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Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii
Journal Pre-proof Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii
Zonghang Zhang, Yiqiu Fu, Haoyu Guo, Xiumei Zhang PII:
S0044-8486(20)30555-X
DOI:
https://doi.org/10.1016/j.aquaculture.2020.736088
Reference:
AQUA 736088
To appear in:
Aquaculture
Received date:
20 February 2020
Revised date:
19 August 2020
Accepted date:
19 October 2020
Please cite this article as: Z. Zhang, Y. Fu, H. Guo, et al., Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii, Aquaculture (2018), https://doi.org/10.1016/j.aquaculture.2020.736088
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© 2018 Published by Elsevier.
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Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii
Zonghang Zhanga, Yiqiu Fua, Haoyu Guob,c, Xiumei Zhangb,c,* [email protected] a
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China,
Qingdao 266003, China
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b
c
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Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National
Corresponding author at: Fisheries College, Zhejiang Ocean University, No.1, Haida South
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*
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Laboratory for Marine Science and Technology, Qingdao 266237, China
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Road, Lincheng Changzhi Island, Zhoushan, China
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Abstract
The stress response is one of the most important aspects of fish welfare in aquaculture. However, relatively few studies focusing on the effect of environmental enrichment on the fish stress response have been conducted, and their limited results were considerably contradictory. The present study aimed to investigate whether the enrichment type and amount had significant effects on the basal stress level and physiological and behavioral responses to acute stress of juvenile black rockfish Sebastes schlegelii. Fish were reared for eight weeks in environments with two enrichment types and three enrichment amounts (i.e., no
environmental
enrichment
control
(C),
low-amount
plant
enrichment
(PL),
medium-amount plant enrichment (PM), high-amount plant enrichment (PH), low-amount
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structure enrichment (SL), medium-amount structure enrichment (SM), and high-amount structure enrichment (SH)), and subsequently, they were successively subjected to two common acute stressors (i.e., air exposure and confinement) and sampled at 0, 0.5, 1, 3 and 6 h after stress. In general, the plant enrichment groups experienced significantly higher basal stress (indicated by cortisol level and opercular beat rate) than the structure enrichment
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groups. Meanwhile, no enrichment control group and low-amount enrichment groups
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experienced significantly higher basal stress than the other two enrichment amount groups. After being subjected to acute stress, the peak cortisol levels of the high-amount enrichment
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groups were significantly higher than that of the other three enrichment amount groups. The
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PM, PH and SL fish recovered to basal stress levels at 1 h, the C and PL fish recovered at 3 h,
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and the SM and SH fish recovered at 6 h after the stress. Moreover, their basal cortisol levels
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showed strong correlations with peak cortisol levels and recovery time from stress. The correlation between cortisol level and opercular beat rate was also strong. Taken together,
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these results provide the first evidence to show that enrichment type and amount had significant effects on the fish stress response and might have important applications in fish husbandry and welfare. Based on these results, we suggest that it may be optimal for fish basal stress level and development of an adaptive capacity to provide a medium-amount (approximately 50% floor space coverage) of mixed enrichment in aquaculture.
Keywords environmental enrichment; stress response; cortisol; opercular beat rate; Sebastes schlegelii
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1. Introduction Fish in aquaculture are often subjected to several potential stressors. For example, an unnatural high rearing density, sudden changes of the water level, transport, sorting, handling, machine noises and intraspecies aggression (Ashley, 2007; Galhardo and Oliveira, 2009). For fish that are destined to be released into the wild (e.g., stock enhancement and conservation
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programs), they will inevitably experience more natural, unpredictable and heterogeneous
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environments after release, including intraspecies competition, predator pressure and variable environmental factors (Johnsson et al., 2014). These stressors from the aquatic environment
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may induce a series of physiological and behavioral responses, which depend on the fish to
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have the ability to adapt to the specific situation (Ashley, 2007; Galhardo and Oliveira, 2009).
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In general, the fish stress response can be divided into three stages: first, a
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hypothalamus-pituitary-interrenal (HPI) axis hormone response is induced, the main end product of which is cortisol; second, many changes in the blood, tissue and metabolism are
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triggered; and third, the performances at the whole-body level is altered, such as behavioral patterns, survival and growth performance (Barton, 2002; Galhardo and Oliveira, 2009). When stress exceeds the fish adaptive capacity, they have an increased energy expenditure, modifications of normal physiological processes and behavioral phenotypes, lower fish welfare, decreased growth performance and adverse impacts on fish reproduction and survival (Ashley, 2007; Barton, 2002; Huntingford et al., 2006). Thus, how to properly decrease the stress response of fish is a particularly important question in aquaculture industry. Environmental enrichment, namely, increasing the environmental complexity by introducing objects (e.g., plants, physical structures) into rearing water (Zhang et al., 2019;
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Zhang et al., 2020a; Zhang et al., 2020b), is believed to be one of the most useful ways to regulate the physiological status, alter behavioral performance and consequently enhance fish welfare (Näslund and Johnsson, 2016; Newberry, 1995). Many recent studies have verified that environmental enrichment could enhance the fish growth rate and neural plasticity (Batzina and Karakatsouli, 2012; Camara-Ruiz et al., 2019; Salvanes et al., 2013), decrease
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aggressive and anxiety-like behaviors (Barcellos et al., 2018; Batzina and Karakatsouli, 2012;
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Zhang et al., 2020a), and promote survival-related behaviors of the released fish (Braithwaite and Salvanes, 2005; D'Anna et al., 2012; Mes et al., 2019; Ullah et al., 2017; Zhang et al.,
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2019). However, studies on the effect of enrichment on the fish stress response are scarce, and
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the limited results of different studies are sharply contradictory. The studies variably showed
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positive (Barcellos et al., 2009; Batzina et al., 2014; Cogliati et al., 2019; Marcon et al., 2018),
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negative (Boerrigter et al., 2016; Madison et al., 2015; Zubair et al., 2012) or no (Näslund et al., 2013) effects. These large differences may be related to many factors, for example, fish
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species and populations, enrichment types and amounts and other experimental designs. However, scarce studies about the effects of enrichment type and amount, especially the interaction of the two factors, on the fish stress response have been conducted, and the question of which specific enrichment type and amount is optimal for fish needs to be answered. Black rockfish Sebastes schlegelii is widely distributed in the coastal areas of China, Korea and Japan (Xi et al., 2017). In recent years, the aquaculture of black rockfish has been quickly prosperous, especially in northern China, for the main purpose of food supply and stock enhancement (Guo et al., 2017). The traditional rearing environments and production
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processes in the aquaculture industry usually involve many harmful stressors. For example, fish typically experience sorting and transport, and these operations inevitably put fish in the air for a period of time, and the new environment very likely differs from the previous environment in water depth, rearing density, tank size, social environment, and other factors (which can be categorized as confinement). Moreover, the typical rearing tanks usually do not
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contain any environmental modifications. These husbandry characteristics may increase the
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fish basal stress level and damage their normal adaptive ability to the environment (Zhang et al., 2020a). In this study, we aimed to determine how enrichment type and amount affected
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the stress response of juvenile black rockfish and to verify whether enrichment type and
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amount are critical factors in enrichment design. Two commonly occurring stressors in
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aquaculture production (air exposure and confinement) were adopted as acute stress. The core
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hormone of the HPI axis (cortisol) and a commonly used behavioral indicator (the opercular
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beat rate) were used to assess the fish stress level.
2. Materials and methods
We have read the policies related to animal experiments (the ARRIVE and PREPARE guidelines) and have confirmed this study complied. All procedures conducted in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Ocean University of China. 2.1. Fish husbandry The experimental animal and fish husbandry were described in detail in our previous work (Zhang et al., 2020a). In brief, eight hundred and forty black rockfish S. schlegelii juveniles
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(mean wet weight 3.08±0.05 g, mean standard length 4.69±0.03 cm) were randomly distributed in 21 glass tanks (60 cm × 50 cm × 50 cm) in groups of 40 (initial rearing density 1.03±0.02 kg/m3) in seven triplicated treatments. The treatment groups were no environmental enrichment control (C), low-amount enrichment with plastic plants (PL), medium-amount enrichment with plastic plants (PM), high-amount enrichment with plastic
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plants (PH), low-amount enrichment with physical structures (SL), medium-amount
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enrichment with physical structures (SM) and high-amount enrichment with physical structures (SH). In the enriched tanks, different types and amounts of objects were introduced
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as shelters (von Krogh et al., 2010; Xi et al., 2017), while the control tanks had no objects.
