Comparison of copper bioavailability in copper-methionine, nano-copper oxide and copper sulfate additives in the diet of Russian sturgeon Acipenser gueldenstaedtii

Accepted Manuscript Comparison of copper bioavailability in copper-methionine, nanocopper oxide and copper sulfate additives in the diet of Russian sturgeon Acipenser gueldenstaedtii

Hewei Wang, Haoyong Zhu, Xiaodan Wang, Erchao Li, Zhenyu Du, Jianguang Qin, Liqiao Chen PII: DOI: Reference:

S0044-8486(17)31176-6 doi:10.1016/j.aquaculture.2017.09.037 AQUA 632842

To appear in:

aquaculture

Received date: Revised date: Accepted date:

8 June 2017 20 August 2017 25 September 2017

Please cite this article as: Hewei Wang, Haoyong Zhu, Xiaodan Wang, Erchao Li, Zhenyu Du, Jianguang Qin, Liqiao Chen , Comparison of copper bioavailability in coppermethionine, nano-copper oxide and copper sulfate additives in the diet of Russian sturgeon Acipenser gueldenstaedtii. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2017), doi:10.1016/ j.aquaculture.2017.09.037

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ACCEPTED MANUSCRIPT Comparison of copper bioavailability in copper-methionine, nano-copper oxide and copper sulfate additives in the diet of Russian sturgeon Acipenser gueldenstaedtii Hewei Wang a, Haoyong Zhu a, Xiaodan Wang a, Erchao Li a, Zhenyu Du a, Jianguang Qin b,

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Liqiao Chen a,* Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal

College of Science and Engineering, Flinders University, Adelaide, SA 5001, Australia

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University, Shanghai 200241, PR China

* Corresponding author:

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Prof. Liqiao Chen Email: [email protected]; Tel: +86-21-54345354

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ACCEPTED MANUSCRIPT ABSTRACT This study was conducted to evaluate the relative bioavailability of copper sulfate (CuSO4), copper-methionine (CuMet) and nano-copper oxide (CuONano) as a diet additive by comparing growth, Cu status, antioxidant activity, immune response and Cu apparent digestibility in Russian sturgeon Acipenser gueldenstaedtii. The control was a semi-purified basal diet without Cu

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supplementation and the experimental diets were prepared with the forms of CuSO4, CuMet and CuONano, representatively. The Cu contents were 2, 4, 6, 8 and 16 mg/kg in the CuMet and

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CuONano diet groups, and 4, 6 and 16 mg/kg in the CuSO4 diet groups. Sturgeon (9.82 ± 0.08 g)

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were fed with the control diet and 13 experimental diets with Cu in different contents and sources for 8 weeks. The bacterial challenge test and Cu apparent digestibility determination were carried

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out at the end. The highest weight gain (WG), whole body Cu concentration, Cu apparent digestibility, copper-zinc superoxide dismutase (Cu-Zn SOD) activity, total antioxidant capacity,

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lysozyme and immunoglobulin M content were achieved at 6 mg Cu/kg in the CuSO4 diet, and 4 mg Cu/kg in both CuMet and CuONano diets. Sturgeon fed 4 mg Cu/kg in the CuMet diet or the CuONano diet had higher growth rate, tissue Cu deposition, antioxidant and immune capacity than

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fish fed the same Cu content in the CuSO4 diet. At the same time, Cu in the CuMet or CuONano

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diet exhibited higher apparent digestibility and Aeromonas hydrophila resistance. When the Cu content was standardized in the form of CuSO4, the relative Cu bioavailability was 153% - 168%

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in the CuMet diet and 172% - 202% in the CuONano diet based on weight gain, hepatic Cu-Zn SOD activity, and Cu concentrations in the whole body and vertebra. The optimal dietary Cu

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requirements was about 5 mg/kg when CuMet or CuONano was used, and increased to 8 mg/kg when CuSO4 was used. This study indicates that Cu in the forms of CuMet and CuONano is 1.5 2 times more bioavailable than in the form of CuSO4 for the dietary Cu requirements of Russian sturgeon. Keywords: Acipenser gueldenstaedtii Copper source Physiological condition Copper status Relative bioavailability

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ACCEPTED MANUSCRIPT 1. Introduction Copper (Cu) is an essential trace mineral element for all animals including fish. Cu works as a cofactor of specific proteins and enzymes such as ceruloplasmin, superoxide dismutase, cytochrome oxidase, lysyl oxidase, dopamine hydroxylase and tyrosinase (Watanabe, et al., 1997). These copper-containing enzymes participate in a series of biological processes including

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scavenging free radicals, electron transport, and syntheses of hemoglobin and collagen (Lall, 2002). Fish can obtain Cu from both diet and water, but the former is the major way of Cu

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acquisition (Kamunde, et al., 2002). In aquaculture, Cu must be supplemented to a practical diet

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because Cu in the dietary ingredients such as fish meal and plant protein sources are inadequate (Berntssen, et al., 1999; Mondal, et al., 2008; Richards, 1997). Cu deficiency could reduce the

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growth of grouper Epinephelus malabaricus (Lin, et al., 2008) and decrease the activities of cytochrome C oxidase in heart and copper-zinc superoxide dismutase in liver (Cu-Zn SOD) of

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channel catfish Ictalurus punctatus (Gatlin and Wilson, 1986). Alkaline phosphatase (AKP) and acid phosphatase (ACP) activities were reduced when blunt snout bream Megalobrama amblycephala fed a diet with Cu deficiency. However, excessive dietary Cu can also slow weight

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gain in rainbow trout Sulmo gairdneri (Julshamn, et al., 1988) and tilapia Oreochromis niloticus ×

in an optimal range.

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O. aureus (Shiau and Ning, 2003). Therefore, in aquaculture feed, the Cu content must be present

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Bioavailability is defined as the degree to which an ingested nutrient in a particular source is absorbed in a form that can be utilized by the animal (Ammerman, 1995). One of the factors that

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affect Cu bioavailability in the diet is the form of Cu compounds (Apines-Amar, et al., 2004; Fairweathertait and Hurrell, 1996; Richards, 1997). Cu inorganic salts such as Cu sulfate, Cu oxide and Cu chloride are traditional dietary Cu additives, and chelated Cu or nanoform Cu is a novel form of dietary Cu additives. Some authors have suggested that the bioavailability of organic chelated Cu is higher than Cu inorganic salts in chick (Mondal, et al., 2008), pig (Veum, et al., 2004), lamb (Senthilkumar, et al., 2008) and fish such as rainbow trout Oncorhynchus mykiss (Apines-Amar, et al., 2004; Apines, et al., 2003), grouper (Lin, et al., 2010) and channel catfish (Paripatananont and Lovell, 1995a). On the contrary, the availability of copper–lysine is only two-thirds of the inorganic salt in lambs (Pott, et al., 1994). Studies using nanoform Cu as dietary 3

