Effects of Replacing Fish Meal by Plant Protein Sources in Fish Feed on Serum and Muscle Cholesterol Levels, Cholesterol Metabolism-Related Enzyme Activity and Gene Expression of Fish – A Review

Research indicates that substituting fish meals with plant protein sources generally reduces total cholesterol levels in fish. Liu et al. (2021) conducted a study indicating that substituting fish meals completely with soybean meals in juvenile redlip mullets did not increase total cholesterol levels. A comparable trend was noted in Nile tilapia (Mahmoud et al., 2014), indicating that replacing fish meal with soybean meal did not negatively affect serum cholesterol levels. Yaghoubi et al. (2016) observed an increase in serum total cholesterol in silvery-black porgy (Sparidentex hasta) juveniles when fed diets with higher levels of soybean meal substituting fish meal, suggesting that plant-based diets may affect lipid metabolism differently compared to marine-based diets. Hansen et al. (2007) observed a significant reduction in T-CHO levels after replacing fish meal with plant proteins in Atlantic cod. The research conducted by Shen et al. (2020) also revealed that substituting a fraction of fish meal with cottonseed protein concentrate markedly decreased T-CHO levels in juvenile golden pompano. Research indicates that non-conventional plant proteins, such as sesame oil cake and rapeseed meal, lead to lower cholesterol levels compared to fish meal diets (Dossou et al., 2018; Jahanbakhshi and Hedayati, 2013). The findings indicate that plant proteins may decrease dietary lipid content, leading to reduced blood cholesterol levels. The source of plant protein employed may provide differing effects depending on the fish species and dietary composition. Rahmdel et al. (2018) observed that the partial substitution of fish meal with sunflower meal markedly increased overall cholesterol levels in common carp. Table 3 demonstrates the effects of replacing fish meal with different plant proteins on specific fish species.

Figure 1.

Step-by-step summary of cholesterol anabolism

LDL-C and VLDL-C are critical biomarkers of cardiovascular health in fish, with elevated levels often indicating impaired health or dietary inadequacies. Studies demonstrate that replacing fish meal with plant proteins often leads to reduced levels of LDL-C and VLDL-C. The study by Carneiro et al. (2020) revealed that substituting fish meal with Chlorella sp. meal resulted in reduced LDL-C and VLDL-C levels in zebrafish, suggesting a potential lipid-lowering effect of plant-derived proteins. Liu et al. (2021) revealed a comparable result when soybean meal substituted fish meal in juvenile redlip mullet, resulting in reduced LDL-C and VLDL-C levels. Zhang et al. (2019 a) demonstrated that juvenile rice field eels consuming diets containing soy protein concentrate instead of fish meal exhibited reduced LDL-C levels. Comparable results were observed in sea bass and rainbow trout in the studies conducted by Bonvini et al. (2018) and Lazzarotto et al. (2018), respectively. Studies demonstrate that soybean meal and various plant protein concentrates reduce LDL-C levels in tilapia and carp (Iqbal et al., 2022 b; Ren et al., 2017). The observed reductions are likely attributable to the lower fat and cholesterol content found in plant-based diets in contrast to fish meals, which are inherently high in cholesterol. Most studies indicate that plant-based protein diets can reduce LDL-C levels; however, the extent of reduction may differ depending on the kind and quantity of plant protein consumed. Deng et al. (2017) demonstrated that soy protein significantly decreased LDL-C levels in tilapia, whereas proteins such as rubber seed meal exhibited a less marked impact. Research on very low-density lipoprotein (VLDL-C) is limited compared to other lipid metrics; however, various studies indicate that substituting fish meal with plant proteins has a negligible effect on VLDL-C levels. Peng et al. (2020) demonstrated that replacing soy protein in carp diets resulted in considerable reductions in VLDL-C levels. López et al. (2015) reported related findings in tilapia, indicating that VLDL-C levels remained constant but total cholesterol and LDL-C markedly diminished with the incorporation of soy protein. Lazzarotto et al. (2018) and Palomba et al. (2022) revealed that the partial substitution of fish meal with plant proteins, such as corn gluten meal, led to negligible and insignificant reductions in VLDL-C levels in rainbow trout. Rahmdel et al. (2018) indicated that plant proteins, such as sunflower meal, may lead to a slight spike in VLDLC levels in fish, although the underlying mechanism is not well understood. These researches indicate that while VLDL-C may demonstrate a decreased response to dietary changes relative to other lipid characteristics, it might still experience moderate decreases after the incorporation of fish meal. Further investigation is required to assess the impact of different plant proteins on VLDL-C and its broader consequences for fish health and lipid transport. Table 4 summarizes some research on the effects of replacing fish meal with different plant proteins on LDL-C and VLDL-C levels in specific fish species.

