As the most economical energy source in aquatic feeds, starch can effectively reduce dietary protein and lipids (Zhou et al., 2022). Moreover, starch plays a prominent role in feed processing, especially for extruded floating pellets, which are typically used as stabilizers and swelling agents (Hemre et al., 2002). Hence, starch is widely used in various practical diet ingredients. However, the ability of fish to utilize starch is limited and related to the fish species. Compared with omnivorous and herbivorous fish, carnivorous fish have a lower ability to make use of dietary carbohydrates. Excessive dietary carbohydrates generally cause persistent glycogen deposition (Wu et al., 2016; Lin et al., 2018; Ma et al., 2019), induce an immune response (Hu et al., 2017; Cheng et al., 2020; Borin-Crivellenti et al., 2021) and further impair the growth and health status of cultured carnivorous fish. Therefore, studies on how to alleviate hepatic glycogen deposition caused by high dietary carbohydrates, which may contribute to coping with the nutritional–technological conundrum in manufacturing aquafeeds of carnivorous fish, should receive much attention.

Largemouth bass, Micropterus salmoides, has been widely cultured in China and has become one of most commercially important carnivorous species for freshwater culture (Li et al., 2021; Liang et al., 2022), and its production increased rapidly from 175 thousand tons in 2009 to approximately 702,093 tons in 2021 (Hou et al., 2023). In recent years, M. salmoides fed a commercial diet instead of fresh/thawed raw fish has been widely accepted by farmers in Zhejiang Province, for which the output of M. salmoides, which exceeds 95% of M. salmoides aquafarms using extruded floating feed, is ranked 2nd in China. However, M. salmoides shows poor utilization of dietary carbohydrates (Ma et al., 2019; Chen et al., 2023), and a starch-rich diet may lead to abnormal hepatic glycogen reservation and lipid metabolism and further impair the health status and growth performance of the fish.

Phytosterols (PSs), which include plant sterols and stanols, are steroid compounds similar to cholesterol that occur in plants and are often enriched in seeds and seed oils. Many investigators have demonstrated the cholesterol-lowering effects of phytosterols in chickens (Zhao et al., 2019), broilers (Naji et al., 2014; Ding et al., 2021) and rats (Marineli et al., 2012). In addition, it has been illustrated in previous reports that phytosterols in the diet could accelerate the growth rate and/or improve the meat quality of broilers and ducks (Wu et al., 2012; Naji et al., 2014). Recently, phytosterols have received increasing attention in the field of antioxidants (Trautwein and Demonty, 2007; Xie et al., 2015). However, little is known about the effects of dietary phytosterols on fish. The use of vegetable oils in feeds for farmed Atlantic salmon (Salmo salar) has increased in recent years (Liland et al., 2013). Phytosterols may also affect fish health through potential effects on cholesterol metabolism and liver lipid levels (Couto et al., 2014; He et al., 2022; Reyes-Becerril et al., 2022).

To date, limited reports are available regarding the effects of dietary phytosterol inclusion on M. salmoides. Therefore, the objective of the present study was to evaluate the effects of phytosterol supplementation in a high-starch diet on the growth performance and glucose and lipid metabolism response of juvenile M. salmoides. It is assumed that the findings of the present study will provide a basis for alleviating hepatic glycogen deposition and improving antioxidant status, which might contribute to more efficient formulation of economical diets for M. salmoides.

