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Review

Cottonseed Protein as an Alternative Feed Ingredient for Fish: Nutritional Metabolism and Physiological Implications

by
Yue Hu
1,
Yang Xie
1,
Youdi Tang
1,
Jiarui Liu
1,
Esau Mbokane
2,
Rana Al-Sayed Dawood
3,
Jie Luo
1,
Debing Li
1,* and
Quanquan Cao
1,*
1
College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
2
Aquaculture Research Unit, School of Agricultural and Environmental Sciences, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa
3
Faculty of Science, Damanhour University, El-Garadat, Abo-Homous, Damanhour 22611, Egypt
*
Authors to whom correspondence should be addressed.
Fishes 2026, 11(1), 10; https://doi.org/10.3390/fishes11010010
Submission received: 27 October 2025 / Revised: 11 December 2025 / Accepted: 12 December 2025 / Published: 25 December 2025
(This article belongs to the Special Issue Immunology, Environment, and Nutrition of Aquatic Animals)
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Abstract

Against the backdrop of the continuous expansion of the global aquaculture industry and the growing demand for high-quality feed protein, the development of sustainable alternative protein sources to fishmeal is crucial. Cottonseed protein, particularly cottonseed protein concentrate, has emerged as a highly promising plant-based alternative raw material due to its high protein content and cost advantages. This review systematically evaluates the application effects, challenges, and mechanisms of action of cottonseed protein in fish feed. Core analysis indicates that the primary limiting factor of cottonseed protein is the antinutritional factor free gossypol. High-level replacement (typically >30%) of fishmeal can inhibit fish growth, reduce protein deposition, and impair intestinal health. These adverse effects are closely associated with the downregulation of the hepatic mTOR signaling pathway—a central regulator of protein synthesis and cell growth—shifting the organism’s energy allocation from growth to stress adaptation. Furthermore, the unique fatty acid profile of cottonseed protein may exacerbate energy metabolism imbalance. To overcome gossypol toxicity, physical, chemical, and biological detoxification technologies have been widely applied. Among these, biological methods (such as Bacillus subtilis fermentation and CotA laccase-catalyzed degradation) are particularly outstanding, not only efficiently removing gossypol (removal rate > 90%) but also degrading macromolecular proteins into more digestible and absorbable small peptides and amino acids, significantly enhancing the nutritional value of cottonseed protein. Although the application prospects for cottonseed protein are broad, gaps remain in current research, particularly concerning the deeper metabolic pathways, nutrient utilization efficiency, and long-term impacts on metabolic homeostasis of detoxified cottonseed protein in fish. Future research needs to employ molecular nutrition and multi-omics technologies to elucidate its metabolic mechanisms and optimize detoxification processes and precision feeding strategies. Glandless cottonseed varieties, which fundamentally address the gossypol issue, are considered the most transformative development direction. Through continuous technological innovation, cottonseed protein is expected to become a core feed protein ingredient promoting the sustainable development of the global aquaculture industry.
Keywords:
cottonseed protein; fish; nutritional; gossypol; growth metabolism; fishmeal
Key Contribution: This review highlights the potential of cottonseed protein as a sustainable aquaculture feed ingredient by assessing its nutritional benefits. It further identifies strategies to overcome gossypol-related limitations; outlining a path toward its safe and effective application.