High-density lipoprotein (HDL-C), frequently referred to as “beneficial” cholesterol, plays a crucial role in lipid transport and overall metabolic health. Many researches yield ambiguous results concerning HDL-C levels after replacing fish meal with plant proteins. HDL cholesterol, known for its beneficial attributes, demonstrates varied reactions to the substitution of plant protein in multiple investigations. Zhao et al. (2021 b) demonstrated that incorporating concentrated dephenolization cottonseed protein into the diet of Oncorhynchus mykiss (rainbow trout) did not significantly impact HDL-C levels, although it did reduce LDL and total cholesterol levels. Lazzarotto et al. (2018) and Palomba et al. (2022) conducted research that revealed no significant alterations in HDL-C levels when plant proteins substituted fish meal in the diets of rainbow trout.

Conversely, Shen et al. (2020) reported a notable decrease in HDL-C levels in juvenile golden pompano following the partial substitution of fish meal with cottonseed protein concentrate. Zhao et al. (2021 a) reported a modest reduction in HDL-C levels in hybrid groupers when fish meal was substituted with rendered extruded soybean meal, suggesting that HDL-C responses may vary based on the specific type of plant protein utilized. In certain instances, HDL-C levels were elevated following the substitution of plant protein. Zhang et al. (2019 a) investigated the rice field eel and found that soy protein concentrates elevated HDL-C levels, which is beneficial for the welfare of fish. The differences may be due to species-specific responses to the inclusion of plant proteins or the specific types of plant proteins utilized, as certain proteins contain antinutritional factors that could hinder lipid metabolism. Further research is necessary to elucidate the effects of plant protein-based diets on lipid metabolism and overall fish health. Table 5 presents a summary of research regarding the impact of substituting fish meal with various plant proteins on the HDLC levels in select aquatic species.

Triglycerides (TG) represent a significant lipid parameter that is affected by dietary intake. Multiple studies indicate that substituting fish meal with plant protein correlates with reduced triglyceride levels. Research suggests that plant-based diets often lead to altered lipid profiles in fish, including changes in serum triglyceride (TG) levels. In many cases, when fish meal is replaced with plant protein sources such as soybean meal, corn gluten meal, or rapeseed meal, the TG levels may decrease due to the absence of marine-derived omega-3 fatty acids and cholesterol. Studies by Gatlin III et al. (2007) and Zhou et al. (2017) indicate that these changes may be due to the reduced bioavailability of these essential lipids in plant proteins, which can result in compromised lipid metabolism. Deng et al. (2017) found that substituting fish meal with rubber seed meal in tilapia diets led to a notable decrease in serum triglycerides, suggesting a lipid-lowering impact of plant-based components. This change might be linked to the absence of marine lipids, which are abundant in fish meal, and the possible lipid-modulating properties of certain plant-derived ingredients (Cai et al., 2022; Sarker et al., 2018). López et al. (2015) found that substituting fish meal with soy protein concentrate and taurine in totoaba juveniles did not significantly affect triglyceride levels, indicating that the choice of plant protein and the addition of essential amino acids like taurine can influence blood lipid profiles. Comparable findings were observed in studies involving juvenile blunt snout bream (Ahmed et al., 2019) and juvenile turbot (Nagel et al., 2012). This suggests that the inclusion of essential amino acids such as taurine can mitigate some of the lipid profile changes associated with plant-based feeds. Table 5 presents a summary of research regarding the impact of substituting fish meal with various plant proteins on the triglyceride levels of certain fish species.

Not all plant proteins produce identical impacts on blood cholesterol levels. The substitution of fish meal with plant proteins in fish feed has demonstrated inconsistent impacts on fish blood cholesterol levels. Although numerous research suggests that plant protein diets decrease total cholesterol, LDL-C, and triglycerides, the impact on HDL-C and VLDL-C remains inconsistent. The results are affected by the kind of plant protein employed, the degree of fish meal substitution, and the particular kind of fish examined. Bonvini et al. (2018) discovered that soybean meal generally elevated total cholesterol and LDL in sea bass, whereas Shen et al. (2020) noted decreased cholesterol levels using cottonseed protein concentrate in golden pompano. Zhang et al. (2019 a) observed a beneficial elevation in HDL-C levels when soy protein concentrates substituted fish meal in rice field eel, indicating that plant proteins vary in their capacity to influence cholesterol and lipid metabolism in fish. Furthermore, Hansen et al. (2007) and Mahmoud et al. (2014) emphasized the necessity of equilibrating plant-based protein sources with vital elements like methionine and taurine, which are generally prevalent in fish meal but are possibly lacking in plant proteins. These amino acids are essential for sustaining satisfactory lipid metabolism and averting adverse changes in cholesterol levels.

