The basal diet was based on soybean meal as carbohydrate sources and fish meal as protein sources. Five isonitrogenous (38%) and isolipidic (6.6%) experimental diets ( Table 1 ) were formulated and supplemented with 0 (Diet 1), 500 (Diet 2), 1,000 (Diet 3), 1,500 (Diet 4), and 2,000 (Diet 5) mg/kg fucoidan (purity ≥98%, Xi ‘an Risen Biotechnology Co., LTD., China). The selected dosages of fucoidan were based on the findings of earlier studies (Sony et al., 2019; Cui et al., 2020). All feed ingredients were crushed and sifted by a 60-mesh sieve, weighed, and mixed by a stepwise expansion method to make pellet feed with a diameter of 1 mm, which was dried naturally outside in a drafty place that is not exposed to direct sunlight and then stored at −20°C until feeding.

Common carp juveniles were purchased from an aquafarm (Chongqing, China) and acclimated for 14 days in a cement breeding pond (6 m × 6 m × 0.8 m) feeding with the commercial feed (Tongwei Co. LTD., China) twice a day. After acclimation to the trial conditions, 450 healthy fish (35.83 ± 0.24 g) were randomly assigned to 15 cement breeding ponds (2.1 m × 1.2 m × 0.8 m), each with 30 fish species. The feeding trial was conducted outdoors for 8 weeks in the aquaculture base of Southwest University (Chongqing, China). Fish in triplicate groups were fed the respective experimental diets to apparent satiation twice daily at 8:00 and 17:00. Meanwhile, the feed intake and mortality of fish in each pond were recorded. During the trial, the dissolved oxygen was ≥6.5 mg/L and the water temperature ranged from 25 to 28°C. The total ammonia nitrogen and nitrites remained< 0.05 mg/L, and the pH was kept at 7.4–8.2. The natural photoperiod was applied.

At the end of the trial, carps were starved for 24 h, weighed and dissected, and then counted to calculate growth index. Twelve fish were caught randomly from each pond, and anesthetized by applying eugenol for sampling. Among them, six fish were selected for blood collection purposes; their body length and weight (body, viscera, and liver) were then recorded for the purpose of computing the somatic indices. The blood of three fish was drawn from their caudal vein using sterile disposable syringes (2 ml), rinsed with heparin sodium solution. After the centrifugation (3,500 rpm, 15 min, 4°C) of the sample, the plasma was separated and stored at −80°C for biochemical index detection. Non-heparinized blood of 3 fish was centrifuged (3,500 rpm, 15 min, 4°C) to obtain the serum for antibacterial activity test. Three fish from each pond were sacrificed to obtain liver and intestine collected in an ultralow-temperature refrigerator for analysis of enzyme activity. Moreover, three hepatic tissues from each pond were placed in 2-ml centrifuge tubes containing Bouin’s solution for micromorphological analysis. After 24 h of fixation, the samples were washed and stored using 70% ethanol solution until the preparation of tissue section.

The assay of plasma biochemical indices in this study comprised alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin (TB), urea nitrogen (UN), creatinine (Cr), glucose (Glu), triglycerides (TG), and total cholesterol (TC), which were all measured by using a fully automated biochemical analyzer (Chemray 240, Shenzhen, China).

The content of interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-10 (IL-10), complement 3 (C3), lysozyme (LYZ), and immunoglobulin M (IgM) in the plasma; the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPX), lipid peroxide (LPO), and malonaldehyde (MDA) in the liver; and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lipase (LPS), amylase (AMS), and trypsin (TRY) in the intestine were determined by utilizing the commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The above kits were carried out in accordance with the manufacturer’s specifications.

The serum from non-heparinized blood was used for antibacterial activity test in vitro. For this purpose, Aeromonas hydrophila (BBAh1) stored in the Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education) (Li et al., 2019) was grown in tryptic soy broth for 24 h at 30°C. Bacterial cells were centrifuged and suspended in sterile PBS and adjusted to an optical density of 0.5 at 546 nm. The diluted bacterial suspension was mixed with serum homogenates for incubation at 37°C for 1 h. After incubation, the number of viable bacterial cells was counted (Eslami et al., 2022).