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Enrichment types included plastic plants (height 10 cm, projected area approximately 8 cm ×
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9 cm = 72 cm2; Fig. 1) and physical structures (height approximately 10 cm, floor space 12
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cm × 6 cm = 72 cm2; Fig. 1). For enrichment amount, 10, 20 and 30 objects corresponded to low-, medium- and high-amount enrichment, respectively. The experimental tanks were part
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of an indoor flow-through seawater system. The water flow rate was 3.5 L/min (exchange rate 4,200%) and the water depth was maintained at 40 cm. The fish were acclimated to the glass tanks for three weeks before the formal experiment, and subsequently they were maintained under experimental conditions for eight weeks. The fish were fed commercial floating pellets (Kaido Brand, Santong Bioengineering Co. Ltd., Anqiu, China) by hand once daily at a restricted feeding rate (2.5% fish body weight daily), since this feeding strategy was proposed to be proper for the growth, physiology and welfare of juvenile rockfish (Guo et al., 2017; Zhang et al., 2020a). All tanks, plastic plants and physical structures were cleaned once weekly. The photoperiod followed the natural day-light cycle. Water temperature
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(25.9±0.18°C), dissolved oxygen (6.68±0.04 mg/L; 97.43±0.25% saturation), salinity (29.73±0.16‰), pH (7.12±0.02), total ammonia-nitrogen (0.236±0.0138 mg/L) and nitrite-nitrogen (0.012±0.0005 mg/L) were continuously monitored. No fish died during the rearing period. 2.2 Experimental design
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After the eight-week experimental period, the fish were subjected to standard stress response
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trials. In this study, we used air exposure (Boerrigter et al., 2016; Madison et al., 2015; Pounder et al., 2016) and confinement (Marcon et al., 2018; Näslund et al., 2013) as stressors,
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which are common methods used in such trials and do not cause any physical harm to the fish.
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Twenty four fish from each tank were quickly netted and subjected to one-minute air
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emersion by holding them in a plastic basin, and subsequently these fish were randomly and
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equally distributed into four well-circulated small glass tanks (40 cm × 30 cm × 30 cm; water level was maintained at 20 cm; six cobbles (diameter 3 cm) and one air stone were introduced
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into each tank) to be allowed to recover from the stress. After 0.5, 1, 3 and 6 h, six fish from one of the four small tanks were sampled, euthanized with an overdose of anesthetic (tricaine methanesulfonate, MS-222, 100 mg/L), weighed (precision 0.01 g) and measured (precision 0.01 cm), and then three visceral masses (due to fish were too small to draw blood) were extracted for measuring the cortisol level (Guo et al., 2017). All sampling procedures were completed within one minute. Ten minutes before each sampling time point (i.e., 0.5, 1, 3 and 6 h after the stress), one 10 min video was filmed by a camera positioned in front of the small tank to determine the behavioral response. Additionally, two days before the stress response trials, one 10 min video of each rearing tank was also filmed (approximately 14:00-15:00),
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and three fish of each tank were randomly sampled and dissected to determine basal behavioral and physiological stress levels (Zhang et al., 2020a). All samples were frozen in liquid nitrogen, and then stored at -80°C until analysis. The remaining fish in the rearing glass tanks were used in another experiment (Zhang et al., 2020a). 2.3. Behavioral study
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In this study, we used the opercular beat rate to evaluate the behavioral response to stress,
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which has been verified to be a valid indicator in other fish species (Braithwaite and Salvanes, 2005; Pounder et al., 2016). Before starting filming, several digital video cameras
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(HDR-AS100V and HDR-XR550, Sony Corporation, Tokyo, Japan; Honor 6X, HuaWei
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Technologies Co. Ltd., Shenzhen, China; iPad Air 2, iPad mini 2, and iPhone 6s plus, Apple
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Incorporation, Cupertino, USA) were fixed along the front side of each tank simultaneously,
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and the distance between tank and camera was approximately 30 cm. Then, the experimenters left the fish, and a 10 min video was filmed. Approximately 10 min later, the experimenters
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came back and turned off the cameras. To eliminate the possible disturbance of the observed fish, the first 3 min and last 2 min of each 10 min video were cut off, and only the middle 5 min was analyzed. The opercular beat rate was measured as the number of opercular beats per minute (Pounder et al., 2016). The observer who quantified the fish behavior was blind to the treatments. 2.4. Cortisol level determination The visceral mass samples were homogenized in cold PBS (9×weight, pH 7.4) and centrifuged at 3000 rpm for 20 minutes in a refrigerated centrifuge (4°C). The supernatants were collected and used for analysis. A commercial Elisa Assay Kit (H094-1-2, Nanjing
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Jiancheng Bioengineering Institute, Nanjing, China), which was proved to have good validation (Zhang et al., 2019; Zhang et al., 2020a; Zhang et al., 2020b), was used to measure the cortisol concentrations according to the manufacturer’s guidelines (for more details about the experimental principle and detailed procedures of this cortisol determination kit, see the Supplementary Material). The intra-assay coefficient of variation was less than 10% and high
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correlations were found between the standard curves (R2 > 0.99).
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2.5. Data analysis
First, we duplicated the data from the control treatment to yield data representative of two
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treatments: no plant enrichment and no structure enrichment. This allowed us to simulate a 2
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(enrichment type: plant and structure) × 4 (enrichment amount: no, low, medium and high)
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factorial design. Then, the main and interaction effects of the two factors were tested by
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two-way ANOVA followed by Duncan’s multiple-range post hoc test. Normality of the data was tested with Shapiro-Wilk’s test and homogeneity of variance of the data was tested with
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Levene’s test prior to ANOVA. To determine the recovery time from stress, one-way ANOVA followed by Duncan’s multiple-range post hoc test were used to detect the differences of cortisol level or opercular beat rate among the five time points (i.e., 0, 0.5, 1, 3 and 6 h after stress) within each treatment (i.e., C, PL, PM, PH, SL, SM or SH treatment). To clarify the relationships between the basal stress level and the stressed status or cortisol level and opercular beat rate, several fitting methods (polynomial, exponential and linear) were performed with the pooled data, and the best fitting curve is presented in the figures. For all parameters in this study, the whole tank was regarded as the statistical unit. All statistical analyses were made using SPSS 17.0 for windows. Differences were considered to be
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significant at a probability level of 0.05. All values in the text and figures were presented as the means ± S.E.
3. Results 3.1. Basal stress level
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This was the second of two complementary studies designed to explore how enrichment
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type and amount affected the growth performance, behavioral phenotype and physiological status of black rockfish. Here, we mainly present the results of the fish stress response,
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whereas the growth data could be found in the companion paper (Zhang et al., 2020a).
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Two-way ANOVA showed that significant main and interaction effects of enrichment type
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and enrichment amount on basal cortisol level were observed (Fig. 2a). Overall, the plant
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enrichment groups had a significantly higher basal cortisol level than did the structure enrichment groups, and meanwhile, the no enrichment control group and the low-amount
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enrichment groups had significantly higher basal cortisol levels than did the other two enrichment amount groups (Fig. 2a). The trend of the basal opercular beat rate was similar to the trend of the basal cortisol level (Fig. 2b). 3.2. Physiological and behavioral responses to stress Peak stress levels of all treatments occurred at 0.5 h after stress (Table 1; Fig. 5). At this time point, the cortisol levels of the plant enrichment groups were significantly lower compared to that of the structure enrichment groups, and the high-amount enrichment groups produced the highest cortisol levels among the four enrichment level groups (Fig. 3a). At 1 h after stress, the no enrichment control group had the highest cortisol level, while at 3 h after
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stress, the high-amount enrichment groups had the highest cortisol levels among the four enrichment amount groups (Fig. 3bc). At 6 h after stress, the plant enrichment groups produced significantly higher cortisol levels than did the structure enrichment groups. Meanwhile, the no enrichment control group and the low-amount enrichment groups had significantly higher cortisol levels than did the other two enrichment amount groups (Fig. 3d),
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which was similar to the trend in basal cortisol levels (Fig. 2a). For behavioral responses, the
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changing trend of the opercular beat rate at 1 h after stress was similar to that of the cortisol
3.3. Time course of recovery from stress
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points (i.e., 0.5, 3 and 6 h after stress) (Fig. 4).
