Effects of dietary Zn on growth performance, antioxidant responses, and sperm motility of adult blunt snout bream, Megalobrama amblycephala

    Effects of dietary Zn on growth performance, antioxidant responses, and sperm motility of adult blunt snout bream, Megalobrama amblycephala Ming Jiang, Fan Wu, Feng Huang, Hua Wen, Wei Liu, Juan Tian, Changgeng Yang, Weiming Wang PII: DOI: Reference:

S0044-8486(16)30329-5 doi: 10.1016/j.aquaculture.2016.06.025 AQUA 632200

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

14 April 2016 14 June 2016 15 June 2016

Please cite this article as: Jiang, Ming, Wu, Fan, Huang, Feng, Wen, Hua, Liu, Wei, Tian, Juan, Yang, Changgeng, Wang, Weiming, Effects of dietary Zn on growth performance, antioxidant responses, and sperm motility of adult blunt snout bream, Megalobrama amblycephala, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.06.025

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ACCEPTED MANUSCRIPT Effects of dietary Zn on growth performance, antioxidant responses, and sperm motility of adult blunt snout bream, Megalobrama amblycephala ,

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a, b

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Ming Jiang a,b , Fan Wub, Feng Huang, Hua Wenb, *, Wei Liub, Juan Tian Changgeng Yangb, Weiming Wanga

College of College of Fisheries, Huazhong Agricultural University, Freshwater

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Aquaculture Collaborative Innovative Centre of Hubei Province, Wuhan, Hubei, 430070, China b

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences,

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Wuhan, Hubei, 430223, China

These authors contributed equally to the paper.

*

Corresponding authors:

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

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Hua WEN

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Key Laboratory of Freshwater Biodiversity Conservation and Utilization of Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of

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Fishery Sciences, Wuhan, Hubei, 430223, China. Tel.: +86 027 8178 0258;

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E-mail addresses: [email protected].

ACCEPTED MANUSCRIPT Abstract A growth experiment was conducted to estimate the effects of dietary Zn on

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growth, antioxidant responses, and sperm motility of adult blunt snout bream,

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Megalobrama amblycephala. Six experimental diets were formulated, each containing

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graded levels of Zn (0, 20, 40, 80, 160, and 320 mg/kg, respectively) supplied as ZnSO4·7H2O, providing the actual dietary Zn values of 7.8, 32.7, 50.3, 87.2, 165.4, and 328.5 mg/kg diet, respectively. Each diet was assigned to three replicate groups of

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15 fish (mean initial body weight: 128.6±0.7 g) for 12 weeks. Results showed that weight gain (WG) and whole body Zn content increased linearly with increasing

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dietary Zn levels, then remained nearly unchanged when Zn levels reached the 50.3 and 87.2 mg/kg diets, respectively. Fish fed with the control diet had a significantly higher feed conversion rate than those fed with Zn supplemented diets (P<0.05).

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Dietary Zn had no significant effects on crude protein of the fish body (P>0.05), but

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had a significant effect on moisture and lipid and ash content (P<0.05). Dietary Zn had significant effects on hepatic malonaldehyde content, superoxide dismutase,

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catalase, total antioxidant capacity, and glutathione peroxidase activity, but had no effect on serum glucose, high-density lipoprotein cholesterol, or total cholesterol content. Serum alkaline phosphatase activity increased significantly (P<0.05),

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whereas triglyceride content decreased significantly with the increase in dietary Zn levels (P>0.05). Testis histological sections showed few leydig cells in the control group. Sperm motility parameters were also affected by dietary Zn. A broken-line regression analysis showed that the optimum dietary Zn requirement of blunt snout bream (approx.128 g) was 52.1 mg/kg for maximum WG and 86.2 mg/kg for maximum whole body Zn content. Insufficient Zn inhibits the growth and testicular development of adult blunt snout bream. However, superfluous Zn (328.5 mg/kg) in the diet decreases the antioxidant function and sperm motility. Keywords: zinc requirement; blunt snout bream; growth performance; sperm motility; antioxidant responses

ACCEPTED MANUSCRIPT Introduction Aquaculture feeds can play a major role in determining the quality and potential environmental impact of finfish, particularly in intensive farming operations (Albert

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and Forster, 2003). The higher the intensity and scale of production, the greater the required nutrient inputs and consequent risk of negative environmental impacts

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emerging through water use and effluent discharge from the aquaculture facility (Raczyńska et al., 2012). Therefore, approaches should be taken for minimizing or reducing the potential negative environmental impacts of farm effluents related to

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feed (Jegatheesan et al., 2011). In general, dissolved inorganic heavy metals have major impacts on the environment. The discharge of high levels of metals into aquatic

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ecosystems might result in selective elimination of the most sensitive life stages of vulnerable fish species (Bervoets et al., 2005). Among the heavy metals, zinc (Zn) is

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an essential trace element that functions as a cofactor in several enzyme systems

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(NRC, 2011). It is required for normal growth, development, and function in animal species (Watanabe et al., 1997, NRC, 2011). However, Zn is one of the most

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dangerous trace metals for aquatic animals (Kuz Mina et al., 2011). Therefore, it is important to evaluate accurate dietary Zn requirements of fish species to decrease the inputs of Zn into the aquatic environment (Buentello et al., 2009, Maage and

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Julshamn, 1993).

According to a review of the research (Prabhu et al., 2014), the Zn requirements for fish species (13 freshwater and 12 sea water) ranged from 15 to 240 mg/kg of the diet. For example, juvenile yellow catfish (Pelteobagrus fulvidraco) require 17.12–20.86 mg/kg (Luo et al., 2011), channel catfish (Ictalurus punctatus) need 20 mg/kg (Gatlin III and Wilson, 1983), Nile tilapia (Oreochromis niloticus) require 37.2–52.1 mg/kg (Huang et al., 2015), and grass carp (Ctenopharyngodon idella) need 55.1 mg/kg . Blunt snout bream (Megalobrama amblycephala), commonly known as Wuchang bream, have been recognized as a main aquaculture species in the Chinese freshwater polyculture system because of its excellent flesh quality, rapid growth performance, high larval survival rate, high disease resistance, and high economic

ACCEPTED MANUSCRIPT value (Zhou et al., 2008). In 2014, Blunt snout bream production reached about 0.74 million ton which ranks seventh in total freshwater fish production in China (Bureau of Fisheries of the Ministry of Agriculture, 2015). Thus, the development of a

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cost-effective artificial feed to promote optimum growth and diet utilization is utmost importance for production of this fish species. In the last decade, three studies have

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reported a wide range in recommended dietary Zn for blunt snout bream at 20, 32.6 and 184.8 mg/kg, respectively (Zhu and Yang., 1998; Jiang et al., 2015; Liu et al., 2014). The differences in recommended amounts have created confusion in

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formulation of a diet for this fish. One possible explanation is there are large differences in dietary Zn requirements among the different life stage for this fish

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species. Therefore, the purpose of this experiment was to evaluate the effects of dietary Zn on growth performance of adult blunt snout bream fed different Zn diets

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for 12 weeks. Meanwhile，Zn supplementation was found to significantly increase

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semen volume, sperm motility, and the percentage of normal sperm morphology(Zhao et al., 2016). Consequently, effects of dietary Zn on sperm motility of adult blunt

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snout bream were also detected.

