2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China;
3 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China;
4 College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China
Atlantic salmon (Salmo salar) is one of the most economically important fish utilized for aquaculture, which were introduced into China using RAS in 2010 due to their high nutritional and economic value. However, Atlantic salmon reared in RAS experience smaller size when compared to fish reared in sea cage. In order to pursuit more economic value, the stocking density of fish reared in RAS is higher than sea cage. High stocking density may lead to poor water quality, furthermore, high stocking density can result in heterogeneous growth rates, final weights and feed intakes (Schram et al., 2006; Yarahmadi et al., 2016). RAS is a relatively close-system, which is characterized by product safely, environmental friendliness and resource efficiency (Martins et al., 2010). The breeding space of RAS is limited, so the fish need to remove to more ponds from time to time to reduce the density. However, the optical density of Atlantic salmon in RAS is still in doubt.
Stocking density is usually considered as an important factor that can determine the productivity of Atlantic salmon (Liu et al., 2015). Crowded Atlantic salmon rearing condition is considered a chronic stressor that can impede fish growth and lead to elevate stress of physiology (Montero et al., 1999). The increased stocking density also could induce stress response, which is harmful for fish health (Ellis et al., 2002). Stressful situations affected both physiology and immunity, and cause lower growth rate and higher incidence of fish diseases (Tort, 2011; Nardocci et al., 2014). Furthermore, the hormone, chemical elements might accumulate with increasing density in RAS. Among chemical elements, cortisol plays an essential role in maintenance of growth, immunity and energy balance, when teleost are exposed to exogenous stresses (Hori et al., 2010). Cortisol is also widely considered as a biomarker of stress to fish. In many fish such as Senegalese sole (Solea senegalensis), rainbow trout, high stocking density could increase plasma cortisol significantly (Laursen et al., 2013; López-Patiño et al., 2013). In RAS, plasma cortisol level also could be affected by stocking density, such as the cortisol concentration is high in the RAS when the density of common carp increased (Schram et al., 2006; Li et al., 2013). In addition, thyroid hormones (TH) and growth hormone (GH) are important regulators of growth and their plasma contents which are correlated with growth rate. In fishes, T3 and GH have been shown to stimulate hepatic IGF-I to regulate growth in tilapia, sea bream et al (Schmid et al., 2003; Leung et al., 2008).
In order to evaluate the impact of Atlantic salmon stocking density on the growth, immunity and physiology in RAS, thyroid hormones, cortisol, glutamic oxalacetic transaminase (GOT), glutamicpyruvic transaminase (GPT) and lysozyme were detected. And we hope that would help us to find the optimum stocking density of Atlantic salmon reared in RAS.2 MATERIAL AND METHOD 2.1 Fish stocking
About 5 872 of Atlantic salmon with an average body mass of 1 413.1±52.3 g were purchased from Shandong Oriental Ocean Sci-Tech Co., Ltd. Shandong Province, China. The fish were distributed to the 5 experimental RAS tanks (6 m×6 m×3 m) randomly. The initial density of each tank is, A: 24 kg/m3; B: 21 kg/m3; C: 15 kg/m3; D: 9 kg/m3 and E: 6 kg/m3. Experimental trials were conducted for 220 days. During the experimental period, water temperature was maintained at 15.0±0.7℃, pH 7.2– 7.5, total ammonia-nitrogen < 0.25 mg/L, salinity between 28.0±0.5. Water was supplied to each tank at a rate of approximately 50 L/min with liquid oxygen to maintain dissolved oxygen between 130±10% saturation in the tank. Illumination was continuous and provided by one fluorescent tube light per tank with light intensity of 60±10 lux on the surface of water. Fish were fed with a commercial salmon diet (Skretting, Norway) containing 42% protein and 22% fat at satiation level three times a day by fish automatic feeder (at about 6:00 AM, 14:00 PM and 20:00 PM) during the period of light-manipulation. The stocking density variations were showed in Table 1. All procedures described in this study were reviewed and approved by the ethical committee of Institute of Oceanology, Chinese Academy of Sciences.2.2 Sampling procedure
The experiment was conducted over seven months, 30 fish per tank were sampled every month after being euthanized, with 0.05% MS-222 in seawater. Body weight and length were recorded, and 3 mL blood of fish (n=15) was collected from the caudal vein. Blood was centrifuged and plasma was stored at -80℃.
The relative weight gain (RWG) was calculated as: RWG=W2/W1×100%, where W1 and W2 were the average individual weight at days T1 and T2. Specific growth rate (SGR) was calculated using the equation: SGR=lnW2–lnW1/T2–T1. Feed conversion ratio (FCR) was calculated as: feed consumption/weight gain. All procedures described in this study were reviewed and approved by the ethical committee of Institute of Oceanology, Chinese Academy of Sciences. All procedures described in this study were reviewed and approved by the ethical committee of Institute of Oceanology, Chinese Academy of Sciences.2.3 RNA extraction, preparation of first strand cDNA and quantitative real-time PCR
Total RNA was extracted from Atlantic salmon liver, spleen and kidney, using Total RNA Extraction Kit (n=5) (BioFlux, Beijing, China), according to the manufacturer's instructions. All total RNA were dissolved into 20 μL RNAase free water. The first stand cDNA synthesized and according to reference (Chi et al., 2017).
