Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii: Growth, behavior and physiology

Journal Pre-proof Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii: Growth, behavior and physiology

Zonghang Zhang, Qingqing Bai, Xiuwen Xu, Haoyu Guo, Xiumei Zhang PII:

S0044-8486(19)30740-9

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734782

Reference:

AQUA 734782

To appear in:

aquaculture

Received date:

28 March 2019

Revised date:

23 October 2019

Accepted date:

25 November 2019

Please cite this article as: Z. Zhang, Q. Bai, X. Xu, et al., Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii: Growth, behavior and physiology, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734782

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© 2019 Published by Elsevier.

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Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii: growth, behavior and physiology Zonghang Zhanga, Qingqing Baia, Xiuwen Xua, Haoyu Guob, Xiumei Zhangb,c,* a

The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China,

Qingdao 266003, China

Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao

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c

Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China

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b

Corresponding author: Xiumei Zhang

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*

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National Laboratory for Marine Science and Technology, Qingdao 266237, China

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Postal address: Fisheries College, Zhejiang Ocean University, No.1, Haida South Road,

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Lincheng Changzhi Island, Zhoushan, China E-mail: [email protected]

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Tel: +86 0580 2089333

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Abstract Environmental enrichment is a promising way to enhance fish welfare in aquaculture. However, the observed effects of enrichment on fish growth, behavior and physiology vary widely among studies, and few studies have focused on the quantification of enrichment. The present study aimed to investigate whether enrichment type and level significantly affect the

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growth performance, aggressive behavior, cortisol level and brain monoaminergic activities of

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juveniles of the black rockfish Sebastes schlegelii. Juveniles were reared for eight weeks under different combinations of enrichment type and enrichment level: no environmental

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enrichment (C), low-level plant enrichment (PL), medium-level plant enrichment (PM),

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high-level plant enrichment (PH), low-level structure enrichment (SL), medium-level

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structure enrichment (SM) and high-level structure enrichment (SH). Subsequently,

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behavioral and physiological parameters were determined. In general, the growth performance, feed intake and food conversion efficiency of C, PM, PH and SL fish were

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relatively higher than those of fish from the other treatments. Low-level plant treatment produced the highest levels of aggressive behavior, cortisol and brain serotonergic system activity, whereas the medium- and high-level structure treatments yielded the lowest levels of these stress-related behavioral and physiological indicators. C fish had significantly higher stress levels than PM, PH and SL fish. No significant difference among treatments was observed in the condition factor, coefficient of weight variation, locomotor activity, growth hormone level or brain dopaminergic system activity. Taken together, these results provide the first evidence to show that the type and level of environmental enrichment have interaction effects on fish growth performance, behavioral phenotype (especially aggressive behavior) 2

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and stress-related physiological processes. Since the control fish had significantly higher stress levels than the fish exposed to medium- and high-level plant enrichment and low-level structure enrichment, we suggest that enriching approximately 50% basal area coverage with objects might be optimal for enhancing fish welfare. Moreover, a possible conceptual model is presented to interpret the effects of enrichment on fish welfare.

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Keywords: environmental enrichment; fish welfare; stress; growth; Sebastes schlegelii

1. Introduction

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With the rapid growth of aquaculture during recent decades, fish welfare has become of

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increasing scientific and public concern (Ashley, 2007; Huntingford et al., 2006). Biologists

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define fish welfare in different ways; most definitions can be categorized into feelings-based,

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function-based and nature-based welfare (Huntingford et al., 2006). However, regardless of the definition adopted, advocates of fish welfare agree that captive fish should maintain

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normal behaviors and a healthy physiological status, be able to adapt to environmental changes, and express specific biological functions. Due to the many stress factors in the aquaculture industry, such as intraspecies aggression, abnormal rearing density, machine noise and regular management, an increasing number of studies have concentrated on fish behavior and physiology from the perspective of fish welfare and have stressed the importance of decreasing fish aggressive behavior and physiological stress response rather than focusing only on maximizing the growth rate and production yield (Ashley, 2007; Johnsson et al., 2014; Näslund and Johnsson, 2016). Several actions have been proposed to enhance fish welfare (Huntingford et al., 2006). 3

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Environmental enrichment, a method to increase environmental complexity by introducing objects (e.g., plants, cobble, physical structures) into the rearing water, is believed to be one of the most valuable and promising methods (Näslund and Johnsson, 2016; Newberry, 1995). Many recent studies have verified that environmental enrichment can enhance fish growth rate (Crank et al., 2019; Kientz and Barnes, 2016; Kientz et al., 2018; Rosburg et al., 2019;

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White at al., 2019) and neural plasticity (Salvanes et al., 2013), decrease aggressive and

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anxiety-like behaviors (Barcellos et al., 2018; Batzina and Karakatsouli, 2012; Näslund et al., 2013), blunt physiological stress responses (Giacomini et al., 2016; Marcon et al., 2018;

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Rosengren et al., 2016), reduce maintenance metabolism (Millidine et al., 2006) and promote

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the survival-related behaviors of released fish (e.g., swimming agility: Ahlbeck Bergendahl et

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al., 2017; competitive ability: Berejikian et al., 2000; foraging behavior: Brown et al., 2003,

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Rodewald et al., 2011; anti-predator response: Ullah et al., 2017; behavioral flexibility: Braithwaite and Salvanes, 2005; survival rate: D'Anna et al., 2012, Roberts et al., 2014).