ACCEPTED MANUSCRIPT additives are scarce, but some researchers have demonstrated that Cu nanoparticles show a better absorption and biological effects than inorganic Cu in piglets and red sea bream Pagrus major (El Basuini, et al., 2016; Gonzaleseguia, et al., 2009), and the chelate trace elements may protect metal ions against anti-nutritional factors present in practical diets (Rider, et al., 2010). Another study suggests that chelating minerals can be absorbed in an intact form (Ashmead, 1992). The

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small size particle of nanomaterials can make nano-nutrients easier for fish absorption and nanomaterials can be absorbed by special endocytosis and through cell bypass pathways

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(Bellmann, et al., 2015). Therefore, higher bioavailability of chelate and nanoform of Cu may be due to their higher absorption or additional biological effects compared to inorganic Cu salts, but

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the specific reason is not clear (Ashmead, 1992; Spears, 1989). The choice of Cu compound with a high utilization rate in fish diet can reduce the amount of dietary Cu added and the level of Cu

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excretion in feces.

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China has become the world’s largest producer of sturgeon, and the Russian sturgeon Acipenser gueldenstaedtii is the major sturgeon species for aquaculture in China (China Fishery Statistical Yearbook, 2014; FAO-Food and Agriculture Organization of the United Nations, 2014; Wei, et al.,

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2011). Until now, most research on sturgeon nutrition has focused on the requirements of protein,

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lipid and carbohydrate (Hung, et al., 1989; Sener, et al., 2005; Stuart and Hung, 1989). However, the knowledge on the mineral nutrition of Russian sturgeon is inadequate. Our previous study has

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identified optimal dietary Cu requirement of Russian sturgeon (Wang, et al., 2015). This study was a continuation of the previous study and aimed to evaluate the bioavailability of dietary copper to

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fish in various Cu compounds including copper methionine (CuMet), nano-copper oxide (CuONano) and copper sulfate (CuSO4) based on the measurements of fish growth, copper status, antioxidant activities, immune responses and Cu apparent digestibility. 2. Materials and methods 2.1. Experimental diets Basal diet was used as the control and its formulation and composition (AOAC, 1995) are shown in Table 1. Casein and gelatin were used as the dietary protein sources and fish oil and soybean oil were used as the main dietary lipid. Corn starch was used as the carbohydrate source. 4

ACCEPTED MANUSCRIPT Copper sulphate CuSO4·5H2O (CuSO4) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), copper methionine (CuMet) (Cu content ≥17%, Xiya Regent Co., Ltd., China) and nano-copper oxide (CuONano) (Particle size 40 nm, Aladin Chemistry Co., Ltd., Shanghai, China) were used as dietary Cu additives. The CuMet was added to the basal diet at 2, 4, 6, 8, 16 mg Cu/kg diet and the analyzed values were 2.46, 4.54, 6.42, 8.52 and 16.38 mg Cu/kg diet,

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respectively. The CuONano was added to the basal diet at 2, 4, 6, 8, 16 mg Cu/kg diet and the analyzed values were 2.54, 4.29, 6.32, 8.44 and 16.25 mg Cu/kg diet, respectively. The CuSO4 was

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only added to the basal diet at 4, 6 and 16 mg Cu/kg diet whereas 2 and 8 mg Cu/kg were not used because the former is too much below the optimal level and the latter is too close to the optimal

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level when CuSO4 is used as the Cu source in the diet (Wang, et al., 2015). The analyzed values were 4.36, 6.34 and 16.15 mg Cu/kg diet, respectively. Dietary Cu concentration was analyzed

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using an inductively-coupled plasma-atomic emission spectrophotometer. Diets were processed into 2.5 mm diameter strips, air dried, ground and sieved to appropriate size and stored at −20 °C

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before feeding.

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2.2. Experimental procedure

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Russian sturgeon juveniles were obtained from a farm in Quzhou, China. Before the experiment, fish were acclimated to the laboratory condition for two weeks in two circular fibreglass tanks (diameter: 2 m, height: 0.51 m) in a flow-through system with continuous aeration, and fed with

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the basal diet during acclimation.

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After acclimation, fish were fasted for 24 h. Healthy sturgeon (9.82 ± 0.08 g) were randomly assigned to 42 aquariums (40×45×100 cm) in a flow-through system at a density of 30 fish per aquarium. Each diet was fed to fish in three randomly chosen aquaria. The diet was divided into two equal meals fed at 08:30 and 16:30 h by hand for 8 weeks. Fish were daily fed at 3% of their body weight. Fish were weighed once every 2 weeks and the daily ration was adjusted according to weight gain. Feed intake was completed within 2-3 min after delivery, and thus the Cu leaching to water was negligible. Excess feed and feces were siphoned daily. The Cu concentration in rearing water was 0.8 - 1.2 μg/L throughout the experiment. During the experimental period, the water temperature was 19.5 - 22.0 °C, dissolved O2 7 mg/L, pH 7.2 and ammonia 0.1 mg/L. A 5

ACCEPTED MANUSCRIPT photoperiod of 12 h light (08:00 to 20:00 h) and 12 h dark was used during the study. 2.3. Sample collection and analysis Growth performance of the juvenile Russian sturgeon was evaluated using weight gain (WG), feed efficiency (FE), survival, condition factor (CF), hepatosomatic index (HSI) and

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viscerosomatic index (VSI) and calculated as in the following: Weight gain (WG, %) = 100 × [(final body weight− initial body weight)/initial body weight].

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Feed efficiency (FE) = (final body weight − initial body weight)/feed intake. Survival (%) =100 × (final amount of fish)/(initial amount of fish).

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Hepatosomatic index (HSI, %) = (hepatic weight/body weight) × 100.

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Viscerosomatic index (VSI, %) = (viscera weight/body weight) × 100. Condition factor (CF) = (body weight/body length3) × 100.