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level, but no significant main or interaction effects were detected at the other sampling time
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Although all of the treatments had the same peak time point (0.5 h after stress), the recovery
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time points were different (Table 1; Fig. 5). In the C and PL fish, the cortisol levels declined to values still significantly higher than the basal levels at 1 h and recovered to basal levels at 3
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h after stress. In the PM, PH and SL fish, cortisol levels dropped to basal levels at 1 h after stress. In the SM and SH fish, cortisol levels remained elevated at 1 h, declined to values that were significantly lower than peak levels while still significantly higher than basal levels at 3 h and recovered to basal levels at 6 h after stress (Table 1; Fig. 5a). The trend of the opercular beat rate was similar to that of the cortisol level (Table 1; Fig. 5b). 3.4. Relationships among physiology, behavior and recovery ability Regardless of enrichment treatment and sampling time point, the correlations between basal cortisol level and peak cortisol level; basal cortisol level and recovery time from stress; basal cortisol level and basal opercular beat rate; and stressed cortisol level and stressed opercular
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beat rate, were very high (Fig. 6). However, the best fitting model differed among the above relationships. Exponential fit and polynomial fit were the best for basal cortisol level and peak cortisol level (Fig. 6a), and basal cortisol level and recovery time (Fig. 6b), respectively. Linear fits provided the best fit for the relationships between basal cortisol level and basal opercular beat rate, and between stressed cortisol level and stressed opercular beat rate (Fig.
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6cd).
4. Discussion
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The main results of this study showed that: 1) Plant enrichment induced significantly higher
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basal stress (indicated by the cortisol level and opercular beat rate) than did structure
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enrichment. Meanwhile, the no enrichment control group and low-amount enrichment groups
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experienced significantly higher basal stress than did the other two enrichment amount groups. 2) After being subjected to acute stress, the peak cortisol levels of the high-amount
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enrichment groups were significantly higher than that of the other three enrichment amount groups. 3) The PM, PH and SL fish recovered to their basal stress level at 1 h, the C and PL fish recovered at 3 h, while the SM and SH fish recovered at 6 h after stress. 4) The basal cortisol levels showed high correlations with peak cortisol levels and recovery time. Meanwhile, the correlation between cortisol level and opercular beat rate was also high. The effect of environmental enrichment on fish basal stress level was considerably different in previous studies, i.e., positive (Barcellos et al., 2009; Cogliati et al., 2019; Näslund et al., 2013), negative (von Krogh et al., 2010) or no (Boerrigter et al., 2016; Madison et al., 2015; Rosengren et al., 2016; Wilkes et al., 2012) effects. Our results showed that enrichment type
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and amount may be key factors affecting fish physiological stress. As we discussed in our previous work, the reasons for the different basal stress levels could be the discrepancy of intraspecies aggression among treatments (Zhang et al., 2020a). Introducing enough objects into the water (represented by the medium- and high-amount enrichment groups) could provide more shelters, restrict the territorial range, obstruct visual contact, and consequently
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decrease aggression and the basal stress level (Näslund and Johnsson, 2016). In contrast,
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introducing few objects (represented by the low-amount enrichment group) may induce more aggression and higher cortisol levels since the object is now a resource for competition and
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not a shelter (Barley and Coleman, 2010). Obviously, in this study, the physical structures had
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more closed structure compared to plastic plants (Fig. 1), and this trait meant that they could
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divide the water space and territory and restrict visual range more efficiently. Therefore, the
enrichment groups.
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structure enrichment groups presented with lower basal cortisol levels than did the plant
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The peak value and recovery duration of cortisol are two commonly used parameters for evaluating fish stress responses. Because of the large discrepancies among fish species, enrichment design, stressor type, sampling time point and other experimental designs, previous studies often obtained sharply contrasting results and conclusions. For example, Barcellos et al. (2009) first reported that the whole-body cortisol concentration of stressed jundiá Rhamdia quelen recovered to the basal level within 12 h when they were reared in white tanks with shelter, while it needed 24 h when they were reared without shelter (Barcellos et al., 2009). Other studies also found enrichment appeared to blunt the hormone response in zebrafish Danio rerio (Giacomini et al., 2016), Atlantic salmon Salmo salar
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(Rosengren et al., 2016) and Chinook salmon Oncorhynchus tshawytscha (Cogliati et al., 2019) following a stressor. However, Boerrigter et al. (2016) reported that the endocrine response of African catfish Clarias gariepinus to air exposure was stronger when providing PVC-tubes under high-density conditions (Boerrigter et al., 2016), and a similar phenomenon was also found in other fish species (Batzina et al., 2014; Madison et al., 2015; Zubair et al.,
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2012). Moreover, there was one study that showed that enrichment had no effect on cortisol
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levels after confinement for 30 min in Atlantic salmon (Näslund et al., 2013). In the present study, the peak cortisol levels of the high-amount enrichment groups were significantly higher
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than that of the other three enrichment amount groups. The PM, PH and SL fish recovered to
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basal cortisol levels at 1 h, the C and PL fish recovered at 3 h, while the SM and SH fish
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recovered at 6 h after stress. A possible explanation is proposed to translate these novel
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results. For the C and PL treatments, the fish maintained a higher aggression and basal stress level (Zhang et al., 2020a), and such a chronically elevated cortisol level may damage the
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sensitivity of their cortisol response to acute stress. This phenomenon is called acclimation and/or desensitization (Barton, 2002; Gilmour et al., 2005; Øverli et al., 1999). For example, Barton et al. (2005) reported that mild but chronic stress (i.e., a high rearing density) could produce higher plasma cortisol levels (even though not significant) and consequently result in suppression of the fish physiological response and adaptive capacity following acute stress (Barton et al., 2005). For the SM and SH treatments, the fish might feel safe and protected, and maintain significantly lower basal cortisol levels. However, when they suffered from acute stress, the sharp contrast between the former comfortable and latter challenging environments might make them feel anxiety and stress, and consequently need more time to
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recover to basal status. For the PM, PH and SL treatments, the fish experienced moderate basal stress, which neither harmed their normal responsive ability to stress or put them into an extremely perplexed condition, and thus they could recover at a faster speed. The high correlations between both basal cortisol level and peak cortisol level and basal cortisol level and recovery time strengthened our above explanations (Fig. 6ab). Interestingly, the two
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fitting curves were different in their best fitting model (exponential fit vs. polynomial fit).
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With the increased basal cortisol level, the peak cortisol level decreased continuously while the recovery time first decreased and then increased, which was presented as a U-shaped
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curve. From this, we can infer that considering fish welfare, a moderate basal stress level
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fish normal physiological functions.
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should be adopted, and in this view, the medium-amount enrichment seemed to be optimal for
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The opercular beat rate is often used as a noninvasive indicator of fish stress level (Braithwaite and Salvanes, 2005; Pounder et al., 2016). In this study, the body size of the fish
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for measuring opercular beat rate was 6.74±0.06 cm (standard length) and 10.33±0.25 g (wet weight), and this fish size was large enough to observe the opercular/buccal movements. In normal conditions, the black rockfish is very sedentary. This behavioral laziness also allows us to observe their behaviors easily. Actually, the published papers all mainly used sedentary or large fish as their target species to count the opercular beat rate. In other words, this behavioral parameter is more suitable for sedentary/large fish (e.g., rainbow trout (Pounder et al., 2016), cod (Braithwaite and Salvanes, 2005)), but not active/small fish (e.g., zebrafish, guppy). In contrast, the swimming behavior or locomotor activity may be more proper for active fish, but not sedentary fish. In fact, except for opercular beat rate, another behavioral
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parameter (i.e., locomotor activity) was also determined in this study. However, the results showed no significant differences among treatments at either 0, 0.5, 1, 3, or 6 h after stress (Fig. A1). For each group, although the locomotor activity at 0.5 h after stress was significantly lower than that at other sampling time points, the fish from all treatment groups recovered to the basal level of locomotor activity at 1 h after stress (Fig. A2). These results
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indicated that the locomotor activity may be not a good parameter to evaluate the stress levels
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of black rockfish. The results of the opercular beat rate and locomotor activity may imply that the proper behavioral parameter for evaluating specific biological processes (in this study, the
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stress level) is closely related to fish species. Moreover, the special head features of the black
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rockfish (i.e., their head, opercular and mouth are large) allow us to detect the
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opercular/buccal beat rate relatively easier than in other species (Fig. A3). In brief, the body
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size, sedentary personality and morphological features make the observations and determinations of opercular beat rate possible in black rockfish. Our results showed that the
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main changing trend of the opercular beat rate was similar to the trend of the basal cortisol level (Fig. 5). Meanwhile, a high correlation between cortisol level and opercular beat rate was also observed (Fig. 6cd), which further reinforced our main results and conclusions. However, the opercular beat rate at specific sampling time points (i.e., 0.5, 3 and 6 h) after stress were not significantly different among treatments. This discrepancy indicated that this specific behavioral parameter could partly reflect fish stress status, but its sensitivity was lower than physiological parameters (e.g., cortisol for evaluating stress level). It is proper to combine two cohorts of parameters to assess fish stress. Furthermore, there is one thing we should particularly note: we only determined the intermediate 5 min of each video (to
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eliminate the possible disturbance for fish), but the initial minutes may be the most/more critical phase. It would, therefore, be interesting in future studies to include the initial behaviors in the analysis (maybe by continuously filming for 30/60 mins after stress).
