In this study, cottonseed protein concentrate purchased from Xinjiang Jinlan Plant Protein Co., Ltd (Shihezi, China) was obtained through a series of processes including mild temperature extraction, depigmentation, and controlled reduction of carbohydrate content from cottonseed meal (16). CPC is characterized by 61.51% crude protein, 2.36% crude lipid, 0.85% methionine, 2.47% lysine, and 230 mg/kg free gossypol.

Four isoproteic and isolipidic diets supplemented with CPC, substituting either 0%, 20%, 40%, or 60% of fishmeal (FM) protein, were designed and applied in the four experiment groups, named CPC0, CPC1, CPC2 and CPC3, respectively. The formulated dietary crude protein level (54%) and crude lipid level (12%) were the same as those in commercial feeds and the study of Zhao et al. (9). The formulation and proximate composition of the experimental diets are exhibited in Table 1 . The feed production process was consistent with previous studies in our laboratory (9, 27). The manufactured 1.5 mm pellets were stored in a drying oven at 60 °C for 3 h, and then naturally air dried. The dietary pellets were kept at -20 °C for later trials.

Pikeperch were sourced from the Hulan Experimental Station, Heilongjiang Fisheries Research Institute (Harbin, China). After transportation to the recirculating aquaculture operation room of the Heilongjiang Fisheries Research Institute, the fish were fed with an even mixture of the four diets for 2 weeks to adapt to the experimental diets and culture environment. Through the 2-week acclimation, a total of 360 juvenile pikeperch (3.55 ± 0.01 g) were selected and randomly distributed into 12 indoor fiberglass aquariums with a length, wide, height, and water capacity of 0.6 m, 0.45 m, 1.0 m, and 270 L, respectively. Three replicates (aquariums) were arranged per treatment and each aquarium contained 30 fish.

The feeding experiment was executed in the water-recirculating aquaculture facilities of the Fish Nutrition and Feed Department with a feeding period of 8 weeks. Throughout the 8-week experiment, all the fish were hand-fed twice daily at 7:00 and 17:00 to apparent satiation. The residual feeds and feces waste were siphoned out by observing the food intake, and one-third of the circulating water was renewed twice a week. During the feeding trial, an air compressor was utilized to guarantee that the dissolved oxygen in the aquarium was above 6 mg/L. Furthermore, the water temperature was maintained at 23 ± 1°C, pH was 7.7 to 7.9, and the natural photoperiod was implemented.

When the feeding trial was finished, all the pikeperch were counted and weighed together after 24 hours of fasting to calculate the survival rate (SR), mass gain rate (MGR), specific growth rate (SGR), and feeding efficiency ratio (FER). Six fish per aquarium were randomly selected and anesthetized with MS-222 (Ethyl m-aminobenzoate mesylate, 100 mg/L) (28) to individually measure body mass, body length, visceral mass, and liver mass to calculate the condition factor (CF), viscerosomatic index (VSI), and hepatosomatic index (HSI), respectively.

After collecting the growth data, the aforementioned (in section 2.3) six chosen fish per aquarium were dissected to obtain the intact intestine samples and intestinal contents. Of the six intestine samples, four of them were fast-frozen in liquid nitrogen, and stored at -80°C for RNA extraction and enzyme activity measurement. For the other two intestine samples, anterior intestines were fixed with 4% paraformaldehyde fixative to observe the tissue structure. The intestinal contents were collected with two biological replicates in each aquarium, and the intestinal contents of three pikeperch were mixed as a sample for each biological replicate. Then, the samples were fast-frozen in liquid nitrogen and stored at −80 °C for analysis of intestinal microflora composition. Furthermore, six fish were randomly selected from each aquarium and stored at -20 °C for proximate body composition.

All data were analyzed using the statistical software SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The data were subjected to a one-way analysis of variance (ANOVA). When there were significant differences (P < 0.05), the group means were further compared using Duncan’s multiple-range test. The difference was viewed as significant when P < 0.05. The experimental data were presented as the means ± standard deviation (means ± SD).

As shown in Table 3 , there were no significant effects of dietary CPC levels on SR, MGR, and SGR (P > 0.05), but the lower values of MGR and SGR in the CPC substitution groups were observed compared to the CPC0 group. With regard to the CPC substitution groups, the values of MGR and SGR tended to increase with increasing CPC substitution ratios (P > 0.05). In contrast, FER among all groups tended to be significantly elevated, and the highest FER was observed in the CPC3 group (P < 0.05).