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However, negative or no effects of enrichment have been observed in some studies (Berejikian et al., 1999; Boerrigter et al., 2016; Brockmark et al., 2007; Brockmark et al., 2010; Crank et al., 2017; Fast et al., 2008; von Krogh et al., 2010; White et al., 2018; Wilkes et al., 2012). These large differences may not be surprising considering the wide ranges of fish species and populations, enrichment types and levels, and experimental designs across the different studies. To the best of our knowledge, scarce studies have examined the effects of enrichment type and level and their interaction on fish welfare. Identifying the types and levels of enrichment that are optimal for specific fish species is important for enrichment design. 4

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The black rockfish Sebastes schlegelii, a viviparous teleost, is widely distributed in the coastal areas of China, Korea and Japan (Xi et al., 2017a). In recent years, aquaculture of the black rockfish in China, especially in northern China, has rapidly developed, primarily as a source of food and for stock enhancement (Guo et al., 2017). However, the traditional rearing environments in aquaculture do not typically include environmental modifications, which

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may harm fish welfare considering the fish often maintain territories in the wild and express

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severe aggressiveness and cannibalism during their early life stages (Xi et al., 2017a; Xi et al., 2017b). In this study, we aimed to determine how enrichment type and level affect the growth

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performance, behavior and stress-related physiological status of juvenile black rockfish. The

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core hormone of the hypothalamic-pituitary-interrenal tissue (HPI) axis (i.e., cortisol) and

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brain serotonergic and dopaminergic system activities were used to evaluate stress levels,

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2. Materials and methods

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which are closely related to fish welfare.

We have read the policies relating to animal experiments and confirmed this study complied (ARRIVE guidelines; EU Directive 2010/63/EU for animal experiments). All procedures performed in this study were approved by the Institutional Animal Care and Use Committee of Ocean University of China. 2.1. Animals and experimental design Juveniles of the black rockfish S. schlegelii were obtained from a local commercial hatchery and acclimated to experimental conditions for three weeks in glass tanks. Eight hundred and forty fish (mean body length 4.69±0.03 cm, mean body weight 3.08±0.05 g) 5

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were randomly distributed in 21 glass tanks (60 cm × 50 cm × 50 cm) in groups of 40 (initial rearing density 333 ind/m3 or 1.03±0.02 kg/m3) in seven treatments in triplicate (no environmental enrichment control, low-level enrichment with plastic plants, medium-level enrichment with plastic plants, high-level enrichment with plastic plants, low-level enrichment with physical structures, medium-level enrichment with physical structures and

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high-level enrichment with physical structures; C, PL, PM, PH, SL, SM and SH, respectively). In the enriched tanks, different types and numbers of objects (plastic plant dimensions: height

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10 cm, projected area approximately 8 cm × 9 cm = 72 cm2; physical structure dimensions:

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height approximately 10 cm, basal area 12 cm × 6 cm = 72 cm2; Fig. 1) were introduced as

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shelters (von Krogh et al., 2010; Xi et al., 2017a), whereas the control tank had no objects on

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the barren, glass bottom. The details of the treatments in the different tanks are shown in

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Table 1. The objects were chosen because their materials do not chemically interact with water and because they are compatible with the natural habitat structure and color of the fish

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(Batzina and Karakatsouli, 2012).

The experimental tanks were part of an indoor flow-through seawater system equipped with mechanical and biological filters, and each tank was provided with an air stone to ensure high dissolved oxygen. The water flow rate was 3.5 L/min, and the water depth was maintained at 40 cm. The fish were maintained under the experimental conditions for eight weeks (from 12/8/2018 to 6/10/2018), since several authors found that this rearing duration was sufficient to evaluate the growth, behavior and physiology of fish in aquaculture (Boerrigter et al., 2016; Guo et al., 2017; Kientz and Barnes, 2016; White et al., 2018; White et al., 2019). The fish were fed commercial dry pellets (moisture, ≤ 10.0%; crude protein, ≥ 6

Journal Pre-proof 48.0%; crude lipid, ≥ 9.0%; crude ash, ≤ 17.0%; crude fiber, ≤ 2.0%; total phosphorus, 1.5-3.0%; lysine, ≥ 2.5%) by hand once daily (09:00 a.m.) at a restricted feeding rate (2.5% of fish body weight per day), since this feeding strategy was verified to be proper and sufficient for the growth of juvenile rockfish (Guo et al., 2017). The feces and residual fodders were siphoned once daily before feeding. The fish were individually weighed every two weeks to

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adjust food quantity. The adjacent sides of the tanks were masked with plastic boards to

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ensure visual separation. All tank sides, plastic plants and physical structures were gently cleaned once weekly. The photoperiod followed the natural day-light cycle. Water

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physicochemical characteristics were continuously monitored: temperature, 25.9±0.18°C;

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dissolved oxygen, 6.68±0.04 mg/L (97.43±0.25% saturation); salinity, 29.73±0.16; pH,

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7.12±0.02; total ammonia-nitrogen, 0.236±0.0138 mg/L; and nitrite-nitrogen, 0.012±0.0005

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mg/L. No mortality was observed during the whole rearing period. 2.2. Behavioral observations

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During the rearing period, behaviors were recorded at weeks two, four, six and eight from the front side of each tank. For each week, two 60 min videos were filmed (10:00-11:00 and 14:00-15:00) to determine the aggressive behavior of the global tank; in total, eight videos were recorded for each tank. In addition, because the clarify of the videos did not allow us to examine the more subtle behaviors, one additional 10 min video per tank was recorded each week that targeted a randomly selected local area (at approximately 14:00-15:00) to determine the locomotor activity and opercular beat rate of the randomly sampled fish. At least five fish were included in the video frame, and a total of four videos per tank were recorded. To eliminate the possible disturbance to the observed fish, only the middle 10 min of each 7

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60 min video was analyzed to determine the aggressive behavior, which was quantified by counting the number of instances of chasing, nipping and biting among the fish (Batzina and Karakatsouli, 2012). Similarly, only the middle 5 min of each 10 min video was analyzed to determine the locomotor activity and opercular beat rate. As black rockfish exhibit periods with little swimming activity, “time spent moving” (Øverli et al., 2002) may be a poor

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indicator of activity level in this fish species (von Krogh et al., 2010). Thus, in the present

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study, locomotor activity was defined as the number of turns (with one turn defined as any change in direction exceeding 90 degrees) and movements (with one movement defined as

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any advance or retreat exceeding one body length) per minute. The opercular beat rate was

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measured as the number of opercular beats per minute (Pounder et al., 2016).