SREBPs are critical transcription factors that regulate the expression of genes involved in lipid and cholesterol metabolism. SREBPs exist in two main isoforms: SREBP-1, which primarily controls fatty acid and triglyceride synthesis, and SREBP-2, which regulates cholesterol biosynthesis and uptake. Replacing fish meal with plant protein sources typically leads to increased expression of SREBPs, particularly SREBP-2, as fish attempt to compensate for the lower dietary cholesterol provided by plant proteins. This upregulation activates genes involved in cholesterol biosynthesis, such as HMG-CoA reductase (HMGCR), to maintain cholesterol homeostasis. Bou et al. (2017) found that replacing fish meal with soy protein concentrate in Atlantic salmon (Salmo salar) diets led to the upregulation of SREBP-2 expression. This upregulation was linked to the lower dietary cholesterol in plant-based diets, which triggered the activation of cholesterol biosynthesis genes, including HMGCR.

In a study on Atlantic salmon (Salmo salar), Caballero-Solares et al. (2018) demonstrated that diets containing plant protein induced an increase in SREBP-2 activity. Zhu et al. (2020) reported that feeding plant protein to rainbow trout (Oncorhynchus mykiss) resulted in an increased expression of SREBP-2 and its target genes, thereby increasing SREBP-2 enzyme activity. Mellery et al. (2015) demonstrated that replacing fish meal with plant protein sources, including soy protein and rapeseed protein, in rainbow trout diets significantly increased SREBP-2 expression in the liver. Liland (2011) showed replacing fish meal with plant proteins and vegetables oils in Atlantic salmon (Salmo salar) diets led to increased expression of SREBP-2 and its downstream genes, including HMGCR. Much research with similar results is shown in Table 6. This was interpreted as a compensatory mechanism to cope with the lower dietary cholesterol provided by plant proteins (Caballero-Solares et al., 2018; Liland, 2011; Mellery et al., 2015; Zhu et al., 2020).

While SREBP-2 primarily regulates cholesterol metabolism, SREBP-1 plays a crucial role in controlling fatty acid synthesis. Plant-based diets, which are often deficient in essential fatty acids found in fish meal, can lead to the upregulation of SREBP-1 as fish attempt to synthesize the necessary fatty acids endogenously. In a study by Morais et al. (2011), Atlantic salmon fed plant protein-based diets showed increased SREBP-1 expression, leading to higher rates of fatty acid synthesis. This was linked to the lower availability of omega-3 fatty acids in plant-based diets, which prompted the fish to up-regulate SREBP-1 to synthesize fatty acids. In Atlantic salmon, diets containing protein-based diets resulted in increased SREBP-1 expression, driving the synthesis of fatty acids to compensate for the lower lipid content of plant-based diets (Caballero-Solares et al., 2017).

SREBPs are regulated by a feedback mechanism based on intracellular cholesterol and fatty acid levels. When dietary cholesterol is low, as in plant-based diets, SREBP-2 is cleaved and translocated to the nucleus, where it activates genes involved in cholesterol biosynthesis, such as HMGCR (Kortner et al., 2013; Ranasinghe et al., 2022). Similarly, when fatty acids are deficient, SREBP-1 is activated to enhance fatty acid synthesis. Phytosterols, present in many plant proteins, also affect SREBP regulation. These sterols compete with cholesterol for absorption, further reducing cholesterol availability and enhancing the activation of SREBP-2. Summary of research regarding the impact of substituting fish meal with various plant proteins on SREBPs of certain fish species is shown in Table 8.

