conditions for 1 week. Thereafter, fish were randomly allocated to 9 tanks (500 L), 40 fish per tank, 3 tanks per treatment. Water temperature, dissolved oxygen and pH were monitored daily and maintained at 25.2 1.2 °C, 1 7.43 0.13 mg L and 7.43 0.13, respectively. Continuous aeration was provided to each tank through an air stone connected to a central air compressor. During the rearing trial (7 weeks), the fish were hand-fed with experimental diets to apparent satiation three times a day at 7:30, 12:30 and 17:30 as described elsewhere (Ebrahimi Table 1 Dietary formulations and proximate composition of the experimental diets (g kg 1) Ingredient

Control

5 g kg

Fish meal Wheat flour Soybean meal Gluten Soybean oil Fish oil Mineral premix1 Vitamin premix1 Binder2 Antifungi3 Antioxidant4 Raftiline, IPS5

400 210 135 55 60 60 30 20 20 5 5 0

398 208 134 54 59 59 30 20 20 5 5 5

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10 g kg

896.0 382.3 102.2 34.0 17.58

397 205 132 52 58 58 30 20 20 5 5 10

897.8 383.1 103.8 35.0 17.62

899.0 384.0 103.1 35.0 17.61

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Premix detailed by Jalali et al. (2009). Amet binder TM, Mehr Taban-e- Yazd, Iran. 3 ToxiBan antifungal (Vet-A-Mix, Shenan- doah, IA). 4 Butylated hydroxytoluene (BHT) (Merck, Germany). 5 Raffinerie Tirlemontoise, Tienen, Belgium (inulin > 930 g kg 1). 2

content

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Aquaculture Nutrition, 21; 242–247 ª 2014 John wiley & Sons Ltd

Growth performance parameters: weight gain (WG) (%), specific growth rates (SGR), feed conversion ratio (FCR), condition factor (CF) and survival rate were calculated according to the following formulas: weight gain = W2 W1, specific growth rate (SGR) = 100 [ln W2 ln W1]/T, condition factor (CF) = 100 9 (body weight; g)/(body length; cm)3 and feed conversion ratio (FCR) = FO/WG. W1 is initial weight (g), W2 is final weight (g), T is time (days), FO is feed offered (g) and WG is weight gain (g), survival = (Nf/N0)*100; where N0 is initial number of fish and Nf is final number of fish.

Chemical analysis of triplicate samples of the diets and three fish carcasses samples from each treatment were determined according to AOAC (1990). Gross energy was calculated using the conversion factors of 23.6, 39.5 and 17.0 kJ g 1 for protein, lipid and nitrogen-free extract (NFE), respectively, as described elsewhere (Brett & Groves 1979).

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Proximate analysis Dry matter Crude protein1 Crude lipid1 Ash1 Gross Energy (MJ kg 1)

et al. 2012). Uneaten feed was collected 1 h after feeding and dried at 60 °C.

After 7 weeks of feeding, 3 fish (starved for 24 h) were sampled from each tank for enzymatic analysis. Starvation prior to sampling was carried out according to Yanbo and Zirong (2006). The entire intestines were sampled and rinsed with cold distilled water at 4 °C (Yanbo & Zirong 2006). Thereafter, the intestinal samples were thoroughly homogenization in phosphate-buffered saline (pH 7.5; PBS) and centrifuged at 15 000 g for 15 min at 4 °C. Supernatants were thereafter transferred to new tubes and stored at 80 °C until further analysis. The total protein contents of the supernatants were determined as described previously (Bernfeld 1951) using bovine serum albumin as a standard. Protease activity was measured as described by Hidalgo et al. (1999) using casein hydrolysis at pH 8. Amylase activity was quantified using starch as a substrate at optical density at 540 nm (Bernfeld 1951), and lipase activity was determined by the measurement of fatty acids released following enzymatic hydrolysis of triglycerides in a stabilized emulsion of olive oil (Borlongan 1990; Yanbo & Zirong 2006). Digestive enzyme activities (protease, amylase and

lipase) are presented as specific activity (U mg tein min 1).

