Effects of Poria cocos polysaccharide on growth performance, physiological parameters, and lipid metabolism of spotted sea bass Lateolabrax maculatus*

Jing LU, Zhangfan HUANG, Youling YE, Anle XU, Zhongbao LI,**

1 Fisheries College, Jimei University, Xiamen 361021, China

2 Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment, Xiamen 361021, China

Abstract The aquaculture industry has developed significantly over the past few decades and has had a substantial impact on the global food supply and marine fisheries resources.However, some problems arise behind the scenes due to excessive intensive farming, such as slow animal growth, frequent disease,and lipid metabolism disorders.These problems have limited the sustainable development of the aquaculture industry, and a continuable solution is required.The use of fungal polysaccharide appears to provide a solution to these problems.Therefore, different supplemented levels of Poria cocos polysaccharide (PCP) (0, 0.4, 0.8, 1.2, 1.6, and 2.0 g/kg, respectively) were fed to spotted sea bass(Lateolabrax maculatus) in similar size (30.28±0.18 g) in current study.The effects of PCP on growth,physiological parameters, and lipid metabolism of spotted sea bass were investigated after a 4-week rearing period.Results showed, fish with PCP intake presented a significantly higher weight gain, specific growth rate, and a significantly lower feed conversion ratio.Significantly higher trypsin activity in liver and intestine were observed in fish with PCP intake.The superoxide dismutase activity in serum and liver of fish with PCP intake were significantly improved, while significantly higher serum total antioxidant capacity and hepatic catalase activity were also observed.However, no significant differences in lysozyme and alkaline phosphatase activity were evident among groups.Fish with PCP intake showed a significantly lower total cholesterol, but no noteworthy change in triglyceride and lipid-metabolismrelated genes expression were observed among groups.Results indicated that intake of PCP has a positive effect on growth and antioxidant capacity of spotted sea bass, but seems to have a limited effect on the non-specific immunity and lipid metabolism of spotted sea bass.Based on the regression analysis results,1.4 g/kg of PCP is the optimal dose for spotted sea bass in size (30.28±0.18 g).

Keyword: spotted sea bass; Poria cocos; polysaccharide; growth; lipid metabolism

1 INTRODUCTION

The aquaculture industry has developed significantly over the past few decades and has had a substantial impact on the global food supply and marine fisheries resources (Food and Agriculture Organization of the United Nations, 2020; Gentry et al., 2020; Naylor et al., 2021).However, with its rapid intensification, some problems arise.For example, diseases of aquatic animals are characterized progressively as complicated (Bondad-Reantaso et al., 2005).In the past few decades,antibiotics and chemotherapeutics have been widely used for preventing various infections, which enhance the survival and growth performance of farming species (Liu et al., 2017).However, longterm use or even abuse of antibiotics can lead to several new problems such as pathogenic drug resistance, environmental pollution and drug residues (Watts et al., 2017).Besides, fish meal maintains high prices due to the expansion of aquaculture and the recession of marine fishery resources (Tacon and Metian, 2008).In order to reduce costs, increasing lipid levels in aquatic feed has been adopted to spare proteins and increase the weight gain of animals (Tocher, 2003).Nevertheless, the increase in lipid intake would result in animals’ lipid metabolism disorders, and thus cause animal appetite loss, lipid deposition, and even fatty liver syndrome (Du et al., 2005; Gao et al., 2011).In addition, the protein-sparing effect of lipid may be covered due to the lipid metabolism disorders of animals (Regost et al., 2001).These problems have limited the development of the aquaculture industry largely, and a sustainable solution was urgently required.

In recent years, it has been realized that regulating the function of the organism itself is key to improving metabolism disorders in aquatic animals and controlling diseases.Therefore, modern aquaculture has focused on the feed additives using,which has been proven effective in improving various performances of animals and yielded a great achievement (Dawood et al., 2018).Among various feed additives, polysaccharide is one of the promising one.Researchers now have noted that polysaccharides have a variety of bioactivities; not only in terms of enhancing growth performance and non-specific immunity, but also in terms of improving the antioxidant capacity and lipid metabolism in animals (Mohan et al., 2019; Wu et al., 2019; Yuan et al., 2019; Chen et al., 2021; Liu et al., 2021).Consequently, a number of studies in aquaculture have now focused on the use of polysaccharides, and they have been applied in the culturing of sea cucumberCucumariafrondosa(Wei et al., 2015), white shrimpLitopenaeusvannamei(Angela et al., 2020), Nile tilapiaOreochromis niloticus(Farag et al., 2022), and other important economic aquatic animals.These studies suggest that the use of polysaccharides has yielded a great deal of positive results.