Previous studies on partridge shank chickens (Zhao et al., 2019), broilers (Naji et al., 2014; Ding et al., 2021) and rats (Marineli et al., 2012) have indicated that phytosterols can influence growth performance, feed utilization, and lipid metabolism and improve the antioxidant capacity of animals and can also enhance immune responses and antimicrobial activity but exert few effects on growth performance in some fish species (Couto et al., 2014; He et al., 2022; Reyes-Becerril et al., 2022). In addition, phytosterol can alleviate the damage caused by a high-lipid diet (He et al., 2022). The limited starch utilization capacity of M. salmoides has been demonstrated in a number of previous studies, and body weight gain, feed efficiency, and hepatic or intestinal health status could be negatively influenced by excessive dietary carbohydrates (Lin et al., 2018; Li et al., 2019; Ma et al., 2019; Zhang et al., 2020; Huang et al., 2021). To alleviate the negative effects induced by a high-carbohydrate diet, researchers have identified several feed additives, such as bile acid (Yu et al., 2019), resveratrol (Liu et al., 2021), berberine (Xia et al., 2022) and sodium diacetate (Fang et al., 2024). By exploring the effects of phytosterols in M. salmoides diets with a high starch content for the first time, our study revealed that phytosterols could effectively decrease the negative impact on feed utilization efficiency and plasma biochemical indices of M. salmoides induced by a high-starch diet and could also promote weight gain and specific growth ratios of M. salmoides, which is different from reports on large yellow croaker (Larimichthys crocea), gilthead sea bream (Sparus aurata L.) and European sea bream (Dicentrarchus labrax). The difference might be attributed to differences in animal species, size, starch level and dose of phytosterols. In addition, our results revealed that as the dietary phytosterol concentration increased from 0.1% to 0.5%, the FCR and IPFR significantly decreased, which indicated that supplemental phytosterols could promote feed utilization by M. salmoides and that the phytosterol dosage appeared to be significantly associated with these effects. Digestive enzymes are important compounds that mediate nutritional processes, and their activities represent the ability of the organism to digest and absorb nutrients (Lundstedt et al., 2004). The activities of protease and lipase in pyloric plants tended to increase markedly with increasing dietary phytosterol levels, which may have contributed to the recovery of feed utilization efficiency from high starch stress. However, the amylase activities were relatively low and not affected by the dietary treatments. This result was in accordance with previous reports (Li et al., 2020).

The plasma biochemical index is an important indicator for assessing fish health and nutritional status and is involved in many metabolic processes (Fırat et al., 2011). In our study, except for total protein (TP), superoxide dismutase (SOD) and catalase (CAT), the concentrations of six plasma biochemical indices significantly changed after diet treatment. Phytosterols have been thoroughly proven to lower plasma cholesterol in mammals (Laraki et al., 1993; Matvienko et al., 2002; Doornbos et al., 2005; Couto et al., 2015). Our study revealed that phytosterols play a role in lowering the plasma cholesterol and triglyceride levels in M. salmoides fed a high-starch diet, which indicated that phytosterols promoted lipid metabolism, consistent with findings in juvenile large yellow croaker (He et al., 2022), Atlantic salmon (Salmo salar L.) (Liland et al., 2013) and bluntnose black bream (Megalobrama amblycephala) (Zhang et al., 2012). Any change in the physiological status of the fish, from pollution to nutritional stress, can cause changes in liver enzymes. The fish liver is a key organ of numerous vital metabolic reactions related to carbohydrates, lipids, and proteins. AST and ALT play important roles in protein and amino acid metabolism and may be released into the plasma following tissue damage and dysfunction, and they are regarded as indicators of hepatic injury in aquaculture animals (Wang et al., 2020; Zare et al., 2023). Our results revealed that AST and ALT both significantly increased in the high-starch diet group but tended to decrease with increasing phytosterol concentration. With an additive dose of 0.5%, the concentrations of the indices could revert to normal dietary conditions, indicating the potential protection of phytosterols against negative stimulation of the fish body induced by high starch stress. Similar results were found in trials of bile acids (Guo et al., 2020).

Antioxidative systems are one of the main physiological mechanisms for protecting the health of aquatic animals (Castex et al., 2009). Antioxidation eliminates active middle products, especially oxyradicals, to protect cells and molecules from the oxidative damage caused by the attack of radicals. As the first line of defense against oxidative stress (Ighodaro and Akinloye, 2018), SOD and CAT are important antioxidative enzymes that work together to catalyze free radicals. MDA is a generally accepted and widely used biomarker of oxidative stress, notably lipid peroxidation (Tsikas, 2017), and SOD and MDA are often used as effective indicators to objectively reflect the antioxidant status of animals (Shadrack et al., 2022). In our study, the concentrations of SOD and CAT increased with increasing phytosterol addition. Moreover, plasma MDA significantly increased in the high-starch diet group but tended to decrease with increasing phytosterol treatment, which revealed the beneficial effect of phytosterol supplementation on the antioxidant status of M. salmoides. Similar results were found in chickens (Zhao et al., 2019) and large yellow croaker (He et al., 2022). Taken together, the increased antioxidant enzyme activity paralleled the reduced level of MDA resulting from phytosterol supplementation, indicating that dietary phytosterols could improve the antioxidant status of M. salmoides.