Materials and methods

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Experimental diet and diet preparation The basal diet was formulated to contain approximately 30.8% crude protein and 7.5% crude lipids (Table 1). Six experimental diets were prepared by supplementing the basal diet with ZnSO4·7H2O at levels of 0, 20, 40, 80, 160, and 320 mg/kg Zn at the expense of cellulose. For the preparation of the experimental diets, all dry ingredients were finely ground through a 0.3 mm size sieve, weighed accurately (~0.1 g) and mixed thoroughly using a groove-type mixer (CH-50, Changzhou Golden Ball Drying Equipment Co., Ltd., China). All ingredients were thoroughly mixed with pre-weighed oil. Next, the ZnSO4·7H2O dissolved solution was added and mixed thoroughly. The mash was then pressed through a meat grinder (TY-432, Shang Hai Tai Yi Machinery, China) and dried to a moisture content of about 10% using

ACCEPTED MANUSCRIPT electrical fans at room temperature. Then, it was broken into small pieces, and sieved to obtain approximate sizes (~2.0 mm in diameter). The dry pellets were placed in plastic bags and stored at −20 °C until time of feeding.

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Experimental fish and feeding management

The experiment was conducted in an indoor recirculating aquarium system (RAS)

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at the Yangtze River Fisheries Research Institute (Wuhan, China). Fish were obtained from the Yaowan experimental base of the Yangtze River Fisheries Research Institute, CAFS (Jingzhou, Heibei), and were maintained in nine aquaria (500 L each) at the

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experimental base for a 4-week acclimatization. During the acclimatization period, fish were fed the basal diet to facilitate adjustment to the experimental diet and

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experimental conditions.

The fish were fasted for 24 h prior to the experiments. Groups of 15

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uniform-sized fish (mean initial weight: 128.6±0.7 g) were randomly distributed in 18

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aquaria (500 L each). The aquaria were connected and running in an independent RAS. Each experimental diet was assigned to triplicate aquaria and fish were hand-fed to

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apparent satiation three times daily (08:30, 12:30, and 16:40) for 12 weeks. Care was taken to ensure that uneaten food remained in the aquarium after feeding. The amount of feed consumed by the fish in each aquarium was recorded once a week.

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Experimental aquaria were provided with continuous flow of circulating water (about 500 L/h) and continuous aeration to maintain dissolved oxygen levels above 5 mg/L and approximately 30% of the water in each tank was replenished daily at 08:00. The exchanged water was from urban tap water purified by quartz sand and activated carbon. During the experimental period, samples of water to test the Zn concentration in RAS were collected 10 min before and after feeding once a week; temperature ranged from 25 to 33 °C, dissolved oxygen ≥ 5.0 mg/l, pH 7.2±0.20, and total ammonia–nitrogen 0.050±0.011 mg/l. The Zn concentration in rearing water ranged from 1.1 to 5.5 μg Zn/L. The experiment was conducted at ambient temperature (23~34 °C) and subjected to a natural photoperiod. Sample collection and analysis At the end of the feeding trial, all fish were fasted for 24 h prior to final sampling.

ACCEPTED MANUSCRIPT All of the fish were anaesthetized with MS-222 (tricaine methane sulfonate). Then the total number and total body weight of fish from each aquarium were measured. Three fish from each replicate aquarium were randomly collected and stored at −20 °C for

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final whole body analysis. Another three fish per aquarium were randomly selected, and blood samples were obtained by puncture of the caudal vein using a 2-ml syringe.

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Samples were allowed to clot at 4 °C for 4 h and then centrifuged (960×g, 4 °C, 15 min) to obtain serum. After serum samples were collected, fish were dissected on ice to obtain testis samples. Parts of the testis were quickly fixed in 5% paraformaldehyde,

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dehydrated in a graded ethanol series, and embedded in paraffin. Section series of 4 μm were stained with haematoxylin and eosin (H&E). Tissue sections were scanned at

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20x with a light microscope and 10 fields were selected. Three other male fish from each barrel with strong physiques and plump gonads

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were injected with a 0.1 mL luteinizing hormone-releasing hormone A3 solution (10

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μg/mL) at the base of the pectoral fin. After 4 hours, fish were anesthetized with MS-222, and semen was collected by massaging the abdominal parts of the body.

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Sperm were collected directly into Eppendorf tubes. The samples were transported on ice (2-4 °C) to the fish sperm physiology laboratory, Yangtze River Fisheries Research Institute (Wuhan, China). Then, 50 μL of water was placed on a slide, and moved to

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the horizon of an optical microscope. A small amount of sperm was placed into the water (the number of sperm in the field of the microscope was less than 500). Sperm motility was documented for 10 s after activation using a JVC TK –U890EG (Yokohama, Japan) digital camera integrated with a Leica DM 2500 microscope (Wetzlar,

Germany).

Sperm

motility

parameters

were

estimated

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computer-assisted sperm analysis (CASA) systems (FSQAS-2000, Wuhan Qianping image technology Co., Ltd, China). The following sperm motility parameters were analyzed: sperm motility, the percentage of normal sperm morphology, sperm fast moving average curvilinear velocity (VCL), average straight linear velocity (VSL), velocity average path (VAP), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), wobble (WOB), straightness (STR), and mitral annular displacement (MAD).

ACCEPTED MANUSCRIPT For proximate analysis of experimental diets, protein content was determined by measuring nitrogen (N×6.25) levels using the Kjeldahl systematic method following acid digestion with an auto Kjeldahl System (kjelflex K-360, BUCHI Labortechnik

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AG, Flawil, Switzerland). Lipid content was measured by ether extraction using a Soxhlet. Diet moisture levels were measured by drying in an oven at 105 °C for 24 h

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and ash levels were measured following combustion at 550 °C (AOAC, 1995). The Zn content of the diets and whole body were analyzed using flame atomic absorption spectrometry (AA-6300C, Shimadzu Corporation, Kyoto, Japan) (Gatlin III and

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Phillips, 1989). The dietary Zn concentrations in the experimental diets were 7.8 (basal diet), 32.7, 50.3, 87.2, 165.4, and 328.5 mg/kg.