Among various polysaccharides,Poriacocospolysaccharide (PCP) is one of the promising polysaccharides.As an edible and medicinal fungus,P.cocosis widely known by its Chinese name Fuling.It was commonly used in the formulation of nutraceuticals, tea supplements, cosmetics, and functional foods in Asia (Chen et al., 2010).As reported previously,P.cocoshas various beneficial functions to human being or animals, while these positive functions were related to its foremost chemical constituent to a great extent, namely PCP(Sun, 2014; Wu et al., 2014; Roloff et al., 2018; Li et al., 2020).Similar to other polysaccharides, PCP has also been reported to have various bioactivities to human or animals (Meng et al., 2016; Li et al.,2019).For example, Li et al.(2019) illustrated the antitumor activity of PCP in human being.Pu et al.(2019) reported the immunomodulatory effect of PCP in mice.Sun et al.(2019) found that PCP could improve hyperglycemia, hyperlipidemia, and hepatic steatosis in mice.As is well known,oligosaccharides are the constituent units of polysaccharides, while according to the report, the major fraction of PCP is β-1,3-Glucan (Zhang et al.,1997; Sun, 2014).β-1,3-Glucan can activate the complement system and regulate the immune pathway to improve animal immunity, and glucan now is considered an ideal immunostimulant in aquaculture, and therefore PCP can also be used as an immunostimulant (Meena et al., 2013).Meanwhile,based on the antioxidant capacity of PCP itself(Tang et al., 2014), dietary intake of PCP also has the potential to improve the antioxidant capacity of aquatic animals.Moreover, according to Sun et al.(2019), PCP can modulate the gut microbiota of animals, and thus play a role in hyperglycemia,hyperlipidemia, and hepatic steatosis.Therefore,PCP seems to present a possible solution to the problems that have limited the development of the aquaculture industry.However, the current research on PCP is mainly focused on its use in terrestrial animals and human being.Little is known yet about the function and use of PCP in almost all of economic aquatic species.Previous studies have discussed that different use dose and period of polysaccharide, and different species and growth stages of aquatic animals will lead to disparate results (Yin et al., 2021).Furthermore, improper use of polysaccharide would cause immune fatigue and other adverse reactions in aquatic animals (Yu et al.,2022).More research is needed to clarify and determine the function and usage of PCP in aquaculture.

Spotted sea bass,Lateolabraxmaculatus, is one of the commercially important fish in China.It was widely cultured in China and brings considerable economic benefits to farmers due to its characteristic of rapid growth, high-quality meat,and adaptability to different salinity regimes and temperature environments (Chinese Fisheries Bureau of Ministry of Agriculture et al., 2020).However, under the background of market demand and the degree of intensive breeding continue to expand, frequent diseases and slow growth were commonly observed in the culturing process of spotted sea bass as what mentioned before.Moreover,compared with other fish species, spotted sea bass tent to accumulate lipids and occurs lipid metabolism disorder easily as a typical carnivorous fish (Du et al., 2002).Different supplemented levels of PCP were fed to spotted sea bass in similar size(30.28±0.18 g) in current study.The effects of PCP on growth, physiological parameters, and lipid metabolism of spotted sea bass were investigated after a rearing period.Firstly, a pharmacological mechanism ofP.cocosin aquaculture can be more clearly understood by explored the effects of PCP.Moreover, the present study explored the feasibility of PCP application in aquaculture, which enriched the theoretical basis of its use in aquaculture and provided solutions to the problems restricting the development of the aquaculture industry.

2 MATERIAL AND METHOD

2.1 Diet preparation

A basic diet was formulated to contain about 460-g/kg crude protein and 100-g/kg crude lipid to satisfy the nutritional requirement of spotted sea bass (Ai et al., 2004), and manufacture as a sinking pellet feed.In the formulation, fish meal and soybean meal were used as main protein source,while fish oil and soybean oil were used as main lipid source.The composition and nutrient levels of the basal diet (dry matter basis) are illustrated in Table 1.This experiment was designed in a single factor, and the basic diets were gradationally mixed with 0.4, 0.8, 1.2, 1.6, and 2.0 g/kg of PCP,respectively.The basic diet was used as control.PCP is a commercial polysaccharide and was purchased from Xi’an Shengqing Biotechnology Co., Ltd., China.The total sugar and ash of PCP were 52.5% and 0.81%, respectively.