1

pro-

Prior to statistical analysis, normality and homogeneity of variance were checked and percentage data were subjected to arcsine transformation (Zar 1994). All statistical analyses were conducted using SPSS statistical package version 17.0 (SPSS Inc., Chicago, IL, USA). Data were subjected to a one-way ANOVA to test the effect of inulin on the growth performance, survival and digestive enzyme activities of common carp. Mean values were considered significantly different at P < 0.05. When the differences were significant, Duncan’s multiple range test was performed (Zar 1994). Data are presented as mean values SEM.

The growth performance parameters of carp fry fed the experimental diets containing various levels of dietary inulin are presented in Table 2. No significant difference was observed in the initial weight of fish in the different treatments. There were no significant differences between final weights, WG, SGR, CF or FCR of common carp fry fed inulin (5 or 10 g kg 1) and the control treatment (Table 2). Dietary inulin significantly affects the proximate carcass composition of protein and lipid (Table 3). Fish fed 10 g inulin kg 1 showed the lowest protein and the highest lipid content, whereas fish fed the control diet displayed highest carcass protein and the lowest lipid content. Ash and moisture content showed no significant difference between the inulin supplemented groups and the control group. Survival of common carp fry after 7 weeks feeding on the three experimental diets is shown in Fig. 1, and the results revealed that dietary inulin significantly increased survival rates of carp fry. The mortality rates of fry in the

Table 2 Growth performances of common carp fry fed diets contain different levels of dietary inulin. Data represent means from 3 replicates per treatment. Values are presented as the mean SE Control Final weight (g) Weight gain (%) SGR CF FCR

1.42 152 1.88 1.61 2.07

0.07 8.23 0.71 0.02 0.62

5 g kg 1.30 145 1.83 1.34 2.53

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10 g kg

0.05 9.10 0.39 0.04 0.29

1.37 158 1.91 1.38 2.30

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0.19 10.1 0.47 0.01 0.41

Table 3 Proximate carcass composition (g kg 1) of common carp fry fed diets containing different levels of inulin. Data represent means from 3 replicates per treatment. Values in a row with the different superscript letters denote significant difference. Values are presented as the mean SE Control Moisture Crude protein Crude lipid Ash

798 123 48.4 46.2

9.1 3.3b 1.0a 11.2

5 g kg 797 102 74.2 44.0

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10 g kg

17.1 7.0a 2.5b 4.1

796 98.1 84.5 41.0

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2.7 0.1a 1.9c 1.6

Figure 1 Survival rate of common carp fry fed a basal diet (control) and two diets supplemented with inulin after 7 weeks of feeding. Bars assigned with different superscripts are significantly different (P < 0.05); values are presented as the mean SEM.

control and 5 or 10 g kg 1 inulin groups were 29.7 3.2, 15.9 6.4 and 12.3 4.1, respectively. The effect of dietary inulin on the digestive enzyme activities (amylase, lipase and protease) is shown in Fig. 2. Protease activity showed no significant difference between prebiotic fed fish (5 g kg 1, 3.22 0.55 and 10 kg 1, 3.65 0.21 U mg 1 protein min 1) and control group (3.07 0.24 U mg 1 protein min 1) (Fig. 2a). Similar findings, no significant differences, were also noticed between the inulin groups and control with respect to amylase and lipase activities (Fig. 2b,c).

Our results revealed that supplementation of 5 g or 10 g inulin kg 1 had no significant effects on carp fry growth performances. These results are in accordance with that reported by Akrami et al. (2009) and Reza et al. (2009) using inclusion level of 10, 20 and 30 g inulin kg 1 in diet to juvenile rainbow trout (Oncorhynchus mykiss) and juvenile beluga (Huso huso), respectively. On the other

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(a)

(b)

(c)

Figure 2 Digestive enzymes activities of protease (a), amylase (b) and lipase (c) of common carp fry fed a basal diet (control) and two diets supplemented with inulin after 7 weeks of feeding. Data represent the mean SEM. Bars assigned with same superscripts are not significantly different (P > 0.05).

hand, supplementation of inulin in diets fed to Nile tilapia (Oreochromis niloticus) (Ibrahem et al. 2010) and turbot (Psetta maxima) (Mahious et al. 2006) significantly increased growth performance parameters. The contradictory results of inulin supplementation might be due to different dosage levels of inulin, fermentability of inulin by the gut microbiota and different intestinal morphology and gut microbiota (Hoseinifar et al. 2010).