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2.3. Sampling and analytical methods

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At the end of the rearing period, 12 fish from each tank were randomly sampled, euthanized with an overdose of anesthetic (tricaine methanesulfonate, MS-222, 100 mg/L),

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and measured for the body weight (precision 0.01 g) and body length (precision 0.01 cm). Subsequently, the fish were rapidly dissected, and the whole brains were extracted for determining the levels of neurotransmitters, including serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic

(5-HIAA)

and

dopamine

(DA)

and

its

metabolites

3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) (since one brain was not sufficient to determine all the neurotransmitters, we pooled brains in groups of three into individual samples, and thus, n = 4 for each tank). The whole visceral masses were removed for measuring cortisol and growth hormone (GH) levels; this was performed because the juveniles were too small to draw blood; we measured only four visceral masses, i.e., n = 4 for 8

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each tank (Guo et al., 2017). All samples were frozen in liquid nitrogen and then stored at -80°C until analysis. All sampling procedures were completed within one minute. The remaining fish in the tanks were used in another experiment. The brain and visceral mass samples were homogenized in cold PBS (9×weight, pH 7.4) and centrifuged at 3000 rpm for 20 min in a refrigerated centrifuge (4°C). The supernatants

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were collected and used for analysis. The cortisol, GH, 5-HT, 5-HIAA and DA concentrations

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were measured using commercial ELISA Assay Kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the DOPAC and HVA concentrations were measured using

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commercial ELISA Assay Kits (Shanghai Yuanye Bioengineering Institute, Shanghai, China)

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according to the manufacturer’s guidelines. These ELISA Assay Kits have been validated

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previously (Guo et al., 2017; Zhang et al., 2019). The intra-assay coefficient of variation was

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less than 10%, and strong correlations (four-parameter logistic curve regression equations) were found between the standard curves (R2 > 0.99).

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2.4. Calculations and data analysis

Specific Growth Rate (SGR (%) = 100×[ln(BWf)-ln(BWi)]/T; BWf (g) = mean final body weight; BWi (g) = mean initial body weight; T (d) = days of rearing), Weight Gain (WG = (BWf-BWi)/BWi), Coefficient of body weight Variation (CV (%) = 100×SD/BWf; SD = standard deviation) and Food Conversion Efficiency (FCE = (BWf-BWi)/FI; FI (g) = feed intake) were calculated for each whole tank, and the Condition Factor (CF (%) = 100×BWf/BLf3; BLf (cm) = mean final body length) was calculated for each fish. To further evaluate the serotonergic and dopaminergic activities, the ratios of 5-HIAA/5-HT, DOPAC/DA, HVA/DA and (DOPAC+HVA)/DA were calculated as indicators of serotonin 9

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and dopamine turnover rates. Based on our experimental design, the data from the control treatment were duplicated to yield data representative of two treatments: 0% plant enrichment and 0% structure enrichment. This approach allowed us to simulate a 2 (enrichment type: plant and structure) × 4 (enrichment level: 0%, 25%, 50% and 75%) factorial design. Two-way ANOVA was

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performed to compare the main and interaction effects of the two factors (i.e., enrichment

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type and enrichment level). One-way ANOVA was afterwards applied to determine the effects of enrichment level within a specific enrichment type group where tank was included as a

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random factor nested within treatment to account for tank effects, followed by Duncan’s post

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hoc test (Barcellos et al., 2009; Madison et al., 2015; Vindas et al., 2017; Xi et al., 2017a).

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Normality of the data was tested with Shapiro-Wilk’s test, and the homogeneity of variance in

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the data was tested using Levene’s test prior to ANOVA. Nonparametric Kruskal-Wallis test followed by Mann-Whitney U test was applied if our results failed to meet the ANOVA

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assumptions (i.e., normality and homogeneity). In general, the effect of tank was not significant in any of the analyses. To clarify the relationships among the growth, behavior and physiology of the fish, polynomial or linear fitting analysis was performed to pooled data. All statistical analyses were conducted using SPSS 17.0 for windows. Differences were considered significant at a probability level of 0.05 (P < 0.05). All values in the text and figures are presented as the means ± S.E.

3. Results 3.1. Growth performance 10

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Two-way ANOVA revealed that significant main or interaction effects of enrichment type and enrichment level on the final body weight (Type: F1, 16 = 4.293, P = 0.055; Level: F3, 16 = 4.729, P = 0.015; Interaction: F3, 16 = 6.044, P = 0.006), specific growth rate (Type: F1, 16 = 10.501, P = 0.005; Level: F3, 16 = 9.912, P = 0.001; Interaction: F3, 16 = 6.452, P = 0.005), weight gain (Type: F1, 16 = 9.729, P = 0.007; Level: F3, 16 = 9.472, P = 0.001; Interaction: F3, 16

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= 5.924, P = 0.006), feed intake (Type: F1, 16 = 1.420, P = 0.251; Level: F3, 16 = 28.811, P <

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0.001; Interaction: F3, 16 = 29.754, P < 0.001) and food conversion efficiency (Type: F1, 16 = 6.610, P = 0.021; Level: F3, 16 = 3.661, P = 0.035; Interaction: F3, 16 = 4.347, P = 0.020),

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however no significant main or interaction effect on the initial body weight (Type: F1, 16 =

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0.326, P = 0.576; Level: F3, 16 = 0.512, P = 0.680; Interaction: F3, 16 = 0.040, P = 0.989), initial

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body length (Type: F1, 16 = 0.005, P = 0.944; Level: F3, 16 = 0.975, P = 0.429; Interaction: F3, 16

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= 1.432, P = 0.270), final body length (Type: F1, 16 = 1.327, P = 0.266; Level: F3, 16 = 0.975, P = 0.429; Interaction: F3, 16 = 1.432, P = 0.270) or condition factor (Type: F1, 16 = 0.002, P =