Fish meal and soybean meal were first crushed by a pulverizer (SGF130, Tianfan Pharmaceutical Machine Factory, Shanghai, China), and then the ingredients were mixed gradually.Approximately 35% of the dry feed weight of water was added to the feed.The resulting feed was then extruded into 2.5-mm pellets using a feed pelleter with a pressure of 0.8 MPa (CD4×1TS, South China University of Technology, Guangzhou, China).The prepared feedwas dried at a constant temperature of 55 ℃ in an oven and subsequently stored in a freezer at -20 ℃.As shown in Table 1, the moisture content, crude protein content, crude lipid content, and ash content of the feed were measured using the directly drying,Kjeldahl, Soxhlet extraction, and calcination methods, respectively, as described in a previous study (Huang et al., 2021).

Table 1 Formulation and proximate composition of the diets (g/kg, dry matter basis)

2.2 Fish and feeding management

Spotted sea bass were sourced from a commercial farm located in Zhangzhou, Fujian Province, China.Fish were temporarily reared in the 1 200-L tanks for a duration of two weeks.During the temporarily rearing period, fish were manually fed with a basic diet until they reached apparent satiety twice a day (at 7:00 and 17:00).Additionally,a gradual acclimatization process to fresh water was conducted by changing the water after a 30-min interval following the afternoon feeding each day.Every water change resulted in a decrease of salinity approximately 1×10-12, until the salinity level was maintained within the range of 0.5×10-12to 2.0×10-12.Before the rearing process, fish were fasted for 24 h,and anesthetized with eugenol in dosage 150 mg/L(Holloway et al., 2004).Twenty-five fish with accordant initial weight (30.28±0.18 g) were randomly selected and bulk-weighed, then allocated into a 200-L tank.The current experiment was conducted in parallel three times that three of tanks were deemed as a group, and there were six groups in total.Spotted sea bass in each group were given diets with gradient varying supplemented levels of PCP for 28 days.

During the rearing process, water samples were daily collected from the water storage tank before the morning feeding.The temperature, dissolved oxygen, pH, and ammonia-nitrogen concentration of water were measured by a portable salinity meter(AZ8371, AZ, China), oxygen meter (HQ40d,HACH, USA) and photoelectric color comparator(DR900, HACH, USA) as described in the previous study, respectively.The temperature was maintained between 26.5 and 29.5 °C, and the salinity was kept in the range from 0.5×10-12to 2.0×10-12.The pH was sustained between 7.8 and 8.2, and dissolved oxygen kept at approximately 7 mg/L.The ammonia-nitrogen concentration remained below 0.3 mg/L.

2.3 Sample collection

The fish were fasted for 24 h before sample collection.Afterwards, the circulation system was closed down.Most of the water was drained out,and the spotted sea bass in each tank were anesthetized using eugenol as mentioned above.The total number of fish was counted, and the total weight of fish in each tank was measured to calculate the relevant growth parameters.Five fish were selected randomly from each tank and measured the length and bulk-weight.Subsequently,blood samples of these fish were collected through tail vein sampling.Blood samples were slowly transferred to a 1.0-mL sterile centrifugal tube, and allowed to stand for 16 h in 4-°C conditions.After standing, five blood samples were centrifuged for 10 min under the condition of 4 °C and 836×gto separate serum, and serum samples were mixed and divided into two parts, then stored at -80 °C.One part of the serum sample was used to detect serum physiological parameters and biochemical parameters,while the other part was for standby use.After the blood sampling, fish were dissected to collect the liver and intestine.Each liver was weighed.Afterwards, the liver was divided into two parts and quick-frozen in liquid nitrogen, then stored at-80-°C condition, for the detection of hepatic physiological parameters and the expression of lipid metabolism related genes, respectively.The intestines were cleaned by 0.88% ice normal saline,and treated the same as the liver for the detection of digestive enzyme activities.

2.4 Growth performance evaluation

The relevant growth parameters included the final weight (WT), the weight gain (WG), the specific growth rate (SGR), the feed conversion ratio (FCR), the daily feed intake (DFI), the hepatosomatic index (HSI), and the condition factor(CF).The calculation formulas are as follows:

In the formulation:W0andWTare the initial weights (g) and the final weight (g) of fish,respectively;Fis the feed intake (g) during the rearing process;dis the number of rearing days;WLandWBare the liver wet weight and body wet weight (g), respectively;Lis the length of the fish(cm).