Overall, the main results of the present study showed that enrichment type and enrichment
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amount had significant effects on fish basal stress level (indicated by cortisol level and
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opercular beat rate) and physiological and behavioral responses to acute stress (especially peak cortisol value and recovery time from stress). The medium- and high-amount enrichment
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groups had significantly lower basal stress levels, and meanwhile the high-amount enrichment
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groups had significantly higher peak cortisol values after acute stress. Thus, we recommend
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that it may be optimal to enrich with medium-amount objects (in this study, we quantified a
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medium amount as 50% floor space coverage) in the aquaculture industry. The PM, PH and SL fish had the fastest recovery speed among the treatments (showed in this study), and these
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three groups also presented significantly lower aggressive behavior and better growth performance (showed in Zhang et al., 2020a). Based on this finding, we suggest that a mixture of types of enrichment may have more benefits for fish physiology and behavior, considering physical structures and plastic plants each have their own superiorities. However, in this study, we did not evaluate a mixed enrichment treatment; therefore, future research needs to be conducted to validate our suggestion. In this work, we reared fish at the best rearing density, but considering the competitive effect of fish number, it would be interesting to explore the interaction effect of enrichment amount and rearing density. Furthermore, considering the current situation that most of the previous related research papers did not mention the
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enrichment type and amount, or even whether they provided environmental enrichment, we suggest that it is essential to clearly describe such details to increase experimental repeatability and enhance fish welfare.
Author Statement
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No conflict of interest exits in the submission of this manuscript, and manuscript is approved
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by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under
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Acknowledgements
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approved the manuscript that is enclosed.
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consideration for publication elsewhere, in whole or in part. All the authors listed have
This work was supported by funds from the National Natural Science Foundation of China
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(41676153; 31172447) and Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao) (2019-ZY-B02).
Declarations of interest None.
Supplementary data Supplementary material
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Mes D., van Os R., Gorissen M., Ebbesson L.O., Finstad B., Mayer I., Vindas M.A., 2019. Effects of environmental enrichment on forebrain neural plasticity and survival success of stocked Atlantic salmon. J. Exp. Biol. 222, jeb212258. 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. Näslund, J., Johnsson, J.I., 2016. Environmental enrichment for fish in captive environments: effects of physical structures and substrates. Fish Fish. 17, 1-30. Newberry, R.C., 1995. Environmental enrichment: increasing the biological relevance of
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2013. Environmental enrichment promotes neural plasticity and cognitive ability in fish. Proc. R. Soc. B. 280, 20131331.
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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. von Krogh, K., Sørensen, C., Nilsson, G.E., Øverli, Ø., 2010. Forebrain cell proliferation, behavior, and physiology of zebrafish, Danio rerio, kept in enriched or barren environments. Physiol. Behav. 101, 32-39. 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.
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Xi, D., Zhang, X., Lü, H., Zhang, Z., 2017. Cannibalism in juvenile black rockfish, Sebastes schlegelii (Hilgendorf, 1880), reared under controlled conditions. Aquaculture. 479, 682-689. 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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Zhang, Z., Bai, Q., Xu, X., Guo, H., Zhang, X., 2020a. Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii: Growth, behavior and
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physiology. Aquaculture. 518, 734782.
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Zhang, Z., Xu, X., Wang, Y., Zhang, X., 2020b. Effects of environmental enrichment on
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growth performance, aggressive behavior and stress-induced changes in cortisol release and
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substrate on the development of the cortisol stress response in the lake sturgeon, Acipenser fulvescens, Rafinesque (1817). Environ. Biol. Fish. 93, 577-587.
Table 1. The stress response of black rockfish S. schlegelii reared with different environments for eight weeks. Different letters indicate significant differences among the different sampling time points within the same treatment, which were detected by one-way ANOVA. Data are presented as the means ± S.E. (n = 3). Paramet
Treatme
Sampling time point (h after stress)
er
nt
0
0.5
1
3
6
value
0.81±0.01
5.32±0.09
3.37±0.05
0.86±0.02
0.85±0.01
<0.00
Cortisol
C c
a
b
c
c
1
0.85±0.03
5.29±0.05
3.57±0.15
0.83±0.03
0.86±0.03
<0.00
(ng/mg)
PL
P
c
a
b
c
c
1
0.74±0.01
5.45±0.11
0.79±0.02
0.77±0.01
0.76±0.03
<0.00
b
a
b
b
b
1
0.74±0.00
5.48±0.07
0.76±0.01
0.76±0.01
0.78±0.01
<0.00
b
a
b
b
b
1
0.74±0.03
5.64±0.07
0.79±0.01
0.76±0.03
0.77±0.02
<0.00
b
a
b
b
b
1
0.67±0.02
5.56±0.23
5.53±0.24
1.82±0.15
0.69±0.01
<0.00
c
a
a
b
c
1
0.67±0.02
6.19±0.18
5.83±0.15
2.25±0.10
0.69±0.02
<0.00
c
a
a
b
c
1
68.9±1.2c
90.2±1.9a
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63.6±2.8c
63.4±1.0c
78.8±1.3b
63.2±1.5c
PM
PH
SL
69.9±2.8c
91.7±3.1a
62.1±0.6b
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OBR PL (number PM
PH
SL
62.2±1.5b
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per time)
SM
86.7±1.9a
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of beats
61.8±1.0b
60.6±3.0c
78.9±0.6b
re
C
ro
SH
of
SM
89.0±2.2a
<0.00 1 <0.00 63.3±1.4c 1 <0.00
64.9±2.0b
61.0±2.5b
62.8±2.2b 1 <0.00
66.7±1.1b
63.5±3.5b
62.7±2.5b 1 <0.00
84.2±2.3a
62.2±1.2b
59.9±3.5b
60.4±3.4b 1 <0.00
85.9±3.9a
83.2±0.7a
70.4±3.5b
59.9±3.1c 1 <0.00
SH
59.3±2.5c
86.0±2.1a
82.3±0.6a
68.0±1.6b
60.5±3.2c 1
C: control; PL: enriched with low-amount plants; PM: enriched with medium-amount plants; PH: enriched with high-amount plants; SL: enriched with low-amount structures; SM: enriched with medium-amount structures; SH: enriched with high-amount structures; OBR: opercular beat rate.
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Fig. 1. Picture of plastic plant and physical structure used as shelter used for enrichment. Fig. 2. Basal cortisol level and opercular beat rate of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Basal cortisol level; (b) Basal opercular beat rate. Different letters indicate significant differences among enrichment amount groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are presented as the means ± S.E. (n = 3).
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Fig. 3. Cortisol level of black rockfish S. schlegelii during (a) 0.5 h, (b) 1 h, (c) 3 h and (d) 6
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h after stress. Different letters indicate significant differences among enrichment amount
presented as the means ± S.E. (n = 3).
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groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are
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Fig. 4. Opercular beat rate of black rockfish S. schlegelii during (a) 0.5 h, (b) 1 h, (c) 3 h and (d) 6 h after stress. Different letters indicate significant differences among enrichment amount
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groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are presented as the means ± S.E. (n = 3).
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Fig. 5. Recovery time course from stress in black rockfish S. schlegelii reared in different environments for eight weeks. (a) Cortisol level; (b) Opercular beat rate. Data are presented
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as the means ± S.E. (n = 3).