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At the end of the feeding trial, fish were fasted for 24 h and then anesthetized with MS222 at

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150 mg/L before handling. Then total number and weight of fish in each aquarium were counted and weighed to calculate the body weight gain and survival. Subsequently, blood samples were collected in the caudal vessels from 5 fish per aquarium. Serum was separated by centrifugation of

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blood at 1500× g for 10 min at 4 °C using a centrifuge (5804R, Eppendorf, Hamburg, Germany). The serum samples were frozen in liquid nitrogen and then stored at −80 °C for determination of

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ceruloplasmin activity, lysozyme activity (LSZ), complement 3 (C3) and immunoglobulin M (IgM) content. After blood collection, viscera and liver were weighed for calculating the VSI and HSI. The liver was rapidly frozen in liquid nitrogen and then stored at −80 °C for determining, copper-zinc superoxide dismutase (Cu-Zn SOD) activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA). The liver Cu concentration was determined in the pooled sample of 5 fish per aquarium. After liver collection, the vertebra was peeled from the fish and pooled to determine the vertebra Cu concentration. Another 3 fish per aquarium were randomly selected, pooled and then stored in −20 °C to determine the whole-body proximate composition and Cu concentration. 6

ACCEPTED MANUSCRIPT All of the enzyme activities, T-AOC, C3, IgM and MDA content were measured by the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The LSZ activity and IgM content were measured using the immunonephelometric assay (Chen and Ji, 1992; Shan, et al., 2007). Methods for C3 activity analysis included measurement of the increase in turbidity after immunity response of C3 and its increased antibody (Thomas, 1998). Serum ceruloplasmin

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activity was measured using o-dianisidine dihydrochloride as a substrate (Schosinsky, et al., 1974). The analysis of Cu-Zn SOD activity was based on SOD-mediated inhibition of nitrite formation

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from hydroxyammonium in the presence of O2− generators (xanthine and xanthine oxidase) (Elstner and Heupel, 1976). T-AOC was measured by the colorimetric technique as described by

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substance (TBARS) assay (Ohkawa, et al., 1979).

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Miller, et al. (1993). MDA concentration was measured by the thiobarbituric acid–reacting

Cu concentration was measured according to the modified method by Gomez et al. (Gomez, et

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al., 2007; Gonzalvez, et al., 2010) using the inductively coupled plasma-atomic emission spectrophotometer (ICP-OES Optima 5300DV; PerkinElmer Corporation) at the Shanghai Academy of Public Measurement, China. Samples were digested with 10 ml of concentrated nitric

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acid and perchloric acid superior grade (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China)

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solution with 9:1 ratio. The digested residue was dissolved in ultrapure water (18.2 MΩ/cm) to the detectable range of ICP-OES. Analytical wavelength of Cu was 327.39 nm and the limit of

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quantitation (LOQ) of Cu was 0.015 mg/L.

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2.4. Challenge test and apparent digestibility determination of Cu After feeding for 8 weeks, Aeromonas hydrophila obtained from East China Normal University, Shanghai, China were used in the bacterial challenge test. First, the 7-d half lethal concentration (LC50) of A. hydrophila for Russian sturgeon was predetermined. Sixteen Russian sturgeon per tank in triplicate were intraperitoneally injected with 0.2 ml of 1010, 109, 108, 107, 106 and 105 CFU/mL of A. hydrophila. The preliminary trial indicated that the 7-d LC50 was 6.7×107 CFU/mL. Then in the final challenge test, 12 fish were randomly sampled from each treatment aquarium (36 fish from each diet) were challenged with an intraperitoneal injection of 0.2 mL of live A. hydrophila at 6.7×107 CFU/mL for 7 d. At the end, the cumulative fish mortality was used as an 7

ACCEPTED MANUSCRIPT indicator for disease resistance against A. hydrophila. After feeding for 8 weeks, another eight fish randomly sampled from each aquarium (24 fish from each diet) were transferred to 42 aquariums (20×22.5×50 cm) to determine apparent digestibility (AD) of Cu. The 0.1% yttrium oxide (Y2O3) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was used as an external marker and added to the diet previously used in each

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aquarium. After feeding the diet containing the indicator, feces were collected from the third day and the collection period was 7 days. Feces was removed with a siphon tube and pooled on a

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screen mesh. The contents of Cu and Y in the feces were determined with the method described

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above. The AD was analyzed according to the following equation (Maynard and Loosli, 1969): AD (%) = [1 – (M feed × N faeces) / (M faeces × N feed)] × 100 where M was the concentration of the

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marker, and N the concentration of the nutrient.

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2.5. Statistical analysis

The results were presented as means ± SE of three replicates. The data were compared between treatments by one-way analysis of variance (one-way ANOVA) using the SPSS 21.0 statistical

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software. When overall differences were significant (P < 0.05), Tukey’s test was used to compare

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the mean values between individual treatments. Two-way ANOVA was also used to test the effects of dietary Cu sources and Cu levels, and their interactions. Dietary Cu requirement of Russian sturgeon was estimated based on weight gain, Cu-Zn SOD activity, bone and whole-body Cu

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concentration by the broken-line regression method (Robbins, 1986). The linear segments of the

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regression lines below the breakpoints were used to compare the relative bioavailability of Cu from CuMet and CuONano with that from CuSO4 by deriving the ratio of the slopes of the lines (Forbes and Parker, 1977). The data of CuMet and CuONano supplemented with Cu levels at 2 mg and 8 mg Cu/kg diet were only used for fitting regression equations but were not used for analysis of variance. 3. Results 3.1. Growth and body indices The growth, feed utilization and body indices data are presented in Table 2. Within each Cu 8

ACCEPTED MANUSCRIPT source, the maximum FBW, WG and FE were obtained in fish fed diets with 6.34, 4.54 and 4.29 mg Cu/kg from CuSO4, CuMet and CuONano, respectively. No significant differences were found among maximum values of FBW, WG and FE from different Cu sources. The FBW, WG and FE of fish fed diets supplemented with Cu at 0 (control) and 16 mg/kg were significantly lower than other groups. There were no significant differences in fish survival (94.4% - 98.9 %) among all

sources significantly affected FBW, WG and FE except survival.

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groups. Two-way ANOVA indicated that the Cu level and interaction between Cu levels and

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As shown in Table 3, HSI (3.1% - 3.4%), VSI (11.2% - 12.2%) and CF (0.92 - 0.95) had no

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significant differences among all treatments. Two-way ANOVA indicated that the Cu level, Cu source and their interaction did not significantly affect HSI, VSI and CF.

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3.2. Whole body proximate composition

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The composition of whole body proximate nutrients are shown in Table 4. No significant differences in the contents of moisture (77.6% - 78.2%), crude protein (13.1% - 13.8%), crude lipid (4.4% - 4.8%) and ash (2.8% - 3.2%) were observed among all the treatments. Two-way

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ANOVA showed that moisture, crude protein, crude lipid and ash were not significantly affected

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by Cu level, source and their interaction.