SQS, an essential enzyme in cholesterol biosynthesis, has been the focus of several studies examining how plant protein-based diets affect fish cholesterol metabolism and overall health. SQS catalyzes the conversion of farnesyl pyrophosphate into squalene, the first committed step in the sterol biosynthesis pathway. This leads to the production of squalene, a precursor to all sterols, including cholesterol. The pathway continues from squalene, eventually leading to the production of lanosterol, which is then converted into cholesterol through a series of further enzymatic reactions. Lanosterol synthase (LSS), also known as oxidosqualene cyclase (OSC), is responsible for the cyclization of 2,3-oxidosqualene (the oxygenated form of squalene) into lanosterol, a critical intermediate in the multi-step process that ultimately leads to the synthesis of cholesterol. This cyclization reaction is a complex transformation where the linear squalene molecule undergoes structural reorganization into the four-ring structure characteristic of sterols like cholesterol. The production of lanosterol marks the first step in the sterol branch of the mevalonate pathway, which is crucial for cholesterol production. Once lanosterol is synthesized, it undergoes multiple enzymatic modifications, including demethylation and saturation reactions, leading to the formation of cholesterol.

SQS and LSS function downstream of HMG-CoA reductase, which is responsible for producing farnesyl pyrophosphate, the substrate for SQS. Since SQS catalyzes a rate-limiting step in sterol synthesis, it is tightly regulated. When cellular cholesterol levels are high, the activity of SQS is downregulated, thereby reducing cholesterol synthesis. Conversely, under conditions of low cholesterol (for instance when fish are fed plant-based diets with low cholesterol content), SQS expression can increase, promoting endogenous cholesterol production to compensate for reduced dietary intake.

In a study by Turchini and Francis (2009) on rainbow trout (Oncorhynchus mykiss), the replacement of fish meal with soybean meal resulted in a significant reduction in dietary cholesterol intake. This led to an increase in the expression of genes involved in cholesterol synthesis, including SQS and LSS. Similarly, Amritha et al. (2020) observed an upregulation of SQS and LSS in Atlantic salmon (Salmo salar) fed a wheat gluten-based diet. Bendiksen et al. (2011) studied the effects of replacing fish meal with lupin protein concentrate in European sea bass (Dicentrarchus labrax) and reported similar findings. Kemski (2018) explored the expression of SQS and LSS in yellow perch (Perca flavescens), comparing fish meal-based diets with soybean meal diets but observed fish on plant-based diets had significantly elevated SQS and LSS gene expressions. Kemski (2018) and Turchini and Francis (2009) noted that while the fish exhibited upregulated SQS and LSS expressions, they also showed impaired growth and liver health, indicating that endogenous cholesterol biosynthesis might not fully compensate for the lack of dietary cholesterol. Amritha et al. (2020) and Bendiksen et al. (2011) also revealed that despite the increase in SQS and LSS expression, the fish demonstrated reduced cholesterol levels and hepatic fat accumulation. This suggested that while SQS and LSS activities were elevated in response to the low cholesterol content in the diet, the overall lipid metabolism was disrupted, leading to suboptimal health outcomes. This highlights a limitation in the ability of plant-based diets to support cholesterol balance through endogenous synthesis alone.

One of the unique challenges posed by plant-based diets is the presence of phytosterols, which are structurally similar to cholesterol and can compete with cholesterol for absorption in the intestine. Guerreiro et al. (2015) and Sissener et al. (2017) conducted a study on European sea bass and Atlantic salmon, respectively. They observed that when fish meal was replaced with plant protein sources rich in phytosterols, there was reduction in the absorption of cholesterol which led to a compensatory increase in SQS and LSS expressions. However, despite the upregulation of SQS and LSS, cholesterol levels in the fish remained lower than those fed fish meal-based diets, indicating the limitations of endogenous synthesis in maintaining cholesterol homeostasis in the presence of phytosterols. Sissener et al. (2017) provided further insights into the interaction where there were decreased plasma cholesterol levels. Several studies have explored strategies to mitigate the negative effects of plant-based diets on cholesterol metabolism. Yao et al. (2021) investigated the addition of cholesterol supplements and essential fatty acids to plant-based diets in common carp (Cyprinus carpio). They found that supplementing the diet with bile acids significantly reduced the compensatory upregulation of SQS and LSS, and improved overall lipid metabolism and growth performance. This suggests that while plant-based diets lead to increased SQS and LSS expression, appropriate supplementation can help restore balance and reduce the need for endogenous cholesterol synthesis. Summary of research regarding the impact of substituting fish meal with some plant proteins on SQS and LSS of certain fish species are shown in Table 6.