As listed in Table 4 , there was no significant difference in the values of VSI and CF and the moisture content and crude lipids between the substitution groups and CPC0 group (P > 0.05). When compared to the CPC0 group, the HSI value in the CPC2 and CPC3 groups and crude ash content in the CPC3 group was significantly decreased (P < 0.05), while the crude protein content in the CPC1 group was markedly decreased (P < 0.05).

The analysis results of intestinal digestive enzyme activities in the pikeperch are presented in Figure 1 . For AMS activity, no significant differences were observed among all the treatments (P > 0.05). The activity of LPS in the CPC2 group was significantly lower than that in the CPC0 group. Trypsin activity was significantly decreased when the dietary CPC replacement level was above 40% (the CPC2 and CPC3 groups) (P < 0.05).

As shown in Figure 2 , the activities of CAT, SOD, and GSH and T-AOC content tended to decline in the CPC substitution groups compared to the CPC0 group. Therein, significantly lower GSH activity and T-AOC content were observed in the CPC substitution groups (P < 0.05), and significantly lower CAT activity was observed in the CPC1 group (P < 0.05). In terms of the CPC substitution groups, both CAT and GSH activity in the CPC3 group were markedly higher in comparison to the CPC1 group (P < 0.05), whereas there was no significant difference in the CAT and SOD activities between the CPC3 group and CPC0 group (P > 0.05).

As shown in Figure 3 , the HE-stained sections illustrated that dietary CPC substitution appeared to result in poor intestinal morphology for pikeperch. In general, CPC substitution thickened the muscle layer to a certain extent, especially in the CPC3 group according to the HE-stained sections. Additionally, more confluent intestinal villi and more obvious villi vacuolation were observed in the CPC1 and CPC2 groups compared to the CPC0 and CPC3 groups. More particularly, the intestine samples in the CPC3 group had exposed intestinal villi. The CPC3 group had fewer intestinal villi compared to the CPC0 group.

The CPC3 group had significantly increased villus height by approximately 16.37% and 28.80% compared to the CPC0 and CPC1 groups, respectively (P < 0.05). The CPC3 group had significantly decreased villus width by approximately 26.55% compared to the CPC2 group (P < 0.05). No significant difference in layer thickness was observed among all treatments (P > 0.05).

The gene expressions in the intestine of pikeperch in the different dietary CPC substitution groups are presented in Figure 4 . In sum, the intestinal expressions of genes related to the mTOR signal pathway, GH/IGF growth axis, tight junction proteins, and anti-inflammatory cytokine (tgf-β) were significantly downregulated to varying degrees in the CPC-supplemented groups (especially in the CPC1 and CPC2 groups) compared to the CPC0 group (P < 0.05), while pro-inflammatory cytokines (il-1β, il8, tnf-β) were significantly upregulated to varying degrees. No significant difference in the intestinal expression of eif4ebp, ghr, zo2, il-1β, il8, and tnf-β was observed between the CPC0 and CPC3 groups (P > 0.05). From the perspective of the CPC-supplemented groups, a majority of the intestinal expressions of genes, except igf1 and tnf-β in the CPC3 group, were significantly upregulated compared to those in the CPC1 and CPC2 groups (P <0.05).

Numerous research results have established an accepted fact that the substitution of fish meal with cottonseed protein concentrate will not adversely affect the growth and feed utilization of farmed carnivorous fish with a reasonable range of substitution (32, 33). In our study, there was no denying that replacing fish meal with CPC in pikeperch feed weakened the SR and growth indexes to some extent, but no significant differences were found between the CPC substitution groups and the control group. Unexpectedly, the 60% CPC substitution group obtained the most optimal MGR and SGR among the CPC substitution groups, indicating that the fish meal dosage, after CPC substitution, could be reduced to 22%. In a similar study on other farmed fish species in Perciformes, 50% CPC substitution reduced the fish meal level in sea perch feed (34) from 45% to 23% and in pearl gentian grouper (Epinephelus fuscoguttatus♀× Epinephelus lanceolatus♂) (35) feed from 50% to 25%. The results appear to suggest that CPC possesses the potential to substitute a relatively higher level of fish meal in the feed of farmed fish in Perciformes. The FER variation trend in our study also supports this conjecture. We found that the highest FER value was in the 60% CPC substitution group and it was significantly higher than that of the control group. Similar findings were published in a study of black sea bass (36), which reported that the lowest feed conversion rate (inverse of FER) occurred at the 80% CPC substitution level. Since the minimum dietary fish meal level designed in our experiment was 22%, it is not possible to determine whether cottonseed protein concentrate can be used as the main protein source to prepare fish meal-less or low-fish meal pikeperch feed. Further experiments are needed to confirm the potential of cottonseed protein concentrate as an alternative protein source for pikeperch.