Hepatic samples were fixed in Bouin’s solution. Then, the fish tissues were placed in plastic cassettes and processed (gradual dehydration in 70%–100% alcohol, clearing in xylene, and paraffin wax embedding). Five-micron-thick sections were cut on a microtome and then stained with hematoxylin and eosin (HE). Slides were examined with a light microscope (Nikon DXM1200).

The growth performance parameters were calculated as follows (Geetanjali et al., 2020; Jayant et al., 2021):

All of the data were expressed as the mean ± SEM. The data were analyzed by one-way analysis of variance and Tukey’s multiple range test using SPSS 19.0 (IBM, Chicago, USA). Differences were considered significant at p< 0.05. The optimal dietary requirement of fucoidan for juvenile carp was calculated by using broken line regression analysis (Robbins et al., 2006).

The growth performance, feed utilization, and somatic indices of juvenile common carp fed the experimental diets containing different levels of fucoidan were presented in Table 2 . At the end of the 8-week culturing trial, Diet 3, Diet 4, and Diet 5 were higher in FW, WG, and SGR than Diet 1 and Diet 2 (p< 0.05). Moreover, FCR in Diet 3 and Diet 4 was significantly lower than in the other diets (p< 0.05). VSI in Diet 3 and Diet 4 was significantly higher than in Diet 1, and fish fed diet 1,500 had a higher VSI than fish fed diet 500. Nevertheless, there were nearly no significant changes in HI, CF, and SR among all the treatments (p > 0.05). Furthermore, the regression analysis between fucoidan supplementation and WG showed that the optimal dietary content of fucoidan was 1,757 mg/kg ( Figure 1 ), and the regression analysis between fucoidan content and SGR showed that the optimal dietary supplementation of fucoidan was 1,666.67 mg/kg for juvenile common carp ( Figure 2 ). Meanwhile, the survival rate of common carp fed with dietary fucoidan remained unaffected compared to the control group, which was 100% in all diets.

The effects of dietary fucoidan on plasma biochemical parameters of juvenile common carp are presented in Table 3 . The highest levels of plasma ALT, AST, TB, and Glu were found in Diet 5, which were statistically higher than other diets (p< 0.05). Additionally, plasma TP and ALB in Diet 3, Diet 4, and Diet 5 were higher than that in Diet 1 and Diet 2 (p< 0.05), whereas nearly no statistically significant differences were observed in UN, Cr, TG, and TC for any of the treatment (p > 0.05).

Table 4 shows that dietary supplementation with fucoidan containing different levels in common carp impacted the immune parameters in plasma. The values of proinflammatory cytokines IL-6 and IL-1β showed a decreasing trend in Diet 2, Diet 3, and Diet 4, and an increasing trend in Diet 5 (p< 0.05). Conversely, the values of anti-inflammatory cytokine IL-10 showed an increasing trend in Diet 2 and Diet 3, and a decreasing trend in Diet 4 and Diet 5 (p< 0.05). Furthermore, compared with Diet 1, dietary addition of fucoidan significantly improved the C3, LYZ, and IgM content (p< 0.05). Moreover, the highest value of C3 and LYZ appeared in Diet 3, and the highest value of IgM appeared in Diet 4. Quite noticeably, the number of A. hydrophila colonies notably diminished (p< 0.05) in the fish serum feeding Diet 3 (cfu = 226 ± 21), Diet 4 (cfu = 301 ± 12), and Diet 5 (cfu = 309 ± 18) compared to Diet 1 (cfu = 362 ± 17) and Diet 2 (cfu = 343 ± 12) ( Figure 3 ).