3.3. Tissue Cu concentrations, apparent digestibility of Cu and Cu retention

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The Cu concentrations of whole body, liver and vertebra are shown in Table 5. Within each Cu source, there was a trend of significant increase of Cu concentration in the whole body, liver and

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vertebra with the increase of dietary Cu levels. Fish fed the control diet and the diet containing 4.36 mg Cu/kg as CuSO4 had the lowest Cu concentration in whole-body and vertebra, and the liver Cu concentration was significantly lower in control. There was a trend that fish fed diets with CuMet or CuONano had relatively higher tissue Cu concentrations than those fed diets with equal levels of Cu in the CuSO4 form and the difference reached a significant level when the Cu content was supplemented at 4 mg/kg in the diet. The Cu concentration was highest in liver (8.6 - 31.2 mg/kg), second highest in vertebra (3.2 - 9.2 mg/kg), but lowest in whole body (2.9 - 5.5 mg/kg). Two-way ANOVA indicated that the Cu concentration in the whole body, liver and vertebra was 9

ACCEPTED MANUSCRIPT significantly affected by the Cu level and source, but not by their interaction. The three Cu sources showed a similar trend in Cu retention and apparent digestibility (Table 5). The Cu retention gradually reduced with the increase of dietary Cu content. In contrast, Cu apparent digestibility increased with the escalation of dietary Cu. Fish fed the CuMet and CuONano diets had significantly higher Cu retention and apparent digestibility than fed CuSO4

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when the diet was supplemented with Cu at 4 mg/kg. There were no significant differences in Cu retention and apparent digestibility among treatments supplemented with Cu at 6 or 16 mg/kg.

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affected by dietary Cu level, source and their interaction.

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Two-way ANOVA indicated that Cu retention and apparent digestibility were significantly

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3.4. Liver antioxidant parameters and serum ceruloplasmin activity

The liver antioxidant parameters and serum ceruloplasmin activity are shown in Table 6. The

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activities of hepatic Cu-Zn SOD, T-AOC and serum ceruloplasmin activity had a trend of reduction in the control and in fish fed Cu supplemented at 16 mg/kg. The values of hepatic Cu-Zn SOD, T-AOC and serum ceruloplasmin were highest in fish fed the diet containing 6.34, 4.54 and

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4.29 mg Cu/kg from the source of CuSO4 or CuONano. The values of hepatic Cu-Zn SOD and

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T-AOC were significantly higher than those in the control and fish fed the diet supplemented with Cu at 16 mg/kg. The serum ceruloplasmin activity in fish fed the control diet was significantly lower and the MDA content in the control and the diets with Cu supplementation at 16 mg/kg was

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significantly higher than in other treatments. Two-way ANOVA indicated that the liver antioxidant

level.

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parameters and serum ceruloplasmin activity were all significantly affected by the dietary Cu

3.5. Serum immune parameters The serum immune parameters are listed in Table 7. Fish fed diets with Cu deficiency and excess had a tendency to decrease fish immune performance. The lysozyme activity was higher in fish fed diets supplemented with 4 and 6 mg Cu/kg and was significantly higher than in the control and other fish fed supplemented Cu at 16 mg/kg in the source of CuSO4 or CuONano, and there were no significant differences among diets supplemented with 4 and 6 mg Cu/kg. The control 10

ACCEPTED MANUSCRIPT group had a significantly lower C3 content than the groups supplemented 4 and 6 mg Cu/kg. Fish fed diets supplemented with Cu at 6.34, 4.54 and 4.29 mg Cu/kg from sources of CuSO4, CuMet and CuONano had the highest IgM content, and were significantly higher than in the control and fish fed 16.15 mg Cu/kg in the form of CuSO4. 3.6. Dietary copper requirement and relative bioavailability analyses

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Analysis by broken-line regression on WG, hepatic Cu-Zn SOD activity, whole-body Cu

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concentration (Fig. 1) indicated that the optimal dietary Cu requirement of juvenile Russian sturgeon was 6.55, 6.61 and 7.15 mg Cu/kg for CuSO4, 4.44, 4.66 and 4.77 mg Cu/kg for CuMet

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and 4.35, 4.31 and 4.43 mg Cu/kg for CuONano. Compared with CuSO4 (100%), the relative bioavailability (RBV) of CuMet was 153%, 158% and 168% based on WG, hepatic Cu-Zn SOD

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activity and whole-body Cu concentration, and RBVs of CuONano were 172%, 186% and 188%.

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3.7. Challenge test

The cumulative mortalities of the challenge test are shown in Fig. 2. The results showed that

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diets supplemented with Cu at 0 and 16 mg/kg reduced the resistance against A. hydrophila

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infection in juvenile Russian sturgeon. The cumulative mortality of fish fed the diet with 6.34 mg Cu/kg from CuSO4, 4.54 and 6.32 mg Cu/kg from CuMet and CuONano were significantly lower than in the control. There were no significant differences in the cumulative mortality among all Cu

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4. Discussion

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supplemented groups.

The effects of dietary Cu deficiency and excess on the growth, Cu deposition in tissue and antioxidant capacity of Russian sturgeon have been investigated in our previous study (Wang, et al., 2015). The focus of the current study was to compare the relative bioavailability (RBV) of dietary Cu in the forms of CuMet, CuONano and CuSO4 in Russian sturgeon. In this study, the maximum FBW and WG were found in sturgeon fed the diets with 6.34 mg Cu/kg as CuSO4, 4.54 mg Cu/kg as CuMet and 4.29 mg Cu/kg as CuONano. Meanwhile, fish fed the diets with 6.34, 4.54 and 4.29 mg Cu/kg from CuSO4, CuMet and CuONano had higher FE than in other groups. However, the maximum WG of fish was not significantly different between Cu sources, indicating 11

ACCEPTED MANUSCRIPT that CuMet and CuONano did not add obvious growth advantage in Russian sturgeon. The optimal dietary Cu requirement was 4.44 mg/kg for CuMet and 4.35 mg/kg for CuONano by broken-line analysis on weight gain. However, the optimal Cu requirements was 6.55 mg/kg for CuSO4 which agrees with our previous finding (Wang, et al., 2015). These results suggest that CuMet and CuONano can promote growth more effectively than CuSO4 for meeting the dietary Cu

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requirement of Russian sturgeon and can reduce the amount of dietary Cu additive. Same as our study, amino acid-chelate Zn, Mn and Cu in rainbow trout (Apines-Amar, et al., 2004), chelated

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Zn in channel catfish (Paripatananont and Lovell, 1995a) and nano-copper in red sea bream (El

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Basuini, et al., 2016) also showed a better effect of growth promotion than with inorganic salts. In the present study, Cu in the form of CuMet and CuONano showed more effective growth

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promotion than in CuSO4 due to improved Cu absorption and deposition efficiency when the dietary Cu content did not exceed the optimal Cu requirement of Russian sturgeon. In previous

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study, Cu in the intestine was absorbed by epithelial cells in the form of Cu ions which could form insoluble or indigestible complexes with endogenous inhibitors (tricalcium phosphate, phytic acid and fiber) to prevent Cu absorption (Kim, et al., 2008). Metal chelates owning to stable structure

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may release an element in its ionic form at the site of absorption or is readily absorbed as an intact

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chelate, which greatly enhances the adsorption by preventing element conversion to an insoluble form (Scott, et al., 1982). Particle size is a key parameter, as uptake efficiency increases with the

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decrease of particle diameter (Florence, et al., 1995). Nanoparticle is easier to absorb as small particle size of copper could be absorbed by organisms as an intact nanoparticle (Florence, et al.,

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1995; Win and Feng, 2005).