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Activity of liver catalase, total superoxide dismutase, and glutathione peroxidase, along with liver malondialdehyde content and total antioxidant capacity were

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analyzed by colorimetry using diagnostic reagent kits from the Nanjing Jiancheng

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Bioengineering Institute (China). Activity of alkaline phosphatase(ALP), along with the serum glucose(GLU), triglyceride(TG), total cholesterol (TCHO) and high-density

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lipoprotein cholesterol(HDL-C )content were analyzed using an automatic biochemical analyzer (Sysmex-800, Sysmex Corporation, Kobe, Japan) and commercial diagnostic reagent kits (Sysmex Wuxi Co., Lt., Wuxi, China).

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Statistical analysis

The results were presented as mean ± SD of three replicates. All data were subjected to one-way analysis of variance (ANOVA). When overall differences were significant (P<0.05), Tukey's test was used to compare the mean values among the treatments. Statistical analyses were performed using SPSS 18.0 for Windows (SPSS, Chicago, IL, USA). The dietary Zn requirement of blunt snout bream was estimated by broken-line regression analysis (Robbins et al., 2006) based on weight gain. Results Growth performance The percent of weight gain (WG, %) increased with increasing dietary Zn levels from 7.8 to 50.3 mg/kg, and then remained nearly unchanged with further increases in dietary Zn (Table 2). The relationship between WG and dietary Zn levels can be

ACCEPTED MANUSCRIPT expressed by a broken-line regression model, which indicated that the optimal dietary Zn content for maximum growth performance of blunt snout bream was 52.1 mg/kg (Fig. 1). The feed conversion ratio (FCR) in the 7.8 mg/kg groups was significantly

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higher than that in other groups (P<0.05). Fish fed the un-supplemented Zn diet had the lowest viscerosomatic index (VSI), significantly lower than that of fish fed

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Zn-supplemented diets (except for the 328.5 mg/kg group) (P<0.05). Condition factor (CF) and Hepatosomatic index (HSI) showed no significant differences among the

Proximate composition of fish body

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treatments. All groups had a 100% survival rate.

Dietary Zn had no significant effects on crude protein of the fish body (P>0.05),

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but had a significant effect on moisture, as well as lipid and ash content (P<0.05) (Table 3). Fish fed the un-supplemented Zn diet had the highest moisture,

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significantly higher than that of fish fed Zn-supplemented diets (P<0.05). Lipid

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content of fish fed the 87.2 mg/kg Zn diet was significantly higher than that of fish fed the 7.8 or 328.5 mg/kg Zn diets (P<0.05). Ash content of fish fed

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Zn-supplemented diets was significantly higher than that of fish fed the un-supplemented Zn diets (P<0.05). Zn content in the fish body increased with increasing dietary Zn levels from 7.8 to 87.5 mg/kg, and then saturated with further

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increases in dietary Zn. The relationship between whole body Zn and dietary Zn levels can also be expressed by a broken-line regression model, which indicated that the optimal dietary Zn content for maximum body Zn accumulation of blunt snout bream was 86.2 mg/kg (Fig. 2). Serum biochemical indices Dietary Zn had no significant effects on serum glucose (GLU), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TCHO) content (P>0.05), but Zn did significantly affect alkaline phosphatase (ALP) activity and triglyceride (TG) content (P<0.05) (Table 4). ALP activity of fish fed the 7.8 and 32.7 mg/kg Zn diet was significantly lower than that of fish fed the other diets (P<0.05), though there were no significant differences among groups (50.3–328.5 mg/kg Zn). TG content of fish fed the 7.8, or 32.7 mg/kg Zn diet was significantly higher than that of fish fed

ACCEPTED MANUSCRIPT the 87.2, 165.4, or 328.5 mg/kg Zn diets (P<0.05). Liver antioxidant status Dietary Zn levels had significant effects on the liver antioxidant status of blunt

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snout bream (Table 5). Liver malondialdehyde (MDA) content of fish fed the 7.8 mg/kg Zn diet was significantly higher than that of fish fed the other diets (P<0.05).

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Liver total antioxidant capacity (T-AOC), activity of total superoxide dismutase (T-SOD), and glutathione peroxidase showed a similar trend of an initial increase followed by a decrease; they were all highest in the 87.2 mg/kg group. Fish fed the

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7.8 mg/kg Zn diet had the lowest activity of catalase (CAT), while there were no significance differences among the fish fed Zn-supplemented diets.

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Testis histology and sperm motility

As shown in the histotomy of Figure 3, mature spermatozoa were visible in each

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group. The 7.8 and 32.7 mg/kg groups were lightly stained with hematoxylin, while

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the other four groups were darkly stained. Mesenchyme were relatively shriveled in the 7.8 mg/kg Zn-treated group; however, the opposite phenomenon was exhibited in

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the 50.3, 87.2, 165.4, and 328.5 mg/kg groups. The mesenchyme of these latter groups were plump, the nucleolus of the leydig cells were clear, and the nuclei were big and round.

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The effects of dietary Zn on sperm motility of blunt snout bream were shown in Table 6. The data for Zn-treated groups could be summarized as follows: the sperm viability and normal morphological rate of the control group were lowest, as well as the percentage of fast-moving sperm, with increases in Zn concentration. The stated parameters gradually rose and then subsequently declined. In terms of sperm viability, the 50.3, 87.2, 165.4, and 328.5 mg/kg groups were distinctly higher than the controls (P<0.05). Furthermore, the normal morphological rate and fast-moving sperm of groups 50.3, 87.2, and 165.4 mg/kg were distinctly higher than that of other groups. All data were statistically significant (P<0.05). Regarding sperm motility, it was shown that Zn did not influence the action of WOB and STR. However, VCL, VSL, VAP, ALH, BCF, LIN, and MAD were seriously affected by Zn (P<0.05). With the increase of dietary Zn, sperm VCL, VSL,

ACCEPTED MANUSCRIPT VAP, ALH, LIN, and MAD significantly increased and then subsequently decreased (P<0.05). The increase of Zn concentration induced a reduction of BCF, with the 50.3, 87.2, 165.4, and 328.5 mg/kg groups obviously lower than that of other groups

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(P<0.05).