Fig. 6. Regression curves of relationships among physiology, behavior and recovery ability based on pooled data. (a) Exponential fit of basal cortisol level and peak cortisol level (n = 21); (b) Polynomial fit of basal cortisol level and recovery time from stress (n = 21); (c) Linear fit of basal cortisol level and basal opercular beat rate (n = 21); (4) Linear fit of stressed cortisol level and stressed opercular beat rate (n = 84).
Highlights 1. Control group and low-amount enrichment groups had significantly higher basal stress than medium- and high-amount enrichment groups.
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2. Peak cortisol levels of high-amount enrichment groups were significantly higher than that of other enrichment amount groups. 3. Recovery time after stress of medium-amount plant, high-amount plant and low-amount structure fish were the shortest among treatments. 4. Basal cortisol level showed high correlations with peak cortisol level and recovery
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time.
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na
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re
-p
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5. Correlation between cortisol level and opercular beat rate was high.
Zonghang Zhang, Yiqiu Fu, Haoyu Guo, Xiumei Zhang PII:
S0044-8486(20)30555-X
DOI:
https://doi.org/10.1016/j.aquaculture.2020.736088
Reference:
AQUA 736088
To appear in:
Aquaculture
Received date:
20 February 2020
Revised date:
19 August 2020
Accepted date:
19 October 2020
Please cite this article as: Z. Zhang, Y. Fu, H. Guo, et al., Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii, Aquaculture (2018), https://doi.org/10.1016/j.aquaculture.2020.736088
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© 2018 Published by Elsevier.
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Effect of environmental enrichment on the stress response of juvenile black rockfish Sebastes schlegelii
Zonghang Zhanga, Yiqiu Fua, Haoyu Guob,c, Xiumei Zhangb,c,* [email protected] a
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China,
Qingdao 266003, China
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b
c
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Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National
Corresponding author at: Fisheries College, Zhejiang Ocean University, No.1, Haida South
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*
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Laboratory for Marine Science and Technology, Qingdao 266237, China
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Road, Lincheng Changzhi Island, Zhoushan, China
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Abstract
The stress response is one of the most important aspects of fish welfare in aquaculture. However, relatively few studies focusing on the effect of environmental enrichment on the fish stress response have been conducted, and their limited results were considerably contradictory. The present study aimed to investigate whether the enrichment type and amount had significant effects on the basal stress level and physiological and behavioral responses to acute stress of juvenile black rockfish Sebastes schlegelii. Fish were reared for eight weeks in environments with two enrichment types and three enrichment amounts (i.e., no
environmental
enrichment
control
(C),
low-amount
plant
enrichment
(PL),
medium-amount plant enrichment (PM), high-amount plant enrichment (PH), low-amount
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structure enrichment (SL), medium-amount structure enrichment (SM), and high-amount structure enrichment (SH)), and subsequently, they were successively subjected to two common acute stressors (i.e., air exposure and confinement) and sampled at 0, 0.5, 1, 3 and 6 h after stress. In general, the plant enrichment groups experienced significantly higher basal stress (indicated by cortisol level and opercular beat rate) than the structure enrichment
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groups. Meanwhile, no enrichment control group and low-amount enrichment groups
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experienced significantly higher basal stress than the other two enrichment amount groups. After being subjected to acute stress, the peak cortisol levels of the high-amount enrichment
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groups were significantly higher than that of the other three enrichment amount groups. The
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PM, PH and SL fish recovered to basal stress levels at 1 h, the C and PL fish recovered at 3 h,
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and the SM and SH fish recovered at 6 h after the stress. Moreover, their basal cortisol levels
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showed strong correlations with peak cortisol levels and recovery time from stress. The correlation between cortisol level and opercular beat rate was also strong. Taken together,
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these results provide the first evidence to show that enrichment type and amount had significant effects on the fish stress response and might have important applications in fish husbandry and welfare. Based on these results, we suggest that it may be optimal for fish basal stress level and development of an adaptive capacity to provide a medium-amount (approximately 50% floor space coverage) of mixed enrichment in aquaculture.
Keywords environmental enrichment; stress response; cortisol; opercular beat rate; Sebastes schlegelii
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1. Introduction Fish in aquaculture are often subjected to several potential stressors. For example, an unnatural high rearing density, sudden changes of the water level, transport, sorting, handling, machine noises and intraspecies aggression (Ashley, 2007; Galhardo and Oliveira, 2009). For fish that are destined to be released into the wild (e.g., stock enhancement and conservation
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programs), they will inevitably experience more natural, unpredictable and heterogeneous
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environments after release, including intraspecies competition, predator pressure and variable environmental factors (Johnsson et al., 2014). These stressors from the aquatic environment
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may induce a series of physiological and behavioral responses, which depend on the fish to
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have the ability to adapt to the specific situation (Ashley, 2007; Galhardo and Oliveira, 2009).
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In general, the fish stress response can be divided into three stages: first, a
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hypothalamus-pituitary-interrenal (HPI) axis hormone response is induced, the main end product of which is cortisol; second, many changes in the blood, tissue and metabolism are
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triggered; and third, the performances at the whole-body level is altered, such as behavioral patterns, survival and growth performance (Barton, 2002; Galhardo and Oliveira, 2009). When stress exceeds the fish adaptive capacity, they have an increased energy expenditure, modifications of normal physiological processes and behavioral phenotypes, lower fish welfare, decreased growth performance and adverse impacts on fish reproduction and survival (Ashley, 2007; Barton, 2002; Huntingford et al., 2006). Thus, how to properly decrease the stress response of fish is a particularly important question in aquaculture industry. Environmental enrichment, namely, increasing the environmental complexity by introducing objects (e.g., plants, physical structures) into rearing water (Zhang et al., 2019;
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Zhang et al., 2020a; Zhang et al., 2020b), is believed to be one of the most useful ways to regulate the physiological status, alter behavioral performance and consequently enhance fish welfare (Näslund and Johnsson, 2016; Newberry, 1995). Many recent studies have verified that environmental enrichment could enhance the fish growth rate and neural plasticity (Batzina and Karakatsouli, 2012; Camara-Ruiz et al., 2019; Salvanes et al., 2013), decrease
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aggressive and anxiety-like behaviors (Barcellos et al., 2018; Batzina and Karakatsouli, 2012;
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Zhang et al., 2020a), and promote survival-related behaviors of the released fish (Braithwaite and Salvanes, 2005; D'Anna et al., 2012; Mes et al., 2019; Ullah et al., 2017; Zhang et al.,
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2019). However, studies on the effect of enrichment on the fish stress response are scarce, and
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the limited results of different studies are sharply contradictory. The studies variably showed
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positive (Barcellos et al., 2009; Batzina et al., 2014; Cogliati et al., 2019; Marcon et al., 2018),
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negative (Boerrigter et al., 2016; Madison et al., 2015; Zubair et al., 2012) or no (Näslund et al., 2013) effects. These large differences may be related to many factors, for example, fish
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species and populations, enrichment types and amounts and other experimental designs. However, scarce studies about the effects of enrichment type and amount, especially the interaction of the two factors, on the fish stress response have been conducted, and the question of which specific enrichment type and amount is optimal for fish needs to be answered. Black rockfish Sebastes schlegelii is widely distributed in the coastal areas of China, Korea and Japan (Xi et al., 2017). In recent years, the aquaculture of black rockfish has been quickly prosperous, especially in northern China, for the main purpose of food supply and stock enhancement (Guo et al., 2017). The traditional rearing environments and production
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processes in the aquaculture industry usually involve many harmful stressors. For example, fish typically experience sorting and transport, and these operations inevitably put fish in the air for a period of time, and the new environment very likely differs from the previous environment in water depth, rearing density, tank size, social environment, and other factors (which can be categorized as confinement). Moreover, the typical rearing tanks usually do not
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contain any environmental modifications. These husbandry characteristics may increase the
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fish basal stress level and damage their normal adaptive ability to the environment (Zhang et al., 2020a). In this study, we aimed to determine how enrichment type and amount affected
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the stress response of juvenile black rockfish and to verify whether enrichment type and
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amount are critical factors in enrichment design. Two commonly occurring stressors in
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aquaculture production (air exposure and confinement) were adopted as acute stress. The core
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hormone of the HPI axis (cortisol) and a commonly used behavioral indicator (the opercular
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beat rate) were used to assess the fish stress level.