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0.969; Level: F3, 16 = 0.034, P = 0.991; Interaction: F3, 16 = 0.047, P = 0.986) was detected. For plastic plant enrichment, one-way ANOVA showed that after eight weeks of rearing, the control fish had significantly higher final body weight than that of the low-level plant enrichment fish (i.e., PL fish) while had no significant difference compared to the fish reared in environments with medium-level plant enrichment and high-level plant enrichment (i.e., PM and PH fish), and no significant difference on the specific growth rate, weight gain and food conversion efficiency was observed among the four treatments (Fig. 2). As for physical structure enrichment, after eight weeks of rearing, the fish reared in environments with no enrichment and low-level structure enrichment (i.e., C and SL fish) had significantly higher 11

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BW, SGR, WG and FCE than the medium-level structure enrichment fish and high-level structure enrichment fish (i.e., SM and SH fish) (Fig. 2). No significant difference in coefficient of weight variation was observed among the treatments (Kruskal-Wallis test: H6 = 12.518, P = 0.051). 3.2 Behavioral phenotype

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Throughout the rearing period, the levels of aggressive behavior and opercular beat rate

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differed significantly among the treatments (Aggressive behavior: plant enrichment: Kruskal-Wallis test: H3 = 33.924, P < 0.001; structure enrichment: Kruskal-Wallis test: H3 =

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60.753, P < 0.001. Opercular beat rate: plant enrichment: Kruskal-Wallis test: H3 = 10.656, P

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= 0.014; structure enrichment: Duncan test: P < 0.05, n = 12), whereas no significant

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difference in the values of locomotor activity was observed (Kruskal-Wallis test: H6 = 2.264,

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P = 0.894). For plant enrichment, the level of aggressive behavior of C fish was significantly lower than that of PL fish, while was significantly higher than those of PM and PH fish (Fig.

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3a). As for structure enrichment, C fish had the highest while SH fish had the lowest level of aggressive behavior among the fish from the four treatments (Fig. 3a). Furthermore, C and PL fish had significantly higher opercular beat rate than did the fish in the other treatments (i.e., PM, PH, SL, SM and SH fish) (Fig. 3b). 3.3. Physiological status Two-way ANOVA revealed that significant main and interaction effects of enrichment type and enrichment level on the cortisol level (Type: F1, 16 = 41.019, P < 0.001; Level: F3, 16 = 30.284, P < 0.001; Interaction: F3, 16 = 5.454, P = 0.009) and 5-HIAA/5-HT ratio (Type: F1, 16 = 52.136, P < 0.001; Level: F3, 16 = 39.827, P < 0.001; Interaction: F3, 16 = 7.016, P = 0.003), 12

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however no significant main or interaction effect on the growth hormone level (Type: F1, 16 = 4.268, P = 0.055; Level: F3, 16 = 0.183, P = 0.906; Interaction: F3, 16 = 0.532, P = 0.667), 5-HT level (Type: F1, 16 = 0.619, P = 0.443; Level: F3, 16 = 2.427, P = 0.103; Interaction: F3, 16 = 0.120, P = 0.947), DA level (Type: F1, 16 = 0.412, P = 0.530; Level: F3, 16 = 0.342, P = 0.796; Interaction: F3, 16 = 0.604, P = 0.622), DOPAC level (Type: F1, 16 = 1.410, P = 0.252; Level: F3, 0.330, P = 0.804; Interaction: F3, 16 = 0.399, P = 0.756), HVA level (Type: F1, 16 = 0.096, P

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16 =

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= 0.761; Level: F3, 16 = 0.632, P = 0.605; Interaction: F3, 16 = 0.861, P = 0.481), DOPAC/DA ratio (Type: F1, 16 = 1.410, P = 0.252; Level: F3, 16 = 0.063, P = 0.979; Interaction: F3, 16 =

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0.347, P = 0.792), HVA/DA ratio (Type: F1, 16 = 0.003, P = 0.955; Level: F3, 16 = 0.166, P =

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0.918; Interaction: F3, 16 = 0.487, P = 0.696) and (DOPAC+HVA)/DA ratio (Type: F1, 16 =

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0.039, P = 0.847; Level: F3, 16 = 0.061, P = 0.980; Interaction: F3, 16 = 0.442, P = 0.726) was

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observed. Enrichment type had significant main effects on the 5-HIAA level (F1, 16 = 5.480, P = 0.033), but no main effect of enrichment level or interaction effect was found (Level: F3, 16 =

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2.853, P = 0.070; Interaction: F3, 16 = 0.853, P = 0.485). For plant enrichment, the cortisol level of C fish was significantly lower than that of PL fish, while was significantly higher than those of PM and PH fish (Fig. 3c). As for structure enrichment, C fish had the highest while SM and SH fish had the lowest cortisol levels among the fish from the four treatments (Fig. 3c). The trend in 5-HIAA/5-HT ratio was similar to the trend in cortisol level (Fig. 3d). 3.4. Relationships among the growth, behavioral and physiological parameters Regardless of enrichment treatment, the correlations between either the physiological parameters and growth or the behavioral parameters and physiological parameters were high (Fig. 4). Regarding the relationship between cortisol level and body weight, the regression 13

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equation using a polynomial fit provided the best fit to the pooled data; similarly polynomial fits were best for the relationships between cortisol level and specific growth rate, cortisol level and food conversion efficiency, the 5-HIAA/5-HT ratio and body weight, the 5-HIAA/5-HT ratio and specific growth rate and the 5-HIAA/5-HT ratio and food conversion efficiency (Figs. 4a-f). However, the linear fits provided the best fit for the relationships

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between aggression and cortisol level, aggression and the 5-HIAA/5-HT ratio and cortisol

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level and the 5-HIAA/5-HT ratio (Figs. 4g-i).