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Discussion

The Zn requirement of blunt snout bream was determined to be a 52.1 mg/kg of diet based on WG in the present study. This value is different from values estimated in

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previous studies. Zhu and Yang (1998) examined several mineral requirements of blunt snout bream using an orthogonality experiment and suggested that the minimum

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Zn requirement to promote normal growth and prevent Zn deficiency was 20 mg/kg. Jiang et al. (2015) estimated the Zn requirement of blunt snout bream (initial body

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weight 3.6 g) to be 32.6 mg/kg for maximum growth and 47.6 mg/kg for maximum

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fish Zn concentration. Meanwhile, Liu et al. (2014) reported dietary Zn for blunt snout bream (initial body weight 50 g) to be 184.8 mg/kg (based on growth

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performance). One reason for the discrepancy in the requirements among the above studies is likely to be the different ingredients used. Liu et al. (2014) used 20.5% fishmeal in the basal diet, which may have led to high hydroxyapatite content in

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experimental diets. Hydroxyapatite has been shown to inhibit gastrointestinal Zn absorption and hence reduce the availability of dietary Zn in fish (Denstadli et al., 2006, Huang et al., 2010, Porn-Ngam, 1995). In a review paper (Prabhu et al., 2014), the authors concluded that diet type was found to have the most significant impact on the minimum dietary inclusion level of Zn. Meta-analytic estimates from studies using practical diets were about threefolds higher than those derived from studies using semi-purified diets, based on both WG and vertebral Zn as the response criteria. In this study, the basal diet composition and rearing system were the same as described in Jiang et al. (2015), but the dietary Zn requirement of blunt snout bream was found to be 52.1 mg/kg (based on growth performance) in comparison to the 32.6 mg/kg reported by Jiang et al. (2015). Differences in Zn requirements by life stage were found to explain the variations. It is clear that the dietary nutrient requirements of

ACCEPTED MANUSCRIPT broodstock are different from those of rapidly-growing juvenile animals (Izquierdo et al., 2001). In this study, fish were grown to sexual maturity, which may lead to excess dietary Zn to maintain gonadal development, especially for male animals and semen

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production (Altaher et al., 2015).

It has been reported that Zn concentrations in the whole fish body and specific

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tissues are a better indicator of Zn nutritional status and are positively correlated with dietary Zn content (Liang et al., 2012, Luo et al., 2011, Huang et al., 2015). Nevertheless, the relationship varies by species. Luo et al. (2011) reported that the Zn

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content of juvenile yellow catfish showed an increasing trend with increased dietary Zn supplementation. The trend stabilized at high dietary concentrations of Zn. Huang

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et al. (2015) also showed that a trend of an initial increase and then a decrease in the Zn content of liver, muscle, bones, and scales of tilapia. However, Eid and Ghonim

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(1994) observed that the Zn content of the fish body, bones, and scales of tilapia

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increased with increases of dietary Zn, without reaching a constant level. In the current study, Zn concentrations in the fish body of blunt snout bream showed an

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increasing trend and then stabilization at high dietary concentrations of Zn. This result was similar to the result of yellow catfish (Luo et al., 2011). The influence of dietary Zn on fish body composition varies by species. Ogino

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and Yang (1978) and Satoh et al. (1987) suggested that a deficiency of dietary Zn significantly increased moisture content, decreased crude protein and ash content of the fish body. Tan and Mai (2001) indicated that dietary Zn had no significant effects on crude protein, lipid, and ash content of juvenile abalone, Haliotis discus hannai Ino. In this study, dietary Zn had no significant effects on crude protein content of the fish body, but had a significant effect on moisture, as well as lipid and ash content. Zn is a pivotal component of the antioxidant defense network that protects membranes from oxidation (Zago and Oteiza 2001). Zn deficiency induces oxidative damage to cell components in animal models and alterations in antioxidant enzymes and substances (Powell 2000). The beneficial effect of Zn on human and animal health (as well as fish) has been well documented. Feng et al. (2011) showed that the activity of SOD, CAT, and GSH-Px in serum, intestines, the liver, and muscle tissue

ACCEPTED MANUSCRIPT of juvenile Jian carp increased with increasing Zn levels. Hidalgo et al. (2002) indicated that a shortage of dietary Zn decreased the SOD and CAT activity in rainbow trout liver. Huang et al. (2015) demonstrated that significant increases of

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SOD and GSH-Px activity were observed in the serum of tilapia with increasing dietary Zn levels. In the present study, significant increases of T-AOC, CAT, SOD,

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and GSH-Px activity were also observed in fish livers with increasing dietary Zn levels (up to the required level), which is consistent with the results of the above-mentioned studies. However, the SOD and GSH-Px activity, and T-AOC

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decreased when fish were fed a higher Zn diet. The trends of these results were consistent with that of WG, indicating dietary Zn toxicity for blunt snout bream.

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MDA results from lipid peroxidation of polyunsaturated fatty acids and it is a marker for oxidative stress (Devasena et al., 2001). MDA can also reflect the

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antioxidant status of fish. Jiang et al. (2015) reported that dietary Zn supplementation

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significantly decreased MDA levels in the liver of juvenile blunt snout bream. In the present study, liver MDA content of fish decreased with increasing dietary Zn

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supplementation. This indicated that dietary Zn supplementation reduced hepatic oxidative stress of blunt snout bream. Lower MDA levels in the liver were also reported by Onderci et al. (2003) and Kucukbay et al. (2006) in fish receiving optimal

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Zn supplementation. The mechanism by which Zn exerts its antioxidant action is not well defined. However, it has been suggested that Zn can protect membranes from iron-initiated lipid oxidation by occupying negatively charged sites with potential iron binding capacity. The synergistic actions of Zn with lipid and water-soluble antioxidants prevented lipid oxidation (Zago and Oteiza, 2001). In addition, the depletion of Zn may enhance DNA damage by impairing DNA repair mechanisms (Valko et al., 2016). Alkaline phosphatase is a Zn-dependent metalloenzyme. Serum ALP activity was also responsive to dietary Zn levels and is often used as an indicator of animal Zn status (Yousef et al., 2002, Shinde et al., 2013), as well as that of fish. In the present study, Serum ALP activity was significantly improved by Zn supplementation. Similar results were found in tilapia (Wu et al., 2015; Sa et al., 2004), Atlantic salmon (Maage