2. Materials and methods
We have read the policies related to animal experiments (the ARRIVE and PREPARE guidelines) and have confirmed this study complied. All procedures conducted in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Ocean University of China. 2.1. Fish husbandry The experimental animal and fish husbandry were described in detail in our previous work (Zhang et al., 2020a). In brief, eight hundred and forty black rockfish S. schlegelii juveniles
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(mean wet weight 3.08±0.05 g, mean standard length 4.69±0.03 cm) were randomly distributed in 21 glass tanks (60 cm × 50 cm × 50 cm) in groups of 40 (initial rearing density 1.03±0.02 kg/m3) in seven triplicated treatments. The treatment groups were no environmental enrichment control (C), low-amount enrichment with plastic plants (PL), medium-amount enrichment with plastic plants (PM), high-amount enrichment with plastic
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plants (PH), low-amount enrichment with physical structures (SL), medium-amount
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enrichment with physical structures (SM) and high-amount enrichment with physical structures (SH). In the enriched tanks, different types and amounts of objects were introduced
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as shelters (von Krogh et al., 2010; Xi et al., 2017), while the control tanks had no objects.
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Enrichment types included plastic plants (height 10 cm, projected area approximately 8 cm ×
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9 cm = 72 cm2; Fig. 1) and physical structures (height approximately 10 cm, floor space 12
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cm × 6 cm = 72 cm2; Fig. 1). For enrichment amount, 10, 20 and 30 objects corresponded to low-, medium- and high-amount enrichment, respectively. The experimental tanks were part
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of an indoor flow-through seawater system. The water flow rate was 3.5 L/min (exchange rate 4,200%) and the water depth was maintained at 40 cm. The fish were acclimated to the glass tanks for three weeks before the formal experiment, and subsequently they were maintained under experimental conditions for eight weeks. The fish were fed commercial floating pellets (Kaido Brand, Santong Bioengineering Co. Ltd., Anqiu, China) by hand once daily at a restricted feeding rate (2.5% fish body weight daily), since this feeding strategy was proposed to be proper for the growth, physiology and welfare of juvenile rockfish (Guo et al., 2017; Zhang et al., 2020a). All tanks, plastic plants and physical structures were cleaned once weekly. The photoperiod followed the natural day-light cycle. Water temperature
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(25.9±0.18°C), dissolved oxygen (6.68±0.04 mg/L; 97.43±0.25% saturation), salinity (29.73±0.16‰), pH (7.12±0.02), total ammonia-nitrogen (0.236±0.0138 mg/L) and nitrite-nitrogen (0.012±0.0005 mg/L) were continuously monitored. No fish died during the rearing period. 2.2 Experimental design
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After the eight-week experimental period, the fish were subjected to standard stress response
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trials. In this study, we used air exposure (Boerrigter et al., 2016; Madison et al., 2015; Pounder et al., 2016) and confinement (Marcon et al., 2018; Näslund et al., 2013) as stressors,
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which are common methods used in such trials and do not cause any physical harm to the fish.
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Twenty four fish from each tank were quickly netted and subjected to one-minute air
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emersion by holding them in a plastic basin, and subsequently these fish were randomly and
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equally distributed into four well-circulated small glass tanks (40 cm × 30 cm × 30 cm; water level was maintained at 20 cm; six cobbles (diameter 3 cm) and one air stone were introduced
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into each tank) to be allowed to recover from the stress. After 0.5, 1, 3 and 6 h, six fish from one of the four small tanks were sampled, euthanized with an overdose of anesthetic (tricaine methanesulfonate, MS-222, 100 mg/L), weighed (precision 0.01 g) and measured (precision 0.01 cm), and then three visceral masses (due to fish were too small to draw blood) were extracted for measuring the cortisol level (Guo et al., 2017). All sampling procedures were completed within one minute. Ten minutes before each sampling time point (i.e., 0.5, 1, 3 and 6 h after the stress), one 10 min video was filmed by a camera positioned in front of the small tank to determine the behavioral response. Additionally, two days before the stress response trials, one 10 min video of each rearing tank was also filmed (approximately 14:00-15:00),
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and three fish of each tank were randomly sampled and dissected to determine basal behavioral and physiological stress levels (Zhang et al., 2020a). All samples were frozen in liquid nitrogen, and then stored at -80°C until analysis. The remaining fish in the rearing glass tanks were used in another experiment (Zhang et al., 2020a). 2.3. Behavioral study
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In this study, we used the opercular beat rate to evaluate the behavioral response to stress,
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which has been verified to be a valid indicator in other fish species (Braithwaite and Salvanes, 2005; Pounder et al., 2016). Before starting filming, several digital video cameras
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(HDR-AS100V and HDR-XR550, Sony Corporation, Tokyo, Japan; Honor 6X, HuaWei
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Technologies Co. Ltd., Shenzhen, China; iPad Air 2, iPad mini 2, and iPhone 6s plus, Apple
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Incorporation, Cupertino, USA) were fixed along the front side of each tank simultaneously,
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and the distance between tank and camera was approximately 30 cm. Then, the experimenters left the fish, and a 10 min video was filmed. Approximately 10 min later, the experimenters
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came back and turned off the cameras. To eliminate the possible disturbance of the observed fish, the first 3 min and last 2 min of each 10 min video were cut off, and only the middle 5 min was analyzed. The opercular beat rate was measured as the number of opercular beats per minute (Pounder et al., 2016). The observer who quantified the fish behavior was blind to the treatments. 2.4. Cortisol level determination The visceral mass samples were homogenized in cold PBS (9×weight, pH 7.4) and centrifuged at 3000 rpm for 20 minutes in a refrigerated centrifuge (4°C). The supernatants were collected and used for analysis. A commercial Elisa Assay Kit (H094-1-2, Nanjing
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Jiancheng Bioengineering Institute, Nanjing, China), which was proved to have good validation (Zhang et al., 2019; Zhang et al., 2020a; Zhang et al., 2020b), was used to measure the cortisol concentrations according to the manufacturer’s guidelines (for more details about the experimental principle and detailed procedures of this cortisol determination kit, see the Supplementary Material). The intra-assay coefficient of variation was less than 10% and high
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correlations were found between the standard curves (R2 > 0.99).
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2.5. Data analysis
First, we duplicated the data from the control treatment to yield data representative of two
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treatments: no plant enrichment and no structure enrichment. This allowed us to simulate a 2
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(enrichment type: plant and structure) × 4 (enrichment amount: no, low, medium and high)
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factorial design. Then, the main and interaction effects of the two factors were tested by
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two-way ANOVA followed by Duncan’s multiple-range post hoc test. Normality of the data was tested with Shapiro-Wilk’s test and homogeneity of variance of the data was tested with
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Levene’s test prior to ANOVA. To determine the recovery time from stress, one-way ANOVA followed by Duncan’s multiple-range post hoc test were used to detect the differences of cortisol level or opercular beat rate among the five time points (i.e., 0, 0.5, 1, 3 and 6 h after stress) within each treatment (i.e., C, PL, PM, PH, SL, SM or SH treatment). To clarify the relationships between the basal stress level and the stressed status or cortisol level and opercular beat rate, several fitting methods (polynomial, exponential and linear) were performed with the pooled data, and the best fitting curve is presented in the figures. For all parameters in this study, the whole tank was regarded as the statistical unit. All statistical analyses were made using SPSS 17.0 for windows. Differences were considered to be
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significant at a probability level of 0.05. All values in the text and figures were presented as the means ± S.E.
3. Results 3.1. Basal stress level
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This was the second of two complementary studies designed to explore how enrichment
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type and amount affected the growth performance, behavioral phenotype and physiological status of black rockfish. Here, we mainly present the results of the fish stress response,
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whereas the growth data could be found in the companion paper (Zhang et al., 2020a).
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Two-way ANOVA showed that significant main and interaction effects of enrichment type
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and enrichment amount on basal cortisol level were observed (Fig. 2a). Overall, the plant
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enrichment groups had a significantly higher basal cortisol level than did the structure enrichment groups, and meanwhile, the no enrichment control group and the low-amount
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enrichment groups had significantly higher basal cortisol levels than did the other two enrichment amount groups (Fig. 2a). The trend of the basal opercular beat rate was similar to the trend of the basal cortisol level (Fig. 2b). 3.2. Physiological and behavioral responses to stress Peak stress levels of all treatments occurred at 0.5 h after stress (Table 1; Fig. 5). At this time point, the cortisol levels of the plant enrichment groups were significantly lower compared to that of the structure enrichment groups, and the high-amount enrichment groups produced the highest cortisol levels among the four enrichment level groups (Fig. 3a). At 1 h after stress, the no enrichment control group had the highest cortisol level, while at 3 h after
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stress, the high-amount enrichment groups had the highest cortisol levels among the four enrichment amount groups (Fig. 3bc). At 6 h after stress, the plant enrichment groups produced significantly higher cortisol levels than did the structure enrichment groups. Meanwhile, the no enrichment control group and the low-amount enrichment groups had significantly higher cortisol levels than did the other two enrichment amount groups (Fig. 3d),
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which was similar to the trend in basal cortisol levels (Fig. 2a). For behavioral responses, the
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changing trend of the opercular beat rate at 1 h after stress was similar to that of the cortisol
3.3. Time course of recovery from stress
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points (i.e., 0.5, 3 and 6 h after stress) (Fig. 4).