In the whole body, liver and vertebra, Cu concentration and retention rate in CuMet and CuONano were higher than in CuSO4 when fish fed the same level of dietary Cu and the differences were significant between Cu sources when the dietary Cu level was 4 mg/kg. Similarly, the apparent digestibility in fish fed 4 mg Cu/kg of CuMet or CuONano was significantly higher than in fish fed 4 mg Cu/kg of CuSO4. Previous studies have also found that chelated minerals had higher absorption efficiency than inorganic trace minerals in channel catfish (Paripatananont and Lovell, 1997), and nano-additives had higher absorption efficiency than inorganic additives in pigs (Gonzaleseguia, et al., 2009; Li, et al., 2016). The results show that the increase in the Cu apparent 12

ACCEPTED MANUSCRIPT digestibility of CuMet and CuONano effectively increase their retention and growth effect in Russian sturgeon, which is consistent with the reports of chelated organic Cu in chick (Aoyagi and Baker, 1993) and sheep (Pal, et al., 2010), and Nanosize ZnO in pig (Li, et al., 2016). Contrary to our result, in a study on red sea bream there were no significant differences of Cu content in the whole body, when fish fed the same dose (4 mg/kg) of Cu nanoparticles and CuSO4 (El Basuini, et al., 2016). This may be due to the difference in particle size or the chemical form of the Cu used in

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the two studies. In El Basuini’s (2016) study, the Cu powder with a particle size ≤ 75 μm (queried

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according to the commodity information in the article) was used, but the CuO particle size was 40 nm in our study. CuO nanoparticles were more effective in absorption than micron CuO and

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CuSO4 in Caco-2 cells at the same concentration (Gao, 2014). In the present study, no significant differences of Cu retention rate were found when Russian sturgeon fed the diet with different Cu

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sources supplemented at the same amount of 6 or 16 mg/kg. This may be due to the fact that the dietary Cu content ≥ 6 mg/kg has reached or exceeded the requirements of Russian sturgeon and

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fish has removed excessive Cu from the body.

In addition to tissue Cu deposition, antioxidant and immune indicators are commonly used to

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evaluate the long-term Cu nutritional status of animals (Bonham, et al., 2002; Watts, et al., 2001).

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In the present study, the Cu-Zn SOD, T-AOC and ceruloplasmin activity were lower but the MDA content was higher than in the control and the diet supplemented with 16 mg Cu/kg. Cu deficiency

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leads to incomplete structure of Cu-Zn SOD in animals and excess Cu increases free radicals in tissues, which may explain the difference of physiological performance at two extreme Cu

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contents (Prohaska and Brokate, 2001; Schuessel, et al., 2005). Similar to the effect of dietary Cu sources on Russian sturgeon growth and Cu absorption, the antioxidation of fish fed diets with 4.54 mg Cu/kg in CuMet and 4.29 mg Cu/kg in CuONano were equivalent to that of fish fed 6.34 mg Cu/kg in CuSO4, indicating that CuMet and CuONano have a better ability to enhance antioxidant capacity in Russian sturgeon than CuSO4 when the dietary Cu content is within the optimal range of dietary requirement in Russian sturgeon. Cu performs several functions in the immune system through the specific mechanism of action is not clear. Cu deficiency causes impairment of mammalian T cell proliferations, cytokine production, antibody production and susceptibility to disease (Dorton, et al., 2003; Gengelbach and Spears, 1998). The higher lysozyme, 13

ACCEPTED MANUSCRIPT C3 and IgM contents were found in sturgeon fed the diet with Cu content at 4.54 mg/kg in CuMet, 4.29 mg/kg in CuONano and the diet with Cu supplemented at 6 mg Cu/kg where the cumulative mortality was lower than in other groups. There was a similar report that zinc-methionine is more effective in maintaining host defence against Edwardsiella ictaluri (Paripatananont and Lovell, 1995b).

PT

The higher cost of most mineral chelates and nanoparticles relative to inorganic sources has generally limited their use in aquaculture diets (Lin, et al., 2010). But aquaculture must take into

RI

account to balance economic cost and environmental protection for making diets. It is necessary

SC

for aquaculture producers to use highly absorbent dietary Cu to reduce environmental pollution. Due to limited supply of fish meal, protein of a plant source has been increasingly used in

NU

aquaculture feed. Compared with fish meal, plant feed ingredients have a higher content of mineral inhibitors which will reduce the absorption efficiency of the minerals (Bell, 1993; Davies,

MA

1979). In this context, chelated and nanoform Cu are promising alternative to inorganic Cu in fish diet to improve absorption efficiency and utilization rate.

D

In conclusion, CuMet or CuONano showed 1.5 – 2.0 times higher RBV than CuSO4 in

PT E

semi-purified basal diets for meeting the dietary Cu requirements of juvenile Russian sturgeon. The optimal dietary Cu requirement for this fish was about 5 mg/kg when CuMet or CuONano was used, and was 8 mg/kg when CuSO4 was used. These results imply that supplementation of

CE

Cu in Russian sturgeon can be reduced by using chelated or nanoform Cu to replace inorganic Cu,

AC

resulting in high Cu absorption in fish and low waste discharge of Cu to the environment. Acknowledgements

This research was supported by grants from the National ‘Twelfth Five-Year’ Plan for Science & Technology Support (2012BAD25B03), the National Basic Research Program (973Program, No. 2014CB138803), the Special Fund for Agro-scientific Research in the Public Interest (No. 201203065), the National Natural Science Foundation of China (No. 31572629) and partly by the E-Institute of Shanghai Municipal Education Commission (No. E03009).The authors thank Qi Li, Tingting Zhu and other members of the laboratory for their kind assistance.