ACCEPTED MANUSCRIPT and Julshamm, 1993), and grass carp (Liang et al., 2012). Numerous studies have evaluated the effects of Zn supplementation on serum lipids in humans and have demonstrated varying results. In general, Zn supplementation has favourable effects

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on serum lipid parameters (Ranasinghe et al., 2015). In this study, Zn supplementation significantly reduced triglycerides, but had no significant effects on serum

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high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TCHO) content. Jiang et al. (2015) showed that Zn supplementation significantly decreased HDL-C and TG levels in the serum of juvenile blunt snout bream. However, there is little

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information on the effects of Zn supplementation on serum lipids in fish. As shown in testicular histology, leydig cells were barely visible in the 7.8 mg/kg

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group and interstitial tissue was immaturely developed. For the 32.7 mg/kg group, some leydig cells were detected, and there was a positive correlation between the

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cells and dietary Zn, with increase of the dietary Zn. Mesenchymal cells are a type of

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endocrine cell located in the testis, the primary function of which is to secrete androgen, thus stimulating spermatogenesis and maturity (Haider., 2004). Research

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on Pseudosciaena crocea has proven a decisive relationship between the leydig cells in the spermary and sexual maturity. The leydig cells in the spermary are much more developed in sexually-mature fish than in immature fish (Fang et al., 2002). The

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results of this study imply that dietary Zn enhance spermary development by promoting the development of leydig cells. Zn affects the development of the male reproductive system as an indispensable microelement in spermatogenesis and is involved in production, maturity, activation, and capacitation of sperm (David et al., 2002). In this study, sperm motility parameters, such as viability, normal morphological rate, and the percentage of fast-moving sperm, in the control group were all lower than that of the Zn-supplemented groups, which indicated that Zn inactivated sperm motility in the blunt snout bream. As an indispensable microelement for fish, Zn can protect spermic plasmalemma against lipid peroxidation with powerful anti-oxidation. In addition, it is also involved in maintenance of the stability of the cytomembrane and maintaining the viability of sperm. Moreover, Zn plays a crucial role in catalyzing, activating, and

ACCEPTED MANUSCRIPT synthesizing some important enzymes associated with spermatogenesis, such as adenylate cyclase, alkaline phosphatase, and acid phosphatase; thus, spermic activity was directly affected by Zn (Brandão-Neto 1995). Parameters such as VSL, VCL, and

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VAP reflect relative displacement or trajectory distance in unit time, representing spermic motility and acting as vital sperm motility parameters in the research of carp

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(Ravinder et al., 1997), Clarias lazera (Rurangwa et al., 2001), sturgeon (Liu et al., 2007), and other species of fish(Rurangwa et al., 2004). BCF is defined as the frequency of crossing the average path of the sperm. Whipping frequency distinctly

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increased in response to decreased sperm motility. Results in this study showed that BCF in control groups was dramatically higher than that of Zn-treated groups, which

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implied that 7.8 mg/kg Zn is insufficient for maintaining normal spermic activity, leading to decreased motility. Motion mode is reflected by ALH, LIN, and MAD,

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which revealed positive correlation with dietary Zn. The data suggested that

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appropriate Zn contributes to sperm motility. However, when Zn content increases further to 328.5 mg/kg, sperm parameters such as viability, normal morphological rate,

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the percentage of fast-moving sperm, VCL, VSL, VAP, ALH, and MAD showed a downward trend rather than a continuing rise, which indicated that excess Zn has negative effects on sperm quality.

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Conclusion

In conclusion, the present study has demonstrated that the optimal dietary Zn requirement for adult blunt snout bream (approx.128 g) is 52.1 mg/kg based on WG. The liver antioxidant indices and sperm motility of blunt snout bream were also significantly influenced by dietary Zn levels. Acknowledgments This work was supported by grants from China Agriculture Research System (No.CARS-46), and the Special Basic Research Fund for Central Public Research Institutes (No.2013JBFM22).

ACCEPTED MANUSCRIPT References

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Altaher, Y.M., Abdrabo, A.A., 2015. Levels of zinc and copper in seminal plasma of

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Sudanese infertile males. British Journal of Medicine & Medical Research 5, 533-538.

Analysis, 16th edn. AOAC, Arlington, VA.

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AOAC (Association of Official Analytical Chemists), 1995. Official Methods of

Bervoets, L., Knaepkens, G., Eens, M., Blust, R., 2005. Fish community responses to

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metal pollution. Environmental Pollution 138, 338-349.

Buentello, J.A., Goff, J.B., Gatlin, D.M., 2009. Dietary zinc requirement of hybrid

MA

striped bass, Morone chrysops×Morone saxatilis, and bioavailability of two chemically different zinc compounds. Journal of the World Aquaculture Society 40, 687-694.

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Bureau of Fisheries of the Ministry of Agriculture, 2015. Fisheries Statistical

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Yearbook of China, China Agricultural Press, Beijing, China. David, T., Mayer, G. D., Walsh, P. J., Christer, H., 2002. Sexual maturation and

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reproductive zinc physiology in the female squirrelfish. Journal of Experimental Biology 205, 3367-76.

Denstadli, V., Skrede, A., Krogdahl, Å., Sahlstrøm, S., Storebakken, T., 2006. Feed

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intake, growth, feed conversion, digestibility, enzyme activities and intestinal structure in Atlantic salmon (Salmo salar L.) fed graded levels of phytic acid. Aquaculture 256, 365-376. Devasena, T., Lalitha, S., Padma, K., 2001. Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis. Clinica Chimica Acta 308, 155-161. Do Carmo E Sá, M.V., Pezzato, L.E., Ferreira Lima, M.M.B., de Magalhães Padilha, P., 2004. Optimum zinc supplementation level in Nile tilapia Oreochromis niloticus juveniles diets. Aquaculture 238, 385-401. Eid, A.E., Ghonim, S.I., 1994. Dietary zinc requirement of fingerling Oreochromis niloticus. Aquaculture 119, 259-264.

ACCEPTED MANUSCRIPT Fang, Y.G., Ong Y.Z., Zhou, J., Xie, F.Q., Liu J.F., 2002. Mechanism of gonadal precocity in cultured Pseudosciaena crocea: a study of microstructure and submicrostructure of leydig cell and sertoli cell in testis. Journal of Oceanography in

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Taiwan Strait 23, 275-279.

Feng, L., Tan, L.N., Liu, Y., Jiang, J., Jiang, W.D., Hu, K., Li, S.H., Zhou, X.Q., 2011.

