Yngzhen Li, Yingming Yng, Limin Song, Jilin Wng, Yunri Hu, Qin Yng,Peng Cheng, Jinhi Li

a Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, 266071, China

b Tianjin Fisheries Research Institute, Tianjin, 300221, China

c Sangtong Bio-engineering (Weifang) CO.Ltd, Weifang, 261210, China

Keywords:

ABSTRACT

1. Introduction

For aquaculture species, as well as other farmed plants and animals,growth and disease resistance are the main parameters of commercial significance. Genetic improvement of growth performance and disease resistance through selective breeding is acknowledged as an effective and sustainable strategy in aquaculture (Bangera, Ødegård, Præbel,Mortensen, & Nielsen, 2011; Gjedrem, Robinson, & Rye, 2012; Ødegård,Baranski, Gjerde, & Gjedrem, 2011). However, this approach is laborious, costly and time consuming. Alternatively, probiotics as a lasting environmental-friendly solution have been world widely used in aquaculture due to their beneficial impacts on growth performance, health status and feed efficiency. Recently, the roles, administrations and developments of probiotics and its positive impacts on disease control and growth enhancement in aquaculture have been well documented and reviewed (e.g., Dawood & Koshio, 2016; Dawood, Koshio, Abdel-Daim,& Doan, 2019; Kuebutornye, Abarike, & Lu, 2019; Newaj-Fyzul,Al-Harbi, & Austin, 2014; Akhter, Wu, Memon, & Mohsin, 2014;P´erez-S´anchez, Ruiz-Zarzuela, Blas, & Balc´azar, 2014; Wang, Ran,Wang, Zhang, Ding, Yang, Olsen, Ringø, Bindelle, & Zhou, 2019).

In general, for aquatic animals, probiotics can directly enhance the innate immune response, promote digestion and absorption and competitively suppress or exclude pathogenic microorganisms (Akhter,Wu, Memon, & Mohsin, 2015; Balcazar et al., 2006; Nayak, 2010). According to numerous previous studies, it is widely accepted that lactic acid bacteria (e.g., Lactobacillus spp.) and Bacillus spp. are the most commonly used probiotics in aquaculture practices, including fish and invertebrates. Ample evidences showed that L. plantarum and B. subtilis can improve growth performance, strengthen immune responses,modulate activities of antioxidant and digestive enzymes in aquatic species (e.g., review studies by Wang et al., 2019; Akhter et al., 2015;Kuebutornye et al., 2019; Newaj-Fyzul et al., 2014; Ringø, 2020).

Olive flounder (Paralichthys olivaceus) is a commercially important marine flatfish species in China, South Korea and Japan (Hasan, Jang,Lee, Kim, Hur, Lim, Bai, & Kong, 2019a; Li, Yang, Zheng, & Cheng,2019). In recent years, the frequent breakouts of bacterial diseases during grow-out period have resulted in mass mortality and tremendous economic losses in Northeast Asian countries (Kim, Harikrishnan, Kim,Balasundaram, & Heo, 2010; Li et al., 2020; Park, 2019; Xu & Zhang,2014). Previous studies revealed that Lactococcus spp. and Bacillus spp.demonstrated a broad spectrum of antimicrobial activity which could be used to control pathogenic bacteria under experimental rearing environment in olive flounder, and at the same time growth performance could also be enhanced (Heo, Kim, Kim, Hossain, & Kong, 2012; Heo,Kim, Kim, Bai, & Kong, 2013; Kim et al., 2013; Hasan et al., 2018).However, little information is available on the beneficial effects of L. plantarum and B. subtilis during the whole growth period (from juvenile to commercial size) under commercial rearing environment.

Given this, this study was conducted to detect that to what extent the growth performance and health status could be improved by adding L. plantarum and B. subtilis alone to basal diet in olive flounder. A 10-month feeding trial was designed and carried out under a commercial aquaculture production environment. The roles of probiotics on growout survival, growth parameters, immunological indices, antioxidants ability and digestive enzymes activity were investigated. This information will help us fine-tune the probiotics administration strategy for improving quality and productivity in olive flounder.

2. Materials and methods

2.1. Ethics statement

The collection and handling of the animals in the study was approved by the Yellow Sea Fisheries Research Institute’s animal care and use committee. For sampling, all fish were anesthetized using MS-222 to minimize suffering.