The gene expression of Atlantic salmon lysozyme were quantified using SYBR TransStart Top Green qPCR SuperMix Kit (TransGen, Beijing, China) in an eppendorf Mastercycler ep realplex real-time PCR instrument (Eppendorf, German) according to the reference (Chi et al., 2017). The primers used to quantified Atlantic salmon lysozyme gene were described in Table 2. The methods of qPCR were according to (Chi et al., 2017).2.4 Measurement of plasma concentration of enzyme activities
Concentration of plasma T3 and GH were determined by commercially available solid-phase Iodine radioimmunoassay kit, following the method described previously (Carter, 1978).
Lysozyme activity was measured by a turbidimetric assay, a solution of 0.5 mg/mL micrococcus lysodeikticus was prepared. Then add 10 μL of plasma and 250 μL of the above suspension. After that, the reaction was carried out at room temperature (25℃) to measure the absorbance at 450 nm in 0.5 and 4.5 min. Lyophilized egg-white lysozyme was diluted b sodium phosphate buffer (pH 6.2, 0.05 mol/L) to generate a standard curve. All experiments were performed in triplicate.
Plasma GOT and GPT were assayed by the commercial kit purchased from Nanjing jiancheng Bioengineering Institute (Nanjing, China) according to the instructions. The plasma GOP and GPT activity were measured at 37℃ for 1 h.2.5 Statistical analysis
All statistical analysis in this experiment was performed using Spss 20.0. The results presented as mean±SE and compared using one-way ANOVA followed by Tukey's test. All assays in this study were performed in triplicated independently.3 RESULT 3.1 The effect stocking density on the mortality and growth in the RAS
The mortality could be affected by stocking density significantly (Table 3). The mortality was calculated in every separate period, the mortality and stocking density present the positive correlation. The mortality increased with increasing stocking density. And the maximum mortality was appeared at the day 100 (group A, 3.22%), the stocking density reached at 30 kg/m3. However, after day 100 the mortality is decreased in group A.
The body weight and body length also could be affected by stocking density. The results showed that the body weights of Atlantic salmon reared in higher stocking density groups was lower than lower stocking density groups. The differences were first appeared about at day 10, and it got more and more significant over the experiment time (P < 0.05). Meanwhile, the SGR, WGR and FCR under different stocking density were showed in Fig. 1. The WGR of different group were almost maintained at the same level at the first 10 days. Then the differences appeared at 40th day and became more and more significant with increasing density by the end of the experiment. The WGR of high density groups were significant lower than the low density groups (Fig. 1a). The SGR significantly decreased with increasing stocking density. At the first 70 days, the differences of SGR were significant among groups, however, the SGR differences decreased from 100th day (Fig. 1b). The FCR of different group were almost maintained at the same level at the first 40 days. And the differences were appeared at 70th day, the FCR of high density groups were higher than low density groups significantly, and the differences increased over time (Fig. 1c).3.2 The effect of stocking density on the thyroidlive axis of Atlantic salmon reared in RAS
The effect of stocking density on the plasma T3 and GH were showed in Fig. 2. At the first 40 days, the Atlantic salmon plasma T3 were almost maintained at the same level (P > 0.05). However, the plasma T3 of low stocking density groups were significantly higher than that in the high stocking density at 70th day, and then the differences increased over time (Fig. 2a). The significant differences of plasma GH were first appeared at 10th day, the plasma GH of group A were significantly lower than those of other groups. Then the differences increased with the increasing stocking density from 40th day, however, the differences became not significant again from 160th day (Fig. 2b).3.3 The effect of stocking density on the cortisol of Atlantic salmon reared in RAS
The differences of cortisol first appeared at 40th day, the cortisol of high density groups were higher than medium and low density groups significantly (Fig. 3). However, the differences became nonsignificant from 130th day.3.4 The effect of stocking density on the GOT and GPT of Atlantic salmon reared in RAS
The differences of GOT were not significant between different density groups at the early stage of experiment (day 0–70), however, the differences increased with increasing stocking density. The differences of GPT were first appeared at 70th day, and the plasma GPT level of high stocking density group (group A) was higher than those of other groups. Then the differences became non-significant over time (Fig. 4).3.5 The effect of stocking density on the lysozyme of Atlantic salmon reared in RAS
The activities of lysozyme in plasma were showed in Fig. 5a, There are no significant differences between groups. Then the expression level of lysozyme gene in liver, kidney and spleen were detected using RTPCR. The results showed that the differences of lysozyme expression level in liver were first appeared at day 130, and the differences increased over time. The expression level of lysozyme in high density groups was lower than medium and low density groups (Fig. 5b). Contrary to liver, the differences of expression level of lysozyme gene in kidney were first appeared at day 190, and the expression level of lysozyme in high density groups were higher than low density groups (Fig. 5c). There were no differences of expression level of lysozyme in spleen over the expression (Fig. 5d).4 DISCUSSION
Mortality is an important indicator of adaptation to the environment by fish. Studies have demonstrated that high stocking density can result in damage or death of fish (Bolasina et al., 2006; Ashley, 2007). Many studies indicated that high stocking density can lead to damage or death of fish (Laiz-Carrión et al., 2012). In this study, during 220 days experiment, the mortality of high density groups was higher than low stocking density group significantly. And we found that when the stocking density reaches to 30 kg/m3, the mortality increased suddenly and reach a peak. RAS is relative closed system, the metabolic production, toxin and microorganism et al. are easily accumulate and outbreak with the increase stocking density. So we think the stocking density of Atlantic salmon reared in RAS should not exceed 30 kg/m3. In this study, we found that the mortality were increased before day 100, after then the mortality were decreased (group A). We thinks there are two reasons for this phenomenon: first, we think there is threshold density for fish reared in RAS, the mortality mightbe increased before the threshold density, after that the mortality might be decreaed. Second, increasing density will induce high mortality, however, the weak fish died early, so the fish alive after day 100 in the RAS are strong, and has adapted the high density, then the mortality is decreased.