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Influence of dietary zinc on lipid peroxidation, protein oxidation and antioxidant defence of juvenile Jian carp (Cyprinus carpio var. Jian). Aquaculture Nutrition 17, e875-e882.

NU

Gatlin III D.M., Wilson, R.P., 1983. Dietary zinc requirement of fingerling channel catfish. The Journal of nutrition 113, 630-635.

MA

Gatlin III, D.M., Phillips, H.F., 1989. Dietary calcium, phytate and zinc interactions in channel catfish. Aquaculture 79, 259-266.

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Haider, S.G., 2004. Cell biology of leydig cells in the testis. International Review of

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Cytology 233,181-241.

Hidalgo, M.C., Expósito, A., Palma, J.M., de la Higuera, M., 2002. Oxidative stress

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generated by dietary Zn-deficiency: studies in rainbow trout (Oncorhynchus mykiss). The International Journal of Biochemistry & Cell Biology 34, 183-193. Huang, F., Jiang, M., Wen, H., Wu, F., Liu, W., Tian, J., Yang, C.G, 2015. Dietary zinc

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requirement of adult Nile tilapia (Oreochromis niloticus) fed semi-purified diets, and effects on tissue mineral composition and antioxidant responses. Aquaculture 439, 53-59.

Huang, S.C., Chen, S.M., Huang, C.H., 2010. Effects of dietary zinc levels on growth, serum zinc, haematological parameters and tissue trace elements of soft-shelled turtles, Pelodiscus sinensis. Aquaculture Nutrition 16, 284-289. Izquierdo, M.S., ndez-Palacios, H., Tacon, A.G.J., 2001. Effect of broodstock nutrition on reproductive performance of fish. Aquaculture 197, 25–42. Jegatheesan, V., Shu, L., Visvanathan, C., 2011. Aquaculture effluent: impacts and remedies for protecting the environment and human health. Encyclopedia of Environmental Health, 123-135. Jiang, M., Huang, F., Wen, H., Wang W M., Wu, F., Liu, W., Tian, J., Yang, C.G, 2015.

ACCEPTED MANUSCRIPT Effects of dietary zinc on growth, serum biochemical indexes and antioxidant responses of juvenile blunt snout bream, megalobrama amblycephala. Journal of Fishery Sciences of China 22, 1167-1176.

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Kucukbay, Z., Yazlak, H., Sahin, N., Tuzcu, M., Nuri Cakmak, M., Gurdogan, F., Juturu, V., Sahin, K., 2006. Zinc picolinate supplementation decreases oxidative stress

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in rainbow trout (Oncorhynchus mykiss). Aquaculture 257, 465-469. Kuz Mina, V.V., 2011. The influence of zinc and copper on the latency period for feeding and the food uptake in common carp, Cyprinus carpio L. Aquatic Toxicology

NU

102, 73-78.

Liang, J., Yang, H., Liu, Y., Tian, L., Liang, G., 2012. Dietary zinc requirement of

MA

juvenile grass carp (Ctenopharyngodon idella) based on growth and mineralization. Aquaculture Nutrition 18, 380-387.

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Liu H C, Ye Y.T, Cai C.F, Wu T, Chen K.Q, Pu Q.H., 2014. Dietary Zn requirement of

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Megalobrama amblycephala. Journal of Fisheries of China 38: 1522-1529. Liu, L., Linhart, O., Wei, Q.W., Guo, F., Zhang, J.M., Liu J.Y., 2007. Comparative

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study of activating mediums for the cryopreserved sperm of several sturgeons using CASA. Journal of Fisheries of China 31, 711-720. Luo, Z., Tan, X., Zheng, J., Chen, Q., Liu, C., 2011. Quantitative dietary zinc

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requirement of juvenile yellow catfish Pelteobagrus fulvidraco, and effects on hepatic intermediary metabolism and antioxidant responses. Aquaculture 319, 150-155. Maage, A., Julshamn, K., 1993. Assessment of zinc status in juvenile Atlantic salmon (Salmo salar) by measurement of whole body and tissue levels of zinc. Aquaculture 117, 179-191. NRC, 2011. Nutrient requirements of fish and shrimp. National academy press, Washington, D.C. Ogino, C., Yang, G., 1978. Requirement of rainbow trout for dietary zinc. Bulletin of the Japanese Society of Scientific Fisheries 44, 1015-1018. Ogino, C., 1980. Fish nutrition and feed. Taiwan: Fish world magazine, 1980: 275. Onderci, M., Sahin, N., Sahin, K., Kilic, N., 2003. Antioxidant properties of chromium and zinc. Biological Trace Element Research 92, 139-149.

ACCEPTED MANUSCRIPT Porn-Ngam, N., 1995. A study on dietary inhibitors of zinc utilization in rainbow trout (Oncorhychus mykiss). Doctoral thesis. Department of Aquatic Biosciences, Tokyo University of Fisheries, Japan.

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Powell, S.R., 2000. The antioxidant properties of zinc. Journal of Nutrition 130, 1447S-1454S.

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Prabhu, P. A. J., Schrama, J. W., & Kaushik, S.J., 2014. Mineral requirements of fish: a systematic review. Reviews in Aquaculture 6, 1-48.

Raczyńska, M., Machula, S., Choiński, A., Sobkowiak, L., 2012. Influence of the fish

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pond aquaculture effluent discharge on abiotic environmental factors of selected rivers in Northwest Poland. Acta Ecologica Sinica 32, 160-164.

MA

Ranasinghe, P., Wathurapatha, W.S., Ishara, M.H., Jayawardana, R., Galappatthy, P., Katulanda, P., Constantine, G.R., 2015. Effects of zinc supplementation on serum

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lipids: a systematic review and meta-analysis. Nutrition & Metabolism 12, 1-16.

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Ravinder, K., Nasaruddin, K., Majumdar, K. C., Shivaji, S., 1997. Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp

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following short-term storage of semen. Journal of Fish Biology 50, 1309-1328. Rurangwa, E., Kime, D.E., Ollevier, F., Nash, J.P., 2004. The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture 234, 1-28.