Studies have revealed that the application of CPC as a fish meal substitute did not significantly impact the morphology and composition of the bodies of black sea bass (36) and rainbow trout (37). However, other studies have demonstrated that replacing fish meal with CPC showed the opposite. Shen et al. (2019) reported that for Trachinotus ovatus, replacing up to 24% of the fish meal with CPC could significantly increase CF, and replacing up to 36% to 60% of fish meal with CPC could significantly increase the crude protein content. This change in morphology and composition of fish bodies also appears in our study. However, unlike previous studies that focused on significant changes in CF (38) and crude lipid content (19), our current study indicated that significantly lower HSI value and crude ash content and higher crude protein content were observed in the 60% CPC substitution group. A comparison of different studies may ascribe the differentiation to variations in cottonseed protein processing methods, fish species, and culture conditions.

The primary function of the intestine in fish is the digestion and absorption of foreign nutrients, which is directly embodied by morphology and digestive enzyme activity (39). Excellent morphological parameters including appropriate villus height, villus width, and muscle layer thickness can increase the digestive contact area and promote peristalsis (40). Fish digestive enzymes mainly include protease, lipase, and amylase. The activity of these enzymes reflects the digestive capacity of fish, affecting the absorption and utilization of protein, fat, and carbohydrate and the efficiency of energy acquisition (41). Studies on silver sillago (Sillago sihama Forsskál) (42) and large yellow croaker (20) have found that when CPC replaced a higher proportion of fish meal, the villus height, muscle thickness, and digestive enzyme activities tended to decrease. In our study, we also found that intestinal digestive enzymes including trypsin, lipase, and amylase presented a decreasing tendency, and, in particular, trypsin activity in the 60% substitution group was reduced to the lowest level compared to the control group and 20% substitution group. Previous research has indicated that replacing an overproportion of fish meal with plant protein sources will negatively affect the protease activity of the digestive organs of carnivorous fish (43, 44), This is probably due to a reduction in the bioactive substances with the increase in fish meal substitution which weakens the ability of the pancreas and intestinal glands to secrete digestive enzymes. Hence, decreased trypsin activity in the intestine might be an adaptation of juvenile pikeperch to concentrated cottonseed protein in the diet. However, unlike previous studies (20, 42), we found that the villus height in the intestine is significantly increased, suggesting that the intestine of pikeperch can adapt to an increase in CPC substitution by increasing peristaltic function.

A higher proportion of plant protein can aggravate the oxidation stress of fish, thereby causing body damage and affecting the utilization efficiency of the plant protein (45). Concurrently, intestinal inflammation is triggered by the release of inflammatory factors due to oxidative stress (46). The change in antioxidant enzyme (SOD, CAT, etc.) activities and antioxidative substance content (GSH, etc.) could reflect the degree of oxidation to some extent (47). The intestinal immune system locally induces cells to produce pro-inflammatory factors such as interleukin-1 (il-1), il-8, and tumor necrosis factor-α (tnfα), and effector cells to produce anti-inflammatory factors such as transforming growth factor-β (tgf-β). Once the function and quantity of these two parts of cytokines are abnormal, intestinal homeostasis is broken, and the fish is extremely vulnerable to pathogen attack, which can induce a more intense inflammatory response in various tissues (48). Foodborne oxidative stress and inflammation caused by plant protein sources are generally attributed to their high content of anti-nutritional factors (49).