HMGCR is a key enzyme in the mevalonate pathway for cholesterol biosynthesis. HMGCR is the rate-limiting enzyme in cholesterol biosynthesis, and its expression is influenced by dietary cholesterol levels, which are typically lower in plant-based diets than in fish meals. These upregulations activate genes such as low-density lipoprotein receptor (LDLR), liver X receptor (LXR), and ATP-binding cassette transporters (ABC family). Zhu et al. (2018) investigated the impact of replacing fish meal with soy protein concentrate in rainbow trout (Oncorhynchus mykiss) diets. The study found an increase in HMGCR expression in the liver, suggesting enhanced cholesterol biosynthesis. In a study on Nile tilapia, researchers explored the effects of replacing fish meal with soybean meal and corn gluten meal. Liland (2011) found that HMGCR mRNA expression was significantly higher in fish fed plant-based diets compared to those fed traditional fish meal-based diets. A study conducted by Mellery et al. (2015) replaced fish meal with a blend of soy protein and rapeseed protein concentrate in rainbow trout diets. The results demonstrated a significant upregulation of HMGCR expression in the liver tissues of fish fed plant-based diets. The up-regulation of HMGCR was directly linked to the need for increased endogenous cholesterol synthesis, compensating for the reduced cholesterol intake from plant-based diets to maintain homeostatic cholesterol levels (Caballero-Solares et al., 2018). The increase in HMGCR expression in response to plant-based diets can be attributed to several mechanisms. One of the primary regulatory pathways is through sterol regulatory element-binding proteins (SREBPs), particularly SREBP-2, which regulates cholesterol biosynthesis. When dietary cholesterol is low, as in plant-based diets, SREBP-2 is activated and induces the expression of HMGCR to increase cholesterol synthesis (Bou et al., 2017; Zhu et al., 2020). Additionally, the presence of phytosterols in plant-based diets, which compete with cholesterol for absorption, may further exacerbate the need for endogenous cholesterol synthesis. Summary of research on the effect of substituting fish meal with some plant proteins on HMGCR expression of some fish species is shown in Table 7.

LDLR is a critical component of cholesterol homeostasis, and it is responsible for mediating the uptake of cholesterol-containing lipoproteins from the bloodstream into cells. Replacing fish meal with plant protein sources generally results in an upregulation of LDLR expression in fish. Cai et al. (2022) found that gibel carp (Carassius auratus gibelio) diets containing plant proteins exhibited marked upregulation of LDLR expression. This increase in LDLR activity helped maintain cholesterol homeostasis despite the lower dietary cholesterol input. Murashita et al. (2024) replaced fish meal with plant proteins in red sea bream (Pagrus major) diets and observed a significant increase in LDLR expression. Furthermore, in a study on red sea bream, fish fed diets with increasing amounts of fermented rapeseed meal had significantly elevated LDLR mRNA expression (Dossou et al., 2018). All these studies agree with other research in Table 6.

The upregulation of LDLR in fish fed plant-based diets is largely driven by a need to balance cholesterol homeostasis. Elevated LDLR mRNA expression indicates a physiological response to the lower cholesterol levels in the diet (Ye et al., 2019). Low dietary cholesterol levels reduce the feedback inhibition on LDL receptor expression, leading to increased LDLR activity to boost cholesterol uptake (Bou et al., 2017). This is often accompanied by changes in other genes involved in lipid metabolism, such as HMG-CoA reductase (HMGCR), which is upregulated to synthesize cholesterol endogenously (Caballero-Solares et al., 2018; Hussain et al., 2024; Ye et al., 2019). Moreover, the presence of phytosterols further reduces cholesterol availability, triggering a compensatory upregulation of LDLR to increase the efficiency of cholesterol transport from the bloodstream (Ye et al., 2019). Table 8 gives a summary of research on the effect of substituting fish meal with some plant proteins on LDLR of some fish species.