2.5 Digestion parameters analysis

The relevant parameters of digestion included the activity of amylase (AMS), lipase (LPS), and trypsin (TRS).These parameters were detected in the liver and intestine using commercial kits.All kits used for determining these parameters were purchased from the Nanjing Jiancheng Bioengineering Institute (Catalog Nos: AMS: C016-1-1, LPS: A054-2-1, TRS: A080-2).Initially, five samples from each tank were combined and weighed.Subsequently, the samples were homogenized with 10 volumes (w/v)of 0.88% normal saline solution in an ice-water bath(FSH-2A, TAINA, China).An exception is that the samples for detecting TRS were asked to be homogenized with a specified medium in corresponding kits.Following the homogenization process, the samples were subjected to centrifugation under specific conditions as instructed by the kit,resulting in the separation of the supernatant.The resulting supernatant was diluted with normal saline in varying proportions.Prior to the experiment, a pre-experiment was conducted to determine the appropriate concentration for measuring each parameter.Finally, each parameter was detected guided by the instruction of kits, and measured by a colorimetric method in different wave lengths.The changes in parameters were measured using a spectrophotometer (UV-1200, MAPADA, China) or microplate reader (EPHCH2T, BioTek, USA).The prepared samples were allowed to detect relative parameters within 48 hours.

2.6 Physiological parameters analysis

The physiological parameters detected in the present study include can be divided into two parts:non-specific immunity and antioxidant capacity.The relevant parameters of non-specific immunity included the activity of lysozyme (LZM) and alkaline phosphatase (AKP) in serum and liver;however, the relevant parameters of antioxidant capacity covered the serum activity/content of superoxide dismutase (SOD), malonaldehyde(MDA), total antioxidant capacity (T-AOC), and hepatic activity/content of SOD, MDA, catalase(CAT).For the detection process, the serum samples were either used directly or diluted to a specific concentration with 0.88% normal saline, following the instructions.Similarly, the liver samples underwent the same treatment as mentioned above.The sample requirements, detection methods for these parameters, and the source of the commercial kit used remained consistent with the information provided previously (Catalog Nos: LZM: A050-1-1;AKP: A059-1; SOD: A001-3; MDA: A003-1; T-AOC:A015-2-1).

2.7 Biochemical parameters analysis

The serum biochemical parameters that were detected included the level or activity of triglyceride(TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total protein (TP), albumin(ALB), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).The aforementioned parameters were detected in the serum of fish utilizing commercial kits.The sample requirements,detection methods for these parameters, and the source of the kit used remained consistent with the information provided earlier (Catalog No: TG:A110-1-1; TC: A111-1; HDL-C: A112-1-1; LDL-C:A113-1-1; TP: A045-4; ALB: A028-1; ALT: C009-2-1; AST: C010-2-1).

2.8 Analysis of genes related to lipid metabolism expression

The present study focused on the genes associated with lipid metabolism, included fatty acid synthetase (FAS), sterol regulatory element binding protein 1 (SREBP-1), acetyl CoA carboxylase 1(ACC1), acetyl CoA carboxylase 2 (ACC2),peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1 (CPT-1), hormonesensitive lipase (HSL), adipose triacylglycerol lipase(ATGL), and lipoprotein lipase (LPL).The relative expression levels of these genes in the liver were measured using real-time quantitative polymerase chain reaction (RT-qPCR).The detection process can be divided into two steps as follows.

In the first step, five livers of fish per tank were blended together to a single sample.The total RNA was extracted from 10 to 20 mg of each sample by commercial kits (RC101, Nanjing Vazyme Biotech Co., Ltd., China).Throughout the procedure, all consumables and instruments used were kept free from RNase contamination.Subsequently, the integrity of each RNA sample was assessed by conducting 1% agarose gel electrophoresis, while the concentration and purity were determined using a micro spectrophotometer (NANODROP2000,Thermo Scientific, USA).Following successful quality inspection, commercial kits (R323-01,Nanjing Vazyme Biotech Co., Ltd., China) were employed for reverse transcription of RNA to generate cDNA.The obtained cDNA was then stored in a refrigerator at -20 ℃ for further use.

RT-qPCR was conducted using the SYBR Green I chimeric fluorescence method with a commercial kit (Q711-02/03, Nanjing Vazyme Biotech Co., Ltd.,China).Duplicate reactions were carried out for each sample utilizing a fluorescence quantitative PCR instrument (LC480, Roche, Switzerland).For each sample, reactions were prepared in a 96-well plate with a total volume of 20 μL.This was achieved by combining 2 μL of template cDNA, 10 μL of 2× concentrated SYBR Green Mix as the fluorescent intercalating agent, 0.4 μL of forward and reverse primers (10 μmol/L each), and finally adding 7.2 μL of RNase-free ddH2O.The thermal profile for all reactions consisted of an initial predegeneration step at 95 ℃ for 30 s, followed by 45 cycles of denaturation at 95 ℃ for 10 s,annealing at 60 ℃ for 20 s, and extension at 72 ℃for 20 s.Fluorescence levels were monitored at the end of each cycle.In all cases, the dissociation curve analysis demonstrated a single peak,indicating the specificity of the PCR amplification.The primers utilized in this study, which are illustrated in Table 2, were designed based on previous studies conducted on spotted sea bass(Zhang, 2018; Huang, 2019).The β-actin was used as housekeeping genes, and the relative expression of each gene among groups were calculated by 2-ΔΔCtmethod.The calculation formulas are as follows:

In the formula:CtYAandCtYHare theCt of A gene in group Y andCt of housekeeping gene (β-actin) in group Y, respectively, while theCtXAandCtXHare theCt of A gene in group X andCt of housekeeping gene in group X, respectively.