As displayed in Table 5 , antioxidative parameters in the liver were also affected significantly by dietary fucoidan supplementation. Distinctly, the activity of enzymes in antioxidant systems SOD (in Diet 3 and Diet 4), CAT (in Diet 2 to Diet 5), and POD and GPX (in Diet 3 to Diet 5) was significantly increased by feeding fucoidan (p< 0.05). Meanwhile, the content of the peroxidation product LPO (in Diet 3 to Diet 5) and MDA (in Diet 2 to Diet 5) was dramatically decreased by the supplementation of fucoidan (p< 0.05).

The digestive enzyme activity in the intestine was also affected by feeding fucoidan ( Table 6 ). Obviously, all the digestive enzyme activity in Diet 3, Diet 4, and Diet 5 was significantly elevated compared to Diet 1 (p< 0.05). Notably, the activity of LPS in Diet 5 was the highest (p< 0.05), and the activity of AMY and TRY in Diet 3 and Diet 4 was higher than the other Diets (p< 0.05).

The effect of dietary supplementation of fucoidan on the hepatic morphology of juvenile common carp is presented in Figure 4 . Compared to Diet 1 and Diet 5, the hepatic tissues of fish feeding Diet 2, Diet 3, and Diet 4 were obviously more intact: hepatocytes were arranged neatly, with a clear cellular boundary and the nucleus mainly in the cell center, and slight vacuolization was observed. In Diet 5, hepatocytes were arranged irregularly, with a blurred cellular boundary and the nucleus located at the cellular periphery (or even disappeared), cellular swelling, severe vacuolization, and even melanin macrophage and lymphocyte infiltration.

The growth rate (FW, WG, and SGR) of juvenile common carp supplemented with fucoidan were enhanced compared with the control group in the present study. Similarly, fucoidan additive in the diets of rohu (L. rohita) (Mir et al., 2017; Adnan et al., 2018), red sea bream (P. major) (Sony et al., 2019), gible carp (C. auratus) (Cui et al., 2020), and Nile tilapia (O. niloticus) (Abdel et al., 2021) improved growth performance. Notably, the regression analysis between dietary fucoidan supplementation and SGR or WG showed that the optimal dietary content of fucoidan was 1,666.67 mg/kg or 1,757 mg/kg for the growth of juvenile common carp in this study. However, the best recipes of fucoidan in P. major (0.4%) (Sony et al., 2019), C. auratus (30 g/kg) (Cui et al., 2020), and L. rohita (2%) (Mir et al., 2017) are very different. Therefore, a study on the appropriate amount of fucoidan added to different fishes was very important and necessary.

In the present study, dietary fucoidan could significantly improve the immunity of juvenile common carp by downregulating the level of proinflammation factors (IL-6 and IL-1β) and enhancing the specific (IgM) and non-specific (lysozyme and C3) immunity. Analogously, immune cells’ activation and the high expression of IgM, IgG, and IgA in plasma were observed in mice with dietary fucoidan (Makoto et al., 2019). The results of cytokines in this study showed that IL-6 and IL-1b decreased in Diets 2, 3, and 4, and increased in Diet 5. On the other hand, IL-10 increased in Diets 2 and 3, but decreased in Diets 4 and 5. Therefore, we could speculate that fucoidan might regulate the level of inflammation and have a proinflammatory effect at a high dose, which means that the dosage of fucoidan added in the feed should not be that high; otherwise, it will have adverse effects. Obviously, fucoidan could regulate the expression of inflammatory cytokines by activating inflammation-related signaling pathways, such as the NF-kB and eNOS signaling pathways (Wang and Chen, 2015; Caroline et al., 2019). Moreover, the survival rate of fry of L. rohita infected with A. hydrophila was significantly improved, and the content of lysozyme in plasma was also markedly increased (Adnan et al., 2018). Interestingly, we found that plasma lysozyme activity was negatively correlated with serum antimicrobial capacity in this study, which means that lysozyme activity in plasma is an important index to measure the antibacterial ability of the body (Kord et al., 2021).