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Rurangwa, E., Volckaert, F. A., Huyskens, G., Kime, D. E., Ollevier, F., 2001. Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (clarias gariepinus).Theriogenology 55, 751-769. Satoh, S., Takeuchi, T., Watanabe, T., 1987. Availability to rainbow trout of zinc in white fish meal and of various zinc compounds. Nippon Suisan Gakkaishi 53, 595-599. Tacon, A.G.J., Forster, I.P., 2003. Aquafeeds and the environment: policy implications. Aquaculture 226, 181-189. Tan, B., Mai, K., 2001. Zinc methionine and zinc sulfate as sources of dietary zinc for juvenile abalone, Haliotis discus hannai Ino. Aquaculture 192, 67-84. Valko, M., Jomova, K., Rhodes, C.J., Kuča, K., Musílek, K., 2015. Redox- and

ACCEPTED MANUSCRIPT non-redox-metal-induced formation of free radicals and their role in human disease. Archives of Toxicology 90, 1-37. Watanabe, T., Kiron, V., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture

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151, 185-207.

Wu, F., Jiang, M., Wen, H., Liu, W., Tian, J., Yang, C.G., Huang, F., 2015. Dietary

niloticus). Freshwater Fisheries 45, 63-69.

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zinc requirement of genetically improved farmed tilapia (GIFT, Oreochromis

Zago, M.P., Oteiza, P.I., 2001. The antioxidant properties of zinc: interactions with

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iron and antioxidants. Free Radical Biology & Medicine 31, 266-274. Zhao, J., Dong, X., Hu, X., Long, Z., Wang, L., Liu, Q., Sun, B., Wang, Q., Wu, Q., Li,

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L., 2016. Zinc levels in seminal plasma and their correlation with male infertility: A systematic review and meta-analysis. Scientific Reports 6, 22386.

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Zhou, Z., Ren, Z., Zeng, H., Yao, B., 2008. Apparent digestibility of various feedstuffs

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for bluntnose black bream Megalobrama amblycephala Yih. Aquaculture Nutrition 14, 153-165.

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Zhu, Y.Z., Yang, G.H., 1998. Requirement of Megalobrama amblycephala, fingerlings

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for trace elements. Journal of Shanghai Fisheries University 7 (suppl.):123-129.

ACCEPTED MANUSCRIPT Figure Caption Figure 1. Broken-line analysis of weight gain (WG) in relation to dietary zinc (Zn) for blunt snout bream (Megalobrama amblycephala).

content for blunt snout bream (Megalobrama amblycephala).

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Figure 2. Broken-line analysis of the relationship between dietary Zn and whole body Zn

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Figure 3. Histologically-observed testis of blunt snout bream (×200, Sz, spermatozoa; IT, interstitial tissue; LC, leydig cell).

Testis of blunt snout bream fed a diet supplemented with 7.8 mg/kg Zn.

b.

Liver of blunt snout bream fed a diet supplemented with 32.7 mg/kg Zn.

c.

Liver of blunt snout bream fed a diet supplemented with 50.3 mg/kg Zn.

d.

Liver of blunt snout bream fed a diet supplemented with 87.2 mg/kg Zn.

e.

Liver of blunt snout bream fed a diet supplemented with 165.4 mg/kg Zn.

f.

Liver of blunt snout bream fed a diet supplemented with 328.5 mg/kg Zn.

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a.

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Figure 1

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130

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110

y=116.1-0.43(52.1-x) 100

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R2 =0.9009

When x>52.1, (52.1-x)=0

90

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weight gain (%)

120

80 0

50

100

150

200

250

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dietary Zinc level (mg/kg)

300

350

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110

90

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100

y=107.6-0.403(86.2-x) R2 =0.9305

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when x>86.2, (86.2-x)=0

70

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100

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0

150

200

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dietary Zn level (mg/kg)

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Zn content in fish body(μg/g）

120

250

300

350

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Figure 3

ACCEPTED MANUSCRIPT Table 1. Formulation and proximate analysis of the basal diet (on air dry weight basis). ingredient

content /%

proximate composition/%

casein

32

crude protein

glutin

8

crude lipid

dextrine

36

corn oil

3.5

soybean oil

3.5

choline chloride

0.25

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7.5 3.1

moisture

10.1

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ash

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vitamin premix1

1

mineral premix2

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micro-cellulose

30.8

10.75

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Note: 1. Vitamin premix (IU or mg/kg dry diet)：vitamin A 4500 IU；vitamin D 1000 IU； vitamin E 100；vitamin K3 5；thiamine 10；riboflavin 20；pyridoxine 10；cyanocobalamin 0.05；Niacin 25；vitamin C 400；calcium

Zn.

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pantothenate 100；folic acid 5；biotin 1；inositol 500. 2. Mineral premix refers to literature (Ogino 1980), except for

ACCEPTED MANUSCRIPT Table 2. Growth performance and feed utilization of adult blunt snout bream, Megalobrama

50.3

87.2

165.4

328.5

IBW

127.0±2.2a

130.3±2.3a

130.2±2.6a

127.2±5.2a

129.6±3.6a

127.2±2.9a

FBW

252.0±3.2a

266.6±2.4a

282.7±2.2b

273.0±5.4b

279.8±5.5b

274.2±3.2b

WG

98.4±0.7a

104.6±0.6b

117.1±1.0c

114.6±1.0c

115.8±1.0c

115.5±2.2c

FCR

2.16±0.06b

1.93±0.09a

1.88±0.04a

1.92±0.05a

1.95±0.06a

1.90±0.04a

CF

2.21±0.06a

2.30±0.05a

2.25±0.09a

2.28±0.05a

2.22±0.07a

2.30±0.05a

HSI

1.46±0.08a

1.56±0.08a

1.50±0.03a

1.48±0.04a

1.54±0.05a

1.48±0.04a

VSI

9.09±0.53a

10.14±0.47b

10.18±0.72b

10.43±0.24b

10.43±0.28b

9.74±0.52ab

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amblycephala, fed different Zn diets for 12 weeks.*

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*Means in the same line sharing the same superscript letter are not significantly different, as determined by Tukey's test (P > 0.05). IBW (g/fish) = initial mean weight; FBW (g/fish) = final mean

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weight; WG (percent of weight gain, %) = (FBW – IBW)/IBW × 100; FCR (Feed conversion ratio) = g fish intake / (g total final fish weight – g total initial fish weight + g dead fish); CF (condition factor, g/cm3)= (g body weight)/(cm body length)3×100; HSI (hepatosomatic index)=100×(g liver weight)/(g

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body weight); VSI (viscerosomatic index)= 100×(g viscera weight)/(g body weight).