2.2. Diet preparation

The basal diet and experimental diets with probiotics (i.e., Lactobacillus plantarum and Bacillus subtilis) were provided by Sangtong Bioengineering (Weifang) CO., Ltd, China. Briefly, L. plantarum (powder,approximately 1.0 ×10CFU/g) and B. subtilis (powder, approximately 2.0 ×10CFU/g) products were supplemented separately to the basal diet with an additive amount of 5 g per kg basal diet (0.5%). The main ingredients of basal diet were fish meal, shrimp meal, fish oil, fermented soybean protein, yeast powder, vitamin premix and mineral premix (the detailed proportions were not provided by feed manufacturer). Then all the ingredients were manufactured into different sizes of pellet feed under a commercial production procedure with low processing temperature (～45C). Two experimental groups fed basal diet with supplement of L. plantarum (5.0 ×10CFU/g diet) and B. subtilis (1.0 ×10CFU/g diet) respectively, and control group fed with basal diet without probiotics supplement. The proximate compositions of basal diet on dry matter basis provided by feed manufacturer were presented in Table 1.

2.3. Fish grow-out maintenance

The experiments were started on 28th August 2018 and ended on 28th June 2019. Olive flounder juveniles (approximate 4-month old)obtained from flatfish breeding station in Haiyang, Shandong Province,China. 3240 olive flounder juvenile fish were distributed randomly into three groups, each group was randomly assigned to triplicate fiberglass tanks (i.e., 9 tanks), each tank (2.5 m) with 360 individuals. Each tank was equipped with a continuous aeration and flow-through water exchange system. During grow-out period, tanks were supplied with filtered and sterilized sea water and deep well seawater. Water temperature was controlled at 10-24C, the salinity was 28-30, the dissolved oxygen was 6-8 mg/L, the rate of water exchange was 500% per day. Fish were hand-fed to apparent satiation two times daily (08:00 and 20:00). About six months after stocking, fish in each tank were divided equally into two separate tanks (i.e., total 18 tanks). Neither chemical nor antibiotic treatments were used at any time.

2.4. Growth performance and survival

2.5. Sample collection

At the end of the feeding trial, three 24 h starved fish were randomly selected from each initial tank and anesthetized with MS-222 (60 mg/L).Approximately 1 mL of whole blood samples (n =27) were drawn from the caudal vein using syringes then collected in sterile centrifugal tube(1.5 mL). Blood samples were allowed to clot at the room temperature for 1 h. Serum was separated by centrifugation (1500 ×g, 10 min, 4C)and stored at - 80C until use. Thereafter, the fish was immediately dissected out under sterile conditions and fresh liver, stomach and middle intestine tissue samples (n =27 respectively) were collected and then stored at - 80C. Before using, 1 g of thawed tissue samples were homogenized by adding 9 g phosphate buffer solution (PBS, 0.01 M, pH= 7.4) and then centrifuging (1000 × g) for 10 min to separate the supernatant carefully for subsequent analysis.

Table 1 The proximate compositions of basal diet on dry matter basis provided by feed manufacturer.

2.6. The assay of immune parameters, antioxidant ability and digestive enzyme activity

The serum and homogenized liver supernatant samples were used to assay the immune parameters (lysozyme (LYS), acid phosphatase (ACP),immunoglobulin M (IgM), complement protein 3 (C3) and complement protein 4 (C4)) and antioxidant ability (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA)) respectively. The homogenized liver, stomach and intestine supernatant samples were used to assay digestive enzyme (proteinase, amylase and lipase) activity. All the parameters and indices mentioned above were measured using commercial fish Kits (Shanghai Jining Biotechnology Co., Ltd, China)following the manufacturer’s instructions. Briefly, 50 μL diluted samples(1:4) and 100 μL horseradish peroxidase (HRP) conjugated antibodyenzyme reagent were added per well, incubated at 37C for 60 min and then washed with washing solution for 5 times. After drying, tetramethyl benzidine (TMB) chromogenic substrate mixture (A and B) was pipetted into each well and blended gently. After incubation at 37C for 15 min in darkness, the enzymatic reaction was stopped by adding 50 μL stopping buffer. Then the absorbance at 450 nm was assayed using a Rayto RT-6100 microplate reader (Rayto Life Science, Shenzhen,China), thus allowing the estimation of the concentrations of target parameters from a calibration curve obtained under the same assay conditions. Three technical replicates were designed in this experiment.