LXR is a nuclear receptor that plays a central role in regulating cholesterol, lipid metabolism, and inflammatory responses in fish. LXRs regulate the expression of genes involved in cholesterol efflux, such as ATP-binding cassette transporters (ABC family) and fatty acid synthesis genes. LXR regulates cholesterol homeostasis by promoting cholesterol efflux through the upregulation of transporters like ABCA1, ABCG5, and ABCG8, which are responsible for removing excess cholesterol from cells. LXR also regulates lipogenesis by activating genes involved in fatty acid synthesis, including SREBP-1. Given that plant-based diets tend to be lower in cholesterol and higher in phytosterols compared to fish meals, LXR activity is often modulated in response to these dietary changes. Replacing fish meal with plant protein sources can alter the expression of LXR and its target genes due to differences in the sterol and fatty acid content of plant proteins. Fish adapt to these changes by modulating LXR activity to maintain lipid homeostasis. Takakuwa et al. (2023) found that replacing fish meal with plant protein concentrate in red sea bream diets led to an increase in LXR expression. This was accompanied by higher expression of cholesterol transporters, indicating an enhanced cholesterol efflux in response to the reduced cholesterol content of the plant-based diet. Zhu et al. (2020) reported that rainbow trout fed plant protein exhibited increased LXR expression. This upregulation was linked to enhanced cholesterol efflux through the activation of ABCA1 and ABCG5, as the fish adapted to the lower cholesterol levels in the plant-based diet. In a study by Caballero-Solares et al. (2018), Atlantic salmon fed diets containing terrestrial alternatives to fish meal (plant protein) showed increased LXR activity. The upregulation of LXR in these fish was linked to enhanced expression of ABCA1 and other transporters involved in cholesterol removal, suggesting a compensatory mechanism in response to the low cholesterol content of plant-based diets. Bou et al. (2017) investigated the effects of replacing fish meal with soy protein concentrate on LXR expression in Atlantic salmon. The study found that LXR expression was upregulated. This up-regulation led to increased expression of cholesterol efflux transporters, including ABCA1 and ABCG5. Liang et al. (2024) studied the effects of soy protein concentrate-based diets on LXR expression in largemouth bass. The fish exhibited increased LXR expression, which promoted the upregulation of genes involved in cholesterol efflux, including ABCG5 and ABCG8. This response was associated with the higher phytosterol content of the plant-based diet, which competes with cholesterol for absorption, triggering an adaptive response via LXR. In Atlantic salmon, Morais et al. (2011) found that feeding plant-based diets led to increased LXR expression, which activated SREBP-1 and promoted fatty acid synthesis. Table 8 gives a summary of research on the effect of substituting fish meal with some plant proteins on LXR of some fish species.

ABC transporters are a large family of proteins involved in the transport of various molecules across cellular membranes, including lipids, sterols, and xenobiotics. These transporters, especially ABCA1, ABCG1, ABCG5, and ABCG8, play a critical role in cholesterol homeostasis by mediating cholesterol efflux from cells, thus maintaining lipid balance in fish.

ABCA1 and ABCG1 is one of the key transporters involved in the export of cholesterol and phospholipids to lipid-poor apolipoproteins, facilitating the formation of high-density lipoproteins (HDL). This process is crucial for the reverse cholesterol transport pathway, where excess cholesterol is removed from tissues and transported to the liver for excretion. ABCA1 helps prevent cholesterol accumulation in cells, aligning with catabolic processes that reduce cholesterol levels. ABCG1 works alongside ABCA1 to export cholesterol to HDL particles. It also contributes to reverse cholesterol transport, ensuring cholesterol is cleared from peripheral tissues and ultimately eliminated. In fish fed plant-based diets, ABCA1 expression tends to be up-regulated to compensate for the lower dietary cholesterol intake. Bou et al. (2017) reported that replacing fish meal with soy protein concentrate led to an increase in ABCA1 expression in Atlantic salmon. This upregulation was attributed to the reduced availability of dietary cholesterol, which prompted the fish to increase cholesterol efflux to maintain cellular lipid balance. In a study by Caballero-Solares et al. (2018), Atlantic salmon (Salmo salar) fed diets containing plant proteins exhibited a marked increase in ABCA1 expression.