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level, but no significant main or interaction effects were detected at the other sampling time
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Although all of the treatments had the same peak time point (0.5 h after stress), the recovery
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time points were different (Table 1; Fig. 5). In the C and PL fish, the cortisol levels declined to values still significantly higher than the basal levels at 1 h and recovered to basal levels at 3
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h after stress. In the PM, PH and SL fish, cortisol levels dropped to basal levels at 1 h after stress. In the SM and SH fish, cortisol levels remained elevated at 1 h, declined to values that were significantly lower than peak levels while still significantly higher than basal levels at 3 h and recovered to basal levels at 6 h after stress (Table 1; Fig. 5a). The trend of the opercular beat rate was similar to that of the cortisol level (Table 1; Fig. 5b). 3.4. Relationships among physiology, behavior and recovery ability Regardless of enrichment treatment and sampling time point, the correlations between basal cortisol level and peak cortisol level; basal cortisol level and recovery time from stress; basal cortisol level and basal opercular beat rate; and stressed cortisol level and stressed opercular
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beat rate, were very high (Fig. 6). However, the best fitting model differed among the above relationships. Exponential fit and polynomial fit were the best for basal cortisol level and peak cortisol level (Fig. 6a), and basal cortisol level and recovery time (Fig. 6b), respectively. Linear fits provided the best fit for the relationships between basal cortisol level and basal opercular beat rate, and between stressed cortisol level and stressed opercular beat rate (Fig.
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6cd).
4. Discussion
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The main results of this study showed that: 1) Plant enrichment induced significantly higher
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basal stress (indicated by the cortisol level and opercular beat rate) than did structure
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enrichment. Meanwhile, the no enrichment control group and low-amount enrichment groups
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experienced significantly higher basal stress than did the other two enrichment amount groups. 2) After being subjected to acute stress, the peak cortisol levels of the high-amount
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enrichment groups were significantly higher than that of the other three enrichment amount groups. 3) The PM, PH and SL fish recovered to their basal stress level at 1 h, the C and PL fish recovered at 3 h, while the SM and SH fish recovered at 6 h after stress. 4) The basal cortisol levels showed high correlations with peak cortisol levels and recovery time. Meanwhile, the correlation between cortisol level and opercular beat rate was also high. The effect of environmental enrichment on fish basal stress level was considerably different in previous studies, i.e., positive (Barcellos et al., 2009; Cogliati et al., 2019; Näslund et al., 2013), negative (von Krogh et al., 2010) or no (Boerrigter et al., 2016; Madison et al., 2015; Rosengren et al., 2016; Wilkes et al., 2012) effects. Our results showed that enrichment type
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and amount may be key factors affecting fish physiological stress. As we discussed in our previous work, the reasons for the different basal stress levels could be the discrepancy of intraspecies aggression among treatments (Zhang et al., 2020a). Introducing enough objects into the water (represented by the medium- and high-amount enrichment groups) could provide more shelters, restrict the territorial range, obstruct visual contact, and consequently
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decrease aggression and the basal stress level (Näslund and Johnsson, 2016). In contrast,
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introducing few objects (represented by the low-amount enrichment group) may induce more aggression and higher cortisol levels since the object is now a resource for competition and
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not a shelter (Barley and Coleman, 2010). Obviously, in this study, the physical structures had
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more closed structure compared to plastic plants (Fig. 1), and this trait meant that they could
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divide the water space and territory and restrict visual range more efficiently. Therefore, the
enrichment groups.
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structure enrichment groups presented with lower basal cortisol levels than did the plant
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The peak value and recovery duration of cortisol are two commonly used parameters for evaluating fish stress responses. Because of the large discrepancies among fish species, enrichment design, stressor type, sampling time point and other experimental designs, previous studies often obtained sharply contrasting results and conclusions. For example, Barcellos et al. (2009) first reported that the whole-body cortisol concentration of stressed jundiá Rhamdia quelen recovered to the basal level within 12 h when they were reared in white tanks with shelter, while it needed 24 h when they were reared without shelter (Barcellos et al., 2009). Other studies also found enrichment appeared to blunt the hormone response in zebrafish Danio rerio (Giacomini et al., 2016), Atlantic salmon Salmo salar
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(Rosengren et al., 2016) and Chinook salmon Oncorhynchus tshawytscha (Cogliati et al., 2019) following a stressor. However, Boerrigter et al. (2016) reported that the endocrine response of African catfish Clarias gariepinus to air exposure was stronger when providing PVC-tubes under high-density conditions (Boerrigter et al., 2016), and a similar phenomenon was also found in other fish species (Batzina et al., 2014; Madison et al., 2015; Zubair et al.,
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2012). Moreover, there was one study that showed that enrichment had no effect on cortisol
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levels after confinement for 30 min in Atlantic salmon (Näslund et al., 2013). In the present study, the peak cortisol levels of the high-amount enrichment groups were significantly higher
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than that of the other three enrichment amount groups. The PM, PH and SL fish recovered to
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basal cortisol levels at 1 h, the C and PL fish recovered at 3 h, while the SM and SH fish
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recovered at 6 h after stress. A possible explanation is proposed to translate these novel
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results. For the C and PL treatments, the fish maintained a higher aggression and basal stress level (Zhang et al., 2020a), and such a chronically elevated cortisol level may damage the
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sensitivity of their cortisol response to acute stress. This phenomenon is called acclimation and/or desensitization (Barton, 2002; Gilmour et al., 2005; Øverli et al., 1999). For example, Barton et al. (2005) reported that mild but chronic stress (i.e., a high rearing density) could produce higher plasma cortisol levels (even though not significant) and consequently result in suppression of the fish physiological response and adaptive capacity following acute stress (Barton et al., 2005). For the SM and SH treatments, the fish might feel safe and protected, and maintain significantly lower basal cortisol levels. However, when they suffered from acute stress, the sharp contrast between the former comfortable and latter challenging environments might make them feel anxiety and stress, and consequently need more time to
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recover to basal status. For the PM, PH and SL treatments, the fish experienced moderate basal stress, which neither harmed their normal responsive ability to stress or put them into an extremely perplexed condition, and thus they could recover at a faster speed. The high correlations between both basal cortisol level and peak cortisol level and basal cortisol level and recovery time strengthened our above explanations (Fig. 6ab). Interestingly, the two
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fitting curves were different in their best fitting model (exponential fit vs. polynomial fit).
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With the increased basal cortisol level, the peak cortisol level decreased continuously while the recovery time first decreased and then increased, which was presented as a U-shaped
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curve. From this, we can infer that considering fish welfare, a moderate basal stress level
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fish normal physiological functions.