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4. Discussion

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The main results of this study showed that the growth performance, feed intake and food

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conversion efficiency of the PM, PH, SL and C fish were better than those of the fish from the

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other treatments. The PL treatment produced the highest levels of aggressive behavior, cortisol and brain serotonergic system activity, whereas the SM and SH treatments yielded the

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lowest levels of these stress-related behavioral and physiological indicators. To the best of our knowledge, this study is the first to show that enrichment type and level have significant main and interaction effects on fish growth, behavior and physiology. Moreover, in this study, we quantified the enrichment level by considering both the basal area of coverage and the number of objects. This approach may represent a feasible method that can serve as a reference standard for related studies and application in the aquaculture industry. An unnegligible issue is that the glass tanks used in the present study differ to a typical concrete tank or sea net cage. The environmental discrepancy generated by containers may also affect fish, contributing to the variation of growth performance and stress level. However, scarce 14

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studies focusing on the growth performance of rockfish and stress level in a typical rearing environment have been conducted. Some related studies were conducted using small net cages, so it is difficult to compare these growth and physiological results, considering sharp discrepancies in fish body size, rearing density, feeding strategy, water temperature and other experimental designs. Actually, based on our observations, the fish body size in barren glass

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tanks were similar to their counterparts rearing in the typical concrete tanks, at least no

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observed differences were found. Thus, the acquired results in this study could have some representativeness, but still need further investigations.

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Studies on the effect of environmental enrichment on fish aggressive behavior are few and

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have yielded contradictory results. For example, the use of blue and red-brown substrates

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decreased aggressive behavior in the gilthead seabream Sparus aurata (Batzina and

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Karakatsouli, 2012), whereas introducing vertical rod structures had no effect on the rate of aggression in the zebrafish Danio rerio (Wilkes et al., 2012). Furthermore, providing

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PVC-tubes under high-density conditions even increased aggression in the African catfish Clarias gariepinus (Rosengren et al., 2016). Similar discrepancies are evident among other studies (Barley and Coleman, 2010; Barreto et al., 2011; Berejikian, 2005). Our results indicate that enrichment type and level might be critical factors affecting fish aggressiveness. With plastic plants, the introduction of a sufficient number of objects into the rearing environment (represented by the medium- and high-level plant treatments in the present study) might restrict territorial range and visual contact, thereby providing individuals greater opportunities to express territorial behavior and consequently decreasing aggressive behavior towards conspecifics (Näslund and Johnsson, 2016). In contrast, introducing few plastic 15

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plants or other objects (e.g., as in the low-level plant treatment in this study) might promote aggression, since the objects may represent not shelters but resources to compete over (Barley and Coleman, 2010). In terms of physical structures, they provided the fish with more closed structures than did the plastic plants (Fig. 1); this feature allowed the fish to divide water space and territory more efficiently and restricted visual range to a greater extent. Therefore,

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the use of low-level (approximately 25% basal area coverage) structure enrichment was

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almost as effective as medium-level (approximately 50% basal area coverage) plant enrichment. Based on this analysis, it is reasonable that the SL treatment yielded significantly

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lower aggression than the PL treatment but similar levels of aggression as the PM and PH

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treatments. In contrast, the introduction of too many structures (e.g., as in the medium- and

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high-level structure treatments in this study) might provide sufficient shelters for most of the

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juveniles, and consequently, yield the lowest levels of aggression among all the treatments. Physiological stress status is one of the most important and common indicators of fish

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welfare (Ashley, 2007; Huntingford et al., 2006). Fish in aquaculture typically suffer high levels of chronic or acute stress from their aquatic environment or intraspecies interactions, which may induce a variety of physiological processes. The hypothalamic-pituitary-interrenal tissue (HPI) axis and neurotransmitter activities are often considered to be the primary stage of neuroendocrine regulation, subsequently affecting immune function, enzyme activities, blood parameters and other secondary responses, and consequently inducing changes at the whole-organism level, such as behavior, growth and survival (Galhardo and Oliveira, 2009; Mommsen et al., 1999; Sørensen et al., 2013). Brain serotoninergic system activity and plasma/body cortisol levels have been suggested to be fairly accurate and commonly used 16

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indicators of fish stress level and welfare (Batzina et al., 2014; Pounder et al., 2016; Rosengren et al., 2016). Our results showed that the trends in the 5-HIAA/5-HT ratio and cortisol level across treatments were similar to the trend in aggressive behavior (Fig. 3), and close correlations between these parameters were observed (Fig. 4). These results indicated that some or most of the physiological stress might have resulted from conspecific aggression.

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These physiological results corroborate the behavioral results (opercular beat rate and

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aggressive behavior), implying that enrichment of certain types and at certain levels were indeed beneficial for fish welfare. However, dopaminergic system activity (indicated by DA,

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DOPAC, HVA, DOPAC/DA, HVA/DA and (DOPAC+HVA)/DA) did not differ significantly

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among the treatments. This result is consistent with the findings of previous studies showing

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that substrate color did not affect brain dopaminergic activity in general (Batzina et al., 2014;

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Batzina et al., 2014). Moreover, in a previous study examining the effects of skin extract exposure and shelter on the brain monoaminergic activity of the crucian carp Carassius

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carassius, brain dopaminergic activity increased in the telencephalon and decreased in the brain stem following skin extract exposure, whereas shelter did not stimulate such changes (Höglund et al., 2005). This result implied that dopaminergic activity might be more closely related to sensory enrichment (especially that involving olfactory cues) than to structure enrichment. Furthermore, considering that the stress response is a fundamental aspect in fish welfare and positive control is of importance for evaluating fish stress level and putting the results in a realistic perspective, we conducted two other experiments focusing on the copying style and stress response of rockfish (In preparation). In the first experiment, 53 fish (body length: 17

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7.53±0.11 cm; body weight: 9.75±0.27 g) were solely reared in well-circulated glass tanks (40 cm × 30 cm × 30 cm) for seven days (a period which is sufficient to recover from handle stress and recover to normal appetite). This isolate environment completely prohibits any possibility of social interaction with other fish. The main results showed that the average cortisol concentration of isolate fish is 0.653±0.013 ng/mg (0.531-1.015 ng/mg), which is

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significantly lower than that of C (t test: t63 = 5.599, P < 0.001), PL (t test: t63 = 7.191, P <