The ABCG5 and ABCG8 transporters are expressed in the liver and intestines and they are involved in the biliary excretion of cholesterol, a crucial step in cholesterol catabolism (Plosch et al., 2006). The ABCG5 and ABCG8 transporters form a heterodimer that plays a key role in transporting cholesterol and phytosterols across the cellular membrane (Dikkers and Tietge, 2010). These transporters help regulate the excretion of sterols into bile and are particularly important in fish fed plant-based diets due to the higher levels of phytosterols in plant ingredients (Dikkers and Tietge, 2010; Wang et al., 2015). Mellery et al. (2015) showed that rainbow trout fed diets containing soy protein and rapeseed protein exhibited an increase in ABCG5 and ABCG8 expression. Moreover, Liang et al. (2024) found that largemouth bass (Micropterus salmoides) fed diets with soy protein concentrate meal exhibited increased expression of ABCG5 and ABCG8 in the liver. In all these researches, the upregulation was attributed to the higher levels of phytosterols in the plant-based diet, which necessitated the enhanced excretion of excess sterols to maintain lipid balance (Gu et al., 2014; Kopylov et al., 2021; Lifsey et al., 2020; Sissener et al., 2018; Takase and Ushio, 2018; Zhu et al., 2018). Plant-based diets, especially those containing ingredients such as soy, rapeseed, and corn gluten, tend to have higher levels of phytosterols, which can influence the expression of ABC transporters involved in cholesterol and sterol transport (Couto et al., 2014; Gu et al., 2014, 2017; Takakuwa et al., 2023). Phytosterols compete with cholesterol for absorption in the intestine, reducing cholesterol uptake and increasing the need for cholesterol efflux through ABCA1. This leads to the up-regulation of ABCA1 to maintain cellular cholesterol levels. Furthermore, the higher levels of phytosterols in plant-based diets stimulate the expression of ABCG5 and ABCG8, which are responsible for excreting excess phytosterols and cholesterol. This mechanism helps fish maintain lipid homeostasis by preventing the accumulation of excess sterols in the body. Summary of research on the effect of substituting fish meal with some plant proteins on ABC transporters of some fish species is shown in Table 8.

ACAT is responsible for esterifying free cholesterol into cholesterol esters for storage within cells. This enzyme helps regulate intracellular cholesterol levels by storing excess cholesterol in the form of esters. ACAT activity is elevated when fish consume diets rich in cholesterol. This enzyme helps manage excess cholesterol by converting it into a storage form, preventing an overload of free cholesterol in cells. Several studies, such as those by Liang et al. (2024), highlight that plant-based diets rich in soybean or corn gluten led to increased ACAT expression. The rise in ACAT activity is an adaptive mechanism to store the excess free cholesterol as esters, preventing intracellular cholesterol overload. This was observed in fish like juvenile largemouth bass and gilthead sea bream. Fish fed plant proteins need to convert more cholesterol into esters, which reflects an attempt to maintain cellular cholesterol balance (Jiang et al., 2024 b; Valente et al., 2011). However, this increased activity might also indicate lipid storage issues and the onset of fatty liver over time (Katan et al., 2019; Liland, 2011). Studies like those by Deng et al. (2013), Tong et al. (2020) and Koven et al. (2016, 2023) show that cholesterol supplementation improves ACAT activity in fish fed plant proteins, such as soy protein concentrate. The addition of cholesterol helps balance the lipid metabolism disrupted by plant-based diets, ensuring proper lipid storage without excessive sterol accumulation. Cholesterol supplementation can mitigate the negative impacts of plant proteins on ACAT, stabilizing cholesterol esterification and bile acid synthesis. This strategy also reduces metabolic stress, especially in fish species heavily dependent on dietary cholesterol. Research by He et al. (2022) and Takase and Ushio (2018) emphasizes that diets containing phytosterols (naturally present in plant-based diets) suppress ACAT expression. Phytosterols mimic cholesterol, competing with it for metabolism but do not activate ACAT efficiently. This can impair the esterification process, leading to the accumulation of free cholesterol in tissues (He et al., 2022). Some studies, such as Liang et al. (2024) and Yuan et al. (2020), suggest that plant oils cause only moderate changes in ACAT activity. These oils, when combined with plant proteins, maintain lipid metabolism without triggering significant sterol accumulation or suppressing ACAT function. Diets with balanced lipid sources (for example, plant oils mixed with fish oils) reduce the metabolic burden on ACAT and ensure efficient cholesterol regulation without severe disruptions in metabolism (Ge et al., 2023). Studies like Xu et al. (2020) report that taurine supplementation alongside plant proteins helps normalize ACAT activity. Taurine plays a role in bile acid synthesis, improving cholesterol metabolism and alleviating the metabolic stress caused by plant protein diets. Fish species, such as Japanese seabass, benefited from taurine supplementation, maintaining both lipid balance and bile acid synthesis. This strategy shows promise for optimizing plant-based diets in aquaculture. Table 9 presents a summary of research on the effect of substituting fish meal with some plant proteins on ACAT activity in some fish species.

This enzyme catalyzes the first step in the conversion of cholesterol into bile acids in the liver. It plays a critical role in regulating cholesterol levels by facilitating the excretion of cholesterol as bile acids (Li and Chiang, 2009). CYP7A1 activity is influenced by the amount of dietary cholesterol. Higher dietary cholesterol can increase the need for bile acid production, leading to higher activity of CYP7A1 (Caponio et al., 2020). This enzyme helps balance excess dietary cholesterol by converting it to bile acids for excretion (Just et al., 2018).