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In contrast to the positive results of the present study, showing that dietary inulin significantly increased survival of common carp fry compared with the control treatment, dietary inulin has been reported to display no significant effect on survival of beluga juveniles (~ 60 g) (Reza et al. 2009) or juvenile rainbow trout (~ 40 g) (Akrami et al. 2009). Despite these contradictory results, there are numerous reports indicating positive effects of dietary prebiotics on survival rates of cultured organism (Mahious & Ollevier 2005; Staykov et al. 2005; Mahious et al. 2006; Sheikholeslami et al. 2007; Salze et al. 2008; Hoseinifar et al. 2010, 2011a,b; Ibrahem et al. 2010; Soleimani et al. 2012). Based on the fact that production of larvae and fry are still a bottleneck in commercial production, the results of the present study showing increased survival rate in early life stages of common carp by prebiotic is of interest and might be attributed to improved general health, gut microbiota, gut morphology or immune status. However, as the topic has not been clearly elucidated, further studies are needed. Our results showed that dietary inclusion of inulin had no significant effects on digestive enzyme activities of common carp fry. In accordance with our results, Refstie et al. (2006) showed that inulin (75 g kg 1) fed to Atlantic salmon (Salmo salar L.) for 3 weeks caused no significant changes in digestive enzymes activities (trypsin, amylase, alkaline phosphatase and leucine aminopeptidase). Likewise, Anguiano et al. (2013) reported that inclusion of 4 prebiotics [fructooligosaccharide (FOS), Bio-MOS, transgalactooligosaccharide and GroBiotic-A] in diets of hybrid striped bass (Morone chrysops 9 M. saxatilis) and red drum (Sciaenops ocellatus) did not cause significant changes in the activities of digestive enzymes (pepsin, trypsin, chymotrypsin, aminopeptidase, a-amylase, lipase). However, these results are different from those obtained by Soleimani et al. (2012) fed Caspian roach (Rutilus rutilus) fry diets supplemented with fructooligosaccharide (10, 20 and 30 g FOS kg 1) and allogynogenetic crucian carp fed xylooligosaccharide (50, 100 and 200 mg kg 1) (Xu et al. 2009). Although not investigated in the present study, the contradictory results may be attributed to the inability of intestinal microbiota to ferment inulin (Reza et al. 2009; Hoseinifar et al. 2011b) and as a natural consequence the topic merits further investigations. The dietary supplementation of inulin significantly affected the carcass composition especially protein and lipid content of common carp fry. Similar to our finding, significantly lower carcass composition of protein content was observed in red drum (Sciaenops ocellatus) fed 10 g inulin kg 1 compared with the control treatment

(Burr et al. 2009). Changes in protein and lipid content in carcass of common carp fed inulin and the control group may be due to changes in their synthesis, deposition rate in muscle (Abdel-Tawwab et al. 2008). The results of the present study showed that inulin did not affect growth performances and digestive enzymes activities of common carp fry. However, the positive results obtained on survivability of inulin fed fish encourage further research on administration of inulin and other prebiotics in common carp fry studies. Furthermore, evaluations of the mechanisms of action, optimal inclusion levels, effects on the innate immune system, indigenous gut microbiota (autochthonous and allochthonous), gut morphology and challenge studies merits further investigations.

This work was supported by Young Researchers Club, Lahijan Branch. The author wish to thank Orafti company managers (Raffinerie Tirlemontoise, Tienen, Belgium) for their support and provision of inulin and Professor Van Loo and Dr. Mahious for their kind help during the experiment.

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