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0.001), PM (t test: t63 = 3.094, P = 0.003), PH (t test: t63 = 3.308, P = 0.002) and SL (t test: t63 = 3.311, P = 0.002) fish but is statistically similar to that of SM (t test: t63 = 0.659, P = 0.513)

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and SH (t test: t63 = 0.757, P = 0.452) fish (unpublished data). In the second experiment, air

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exposure and confinement were used as acute stressors, and the main results showed that after

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being subjected to stressors, the average cortisol level of control fish peaked at 5.315±0.094

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ng/mg (0.5 h after stress) and recovered to basal level at 3 h after stress (unpublished data). These results indicate that the fish from C, PL, PM, PH and SL treatments truly had some

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social interactions (mainly aggression) and subsequently causing some mild social stress (indicated by significantly higher cortisol level). To our knowledge, the similar phenomenon is commonly observed in some other fish species (Barcellos et al., 2009; Batzina et al., 2014; Näslund et al., 2013), which further strengthen our results. No significant difference in fish growth hormone level was observed among the treatments, suggesting that the observed differences in growth performance might have been mediated by regulatory pathways other than the hypothalamus-pituitary-liver axis. Several studies have shown that high stress levels are inversely associated with feed intake, food conversion efficiency, energy consumption, and consequently, growth performance (Gregory and Wood, 18

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1999; Mommsen et al., 1999). Accordingly, in the present study, the low-level plant enrichment treatment, which yielded the highest levels of aggressive behavior, cortisol and serotoninergic activity, yielded significantly lower final body weight than did the control treatment. Unexpectedly, the treatments with medium- and high-level physical structure enrichment also yielded significantly lower growth and feed performance than the control

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treatment. A possible explanation for this finding involves the relationship between stress

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level and growth performance: as this relationship follows an inverted U-shaped dose-dependent curve (Sørensen et al., 2013), the levels of stress hormones in these

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treatments might have depressed fish growth (Fig. 4). This possibility is partly supported by

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the growth results of the C, PM, PH and SL treatments. Although the C, PM, PH and SL

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treatments yielded similar growth data, the stress level of the C fish was significantly higher

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than those of the fish of the other three treatments, which suggests that the stress hormone level of the C fish was insufficient to unlock the growth loads. From the consideration of fish

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welfare, medium-level (approximately 50% basal area coverage) and high-level (approximately 75% basal area coverage) enrichment with plants and low-level (approximately 25% basal area coverage) enrichment with structures seem to yield the optimal stress levels and growth performance. Based on our analyses of fish growth, stress and welfare, we propose a conceptual model that summarizes the effects of environmental enrichment, especially the effect of enrichment level, on fish welfare (Fig. 5). In this model, fish welfare is evaluated by growth (mainly indicated by body weight and specific growth rate) and stress (indicated by aggression, cortisol level and serotoninergic activity), which were two main parameters affected by enrichment in this study. As enrichment level increases, the level 19

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of physiological stress initially increases rapidly due to intraspecies competition over the shelter resources and then decreases, ultimately reaching its lowest levels, with the trend over time following an inverted U-shape (Fig. 5). Due to the depressing effects of the highest and lowest stress levels on feeding activity and growth performance, the two lowest points of

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growth correspond to the highest and lowest points of stress (Fig. 5).

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Overall, the present study showed that fish from a rearing environment with no enrichment, enriched with plants at a medium-level (approximately 50% basal area coverage) or

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high-level (approximately 75% basal area coverage), or enriched with structures at a

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low-level (approximately 25% basal area coverage) had relatively higher growth performance,

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feed intake and food conversion efficiency than did fish from the other treatments. The

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low-level plant treatment yielded the highest levels of aggressive behavior, cortisol and brain serotonergic system activity; the medium- and high-level structure treatments yielded the

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lowest levels of these stress-related behavioral and physiological indicators. Although growth performance was similar among the control fish and those from the low-level structure, medium-level plant and high-level plant treatments, the control fish had significantly higher stress level than did the fish from these other three treatments. This unexpected result indicates that the stress hormone level of the control fish was insufficient to unlock the growth loads. However, from the perspective of fish welfare, we consider it optimal to enrich the environment with approximately 50% basal area coverage with objects (depend on the “closed” vs. “open” structure of the objects). Taken together, our results provide the first evidence to show that the type and level of environmental enrichment have pronounced 20

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effects on fish growth performance, behavior (especially aggressive behavior) and stress-related physiological processes. For fish farmers and readers with wide academic backgrounds, this study may also provide some useful information. Some fish species (especially carnivorous fishes) hold an aggressive instinct and such social interactions between fish individuals often result in severe cannibalism rate, physical injury and social

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stress, and subsequent higher growth deficits, lower disease resistance ability, which may

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cause large economic losses (for fish farmer) and worrying conditions of fish welfare (for animal protector). Our study showed that simple introduction of proper objects into the

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rearing tanks can effectively solve these problems, i.e., shelters provide visual obstacles and

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sanctuaries for individuals, naturally decrease harmful social interactions (e.g. aggression)

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and social stress, subsequently increase fish appetite and digestive ability and potentially

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improve fish welfare and growth performance. In this design, the proper choices of enrichment type and level are especially important, and we recommend that the physical

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structure and plastic plant are applicable enrichment types and enrichment level can be set as 50% basal area coverage depending on the type of object. Furthermore, in stock enhancement, the traditional simplified rearing environment can cause wild-adaptive ability deficiency and lower survival rate of released fish, and proper enrichment in early hatchery environment can enhance fish neural development, cognitive ability and behavioral flexibility, and therefore, the recommended enrichment type and level in this study may also have potential benefits for fish naturalization (for fishery agency). On the other hand, because of the complexity of the growth-regulatory and physiological processes of fish and the low numbers of enrichment levels and types in our study, we were unable to comprehensively evaluate enrichment effects; 21

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however, we proposed a conceptual model for the estimation of these effects. More complex designs and mathematic model fitting should be utilized in the future to identify the optimal types and levels of enrichment. Additionally, accumulation of food pellets and faeces in enriched environment are main concerns for fish farmers, so the designment of mobile and labor-free enrichment mode may be a fruitful field in the future. Considering the special

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behavioral habits of black rockfish, the above results might be fish species- or life

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stage-dependent, and in the present study, the major influencing factors such as water quality, flow velocity, illumination intensity and human disturbance were strictly controlled in order

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to avoid the interferences from biotic and environmental factors, whereas we might ignore

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and do not exclude several uncontrollable factors, and in general, more intensive researches

Acknowledgments

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should be taken to validate these novel findings.