Several studies, including those by Zhang et al. (2019 b) on Japanese seabass and Zhao et al. (2020) on common carp, report that plant-based diets tend to reduce CYP7A1 expression. This decrease in enzyme activity corresponds to lower bile acid synthesis, which may compromise the ability to metabolize and excrete excess cholesterol. Moreover, accumulation of cholesterol in hepatic tissues potentially leads to metabolic disorders like fatty liver in some species. This suggests that fish meal contains components such as certain amino acids, cholesterol, or other nutrients, that are crucial for maintaining optimal bile acid metabolism. Kortner et al. (2014) found that supplementing plant-based diets with dietary cholesterol helped restore CYP7A1 activity. In this study on Atlantic salmon, supplemental cholesterol (1–1.5%) stimulated the production of bile acids, balancing cholesterol metabolism disrupted by the absence of fish meal. This implies that cholesterol from fish meal plays a vital role in modulating CYP7A1. External cholesterol sources can partially compensate for the effects of plant protein diets on cholesterol metabolism. Wei et al. (2020) studied Amur sturgeon and found that a complete replacement of fish meal with plant protein reduced CYP7A1 expression, which impaired bile acid homeostasis and intestinal health. The study highlighted those changes in bile acid metabolism also affected immune function, demonstrating a complex link between lipid metabolism and immunity. This suggests that bile acids, regulated by CYP7A1, may have secondary effects beyond digestion, particularly in immune modulation and liver function. The study on Nile tilapia by Li et al. (2023) emphasized that diets high in plant protein altered the fish’s cholesterol turnover. The suppression of CYP7A1 led to cholesterol accumulation, negatively affecting lipid metabolism and energy homeostasis. In some cases, the altered metabolism resulted in higher body adiposity and metabolic imbalances. These findings illustrate the importance of bile acid synthesis not only for excreting excess cholesterol but also for maintaining lipid equilibrium within the body. Chen et al. (2024) observed that largemouth bass fed mixed protein diets, including plant proteins, exhibited inhibited CYP7A1 expression. This suppression disrupted bile acid receptor signaling pathways (for example, FXR-RXR pathways), which play a role in cholesterol metabolism feedback mechanisms. The disruption reduced the fish’s ability to regulate cholesterol levels, illustrating the complexity of lipid regulation pathways. In some cases, partial replacement of fish meal with plant proteins was found to mitigate these negative effects. For example, Murashita et al. (2024) reported that mixed diets containing both animal and plant proteins maintained better bile acid metabolism, including CYP7A1 expression, compared to full plant-based diets. This suggests that gradual substitution or inclusion of specific supplements could help balance the metabolic effects. The studies collectively highlight the complex relationship between fish meal, plant proteins, and CYP7A1 expression. Plant protein diets reduce CYP7A1 expression, affecting the synthesis of bile acids and disrupting cholesterol metabolism. Cholesterol supplementation or mixed diets help restore bile acid metabolism, mitigating the negative impacts of fish meal replacement. Long-term disruption of CYP7A1 and bile acid metabolism can lead to fatty liver, metabolic disorders, and compromised immune function in fish. Gradual substitution strategies or additional lipid supplements may offer solutions to balance lipid metabolism in plant-based aquafeeds. CYP7A1 plays a pivotal role in cholesterol metabolism, and its suppression from plant protein diets can lead to various metabolic disturbances. While dietary cholesterol and partial substitution strategies show promise in mitigating these effects, further research is required to optimize plant-based diets for aquaculture, ensuring both growth performance and metabolic health.

While the upregulation of these cholesterol metabolic genes and enzymes in response to plant-based diets helps fish maintain cholesterol and lipid homeostasis, the long-term effects of these diets on fish health must be carefully monitored. Studies have shown that prolonged reliance on plant protein diets may lead to hepatic lipid accumulation and fatty liver disease in some species, as a result of altered lipid metabolism (Bou et al., 2017; Caballero-Solares et al., 2018; Mellery et al., 2015). Careful balancing of lipid content, the inclusion of essential fatty acids, and minimizing the anti-nutritional effects of phytosterols in plant-based diets are important to ensure optimal health and growth in aquaculture species. Table 10 contains research on the effect of substituting fish meal with some plant proteins on CYP7A1 activity in some fish species.