14

ACCEPTED MANUSCRIPT

References

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Lin, Y.H., Shie, Y.Y., Shiau, S.Y., 2008. Dietary copper requirements of juvenile grouper, Epinephelus malabaricus. Aquaculture 274, 161-165. Lin, Y.H., Shih, C.C., Kent, M., Shiau, S.Y., 2010. Dietary copper requirement reevaluation for juvenile grouper, Epinephelus malabaricus, with an organic copper source. Aquaculture 310, 173-177. Maynard, L., Loosli, J., 1969. Animal Nutrition, McGraw-Hill, New York, NY, USA, pp. 613. Miller, N.J., Rice-Evans, C., Davies, M.J., Gopinathan, V., Milner, A., 1993. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci. 84, 407-412. Mondal, M.K., Das, T.K., P, Samanta, C.C., Bairagi, B., 2008. Influence of dietary inorganic and organic copper salt and level of soybean oil on plasma lipids, metabolites and mineral balance of broiler chickens. Anim. Feed Sci. Technol. 139, 212-233. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351-358. Pal, D.T., Gowda, N.K., Prasad, C.S., Amarnath, R., Bharadwaj, U., Suresh, B.G., Sampath, K.T., 2010. Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes. J. Trace Elem. Med Biol. 24, 89-94. Paripatananont, T., Lovell, R.T., 1995a. Chelated zinc reduces the dietary zinc requirement of channel catfish, Ictalurus punctatus. Aquaculture 133, 73-82. Paripatananont, T., Lovell, R.T., 1995b. Responses of channel catfish fed organic and inorganic sources of zinc to Edwardsiella ictaluri challenge. J. Aquat. Anim. Health, 147-154. Paripatananont, T., Lovell, R.T., 1997. Comparative net absorption of chelated and inorganic trace minerals in channel catfish Ictalurus punctatus diets. J. World Aquacult. Soc. 28, 62-67. Pott, E.B., Henry, P.R., Ammerman, C.B., Merritt, A.M., Madison, J.B., Miles, R.D., 1994. Relative bioavailability of copper in a copperlysine complex for chicks and lambs. Anim. Feed Sci. Technol. 45, 193–203. Prohaska, J.R., Brokate, B., 2001. Lower copper, zinc-superoxide dismutase protein but not mRNA in organs of copper-deficient rats. Arch. Biochem. Biophys. 393, 170-176. Richards, M.P., 1997. Trace mineral metabolism in the avian embryo. Poult. Sci. 76, 152-164. Rider, S.A., Davies, S.J., Jha, A.N., Clough, R., Sweetman, J.W., 2010. Bioavailability of co-supplemented organic and inorganic zinc and selenium sources in a white fishmeal-based rainbow trout (Oncorhynchus mykiss) diet. J. Anim. Physiol. Anim. Nutr. 94, 99-110. Robbins, K.R., 1986. A method, SAS program, and example for fitting the broken-line to growth data. Univ. Tenn. Res. Rep. 86-09. Univ. of Tenn. Agric. Exp. Sta., Knoxville. Schosinsky, K.H., Lehmann, H.P., Beeler, M.F., 1974. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin. Chem. 20, 1556-1563. Schuessel, K., Schafer, S., Bayer, T.A., Czech, C., Pradier, L., Muller-Spahn, F., Muller, W.E., Eckert, A., 2005. Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice. Neurobiol. Dis. 18, 89-99. Scott, M.L., Nesheim, M.C., Young, R.J., 1982. Nutrition of the Chicken. Scott, M.L. and Associates, Ithaca, NY. 17

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Sener, E., Yildiz, M., Savas, E., 2005. Effects of dietary lipids on growth and fatty acid composition in Russian sturgeon (Acipenser gueldenstaedtii) juveniles. Turk. J. Vet. Anim. Sci. 29, 1101-1107. Senthilkumar, P., Nagalakshmi, D., Reddy, Y.R., Sudhakar, K., 2008. Effect of different level and source of copper supplementation on immune response and copper dependent enzyme activity in lambs. Trop. Anim. Health Prod. 41, 645-653. Shan, T., Wang, Y., Wang, Y., Liu, J., Xu, Z., 2007. Effect of dietary lactoferrin on the immune functions and serum iron level of weanling piglets. J. Anim. Sci. 85, 2140-2146. Shiau, S.Y., Ning, Y.C., 2003. Estimation of dietary copper requirements of juvenile tilapia, Oreochromis niloticus × O. aureus. Anim. Sci. 77, 287-292. Spears, J.W., 1989. Zinc methionine for ruminants: relative bioavailability of zinc in lambs and effects of growth and performance of growing heifers. J. Anim. Sci. 67, 835-843. Stuart, J.S., Hung, S.S.O., 1989. Growth of juvenile white sturgeon (Acipenser transmontanus) fed different proteins. Aquaculture 76, 303-316. Thomas, L., 1998. Clinical Laboratory Diagnostics, 1st ed. TH-Books Verlagsgesellschaft, Frankfurt, Germany, pp. 667–678. Veum, T.L., Carlson, M.S., Wu, C.W., Bollinger, D.W., Ellersieck, M.R., 2004. Copper proteinate in weanling pig diets for enhancing growth performance and reducing fecal copper excretion compared with copper sulfate. J. Anim. Sci. 82, 1062-1070. Wang, H., Li, E., Zhu, H., Du, Z., Qin, J., Chen, L., 2015. Dietary copper requirement of juvenile Russian sturgeon Acipenser gueldenstaedtii. Aquaculture 454, 118-124. Watanabe, T., Kiron, V., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185-207. Watts, M., Munday, B.L., Burke, C.M., 2001. Immune responses of teleost fish. Aust. Vet. J. 79, 570-574. Wei, Q.W., Zou, Y.C., Li, P., Li, L., 2011. Sturgeon aquaculture in China: progress, strategies and prospects assessed on the basis of nation-wide surveys (2007-2009). J. Appl. Ichthyol. 27, 162-168. Win, K.Y., Feng, S.S., 2005. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 26, 2713-2722.

18

ACCEPTED MANUSCRIPT Table 1

Gelatinb

10

Corn starchc

30

Fish oild

5

Soybean oile

5

Choline chloride

0.5

Taurine

0.5

Monocalcium phosphate

3

Vitamin premixf

1

Mineral premix, copper-freeg

1

Cellulose

4

PT E

Crude protein Crude lipid

43.23 9.74 9.94 3.91 0.54

AC

Copper (mg/kg)

CE

Moisture Ash

D

Proximate composition

RI

40

SC

Casein, vitamin-freea

NU

%

MA

Ingredient

PT

Formulation and proximate composition of the basal diet (% dry matter).

a

Casein, vitamin-free: crude protein 92% (Sigma-Aldrich Trading Co., Ltd., Shanghai, China).

b

Gelatin: Sangon Biotech, Shanghai, China.

c

Corn starch: Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.

d

Fish oil: Xiamen Xinsha Pharmaceutical Co., Ltd., Xiamen, China.

e

Soybean oil: Kerry Oils & Grains Industrial Co., Ltd., Shanghai, China.

f

Vitamin premix (mg/kg diet): thiamin hydrochloride, 50; riboflavin, 200; pyridoxine

hydrochloride, 50; vitamin B12, 0.1; nicotinic acid, 200; calcium pantothenate, 100; folic acid, 20; 19

ACCEPTED MANUSCRIPT biotin, 5; inositol, 800; ascorbic acid, 1000; menadione sodium bisulfite, 10; retinol acetate, 15; cholecalciferol, 10; alpha-tocopherol, 200; cellulose, 7340. g

Mineral premix, copper-free (mg/kg diet): ZnSO47H2O, 100; FeSO47H2O, 700; MnSO4H2O,

80; CoCl26H2O, 20; KI, 8; Na2SeO3, 3; MgSO47H2O, 4000; NaCl, 1000; KCl, 1000; AlCl36H2O,

AC

CE

PT E

D

MA

NU

SC

RI

PT

15; cellulose, 3074.