2.9 Data statistics and analysis

Data were collected in Excel 2019 (Microsoft Corp., USA), and executed normality test(Supplementary Table S1 and Figs.S1-S11) and oneway ANOVA analysis to compare the differences between the dietary treatments analyzed via SPSS Ver 26 (International Business Machines Corp.,USA).If the analysis result showed significant differences, Duncan's multiple range test would be performed.Results were expressed as mean±standard deviation.In order to evaluate a suitable dose of PCP, polynomial regression was used to analyze the parameters which presented a significant difference between each group.The optimal fitting model for each parameter was calculated by SPSS Ver 26.Charts were made by Excel 2019, and the dose of PCP corresponding to the extremum value of each parameter was calculated.

3 RESULT

3.1 Growth performance

The results, as shown in Table 3, indicate that spotted sea bass with PCP intake manifested a better growth performance.Fish fed with 0.4, 1.2, and 1.6 g/kg of PCP were observed a higherWT(P＜0.05), while fed with 1.2 g/kg of PCP presented a higher WG and SGR (P＜0.05).A significant decrease in FCR was observed in the fish fed with 1.2 g/kg of PCP (P＜0.05).Nevertheless, no significant change in DFI was observed among groups(P＞0.05) Meanwhile, there was no significant difference in HSI and CF among groups, too(P＞0.05).

WG, SGR, and FCR were selected to carry out polynomial regression analysis.The analysis results showed that the quadratic regression equation is the optimal regression model for WG and FCR, while the cubic regression equation is the optimal regression model for SGR.The regression equations are shown in Fig.1, whereyandxstand for these parameters and the PCP supplemented level,respectively.The results of regression analysis indicated that the amount of PCP added corresponding to the maximum/minimum WG,SGR, and FCR were 1.358 8, 1.351 5, and 1.341 0 g/kg,respectively.

3.2 Digestive enzyme activity

Fig.1 The regression curve and equations of WG (a), SGR (b), and FCR (c)

Table 2 Information of primer

Table 3 Effects of PCP on growth performance of L.maculatus

Table 4 Effects of PCP on digestive enzyme activity of L.maculatus

The data of digestive enzyme activity are shown in Table 4.Following the dietary PCP inclusion, the activity of TRS and LPS were improved whether in the liver or intestine of fish.As the table shown, the hepatic TRS activity was improved in the dietary 0.8- and 1.6-g/kg PCP group (P＜0.05), while enteric TRS activity was improved in the dietary 0.8-g/kg PCP group (P＜0.05).The hepatic LPS activity was improved in the dietary 0.8-g/kg PCP group(P＜0.05), while enteric LPS activity was improved in the dietary 1.6-g/kg PCP group (P＜0.05).Nevertheless, no variability was observed in the activity of hepatic and enteric AMS among groups(P＞0.05).

Activity of hepatic TRS, enteric TRS, hepatic LPS, and enteric LPS were selected to carry out polynomial regression analysis.The analysis results showed that the quadratic regression equation is the optimal regression model for these parameters.The regression equations are shown in Fig.2, whereyandxstand for these parameters and the PCP supplemented level, respectively.The results of regression analysis indicate that the amount of PCP added corresponding to the maximum activity of hepatic TRS, enteric TRS, hepatic LPS, and enteric LPS were 1.346 5, 1.449 7, 1.242 7, and 1.587 4 g/kg,respectively.

3.3 Physiological parameter

The data of antioxidant capacity and non-specific immunity parameters are shown in Tables 5 and 6,respectively.In the present study, dietary PCP supplementation improved antioxidant capacity of fish to some extent.Compared with the control group, a higher serum SOD activity was observed in the dietary 1.6-g/kg PCP group (P＜0.05).Meanwhile, a higher hepatic SOD activity was also observed in the fish with 1.6-g/kg PCP intake(P＜0.05).An improvement in serum T-AOC was observed in dietary 1.6- and 2.0-g/kg PCP group (P＜0.05), while the hepatic CAT activity was also improved in fish with 1.2- and 1.6-g/kg PCP intake(P＜0.05).However, the content of MDA in serum and liver showed no significant change between groups (P＞0.05).In addition, there was no significant variability in the parameters related to the non-specific immunity of spotted sea bass,including the activity of serum and hepatic LZM and AKP (P＞0.05).