Evidentiary research has indicated that as anti-nutritional factors of cottonseed meal, excessive presence of gossypol in the aquatic feed could cause oxidation homeostasis in the liver or intestine and induce the secretion of inflammatory factors, thus resulting in intestinal inflammation (50–52). The origin of cottonseed protein concentrate is precisely derived from the removal of anti-nutritive gossypol from cottonseed meal (16). Research on largemouth bass found that when the gossypol content of CPC was optimized to 169.36 mg/kg, a 30% substitution of fish meal with gossypol-reduced CPC could significantly increase liver antioxidant activity, upregulate the mRNA expression of liver anti-inflammatory factors, and downregulate the mRNA expression of pro-inflammatory factors compared to the no-gossypol control group (53). Nonetheless, excess CPC in the feed caused the fish intestine to exhibit an inflammatory response (26, 54). Our current results showed that when the gossypol content of the CPC was reduced to 230 mg/kg, the 20% to 60% substitution of fish meal with gossypol-reduced CPC still significantly decreased the intestinal activities of CAT and SOD and contents of GSH and T-AOC, downregulated the mRNA expression of tgf-β, and upregulated the mRNA expression of pro-inflammatory factors (il-1β, il-8, and tnf-α) compared to the control group. This finding may imply that the tolerance of pikeperch to gossypol is relatively weaker than that of largemouth bass to a certain extent. It is noteworthy that among the three CPC substitution groups, increases in the intestinal antioxidant enzyme activities, GSH content, and anti-inflammatory factor expression and downregulation in pro-inflammatory factors were observed with increasing CPC substitution ratios. Combined with existing studies, we speculate that increased contents of prebiotics (such as raffinose and stachyose) with the addition of increasing CPC could partially counteract the negative effects of gossypol (18, 55). In any case, the specific mechanism needs to be further explored.

The intestinal mechanical barrier is a vital defense line against the invasion of pathogens and is mainly composed of epithelial cells and intercellular connections (56). Mechanical damage of intestinal mucosa caused by plant protein sources has been demonstrated in many fishes (31, 57). The intestinal villus height of Cyclopterus lumpus was shortened and the lamina propria width was increased when a mixture of soybean protein concentrate and pea protein concentrate replaced more than 25% of fish meal (58). In the current study, when 20% to 60% fish meal was replaced by CPC, the intestinal mucosa of pikeperch presented with mechanical damage in comparison with the control group, which was characterized by uneven intestinal wall thickness and short and sparse intestinal villi. Similar findings were found in a study on largemouth bass (15), which reported that excessive replacement of fish meal with CPC impaired intestinal structure. The permeability of intestinal tight junctions determines the barrier function of the entire intestinal epithelial cells. Transmembrane proteins, such as occludin and claudins, and peripheral cytoplasmal proteins, such as Zos, form a narrow band structure from the outside to the inside, sealing the cell space and preventing intestinal toxic substances from entering the surrounding tissues (59). Merrifield et al. (60) found that the exposure of intestinal cells to tight junctions was correlated with the height and density of intestinal villi. Analogously, CPC substitution levels of 20% to 60% significantly downregulated the gene expression of occludin-a, zo-2, and claudin-15a compared to the control group, which was consistent with changes in intestinal morphological damage. In accordance with the study in our previous study, the morphology and barrier function of common carp (Cyprinus carpio) intestines were damaged when the amount of CPC replacing fish meal exceeded 50% (61). Notably, the injury degree of the intestinal mechanical barrier was mitigated along with relief from oxidative damage and inflammation with increasing CPC substitution ratios. The adaption mechanism needs to be further explored in the future.

The intestinal biological barrier is mainly composed of normal intestinal flora, which can promote the proliferation of intestinal epithelial cells and strengthen the immune function of fish (9, 62–66). Diverse research studies have pointed out that CPC application in feed directly differentiated intestinal microflora structure in aquatic animals (38, 61, 67). Our study on pikeperch indicated that CPC substitution levels of 20% to 60% distinctly changed the intestinal microflora composition and lessened the bacterial diversity and richness, as shown by the Shannon, ACE, chao, and Simpson indices. The NMDS analysis further revealed that the degree of separation of microflora composition tended to be more obvious with increasing dietary CPC substitution ratios. These results suggest that the dietary CPC substitution ratio could vary the intestinal microflora structure of pikeperch.