20

ACCEPTED MANUSCRIPT Table 2

Experimental diets

Analyzed Cu (mg/kg)

IBW2 (g)

FBW3 (g)

Control

0.54

9.87±0.03

52.6±0.8

CuSO4

4.36

9.86±0.01

57.7±1.1

6.34

9.76±0.03

61.3±0.4

16.15

9.81±0.01

55.9±0.6

4.54

9.81±0.05

62.0±1.1

6.42

9.76±0.03

60.0±1.1

16.38

9.77±0.03

53.7±0.9

4.29

9.85±0.01

62.6±0.7

6.32

9.81±0.03

16.25

9.84±0.03

MA

Growth and feed utilization of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1.

P value

0.111

PT E

Two-way ANOVA P-Source

Source×level

CE

P-Level

433.2±6.2

bcd de

528.3±6.4

abc e

e

de

a

Survival (%)

a

96.7±1.9

485.1±11.1

1.13±0.01

bcd

97.8±1.1

d

1.19±0.01

def

98.9±1.1

470.0±5.4

ab

1.10±0.02

abc

94.4±1.1

531.5±9.0

d

1.23±0.02

f

98.9±1.1

1.16±0.01

cde

97.8±2.2

RI

PT

bc

SC

ab

FE5

1.06±0.02

cd

514.4±11.2 449.8±8.6

ab

1.07±0.01

ab

98.9±1.1

536.3±7.6

d

1.22±0.01

ef

98.9±1.1

1.15±0.01

cde

98.9±1.1

1.05±0.02

a

97.8±2.2

NU

CuONano

a

cde

cd

60.5±1.0

517.5±13.2

52.4±0.7a

432.7±7.5

<0.001

<0.001

<0.001

0.528

0.918

0.840

0.344

0.365

<0.001

<0.001

<0.001

0.364

0.001

0.001

0.001

0.449

D

CuMet

WG4 (%)

a

AC

Means in the same column with different superscript letters are significantly different (P<0.05). 1

Value are presented as the mean ± SE (n=3) of three replicates per treatment.

2

IBW: Initial body weight.

3

FBW: Final body weight.

4

WG: Weight gain.

5

FE: Feed efficiency.

21

ACCEPTED MANUSCRIPT Table 3 The body indices of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1. Analyzed Cu (mg/kg)

HSI2

VSI3

CF4

Control

0.54

3.1±0.1

12.2±0.4

0.93±0.01

CuSO4

4.36

3.4±0.1

11.4±0.3

0.92±0.02

6.34

3.4±0.2

12.2±0.5

0.95±0.01

16.15

3.3±0.2

4.54

3.3±0.1

6.42

3.2±0.1

16.38 4.29 6.32

D

16.25

Two-way ANOVA5

P-Level P-Source×level

RI

0.92±0.01

11.9±0.4

0.92±0.01

3.3±0.1

11.4±0.5

0.92±0.02

3.2±0.1

11.8±0.5

0.93±0.01

3.4±0.1

12.1±0.2

0.93±0.02

3.1±0.1

11.7±0.6

0.93±0.01

0.445

0.614

0.961

0.474

0.401

0.694

0.532

0.215

0.713

0.494

0.843

0.821

SC

11.2±0.4

Values are presented as the mean ± SE (n=3) of one determination per fish, 5 fish per

AC

1

CE

P-Source

PT E

P value

0.94±0.01

NU

CuONano

12.2±0.4

MA

CuMet

PT

Experimental diets

aquarium and three aquariums per treatment. 2

HSI: hepatosomatic index.

3

VSI: viscerosomatic index.

4

CF: condition factor.

22

ACCEPTED MANUSCRIPT Table 4 The whole-body compositions (% live weight) of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1. Crude protein (%)

Crude lipid (%)

Ash (%)

Control

0.54

77.6±0.3

13.4±0.4

4.6±0.1

3.96±0.03

CuSO4

4.36

78.0±0.2

13.1±0.1

4.4±0.0

6.34

77.6±0.3

13.8±0.4

4.7±0.1

3.16±0.14

16.15

78.0±0.4

13.2±0.3

4.54

77.9±0.2

13.7±0.2

6.42

78.2±0.4

16.38

78.0±0.4

4.29

77.9±0.4

6.32

77.8±0.3

16.25

77.9±0.2

2.85±0.05

4.8±0.1

2.97±0.04

13.3±0.2

4.6±0.1

2.98±0.16

13.3±0.2

4.7±0.2

2.81±0.04

13.6±0.4

4.6±0.1

2.88±0.08

13.7±0.2

4.6±0.1

2.85±0.10

13.3±0.2

4.6±0.1

3.00±0.10

0.948

0.617

0.757

0.262

0.823

0.696

0.408

0.850

0.903

0.317

0.970

0.310

0.787

0.370

0.485

0.126

P value Two-way ANOVA2

P-Level Source×level

SC

Values are presented as the mean ± SE (n=3) of three determinations of pooled samples of 3

AC

1

CE

P-Source

RI

4.7±0.1

D

CuONano

2.85±0.05

MA

CuMet

PT

Moisture (%)

NU

Analyzed Cu (mg/kg)

PT E

Experimental diets

fish per aquarium and three aquariums per treatment.

23

ACCEPTED MANUSCRIPT Table 5 Whole-body Cu concentrations, liver Cu concentrations, vertebra Cu concentrations, dietary Cu retention and Cu apparent digestibility (AD) of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1.

0.54

2.9±0.2

CuSO4

4.36

3.5±0.1

6.34

4.4±0.1

16.15

4.9±0.1

4.54

4.5±0.1

6.42

4.6±0.1

16.38

5.4±0.1

4.29

4.6±0.1

6.32

4.8±0.2

16.25

5.5±0.1

P value

a

17.2±1.0

b

23.1±1.0

bcd

28.6±1.3

b

23.1±0.9

b

24.7±0.7

cd

30.0±0.9

a

c

6.5±0.4

de

7.4±0.3

b

b

bc

8.9±0.3

a

49.1±0.6

b

30.8±1.0

bc cd

9.1±0.3

bc

33.7±1.0

bcd d

6.8±0.3

NU

18.5±0.7

31.7±1.0

e

8.6±0.3

MA

b

22.6±0.7

cd

7.3±0.3

cde

7.7±0.4

e

9.2±0.4

cd

27.4±0.5

20.8±0.5

b

33.0±0.8

a

47.6±1.3

c d e

c

29.4±1.1

cd

22.4±0.6

b

33.9±1.6

9.4±0.7

a

49.1±1.1

d

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

0.032

0.121

0.184

<0.001

<0.001

Values are presented as the mean ± SE (n=3) of three determinations of pooled samples of 3 fish per aquarium for whole-body, 5 fish per aquarium for liver and vertebra, and three aquariums per treatment.