2.7. Statistical analysis

Prior to statistical analyses, survival rates were transformed to square-root arcsine values and the normality of all the data and the homogeneity of variances among different groups were analyzed by using Shapiro-Wilk test and Levene’s test respectively. One-way analysis of variance (ANOVA) was performed to determine significant differences in various parameter indices, where significance level was set as Pvalue ＜0.05. All statistical analyses were carried out using SPSS 25.0 software. The results were presented as mean ±standard error (SE).

3. Results

3.1. Growth performance parameters and survival

When fish were fed the diets with L. plantarum or B. subtilis, all the growth parameters at harvest (i.e., HBW, WGR and SGR) were significantly higher than control group (P ＜0.05). Survival rate increased significantly when fish were fed the diets with L. plantarum (P ＜0.05).When fish were fed the diet with B. subtilis, survival rate was a numerically higher than that of fish fed the control diet, but not significant (P＞0.05). Between the two experimental groups, when fish were fed diet with B. subtilis supplementation, the growth parameters and survival rate at harvest were significantly higher than fed with L. plantarum supplementation.

3.2. Immunological indices

The concentrations of LYS, ACP, IgM, C3 and C4 in serum were showed in Table 3. Overall, except ACP and C4 in B. subtilis group, the studied serum immune parameters increased significantly (P ＜0.05) in the both dietary groups supplemented with probiotics after 8-month of feeding. All the serum immune indices in L. plantarum group were significantly higher than in B. subtilis group (P ＜0.05).

3.3. Hepatic antioxidant capacity

The present investigation revealed the potential effects of probiotics on antioxidant enzymatic activities. The contents of CAT, SOD, GSH-Px,T-AOC and MDA in liver were showed in Table 4. To a large extent, fishfed diets with L. plantarum or B. subtilis supplementation exhibited higher antioxidant ability at harvest stage. Specifically, when fish were fed diets with L. plantarum or B. subtilis supplementation, comparing to control group, the CAT and T-AOC contents in liver increased significantly and MDA levels were significantly lower. However, for SOD in L. plantarum supplementation group, though there was an improvement,it is not significant; in B. subtilis supplementation group, this parameter increased significantly. In both experimental groups, the improvement of GSH-Px level was negligible (P ＞0.05). Between the both experimental groups, no significant difference was observed for all the studied parameters, each has its superiority.

Table 2 Initial body weight (IBW), harvest body weight (HBW), weight gain rate (WGR),specific growth rate (SGR) and survival rate (SR) of olive flounder. Values are expressed as mean ±SE. Values with different superscript letters in the same row show significant differences (P ＜0.05).

Table 3 Effects of L. plantarum and B. subtilis supplementation in diets on immune response after a 10-month feeding trial in olive flounder. Values are expressed as mean ± SE (n = 9). Values with different superscript letters in the same row show significant differences (P ＜0.05).

Table 4 Effects of L. plantarum and B. subtilis supplementation in diets on liver antioxidant indices after a 10-month feeding trial in olive flounder. Values are expressed as mean ±SE (n =9). Values with different superscript letters in the same row show significant differences (P ＜0.05).

3.4. Digestive enzyme activity

3.4.1. Protease activity

The effects of dietary probiotics administration on protease activity in different viscera were shown in Fig.1. The gastric and intestine protease activities of both dietary probiotic treatment groups were significantly higher than blank group (P ＜0.05), but not significant between the both groups of probiotics added (P ＞0.05). For the protease activity in liver, B. subtilis supplementation group was significantly higher than other two groups (P ＜0.05); the protease activity in L. plantarum supplementation group was only slightly higher than control group (P ＞0.05).

Fig.1. Protease activities in liver, stomach and intestine after a 10-month dietary L. plantarum or B. subtilis additive feeding trial in olive flounder. Different superscripts indicate significant differences (P ＜0.05) between treatments in the same viscus.