The antioxidant system consisted of enzymes (SOD, CAT, POD, and GPX) and non-enzyme (glutathione and vitamins E and C) components, which jointly regulated the antioxidant level and maintained homeostasis (Sony et al., 2019; Abdel-Daim et al., 2020). In addition, the lipid peroxidation product LPO and its decomposition product MDA could directly reflect the rate and degree of lipid peroxidation in tissues (Mahgoub et al., 2020). In this regard, we evaluated the antioxidant capacity of the liver tissue in juvenile common carp fed with dietary fucoidan. The results showed that feeding with fucoidan significantly increased the activities of SOD, CAT, POD and GPX, and decreased the levels of LPO and MDA in common carp. Similarly, P. fulvidraco fed with dietary fucoidan could increase SOD and CAT activities and decrease MDA content (Yang et al., 2014). It was found that the antioxidant activity of fucoidan usually played a role through molecular pathways, such as MAPK (Kim et al., 2011; 2012), Keap1-Nrf2-ARE (Zhang et al., 2012), PI3K-Akt (Han et al., 2015a; 2015b), and the TLR-NFkB signaling pathway (Asanka et al., 2019).

The liver function could be regulated by using proper feed supplements in aquafeeds (Hemre et al., 2002), which would be reflected in antioxidant levels (Mohammadi et al., 2021), inflammation index (Yang et al., 2021), and the histological structure of the liver (Cui et al., 2020). ALT and AST levels in liver were much higher than those in plasma, but liver cell necrosis could lead to ALT and AST levels that are two times higher than plasma. Therefore, plasma ALT and AST could serve as indices to evaluate the degree of hepatic damage (Josekutty et al., 2013). Previous studies have documented that the activities of plasma ALT and AST restored the normal level in mice fed the diet containing 100 mg/(kg·day) fucoidan after BCG vaccine and lipopolysaccharide-induced immunological liver injury; it suggested that fucoidan could significantly reduce the necrosis of hepatocytes and protect the liver (Xiao et al., 2017). Opposite results in the present study showed that the activity of plasma ALT and AST, and the content of TB and Glu were significantly increased in Diet 5 compared to other diets. It indicated that a higher dose (2,000 mg/kg) of fucoidan may induce liver injury and metabolic disorders. Moreover, hepatic micromorphology can be used to assess the healthy status of the liver. In our study, histological observation of the liver showed that the diet with 500–1,500 mg/kg fucoidan could ameliorate the morphology of hepatic tissue to a certain extent, such as reducing vacuolation, inflammatory response, and improving blood circulation in the liver. These findings clearly show that fucoidan may protect the liver, whose mechanism may be associated with the suppression of hepatocyte apoptosis, intrahepatic inflammation, and antioxidant activity (Ahmad et al., 2022; Xue et al., 2022a), whereas a high dosage of fucoidan, particularly at 2,000 mg/kg, led to the increase of plasma ALT and AST levels, as well as significant pathological changes in liver tissue. Thus, it indicated that the addition of low dosage of fucoidan could ameliorate the hepatic morphology of juvenile common carp, whereas the high dosage probably caused further damage to the liver.

ACP was one of the signature enzymes of lysosomes, and its activity could represent the intensity of intracellular digestion in the intestine (Maritza et al., 2012; Zacarias et al., 2013). AKP could directly participate in the transfer of phosphate groups and play a key regulatory role in metabolism. Furthermore, AKP was also an important immunoreactive enzyme, and its activity was often used as a key indicator to diagnose foreign pathogens and environmental pollution (Jean, 2020; Singh and Lin, 2021). In the present study, the activity of ACP, AKP LPS, AMS, and TRY in the intestine was notably enhanced in groups feeding fucoidan, which indicated that fucoidan could effectively improve the digestive ability of the intestine of juvenile common carp. Similar results were observed in C. auratus feeding fucoidan (Cui et al., 2020). Of course, the intestinal digestive function was closely related to intestinal microbial composition (Wu et al., 2019; Amenyogbe et al., 2022; Chang et al., 2023). Thus, future studies are needed to explore the effect of fucoidan on intestinal microorganisms of common carp.