Fig.2 The regression curves and equations of hepatic TRS activity (a), enteric TRS activity (b), hepatic LPS activity (c),and enteric LPS activity (d)

Table 5 Effects of PCP on antioxidant capacity of L.maculatus

Table 6 Effects of PCP on non-specific immunity of L.maculatus

Activity of serum and hepatic SOD, serum TAOC and activity of hepatic CAT were selected to carry out polynomial regression analysis.The analysis results showed that the cubic regression equation is the optimal regression model for serum SOD activity, while the quadratic regression equation is the optimal regression model for hepatic SOD activity, serum T-AOC, and hepatic CAT activity.The regression equations are shown in Fig.3, whereyandxstand for these parameters and the PCP supplemented level, respectively.The results of regression analysis indicate that the amount of PCP added corresponding to the maximum activity of serum and hepatic SOD, serum T-AOC, and activity of hepatic CAT were 1.555 7,1.319 8, 1.561 7, and 1.173 3 g/kg, respectively.

3.4 Biochemical parameter

The data of serum biochemical parameters are shown in Table 7.Results show that spotted sea bass fed with PCP manifested a lower TC content, and there was a significant decrease in dietary 1.6- and 2.0-g/kg PCP group (P＜0.05).Lower LDL-C content was observed in dietary 1.6-g/kg PCP group(P＜0.05); meanwhile, lower AST activity was observed in dietary 1.2-g/kg PCP group (P＜0.05).However, other serum biochemical, including content/activity of TG, HDL-C, TP, ALB, and ALT,have no significant change among groups (P＞0.05).Overall, these results seem indicate that the effect of PCP on biochemical parameters of spotted sea bass is limited.

3.5 Expression of genes related to lipid metabolism

Fig.3 The regression curves and equations of serum SOD activity (a), hepatic SOD activity (b), serum T-AOC (c), and hepatic CAT activity (d)

The relative expression levels of liver lipid metabolism-related genes are shown in Fig.4.As shown in the figure, whether gene related to the fatty acid synthesis, including FAS, SREBP-1,ACC1, ACC2, or decomposition, including PPAR-α,CPT1, HSL, and LPL, no significant change was observed in the relative expression of these gene (P＞0.05).The only exception is the relative expression of ATGL, and its relative expression was increased in dietary 0.8-, 1.2-, and 1.6-g/kg PCP group (P＜0.05).

4 DISCUSSION

A number of recent studies have illustrated that polysaccharide can improve animals’ performance in several aspects, while some polysaccharides, such as HuangqiAstragalusmembranaceuspolysaccharide,GojiLyciumbarbarumpolysaccharide, and other polysaccharides, have been widely studied in aquaculture now (Wu, 2020; Zhang et al., 2020).Nevertheless, little information about PCP in aquaculture has yet to be reported.Fortunately, the current study indicated that PCP could improve performance in several aspects of spotted sea bass.One of the most important functions of polysaccharides in aquaculture is its growthpromoting effect, while this function has been reported in several typical studies (Sun et al., 2020;Zhang et al., 2020; Yu et al., 2022).Similar to other polysaccharides, the present study illustrated that the intake of PCP can improve the growth performance of spotted sea bass, while about 1.35 g/kg of PCP seems to be an optimal supplemented level according to the regression analysis of WG and SGR.In other previous studies, Ma et al.(2009) and Xian et al.(2018) reported that the intake ofP.cocoscould improve the growth of rea drumSciaenopsocellatusand Amur sturgeonAcipenser schrenckii, respectively.As mentioned in theintroduction, various beneficial functions ofP.cocoscan related to PCP, and these two similar examples support the results of this study to a certain extent.PCP itself does not provide nutrients, and its growthpromoting effect on spotted sea bass can be related to the improvement of feed utilization efficiency.Correspondingly, the FCR in the present study was significantly lower in the dietary PCP group.According to the regression analysis, the dietary 1.34 g/kg of PCP corresponds to the minimum FCR.This PCP addition is consistent with the optimal supplementation level for WG and SGR, and the results further indicated the consistency between feed utilization and the growth of aquatic animals.However, contradictory results were also reported in numerous previous studies.For example, Su et al.(2020) reported that dietary Yu-Ping-Feng polysaccharides had no significant influence on the growth performance of white shrimp; in the discussion, authors thought that this phenomenon was due to the various animal species and the period of feeding trial.Furthermore, Yu et al.(2022) also indicated that the intake of 2 g/kg ofAstragaluspolysaccharides would restrain the growth of Asian seabassLatescalcarifer.As is well known, longterm intake of oligosaccharide will lead to animal’s immune fatigue (Ai et al., 2007; Lin et al., 2011),while oligosaccharide is the constituent unit of polysaccharide.The immune stimulation effect of polysaccharide is closely related to the constituent of oligosaccharide.Therefore, the long-term intake of polysaccharide may also lead to animal immune fatigue, while immune fatigue would limit the growth of the animal.This reason has also been discussed in the report of Yu et al.(2022).In addition, differences in the growth stage of animals,types of polysaccharides, and their composition and purity may lead to different results.However, at present, almost all of the studies related to polysaccharides considered the polysaccharide as a whole, which also leads to the fact that most studies cannot clearly discuss and explain the diverse results.Overall, the effects of polysaccharides on various aspects of aquatic animals may further affect their growth performance, while more research is required to focus on the structure, composition of polysaccharides and the molecular mechanism of each component action, to explain its various positive functions to animals.