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This work was supported by funds from the National Natural Science Foundation of China (41676153; 31172447) and the National Program on Key Basic Research Project (973 Program) (2015CB453302).

Declarations of interest: none.

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Aquacult. 80, 162-167. Madison, B.N., Heath, J.W., Heath, D.D., Bernier, N.J., 2015. Effects of early rearing environment and breeding strategy on social interactions and the hormonal response to stressors in juvenile Chinook salmon. Can. J. Fish. Aquat. Sci. 72, 673-683. Marcon, M., Mocelin, R., Benvenutti, R., Costa, T., Herrmann, A.P., de Oliveira, D.L.,

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Näslund, J., Rosengren, M., Del Villar, D., Gansel, L., Norrgård, J.R., Persson, L., Winkowski, J.J., Kvingedal, E., 2013. Hatchery tank enrichment affects cortisol levels and shelter-seeking in Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. 70, 585-590. Newberry, R.C., 1995. Environmental enrichment: increasing the biological relevance of captive environments. Appl. Anim. Behav. Sci. 44, 229-243. Øverli, Ø., Pottinger, T.G., Carrick, T.R., Øverli, E., Winberg, S., 2002. Differences in behaviour between rainbow trout selected for high- and low-stress responsiveness. J. Exp. Biol. 205, 391-395. Pounder, K.C., Mitchell, J.L., Thomson, J.S., Pottinger, T.G., Buckley, J., Sneddon, L.U., 27

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2016. Does environmental enrichment promote recovery from stress in rainbow trout? Appl. Anim. Behav. Sci. 176, 136-142. Roberts, L.J., Taylor, J., Gough, P.J., Forman, D.W., Garcia De Leaniz, C., 2014. Silver spoons in the rough: can environmental enrichment improve survival of hatchery Atlantic salmon Salmo salar in the wild? J. Fish Biol. 85, 1972-1991.

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salmon parr. Ecol. Freshw. Fish. 20, 569-579.

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Rosburg, A.J., Fletcher, B.L., Barnes, M.E., Treft, C.E., Bursell, B.R., 2019.

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Vertically-suspended environmental enrichment structures improve growth of juvenile

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landlocked fall Chinook salmon. Int. J. Innov. Stud. Aquat. Biol. Fish. 5, 17-24.

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Rosengren, M., Kvingedal, E., Näslund, J., Johnsson, J.I., Sundell, K., 2016. Born to be wild: effects of rearing density and environmental enrichment on stress, welfare, and smolt

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migration in hatchery-reared Atlantic salmon. Can. J. Fish. Aquat. Sci. 74, 396-405. Salvanes, A.G.V., Moberg, O., Ebbesson, L.O., Nilsen, T.O., Jensen, K.H., Braithwaite, V.A., 2013. Environmental enrichment promotes neural plasticity and cognitive ability in fish. Proc. R. Soc. B. 280, 20131331. Sørensen, C., Johansen, I.B., Øverli, Ø., 2013. Neural plasticity and stress coping in teleost fishes. Gen. Comp. Endocr. 181, 25-34. Ullah, I., Zuberi, A., Khan, K.U., Ahmad, S., Thörnqvist, P., Winberg, S., 2017. Effects of enrichment on the development of behaviour in an endangered fish mahseer (Tor putitora). Appl. Anim. Behav. Sci. 186, 93-100. 28

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Vindas, M.A., Gorissen, M., Höglund, E., Flik, G., Tronci, V., Damsgård, B., Thörnqvist, P., Nilsen, T.O., Winberg, S., Øverli, Ø., Ebbesson, L.O.E., 2017. How do individuals cope with stress? Behavioural, physiological and neuronal differences between proactive and reactive coping styles in fish. J. Exp. Biol. 220, 1524-1532. von Krogh, K., Sørensen, C., Nilsson, G.E., Øverli, Ø., 2010. Forebrain cell proliferation,

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Physiol. Behav. 101, 32-39.

White S.C., Krebs E., Huysman N., Voorhees J.M., Barnes M.E., 2018. Addition of vertical

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rearing. J. Mar. Bio. Aquacult. 4, 48-52.

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enrichment structures does not improve growth of three salmonid species during hatchery

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White S.C., Krebs E., Huysman N., Voorhees J.M., Barnes M.E., 2019. Use of suspended

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plastic conduit arrays during Brown Trout and Rainbow Trout rearing in circular tanks. N. Am. J. Aquacult. 81, 101-106.

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Wilkes, L., Owen, S.F., Readman, G.D., Sloman, K.A., Wilson, R.W., 2012. Does structural enrichment for toxicology studies improve zebrafish welfare? Appl. Anim. Behav. Sci. 139, 143-150.

Xi, D., Zhang, X., Lü, H., Zhang, Z., 2017a. Cannibalism in juvenile black rockfish, Sebastes schlegelii (Hilgendorf, 1880), reared under controlled conditions. Aquaculture. 479, 682-689. Xi, D., Zhang, X., Lü, H., Zhang, Z., 2017b. Prediction of cannibalism in juvenile black rockfish, Sebastes schlegelii (Hilgendorf, 1880), based on morphometric characteristics and paired trials. Aquac. Res. 48, 3198-3206. 29

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Zhang, Z., Zhang, X., Li, Z., Zhang, X., 2019. Effects of different levels of environmental enrichment on the sheltering behaviors, brain development and cortisol levels of black

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rockfish Sebastes schlegelii. Appl. Anim. Behav. Sci. 218, 104825.