ACCEPTED MANUSCRIPT Table 3. Proximate composition (% on wet weight basis, means ± SD, n=3) of blunt snout bream, Megalobrama amblycephala, fed different Zn diets for 12 weeks.*

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Dietary Zn levels( mg/kg) 32.7

50.3

87.2

165.4

328.5

moisture%

72.55±0.5b

70.2±1.21a

69.6±0.91a

70.01±0.72a

70.57±0.66a

70.72±0.83a

crude protein %

18.05±0.33a

18.51±0.39a

18.23±0.81a

18.2±0.73a

18.02±1.45a

18.77±0.25a

crude fat %

5.38±0.34a

6.16±0.48bc

6.06±0.34bc

6.50±0.46c

6.12±0.27bc

5.88±0.31ab

ash %

3.49±0.14a

3.74±0.07b

3.99±0.13c

4.07±0.12c

4.08±0.10c

4.16±0.16c

Whole body Zinc /(μg/g)

75.41±3.65a

85.89±2.41b

107.23±5.34d

109.28±6.8d

111.68±5.15d

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7.8

94.8±2.73c

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* Means in the same line sharing a same superscript letter are not significantly different, as determined

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by Tukey's test (P>0.05).

ACCEPTED MANUSCRIPT Table 4. Serum biochemical indices (means ± SD, n=3) of adult blunt snout bream, Megalobrama amblycephala, fed different Zn diets for 12 weeks.*

87.2

ALP

51.33±3.51a

51.00±4.00a

69.00±2.00b

GLU

6.12±0.11a

5.75±0.18a

5.69±0.32a

HDLC

5.19±0.28a

5.12±0.35a

4.88±0.17a

TCHO

6.97±0.22a

7.02±0.45a

7.12±0.18a

TG

5.14±0.41d

4.77±0.24cd

4.53±0.22bc

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50.3

165.4

328.5

71.00±1.73b

67.33±1.53b

66.67±2.08b

5.75±0.23a

5.78±0.21a

5.86±0.29a

4.85±0.43a

4.73±0.18a

4.74±0.32a

6.82±0.43a

7.18±0.33a

6.87±0.36a

4.25±0.08ab

4.29±0.29ab

3.89±0.15a

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Dietary Zn levels(mg/kg)

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* Means in the same line sharing a same superscript letter are not significantly different, as determined

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by Tukey's test (P>0.05). ALP: alkaline phosphatase, U/L; GLU: glucose, mmol/L; HDL-C: high-density lipoprotein cholesterol, mmol/L. TCHO: total cholesterol, mmol/L; TG: triglyceride,

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mmol/L.

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Table 5. Some hepatic antioxidant indices (means ± SD, n=3) of adult blunt snout bream,

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Megalobrama amblycephala, fed different Zn diets for 12 weeks.*

32.7

50.3

87.2

165.4

328.5

MDA

3.08±0.10c

2.89±0.23bc

2.78±0.09b

2.70±0.16ab

2.48±0.06a

2.51±0.05a

T-AOC

0.34±0.02a

0.33±0.03a

0.37±0.02ab

0.57±0.03d

0.41±0.03c

0.40±0.01bc

SOD

88.29±3.80a

99.10±6.94ab

108.66±9.83b

126.77±8.49c

102.30±6.11b

103.07±7.98b

CAT

27.02±1.98a

32.69±1.08b

31.55±2.10b

32.47±2.99b

33.15±1.45b

32.17±2.26b

GSH-PX

17.45±0.84a

18.10±1.22a

20.89±1.59b

22.08±0.99b

21.87±1.22b

18.40±1.45a

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Dietary Zn levels (mg/kg)

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* Means in the same line sharing a same superscript letter are not significantly different, as determined

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by Tukey's test (P>0.05). MDA: malondialdehyde, mmol/mgprot; T-AOC: total antioxidant capacity, U/mgprot; SOD: total superoxide dismutase, U/mgprot; CAT: catalase, U/mgprot; and GSH-Px:

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Table 6. Sperm motility of adult blunt snout bream, Megalobrama amblycephala, fed different Zn diets for 12 weeks.*

32.7

50.3

87.2

165.4

328.5

SV

70.16±2.89a

73.62±0.58ab

93.21±6.82d

86.26±5.11cd

93.39±5.78d

80.33±1.51bc

NS

73.72±4.27a

75.65±3.99ab

82.88±1.71c

81.74±2.85bc

83.63±5.22c

75.02±2.37ab

FMS

51.79±3.10a

61.06±2.62b

75.68±3.67d

75.33±3.38d

75.12±5.03d

68.46±1.54c

VCL

63.01±2.61a

77.44±2.45b

89.68±7.01c

92.05±5.17c

98.62±7.88c

77.92±2.84b

VSL

29.63±1.36a

35.34±1.47a

54.24±1.90bc

57.39±5.39c

52.91±4.39bc

49.39±3.06b

VAP

42.50±2.97a

49.84±5.50b

53.92±4.01b

63.50±3.17c

68.51±2.09c

55.99±5.35b

ALH

1.24±0.11a

2.42±0.06b

3.00±0.281c

3.38±0.21d

2.69±0.07b

2.52±0.17b

BCF

4.71±0.21a

4.44±0.31ab

4.28±0.15bc

4.22±0.21bc

4.19±0.21bc

3.92±0.28c

LIN

43.72±3.57a

50.51±1.93b

50.89±2.54b

51.14±3.59b

48.04±2.02ab

50.83±2.28b

WOB

64.79±3.99a

66.71±6.42a

65.31±3.38a

65.78±4.01a

66.98±1.82a

64.67±3.94a

STR

75.43±2.41a

76.12±1.45a

74.38±3.82a

77.74±2.64a

72.53±2.95a

76.06±5.51a

MAD

42.88±2.83a

44.93±3.50a

52.38±2.95bc

51.85±1.50bc

54.24±3.53c

47.36±4.47ab

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Dietary Zn levels (mg/kg)

* Means in the same line sharing a same superscript letter are not significantly different, as determined by Tukey's test (P>0.05). SV: sperm viability, %; NS: the percentage of normal sperm morphology, %; FMS: fast moving sperm, %; VCL: average curvilinear velocity, μm/s; VSL: average straight linear velocity, um/s; VAP: velocity average path, um/s; ALH: amplitude of lateral head displacement, um; BCF: beat cross frequency, /s; LIN: linearity, %; WOB: wobble, %; STR: straightness, %; MAD: mitral annular displacement, %.

ACCEPTED MANUSCRIPT Highlight 1. Dietary Zn requirement of adult blunt snout bream was 52.1 mg/kg for maximum

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WG and 86.2 mg/kg for maximum whole body Zn content. 2. Dietary Zn had great impacts on liver antioxidant status. 3. The spermary was immature with insufficient Zn, meanwhile, excess Zn has negative effects on sperm quality.