3.4.2. Lipase activity

The effects of dietary probiotics administration on lipase activity in different viscera were shown in Fig.2. Overall, at the end of the feeding trial, dietary of B. subtilis supplementation group showed significantly improvement of lipase activity in all the tested viscera comparing to control group (P ＜0.05). However, compare to control group, though the substantial improvements of lipase activity were detected in L. plantarum supplementation group, significance was only observed in stomach (P ＜0.05). Besides, lipase activity in dietary of B. subtilis supplementation group was substantially (significant in liver and stomach,and insignificant in intestines) higher than L. plantarum group.

3.4.3. Amylase activity

The effects of dietary probiotics administration on amylase activity in different viscera were shown in Fig.3. As can be seen from the figure,in liver, the improvement levels of amylase activity in both treatment groups were negligible (P ＞0.05) compared with control group. In stomach, fish group fed diet supplemented with B. subtilis exhibited the highest amylase activity, which was significantly higher than the followed L. plantarum group which was also significantly higher than control group. In intestines, the B. subtilis dietary group showed the highest amylase activity (significantly higher than control), followed by L. plantarum group with no difference to B. subtilis and control group respectively.

Fig.2. Lipase activities in liver, stomach and intestine after a 10-month dietary L. plantarum or B. subtilis additive feeding trial in olive flounder. Different superscripts indicate significant differences (P ＜0.05) between treatments in the same viscus.

Fig.3. Amylase activities in liver, stomach and intestine after a 10-month dietary L. plantarum or B. subtilis additive feeding trial in olive flounder. Different superscripts indicate significant differences (P ＜0.05) between treatments in the same viscus.

4. Discussion

4.1. Experimental design

The probiotics as a dietary supplement has been proposed as a promising alternative approach for enhancing growth, practicing health management and controlling fish diseases (Gildberg, Mikkelsen, Sandaker, & Ringo, 1997; Nikoskelainen, Ouwehand, Bylund, & Salminen,2001). However, in most of the case studies of fish species, feeding trials were commonly conducted by using juvenile fish or within a limited experimental period (usually no more than two months) or some cases followed challenge tests, for examples listed in a reviewed study by Wang et al. (2019). In this study, for detecting the beneficial effects of probiotics on growth performance, grow-out survival and health status till harvest in olive flounder, a 10-month feeding trial was conducted by separately adding L. plantarum and B. subtilis into commercial pellet feed under a commercial rearing environment. Satisfactory results have been achieved as can be obviously observed from the statistical phenotypic data (Table 2). It was worth mentioning that challenge tests with specific pathogen were not performed in this study, this is mainly because that,during olive flounder grow-out period, numerous kinds of pathogens(including bacteria, viruses and parasites) may suffer, it is impossible and unnecessary to carry out challenge tests for different pathogens. We believe that our harvest survival data is persuasive enough. Harvest survival is a desirable trait which will benefit farmers more.

4.2. Beneficial effects of probiotics on growth in olive flounder

In the present work, the growth performance parameters were significantly enhanced by dietary supplementation with L. plantarum or B. subtilis, especially for B. subtilis, because the improvement levels of harvest body weight (HBW) in this group were significantly higher than L. plantarum supplemented group, which may mean a great superiority of dietary B. subtilis supplementation for growth improvement in olive flounder. Coincidentally, the improvement of activities of digestive enzyme detected in dietary B. subtilis supplementation group were all higher than in L. plantarum supplementation group (Fig.1, Fig.2 and Fig.3), but not all the improvements were significant (varied from viscera to viscera). This can be interpreted as that higher digestive enzyme activities imply a higher digestive ability which improve growth rate in turn. The effect of probiotics on growth improvement positively associated with digestive enzyme activity has been confirmed in lots of previous studies, for example in gilthead sea bream (Suzer et al., 2008),tilapia (Liu et al., 2017), silver pomfret (Gao et al., 2016), Pacific white shrimp (Duan et al., 2017) and as well as in olive flounder (Ye, Wang, Li,& Sun, 2011).