Our results revealed that the abundances of the phyla Firmicutes, Proteobacteria, and Actinobacteriota occupied the first three positions in the microflora community of pikeperch following Bacteroidota, which was in line with our previous study on the intestinal microflora of pikeperch (9). In this study, increasing the dietary CPC substitution ratio slightly increased Firmicutes abundance and significantly decreased Actinobacteriota abundance, but the abundance of Proteobacteria in the 60% substitution group was appreciably increased. Similar results were also observed in the study on golden pompano (18) and grass carp (Ctenopharyngodon idellus) (38). Because an excess of Proteobacteria in the intestine could lead to metabolic disturbances or inflammation (68), an abnormal increase in Proteobacteria abundance may have been a potential risk of CPC being used to replace fish meal in pikeperch feed. Apart from this, our study examined the abundance ratio (F/B ratio) of Firmicutes and Bacteroides. Variation of the F/B ratio can indirectly reflect the metabolic homeostasis and growth state (68). In this study, the F/B value of the 60% CPC substitution group appreciably decreased and was positively correlated with a reduction in final mass and MGR. This suggests that CPC has the potential to promote growth by improving the intestine microflora structure.

At the genus level, Staphylococcus and Lactobacillus, belonging to Firmicutes, and Achromobacter, belonging to Proteobacteria, were the most dominant bacteria genera in our study. Among these, Staphylococcus and Achromobacter were the main bacteria genera in the 60% substitution group, and the abundance of the two genera increased gradually with the increase in CPC substitution. Staphylococcus, among gram-positive bacteria, is the most important infection pathogen (69). Similarly, an increased abundance of Staphylococcus was associated with intestinal disorder in common carp (70). In addition, a considerable decrease in Lactobacillus abundance was detected in the 40% and 60% substitution groups compared to the control group and 20% substitution group. It has been reported that Lactobacillus can regulate the homeostasis of the intestinal microecological environment and change the structure of the host’s intestinal microflora, thus improving the intestinal barrier defense against bacterial diseases (71). Together, the increase in Staphylococcus and the decrease in Lactobacillus caused by exorbitant CPC substitution might be deemed an ignition point for damage to intestinal microflora homeostasis and immune ability in this study.

The GH/IGF-1 axis has been regarded as an effective tool to regulate the growth of fish (72). Hence, variation genes in the GH/IGF-1 axis were mapped with changes in growth performance. As reported in a study on Nile tilapia (Oreochromis niloticus), 28 days of starvation suppressed growth and CF, with a concomitant decline in the expression level of IGF-1 (73). The nutrient composition of feed can also regulate the GH/IGF axis, thereby affecting the growth of fish (74). Evidential research has revealed that the level and source of dietary protein or lipids are tightly linked to GH and IGF-I or their mRNAs in gilthead sea bream (Sparus aurata L.) (75), Nile tilapia (76), and common carp (77). It was further shown that the utilization of alternative plant proteins in fish feed may result in liver GH desensitization and inhibit GHR and liver IGF-I gene expression. For instance, the expression of ghr and igf1 genes in the livers of common carp bream was significantly decreased after partial or total replacement of fish meal by CPC in common carp feed (61). The findings of the current study indicated that the expressions of ghr and igf2 first decreased and then rose with increasing CPC replacement levels in diets, which was basically consistent with the changing trend of the growth indexes. However, igf1 expression declined gradually with increasing CPC replacement levels in the diets. Furthermore, the GH/IGF axis also interferes with the nutritional status of tissues by regulating downstream pathways. Therein, activation of the IGF-phosphoinositol triphosphate (PI3K)-Akt-rapamycin target protein (mTOR) metabolic pathway during growth can effectively enhance protein synthesis in cells (78). To regulate protein metabolism in response to various nutrient quality and environmental conditions, activated TOR can regulate two parallel signaling pathways (4E-BP and S6K) to coordinate the balance between protein synthesis and protein degradation (79). Previous studies in the literature on grass carp (38) and common carp (61) reported that high levels of dietary CPC replacing fish meal depressed protein synthesis by inhibiting the activation of the TOR signaling pathway. However, this study found that the expression of mtor, akt1, rps6k1, and eif4ebp first decreased and then rose as the dietary CPC supplement level increased. Notably, the expressions of the four genes in the 60% substitution group were markedly enhanced compared to those in the 20% and 40% substitution groups. This change was very similar to the trend of crude protein content in the whole fish, which may be due to the fact that activation or shutdown of the TOR signaling pathway positively feeds back into protein synthesis. Meanwhile, this study also established that the impaired protein synthesis caused by the weakening of the TOR pathway is triggered by the decreased expression of igf1. Overall, these findings seem to imply that IGF1 in the GH/IGF axis and the IGF-PI3K-Akt-mTOR pathway is a key breakthrough in improving the growth-promoting effectiveness of CPC when replacing fish meal for pikeperch.