The growth of Atlantic salmon reared in RAS also could be affected by stocking density. The growth of Atlantic salmon reared in RAS can be reduced by high stocking density. The high density groups had lower WGR, SGR and higher FCR, this is similar to the reports in Atlantic salmon reared in sea cage (Geng et al., 2012). However, Hosfeld et al. (2009) found that the growth of Atlantic salmon reared in sea cage could not be affected by stocking density. We think that the salmon reared in RAS are more susceptible to the effects of stocking density. And different stages of salmon might response differently to stocking densities, and fish with different stages might show different adaption level for stocking densities (Greaves and Tuene, 2001). In this study, fish are in the period of rapid growth, and they might be more sensitive to the stocking densities than fish in the period of smoltification.
Thyroid-liver axis is exist in vertebrates universally (Zhang et al., 2014), and thyroid have many functions in promoting growth, metabolism et al. Meanwhile, there has been reported that T3 have a direct stimulatory effect on hepatic IGF-I expression to enhance the GH release (Schmid et al., 2000; Li et al., 2010). Some researchers suggest that the inhibition of growth might be related to the deterioration of water quality caused by high rearing density. However, in this study, the water quality were maintained at same level in all experiment tanks due to the RAS. In this paper, the T3 and GH has been studied, the results showed that the fish plasma T3 in high stocking groups is lower than T3 in low stocking density. So we speculate that high stocking density have negative effect on the growth may due to the lower thyroid hormone in high stocking density. Combined the results of SGR and FCR, we speculated that density stress can prove a drop in circulating T3 level and alter the secrection of TH. An insufficiency in TH will lead to abnormal growth, development, and metabolism in Atlantic salmon exposed to environmental stressors.
The activity of GOT and GPT are considered as monitors of health of fish (Prusty et al., 2011), cause injured hepatocytes can induce a release of GOT and GPT into the blood. In our study, the activity of plasma GOT and GPT were increased with increasing stocking density, which indicated the liver was damaged to a certain extent when fish reared in the high stocking density.
Cortisol is an important hormone to maintain normal physiological function, and cortisol can be as a good indicator of stress in fish (Bonga, 1997). Some researcher thought that cortisol is an indicator of short-term stress in Atlantic salmon (Liu et al., 2015). In the present study, the fish cortisol level in high stocking density groups is higher than that in low density groups significantly. This suggests that Atlantic salmon experience more stress in high density groups (Salas-Leiton et al., 2010). However, the differences of cortisol level were no significant after 100th day. So we think that adaptability of fish under high stocking density would be increase over time. And it might be taken 100 days for Atlantic salmon to adapt high density.
Lysozyme is an enzyme exists in blood and mucosal secretions and has a lytic effect on both Gram-positive and Gram-negative bacterial cell walls. And increased level of plasma lysozyme is considered as a protective mechanism in fish (Ingram, 1980; Costas et al., 2013). So activity of lysozyme usually used as an indicator of non-specific immune function in fish (Ahmad et al., 2016). This study showed there are no significant differences in high stocking density. This indicates that stocking densities may have no significant effects on the non-specific immune, and stocking density is not a key factor for lysozyme activity in adapted Atlantic salmon, which consistent with the founding of Liu et al. (2015).5 CONCLUSION
Growth of Atlantic salmon reared in RAS can be affected by stocking density. The mortality could be affected by stocking density significantly, and 30 kg/ m3 might be the maximum density which RAS can afford in China. Furthermore, many physiological parameters of Atlantic salmon reared in RAS are sensitive to stocking density, such as T3, GH, GOT and GPT et al. All these results showed that stocking density not only has effects on mortality and growth but has effects on health of Atlantic salmon. In addition, according to the results of lysozyme, we think stocking density might have no direct effects on non-specific immune. So we speculated that the high mortality of Atlantic salmon reared in RAS may be induced by the worsened functioning of physiology.
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