Our study revealed that phytosterol decreased plasma glucose in accordance with findings for bile acids and berberine (Guo et al., 2020; Xia et al., 2022), which indicated the potential acceleration function of phytosterol in glucose metabolism. Moreover, the levels of five glucose metabolism-related enzymes in the liver of M. salmoides were affected by the dietary treatments. Glycolysis is an important route of glucose catabolism, and GK, PK and HK are enzymes that play key roles in the regulation of the glycolysis pathway (Enes et al., 2010). Similarly to those in a previous study, the activities of GK and PK increased significantly in plants fed a high-carbohydrate diet, as excess glucose activated PK to produce pyruvic acid (Tan et al., 2010). The low activity of HK in fish fed high starch might harm glucose utilization and induce glucose intolerance in largemouth bass. In our study, the activities of hepatic HK did not significantly increase in the groups fed high dietary starch after 56 days of feeding or in the groups supplemented with phytosterol, which might be attributed to the feedback of excess Glu-6-P produced by continuous glycemia inhibiting activity. This was in agreement with the results reported for Megalobrama amblycephala (Zhou et al., 2013) and salmonids (Walton and Cowey, 1982).

The poor inhibition of gluconeogenesis by dietary carbohydrates is considered one of the reasons for the glucose intolerance of carnivorous fish (Panserat et al., 2000). In our study, the levels of key gluconeogenesis enzymes, including GK, PK and HK, showed similar trends, and higher activities were all recorded in the high-starch group, while experimental fish fed a high-starch diet supplemented with phytosterol had results similar to those of the low-starch group. Moreover, the lowest hepatic GK level was found in fish fed a low-starch diet among the groups. The opposite trend was observed for hepatic gluconeogenesis enzymes, and relatively lower activities of PEK and PEPCK were both found in the low-starch group. The different results could be ascribed primarily to differences in dietary starch levels and dietary phytosterol supplementation levels. The regulation of the gluconeogenic pathway by dietary carbohydrate sources is still not consistent, and the different responses of fish to dietary carbohydrate sources should be further clarified. The muscle and hepatic glycogen contents of juvenile M. salmoides fed the experimental diets for 8 weeks were also recorded. Because of the core role of the liver in glycogen reservation, the hepatic glycogen content was significantly influenced by dietary treatment compared to the muscle glycogen content. With increasing dietary phytosterol content, hepatic glycogen decreased gradually, corresponding to changes in the activities of glucose metabolism-related enzymes and in the concentrations of plasma biochemical indices, which might indicate that supplemental phytosterols could promote glucose utilization and metabolism and alleviate the negative stimulation of continuous glycemia stress in M. salmoide fed a high-starch diet.

In summary, dietary phytosterol supplementation could enhance the growth performance, feed utilization, antioxidant status and glucose metabolism of juvenile largemouth bass (Micropterus salmoides) fed a high-starch diet. In addition, the present study revealed that dietary phytosterol supplementation plays a key role in ameliorating the negative stimulation of continuous glycemia stress induced by high starch. Therefore, further studies should focus on the relationships between phytosterol and glucose metabolism as well as determining the metabolic pathways involved under high starch stress to facilitate better dietary starch utilization to reduce the feed costs of M. salmoide.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The animal study was approved by Institutional Review Board of the Science Technology Department of Zhejiang Province. The study was conducted in accordance with the local legislation and institutional requirements.

QL: Conceptualization, Data curation, Formal Analysis, Investigation, Project administration, Writing – original draft. YH: Data curation, Formal Analysis, Investigation, Methodology, Writing – review & editing. NZ: Investigation, Methodology, Writing – review & editing. YB: Investigation, Software, Writing – review & editing. WS: Investigation, Software, Writing – review & editing. XC: Investigation, Validation, Writing – review & editing. GY: Investigation, Validation, Writing – review & editing. QM: Investigation, Validation, Writing – review & editing. RH: Investigation, Validation, Writing – review & editing. XD: Funding acquisition, Project administration, Supervision, Writing – review & editing. FZ: Data curation, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.