4.3. Beneficial effects of probiotics on immune parameters in olive flounder

In aquaculture, evidence showed that probiotics could modulate innate immunity of fish and further help producing natural resistance and high survival capacity (Gobi et al., 2018; Kiron, 2012; Abraham,Babu, Mondal, & Banerjee, 2007; Balcazar et al., 2006). Aside from the phenotypic survival trait, serum immune parameters, including immune related enzymes (i.e. LYS and ACP), immunoglobulin M and complement proteins (i.e. C3 and C4), as major components of fish humoral immune system were also invested in this study. In particular, LYS is a bacteriolytic enzyme which can also act as an opsonin to activate the complement system (Saurabh & Sahoo, 2008). Further, complement activity may be one of the most important serum factors because it can activate the cellular defenses (Harikrishnan, Balasundaram, & Heo,2010). Our results demonstrated that olive flounder survival rate at harvest were significantly elevated by feeding diet with L. plantarum supplementation, but not that much for B. subtilis supplemented diet(Table 2). This should be ascribed to the significantly higher serum immunological indices in dietary L. plantarum supplementation group than B. subtilis group (Table 3). In another olive flounder study, feeding infected (by Uronema marinum) fish with L. plantarum enriched diet resulted in significantly increased survival rate and lysozyme activity than the fish fed with B. subtilis supplementation diet (Harikrishnan,Kim, Kim, Balasundaram, & Heo, 2011). Therefore, this may conclude that, compared with B. subtilis, L. plantarum supplemented diet would be more beneficial for enhancing health status in olive flounder.

4.4. Beneficial effects of probiotics on antioxidant capacity in olive flounder

In fish, SOD, CAT and GSH-Px are the common antioxidant enzymes(Giulio, Habig, & Gallagher, 1993) which can remove excessive damaging reactive oxygen species (ROS) and protect the cells from ROS induced damage by lipid peroxidation (Freeman & Crapo, 1957; Messaoudi, Barhoumi, Said, & Kerken, 2009), and the lipid peroxidation is usually assessed by measuring the content of MDA (Uchiyama & Mihara,1978), the lower the better. The T-AOC level is an important indicator of the responses to oxidative stress (Li et al., 2012). Herein, fish fed diets supplemented with L. plantarum or B. subtilis had elevated the activities of SOD, CAT, GSH-Px and T-AOC and lowered contents of MDA in the liver. The results demonstrated a beneficial effect of L. plantarum and B. subtilis on the antioxidant capacity of olive flounder. Similar results have been reported that when dietary supplemented or water supplied with lactic acid bacteria and Bacillus spp. (compound or single and live or heat-killed), the antioxidant capacity was improved largely to some extent in olive flounder (Beck et al., 2016, 2015; Cha, Rahimnejad,Yang, Kim, & Lee, 2013; Hasan, Jang, Lee, Lee, et al., 2019, 2018; Heo et al., 2013; Kim et al., 2013; Lee et al., 2019, 2013; Nguyen, Park, &Kim, 2017; Taoka et al., 2006; Ye et al., 2011). Thus, the enhanced liver function with a well antioxidant defense system may be another explanation for the healthy growth performance of olive flounder. However,the correlation and mechanism of the simultaneous improvement of antioxidant status and immune response need to be investigated further.

5. Conclusion

In summary, the dietary supplemented L. plantarum or B. subtilis could chance growth performance and confer survival benefit of olive flounder. These beneficial effects can be attributed to the increased innate immunity indices, antioxidant status and digestive enzyme activities. According to our results, the separate supplementation of L. plantarum and B. subtilis in diet is more beneficial for disease resistance (high survival) and growth performance respectively. Hence, the beneficial responses of compounded supplementations of L. plantarum and B. subtilis are expected in olive flounder aquaculture. This may be of great interest to fish farming industries, feed and additive manufacturers and even aquaculture researchers.

CRediT authorship contribution statement

Yangzhen Li: Methodology, Conceptualization, Formal analysis,Writing - original draft. Yingming Yang: Resources, Investigation.Limin Song: Testing and assaying, Data curation, Funding acquisition,Supervision, Writing - review & editing. Jialin Wang: Resources,Investigation. Yuanri Hu: Resources, Investigation, Validation. Qian Yang: Resources, Investigation. Peng Cheng: Resources. Jianhai Li:Resources.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgements

This work was financially supported by Science and Technology Innovation Project for the Youth of Tianjin Agricultural Development Service Center (ZXKJ201908).