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should be adopted, and in this view, the medium-amount enrichment seemed to be optimal for
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The opercular beat rate is often used as a noninvasive indicator of fish stress level (Braithwaite and Salvanes, 2005; Pounder et al., 2016). In this study, the body size of the fish
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for measuring opercular beat rate was 6.74±0.06 cm (standard length) and 10.33±0.25 g (wet weight), and this fish size was large enough to observe the opercular/buccal movements. In normal conditions, the black rockfish is very sedentary. This behavioral laziness also allows us to observe their behaviors easily. Actually, the published papers all mainly used sedentary or large fish as their target species to count the opercular beat rate. In other words, this behavioral parameter is more suitable for sedentary/large fish (e.g., rainbow trout (Pounder et al., 2016), cod (Braithwaite and Salvanes, 2005)), but not active/small fish (e.g., zebrafish, guppy). In contrast, the swimming behavior or locomotor activity may be more proper for active fish, but not sedentary fish. In fact, except for opercular beat rate, another behavioral
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parameter (i.e., locomotor activity) was also determined in this study. However, the results showed no significant differences among treatments at either 0, 0.5, 1, 3, or 6 h after stress (Fig. A1). For each group, although the locomotor activity at 0.5 h after stress was significantly lower than that at other sampling time points, the fish from all treatment groups recovered to the basal level of locomotor activity at 1 h after stress (Fig. A2). These results
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indicated that the locomotor activity may be not a good parameter to evaluate the stress levels
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of black rockfish. The results of the opercular beat rate and locomotor activity may imply that the proper behavioral parameter for evaluating specific biological processes (in this study, the
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stress level) is closely related to fish species. Moreover, the special head features of the black
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rockfish (i.e., their head, opercular and mouth are large) allow us to detect the
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opercular/buccal beat rate relatively easier than in other species (Fig. A3). In brief, the body
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size, sedentary personality and morphological features make the observations and determinations of opercular beat rate possible in black rockfish. Our results showed that the
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main changing trend of the opercular beat rate was similar to the trend of the basal cortisol level (Fig. 5). Meanwhile, a high correlation between cortisol level and opercular beat rate was also observed (Fig. 6cd), which further reinforced our main results and conclusions. However, the opercular beat rate at specific sampling time points (i.e., 0.5, 3 and 6 h) after stress were not significantly different among treatments. This discrepancy indicated that this specific behavioral parameter could partly reflect fish stress status, but its sensitivity was lower than physiological parameters (e.g., cortisol for evaluating stress level). It is proper to combine two cohorts of parameters to assess fish stress. Furthermore, there is one thing we should particularly note: we only determined the intermediate 5 min of each video (to
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eliminate the possible disturbance for fish), but the initial minutes may be the most/more critical phase. It would, therefore, be interesting in future studies to include the initial behaviors in the analysis (maybe by continuously filming for 30/60 mins after stress).
Overall, the main results of the present study showed that enrichment type and enrichment
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amount had significant effects on fish basal stress level (indicated by cortisol level and
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opercular beat rate) and physiological and behavioral responses to acute stress (especially peak cortisol value and recovery time from stress). The medium- and high-amount enrichment
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groups had significantly lower basal stress levels, and meanwhile the high-amount enrichment
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groups had significantly higher peak cortisol values after acute stress. Thus, we recommend
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that it may be optimal to enrich with medium-amount objects (in this study, we quantified a
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medium amount as 50% floor space coverage) in the aquaculture industry. The PM, PH and SL fish had the fastest recovery speed among the treatments (showed in this study), and these
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three groups also presented significantly lower aggressive behavior and better growth performance (showed in Zhang et al., 2020a). Based on this finding, we suggest that a mixture of types of enrichment may have more benefits for fish physiology and behavior, considering physical structures and plastic plants each have their own superiorities. However, in this study, we did not evaluate a mixed enrichment treatment; therefore, future research needs to be conducted to validate our suggestion. In this work, we reared fish at the best rearing density, but considering the competitive effect of fish number, it would be interesting to explore the interaction effect of enrichment amount and rearing density. Furthermore, considering the current situation that most of the previous related research papers did not mention the
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enrichment type and amount, or even whether they provided environmental enrichment, we suggest that it is essential to clearly describe such details to increase experimental repeatability and enhance fish welfare.
Author Statement
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No conflict of interest exits in the submission of this manuscript, and manuscript is approved
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by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under
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Acknowledgements
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approved the manuscript that is enclosed.
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consideration for publication elsewhere, in whole or in part. All the authors listed have
This work was supported by funds from the National Natural Science Foundation of China
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(41676153; 31172447) and Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao) (2019-ZY-B02).
Declarations of interest None.
Supplementary data Supplementary material
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Table 1. The stress response of black rockfish S. schlegelii reared with different environments for eight weeks. Different letters indicate significant differences among the different sampling time points within the same treatment, which were detected by one-way ANOVA. Data are presented as the means ± S.E. (n = 3). Paramet
Treatme
Sampling time point (h after stress)
er
nt
0
0.5
1
3
6
value
0.81±0.01
5.32±0.09
3.37±0.05
0.86±0.02
0.85±0.01
<0.00
Cortisol
C c
a
b
c
c
1
0.85±0.03
5.29±0.05
3.57±0.15
0.83±0.03
0.86±0.03
<0.00
(ng/mg)
PL
P
c
a
b
c
c
1
0.74±0.01
5.45±0.11
0.79±0.02
0.77±0.01
0.76±0.03
<0.00
b
a
b
b
b
1
0.74±0.00
5.48±0.07
0.76±0.01
0.76±0.01
0.78±0.01
<0.00
b
a
b
b
b
1
0.74±0.03
5.64±0.07
0.79±0.01
0.76±0.03
0.77±0.02
<0.00
b
a
b
b
b
1
0.67±0.02
5.56±0.23
5.53±0.24
1.82±0.15
0.69±0.01
<0.00
c
a
a
b
c
1
0.67±0.02
6.19±0.18
5.83±0.15
2.25±0.10
0.69±0.02
<0.00
c
a
a
b
c
1
68.9±1.2c
90.2±1.9a
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63.6±2.8c
63.4±1.0c
78.8±1.3b
63.2±1.5c
PM
PH
SL
69.9±2.8c
91.7±3.1a
62.1±0.6b
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OBR PL (number PM
PH
SL
62.2±1.5b
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per time)
SM
86.7±1.9a
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of beats
61.8±1.0b
60.6±3.0c
78.9±0.6b
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C
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SH
of
SM
89.0±2.2a
<0.00 1 <0.00 63.3±1.4c 1 <0.00
64.9±2.0b
61.0±2.5b
62.8±2.2b 1 <0.00
66.7±1.1b
63.5±3.5b
62.7±2.5b 1 <0.00
84.2±2.3a
62.2±1.2b
59.9±3.5b
60.4±3.4b 1 <0.00
85.9±3.9a
83.2±0.7a
70.4±3.5b
59.9±3.1c 1 <0.00
SH
59.3±2.5c
86.0±2.1a
82.3±0.6a
68.0±1.6b
60.5±3.2c 1
C: control; PL: enriched with low-amount plants; PM: enriched with medium-amount plants; PH: enriched with high-amount plants; SL: enriched with low-amount structures; SM: enriched with medium-amount structures; SH: enriched with high-amount structures; OBR: opercular beat rate.
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Fig. 1. Picture of plastic plant and physical structure used as shelter used for enrichment. Fig. 2. Basal cortisol level and opercular beat rate of black rockfish S. schlegelii reared in different environments for eight weeks. (a) Basal cortisol level; (b) Basal opercular beat rate. Different letters indicate significant differences among enrichment amount groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are presented as the means ± S.E. (n = 3).
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Fig. 3. Cortisol level of black rockfish S. schlegelii during (a) 0.5 h, (b) 1 h, (c) 3 h and (d) 6
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h after stress. Different letters indicate significant differences among enrichment amount
presented as the means ± S.E. (n = 3).
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groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are
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Fig. 4. Opercular beat rate of black rockfish S. schlegelii during (a) 0.5 h, (b) 1 h, (c) 3 h and (d) 6 h after stress. Different letters indicate significant differences among enrichment amount
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groups, which were detected by Duncan’s multiple-range post hoc test (P < 0.05). Data are presented as the means ± S.E. (n = 3).
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Fig. 5. Recovery time course from stress in black rockfish S. schlegelii reared in different environments for eight weeks. (a) Cortisol level; (b) Opercular beat rate. Data are presented
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as the means ± S.E. (n = 3).
Fig. 6. Regression curves of relationships among physiology, behavior and recovery ability based on pooled data. (a) Exponential fit of basal cortisol level and peak cortisol level (n = 21); (b) Polynomial fit of basal cortisol level and recovery time from stress (n = 21); (c) Linear fit of basal cortisol level and basal opercular beat rate (n = 21); (4) Linear fit of stressed cortisol level and stressed opercular beat rate (n = 84).
Highlights 1. Control group and low-amount enrichment groups had significantly higher basal stress than medium- and high-amount enrichment groups.
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2. Peak cortisol levels of high-amount enrichment groups were significantly higher than that of other enrichment amount groups. 3. Recovery time after stress of medium-amount plant, high-amount plant and low-amount structure fish were the shortest among treatments. 4. Basal cortisol level showed high correlations with peak cortisol level and recovery
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time.
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5. Correlation between cortisol level and opercular beat rate was high.