2

Values are expressed on a dry matter basis.

3

Retention (%) = (final carcass metal content − initial carcass metal content/total metal fed ×100%. 24

e

c

Means in the same column with different superscript letters are significantly different (P<0.05). 1

a

13.5±0.5

21.2±0.6

7.2±0.4

31.2±1.2

d

b

cd

26.4±1.2

143.6±3.4

AD (%)

<0.001

AC

P-Level

4.5±0. 5

Retention3 (%)

<0.001

CE

P-Source

b

24.6±0.9

bc d

a

3.2±0.2

c

b

Two-way ANOVA

P-Source×level

a

D

CuONano

8.6±0.5

PT E

CuMet

a

Vertebra Cu concentration2 (mg/kg)

PT

Control

Liver Cu concentration2 (mg/kg)

RI

Whole-body Cu concentration2 (mg/kg)

SC

Analyzed Cu (mg/kg)

Experimental diets

e

ACCEPTED MANUSCRIPT Table 6 Activity of the Cu-Zn superoxide dismutase (Cu-Zn SOD), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) value in liver and serum ceruloplasm activity of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1. Analyzed Cu (mg/kg)

Cu-Zn SOD (U/mg prot.)

Control

0.54

129.8±3.7

a

5.7±0.6

CuSO4

4.36

146.4±2.5

b

7.9±0.5

6.34

167.3±1.8

c

8.4±0.4

16.15

145.7±2.1

b

4.8±0.3

4.54

168.9±2.1

c

8.2±0.6

6.42

166.5±1.4

c

8.4±0.4

16.38

149.9±3.1

b

4.29

172.8±2.6

c

6.32

163.6±3.4

c

7.8±0.5

16.25

147.5±2.1

b

bc

SC

a

P-Level

8.5±1.0

a

15.0±1.2

a

21.0±1.1

b

16.1±1.2

a

24.9±1.3

a

19.2±1.1

b

19.3±1.1

a

22.0±1.2

a

18.1±1.2

b

41.0±1.2 36.5±1.8 61.8±3.2

a b cde bc

36.5±2.6

ab

53.8±3.2

c

36.5±1.8

bc

39.5±1.8

5.8±0.4

ab

54.2±2.6

17.4±1.2

<0.001

<0.001

<0.001

<0.001

0.580

0.063

0.004

<0.001

<0.001

<0.001

0.018

<0.001

0.610

0.231

0.001

NU

c

5.7±0.5

AC

P-Source×level

CE

P-Source

b

53.1±3.0

34.9±1.5

<0.001

Two-way ANOVA

Ceruloplasmin Activity(U/L)

c

PT E

P value

MDA (nmol/mg prot.)

RI

c

D

CuONano

ab

MA

CuMet

T-AOC (U/mg prot.)

PT

Experimental diets

8.2±0.4

e

bcde bcde de bcd bcd

Means in the same column with different superscript letters are significantly different (P<0.05). 1

Values are presented as the mean ± SE (n=3) of one determination per fish, 3 fish per aquarium and three aquariums per treatment.

25

ACCEPTED MANUSCRIPT Table 7 Activity of the lysozyme, complement 3 (C3) and immunoglobulin M (IgM) content in serum of juvenile Russian sturgeon fed the experimental diets with different sources and levels of copper for 8 weeks1. Experimental diets

Analyzed Cu (mg/kg)

Lysozyme (μg/ml)

Control

0.54

13.5±1.3

CuSO4

4.36

20.7±1.3

6.34

22.2±1.7

16.15

14.5±1.7

4.54

21.2±0.7

6.42

20.7±0.8

16.38

14.6±1.0

4.29

20.8±0.5

6.32

21.6±1.5

16.25

14.3±1.3

a

0.30±0.03

ab

cd

0.14±0.01

b

0.47±0.05

bcd

d

0.13±0.01

b

0.50±0.03

cd

ab

0.12±0.01

ab

0.27±0.03

a

d

0.14±0.01

b

0.51±0.04

d

0.14±0.01

b

0.49±0.02

cd

Two-way ANOVA

P-Level P-Source×level

CE

P-Source

PT E

P value

RI

SC

NU

bcd

PT

0.09±0.01

abc

0.12±0.01

ab

0.33±0.04

abc

d

0.14±0.01

b

0.50±0.05

cd

d

0.14±0.00

b

0.49±0.03

cd

a

0.12±0.01

ab

0.36±0.01

abcd

D

CuONano

IgM (g/L)

a

MA

CuMet

C3 (g/L)

<0.001

0.001

<0.001

0.939

0.610

0.461

<0.001

0.003

<0.001

0.934

0.924

0.728

1

AC

Means in the same column with different superscript letters are significantly different (P<0.05). Values are presented as the mean ± SE (n=3) of one determination per fish, 3 fish per aquarium and 3 aquariums per treatment.

26

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 1. Regression analysis of weight gain (WG) (A), hepatic Cu-Zn SOD activity (B), whole body Cu concentration (C) on actual dietary copper concentration. The values of breakpoints (Bkpt) in the lines indicate the optimal dietary copper content for Russian sturgeon juveniles. The linear segments of the regression lines below the breakpoints were used to compare the relative bioavailability (RBV) and the RBV was computed as the ratio of the slope of CuMet or CuONano regression line to the slope of CuSO4 regression line by multiplying 100.

27

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig. 2. Effects of dietary copper sources and levels on cumulative mortality of juvenile Russian sturgeon after infection with A. hydrophila. Means sharing different superscript letters are significantly different (P<0.05).

28

ACCEPTED MANUSCRIPT Highlights: 

Relative bioavailability of copper sulfate (CuSO4), copper methionine (CuMet) and nano-copper oxide (CuONano) as a diet additive in juvenile Russian sturgeon was studied.



Cu in the forms of CuMet and CuONano has a higher apparent digestibility and retention rate than in the form of CuSO4 for Russian sturgeon. Cu in the forms of CuMet and CuONano has better influence on growth, antioxidant capacity

PT



and immune function of Russian sturgeon than in the form of CuSO4.

RI

Cu in the forms of CuMet and CuONano oxide is 1.5 – 2 times more bioavailable than in the

CE

PT E

D

MA

NU

SC

form of CuSO4 for meeting the dietary Cu requirements of Russian sturgeon.

AC



29