Table 7 Effects of PCP on serum biochemical parameters of L.maculatus

Fig.4 The relative expressions of FAS (a), SREBP-1 (b), ACC1 (c), ACC2 (d), PPAR-α (e), CPT1 (f), HSL (g), ATGL (h),and LPL (i) in the liver, respectively

The improvement of fish feed utilization efficiency is commonly considered to be related to higher digestive enzyme activity.In most studies,the enhancement in digestive enzyme activity is consistent with the improvement in feed utilization efficiency and growth performance (Najdegerami et al., 2017; Zheng et al., 2018; Huang et al., 2020).The present study also indicated that spotted sea bass fed with PCP manifested higher TRS and LPS activity, while dietary 1.41 g/kg of PCP is the optimal addition for the digestion of spotted sea bass(average value of regression analysis results of digestive parameters).This optimal supplementation level also corresponds to the regression analysis results of WG, SGR, and FCR.P.cocoswas considered to have the effect of strengthening the spleen and stomach that Ran et al.(2015) have also reported thatP.cocosimproved gastrointestinal function of mice, while this effect is greatly determined by the PCP.Mohan et al.(2022) pointed out that polysaccharide stimulates the population of endogenous probiotic bacteria in the intestines of animals, followed by increased secretion of exogenous digestive enzymes, which leads to better nutrient digestion and storage in tissues.This opinion is widely accepted in aquaculture now.However, the lack of intestinal microbiota analysis is one of the shortcomings of this experiment, which also leads to our inability to analyze the correlation between digestive enzyme activity and intestinal microbiota structure of spotted sea bass.On the other hand, the activity of hepatic TRS and LPS was observed a significant increase in spotted sea bass fed with PCP.This result seems to reveal that PCP also could stimulate the secretion of digestive enzymes in the liver.At this level, polysaccharides may also regulate the secretion and activity of digestive enzymes through related signaling pathways.However, not only PCP, but also little research is available on the digestion-promoting effect of polysaccharides.Although the digestionpromoting effect has been reported in previous studies, various composition and structure of polysaccharides would also lead to different results.

The antioxidant capacity and non-specific immunity play a vital role in fish body health, and they also affect the growth of fish to a great extent.In the past few decades, the positive effects of several polysaccharides on fish non-specific immunity and antioxidant capacity have been reported in studies (Rajendran et al., 2016; Safavi et al., 2019; Shalini et al., 2019), while a few studies have reported that PCP can improve the antioxidant capacity and non-specific immunity of aquatic animals (Qin et al., 2006; Wang et al., 2010, 2011).Different polysaccharides seem to have similar mechanisms of action, and Sun (2014) and Ríos(2011) have discussed the anti-inflammatory activity,antioxidant activity, and immunostimulatory activity of PCP in previous.In the present study, the antioxidant capacity of spotted sea bass fed with PCP was improved; however, no positive effect was observed in non-specific immunity of fish fed with PCP.The observed enhancement in the fish’s antioxidant capacity may be attributed to the inherent antioxidant properties of polysaccharides(Meiet al., 2017).The inherent antioxidant properties of PCP enable it to function as an antioxidant in feed, reducing feed toxicity and thereby minimizing oxidative stress in fish.For instance, PCP supplemented in feed may slow down even prevent the oxidation of fish oil.Additionally,PCP may exert its beneficial effects by inhibiting the growth of detrimental microorganisms during storage and preventing the release of toxic metabolites produced by fungi (Tanet al., 2004).This mechanism further contributes to the reduction of oxidative damage in fish.According to the result of regression analysis, dietary 1.40 g/kg of PCP is the optimal addition for the antioxidant capacity of spotted sea bass (average value of regression analysis results of antioxidant capacity parameters).This value also coincides with other regression analysis results.On the other hand, the immunostimulatory activity of PCP can be due to the effects of oligosaccharide.However, the result in the current study was contrary to other studies.As mentioned above, the long-term intake of polysaccharide will lead to animal immune fatigue.Consequently, more appropriate dosage and usage of PCP needs to be further explored.Besides, the purity of polysaccharide might also be a critical factor to influence the non-specific immunity of fish.Based on the immunity-promoting effect of oligosaccharide, polysaccharide still has a great potential to improve the immunity of aquatic animals.A bacteria/oxidation challenge was needed to directly illustrate the effects of PCP on nonspecific immunity and antioxidant capacity of several aquatic animals.