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Table 1 Parameters of the experimental treatments. Parameter

Treatment PL

PM

PH

SL

SM

SH

NO

0

10

20

30

10

20

30

NF

40

40

40

40

40

40

40

NO/NF (%)

0

25

50

75

25

50

75

BAO (cm2)

0

720

1440

2160

720

1440

2160

BAT (cm2)

3000

3000

3000

3000

3000

3000

3000

BAO/BAT (%)

0

24

48

72

24

48

72

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ro

of

C

C: control; PL: low-level enrichment with plants; PM: medium-level enrichment with plants; PH: high-level enrichment with

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plants; SL: low-level enrichment with structures; SM: medium-level enrichment with structures; SH: high-level enrichment with

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structures; NO: Number of Objects; NF: Number of Fish; BAO: Basal Area of Objects; BAT: Basal Area of Tank.

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Fig. 1. Photographs of the plastic plants and physical structures used in this experiment.

Fig. 2. Growth performance of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Body weight (n = 3); (b) Specific growth rate (n = 3); (c) Weight gain (n = 3); (d) Food conversion efficiency (n = 3). PE: Plastic plant enrichment; SE: Physical

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structure enrichment. Different capital letters indicate significant differences among physical

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structure enrichment treatments and different small letters indicate significant differences

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among plastic plant enrichment treatments. Data are presented as means ± S.E.

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Fig. 3. Behavioral and physiological parameters of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Aggressive behavior (n = 24); (b) Opercular beat

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rate (n = 12); (c) Cortisol level (n = 3); (d) Serotonergic activity (n = 3). PE: Plastic plant

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enrichment; SE: Physical structure enrichment. Different capital letters indicate significant differences among physical structure enrichment treatments and different small letters indicate significant differences among plastic plant enrichment treatments. Data are presented as means ± S.E.

Fig. 4. Regression lines of relationships among growth, behavioral and physiological parameters based on pooled data. (a) Polynomial fit of the cortisol level and body weight. (b) Polynomial fit of the cortisol level and specific growth rate. (c) Polynomial fit of the cortisol level and food conversion efficiency. (d) Polynomial fit of the 5-HIAA/5-HT ratio and body

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weight. (e) Polynomial fit of the 5-HIAA/5-HT ratio and specific growth rate. (f) Polynomial fit of the 5-HIAA/5-HT ratio and food conversion efficiency. (g) Linear fit of aggression and the cortisol level. (h) Linear fit of aggression and the 5-HIAA/5-HT ratio. (i) Linear fit of the cortisol level and the 5-HIAA/5-HT ratio.

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Fig. 5. A conceptual model of the effects of environmental enrichment on fish growth, stress

ro

and welfare. The red curve describes the possible change in fish stress level (which may be indicated by levels of aggression, cortisol and serotoninergic activity) with enrichment level

-p

increasing. The black curve describes the possible change in fish growth performance with

re

enrichment level increasing. Our data indicate that fish welfare may be indicated by the two

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main parameters of stress and growth; shelters with more “open” structures may shift the

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the opposite direction.

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curve toward the right, whereas shelters with more “closed” structures may shift the curve in

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Fig. 1. Photographs of the plastic plants and physical structures used in this experiment.

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Fig. 2. Growth performance of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Body weight (n = 3); (b) Specific growth rate (n = 3); (c) Weight gain (n

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= 3); (d) Food conversion efficiency (n = 3). PE: Plastic plant enrichment; SE: Physical structure enrichment. Different capital letters indicate significant differences among physical

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structure enrichment treatments and different small letters indicate significant differences among plastic plant enrichment treatments. Data are presented as means ± S.E.

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Fig. 3. Behavioral and physiological parameters of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Aggressive behavior (n = 24); (b) Opercular beat

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rate (n = 12); (c) Cortisol level (n = 3); (d) Serotonergic activity (n = 3). PE: Plastic plant enrichment; SE: Physical structure enrichment. Different capital letters indicate significant

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differences among physical structure enrichment treatments and different small letters indicate significant differences among plastic plant enrichment treatments. Data are presented as means ± S.E.

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Fig. 4. Regression lines of relationships among growth, behavioral and physiological parameters based on pooled data. (a) Polynomial fit of the cortisol level and body weight. (b)

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Polynomial fit of the cortisol level and specific growth rate. (c) Polynomial fit of the cortisol level and food conversion efficiency. (d) Polynomial fit of the 5-HIAA/5-HT ratio and body weight. (e) Polynomial fit of the 5-HIAA/5-HT ratio and specific growth rate. (f) Polynomial fit of the 5-HIAA/5-HT ratio and food conversion efficiency. (g) Linear fit of aggression and the cortisol level. (h) Linear fit of aggression and the 5-HIAA/5-HT ratio. (i) Linear fit of the cortisol level and the 5-HIAA/5-HT ratio.

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Fig. 5. A conceptual model of the effects of environmental enrichment on fish growth, stress

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and welfare. The red curve describes the possible change in fish stress level (which may be indicated by levels of aggression, cortisol and serotoninergic activity) with enrichment level

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increasing. The black curve describes the possible change in fish growth performance with enrichment level increasing. Our data indicate that fish welfare may be indicated by the two

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main parameters of stress-- and growth; shelters with more “open” structures may shift the curve toward the right, whereas shelters with more “closed” structures may shift the curve in the opposite direction.

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No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. The work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or

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in part. All the authors listed have approved the manuscript that is enclosed.

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Highlights: 1. Environmental enrichment type and level significantly affect the growth, stress-related physiology and aggression of black rockfish. 2. Enrichment with medium-level plants, high-level plants or low-level structures produced better growth performance and moderate stress level.

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3. The optimal enrichment level for fish welfare is approximately 50% basal area coverage.

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4. A conceptual model which outlined the possible effects of enrichment on fish welfare was

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proposed.

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