Nowadays, polysaccharides have also been widely concerned in improving lipid metabolism disorders in animals, and the positive effects of various polysaccharides on lipid metabolism in aquatic animals have been reported in some studies(Tan et al., 2019; Yuan et al., 2019; Zou et al., 2021;Huang et al., 2022a).However, the present study indicated that the impact of PCP on lipid metabolism of spotted sea bass is limited that most of the biochemical parameters and relative expression of gene related to lipid metabolism showed no significant difference between the control group and the dietary PCP group.This result is also conflicting with a previous report that PCP has a hypoglycemic effect on mice and can to a certain extent improve the disorder of lipid metabolism caused by diabetes (Huang et al.,2016b).There are several reasons for the lipid metabolism disorder in fish, such as environmental factors (Chen et al., 2013; Huang et al., 2016a),physiological factors (Hatlen et al., 2005; Zhang et al., 2012), and the most common and important nutritional factors (Hemre et al., 2002; Du et al.,2006; Tocher et al., 2008).On the hand of fish lipid metabolism disorder caused by nutritional factors,Zou et al.(2021) also reported that pomelo fruitlet polysaccharide ameliorates diet-induced nonalcoholic fatty liver disease in hybrid grouper (Epinephelus

lanceolatusmale×Epinephelusfuscoguttatusfemale).In addition, Tan et al.(2019) reported that dietary supplementation ofL.barbarumextract can improve lipid metabolism in fish fed high lipid diets.In these previous studies, polysaccharides improved lipid metabolism of animals by directly regulating the expression of related genes.Meanwhile, polysaccharides seem to improve lipid metabolism by regulating affecting the composition of the intestinal flora.In the study of Yang et al.(2021), it was reported thatL.barbarumpolysaccharide was able to modulate the gut microbiota to prevent obesity.Furthermore,polysaccharides may improve lipid metabolism by regulating the bile acid metabolic pathway, but the research in this area is limited and needs to be further explored.Based on these factors, the negative result in the present study may be due to the fish in each group did not appear lipid metabolism disorder, and a hyperlipidemia model of fish needs to be built in the follow-up experiment.Furthermore, the purity, structure, and composition of polysaccharides may also affect its effect on lipid metabolism disorders.How polysaccharides are targeted to the signaling pathways related to lipid metabolism and on which structure they are based are still unclear.Based on previous positive results,PCP still has great potential to improve fish lipid metabolism, and its development in this area has great prospects.

In summary, the application of PCP in aquaculture has a great potential, but still has many unclear problems now, such as its optimal dosage used in different fish species, growth stage, and mechanism of action, which need to be explored deeply.In the subsequent experiments, correlation analysis will be conducted based on the analysis of polysaccharide composition and structure and its biological activity function, in order to further clarify the action mechanism of polysaccharide.

5 CONCLUSION

In conclusion, dietary intake of PCP can improve growth performance, digestion, and antioxidant capacity of spotted sea bass.The recommended addition of the PCP for spotted sea bass in size(30.28±0.18) g is 1.4 g/kg.The results reveal the potential for application of PCP to aquaculture practices, and thus PCP could be developed as a dietary supplement.Additionally, based on the aquaculture industry demand-oriented, the development of the PCP industry can also be further stimulated.Convictive studies on the action mechanism of PCP are lacking in this study.Subsequent studies can focus on the optimal dosage of PCP used in different fish species and growth stage, as well as modifications, deeply action mechanisms and negative effects of PCP.

6 DATA AVAILABILITY STATEMENT

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

This experiment was approved by the Animal Ethics Committee of Jimei University (Grant No.JMU202103009).All applicable international,national, and/or institutional guidelines for the care and use of animals were followed by the authors.