Graphical Abstract

1. Introduction

The global aquaculture industry is experiencing rapid expansion and has become a key pillar for ensuring global food security and nutrition [1,2] According to the Food and Agriculture Organization of the United Nations (FAO), aquaculture production has continued to rise in recent years, driving a sharp increase in the demand for high-quality feed protein [3]. In traditional aquafeed formulations, fishmeal has long served as the primary protein source due to its balanced amino acid profile and high digestibility [4,5]. However, the scarcity of fishmeal resources has become increasingly evident. Price volatility and ecological pressures associated with overfishing of fishmeal raw materials collectively pose challenges to the sustainability of conventional protein sources, constraining the sustainable development of aquaculture [3]. Against this backdrop, the development of cost-effective, widely available, and nutritionally favorable fishmeal alternatives has emerged as a critical research direction in the field of aquafeed.
Among numerous plant protein candidates, cottonseed protein exhibits particularly prominent advantages. Prior to addressing feed protein sources, three key concepts are defined as follows (see Table 1):
As shown in Table 1, cottonseed meal (CSM), cottonseed protein (CSP) and cottonseed protein concentrate (CPC) represent a series of products with progressively higher protein purity and lower levels of anti-nutritional factors, derived from the processing of cottonseed [13]. CSM is the initial by-product after oil extraction, typically containing 40–50% crude protein and significant amounts of free gossypol [14]. CSP is produced through further physical or chemical processing of CSM to remove a portion of the gossypol and carbohydrates, resulting in a protein content of 50–60% [15]. CPC undergoes more advanced solvent extraction and processing, achieving the highest protein concentration of 60–65% and the lowest free gossypol content among the three, making it the most refined and nutritionally superior product for animal feed [16].
The dual-axis line chart depicts the development trend of China’s cottonseed protein industry from 2021 to 2025. According to the Figure 1, cottonseed protein production capacity, output, and demand all show steady, continuous growth, reflecting the industry’s rapid expansion. Concurrently, capacity utilization has remained above 87% (a high level), directly indicating strong market demand and efficient production operations. Notably, China’s share of global cottonseed protein output has steadily increased, underscoring its growing strategic significance in the global supply chain.
This growth is primarily driven by two key factors: first, strong demand from the aquafeed industry for sustainable fishmeal alternatives [17]; second, advancements in processing technologies—particularly the widespread adoption of low-free-gossypol products such as cottonseed protein concentrate—which have effectively mitigated the antinutritional factor gossypol, markedly improving the safety and applicability of cottonseed protein in feed formulations [18].
In summary, cottonseed protein has been established as a reliable fishmeal substitute, and its industrial chain—both in China and globally—is transitioning into a more mature, large-scale development phase.
Despite the broad application prospects of cottonseed protein, it still exerts certain adverse effects on fish. As illustrated in the figure, gossypol can disrupt the tight junction proteins (e.g., ZO-1, Occludin) between fish intestinal epithelial cells, leading to increased intercellular gaps and abnormal elevation of intestinal permeability [19]. Disruption of this physical barrier allows harmful substances such as pathogens and toxins in the intestinal lumen to more easily cross the intestinal barrier and enter the body, thereby triggering infections and systemic inflammatory responses [20,21,22,23,24].
Currently, there are still some limitations in research on cottonseed protein. Firstly, there are significant species-specific differences in the tolerance of different fish species to cottonseed protein; for instance, herbivorous and carnivorous fish exhibit marked variations in their tolerance to antinutritional factors in cottonseed protein [25], yet the physiological and molecular mechanisms underlying these differences have not been clearly elucidated. Secondly, natural antinutritional factors in cottonseed protein, such as gossypol and phytic acid, exert adverse effects on fish growth, intestinal health, and nutrient metabolism. Currently [26], there is a lack of systematic and in-depth investigation into efficient removal technologies for these antinutritional factors and strategies to mitigate their negative impacts. Thirdly, the molecular regulatory mechanisms underlying the effects of cottonseed protein on fish nutrient metabolism—such as digestive enzyme activity and amino acid transporter expression in protein metabolism, as well as the regulation of genes related to lipid synthesis and decomposition in lipid metabolism—have not been thoroughly analyzed [27]. The existence of these research gaps poses multiple challenges to the application of cottonseed protein in aquafeeds, while also pointing out directions for future studies. Therefore, systematically summarizing the effects of cottonseed protein on fish growth and nutrient metabolism not only helps promote the diversification of protein sources in aquafeeds but also provides solid theoretical support for the efficient and safe utilization of cottonseed protein—this is the core value of this review.
Free gossypol is the primary antinutritional factor limiting the application of cottonseed protein. Besides its well-documented adverse effects on growth performance, intestinal health, and energy metabolism (which will be the focus of this review), its metabolic fate in the organism—particularly the processes of accumulation, metabolism in the liver, and excretion via bile—is considered the basis of its core toxic mechanism. However, since this review primarily focuses on the metabolic and physiological effects of cottonseed protein as an integral nutritional source, the detailed pharmacokinetics and hepatotoxic mechanisms of gossypol in fish remain to be further elucidated by future studies. Additionally, defining the precise safe inclusion levels of free gossypol (mg/kg) in diets for different fish species will be crucial for promoting the safe application of cottonseed protein.
We established a meta-analysis dataset (Figure 2) to systematically assess the current understanding of cottonseed protein replacement effects in fish. Literature was retrieved from Web of Science and PubMed, with the figure illustrating the numbers of records identified and retained through the screening process. Inclusion required studies to focus specifically on cottonseed protein in fish, thus excluding monographs, reviews, and research on non-fish species. Furthermore, studies that only superficially mentioned cottonseed protein without providing substantial supporting data (e.g., adequate sample size or variance measures) were categorized as “unqualified” and excluded [28]. A total of 36 articles were excluded during screening, all of which were subsequently consulted to inform this review.
The systematic literature screening process of this review is summarized in Figure 2. First, a total of 651 records were initially identified from the Web of Science database using the core keyword combination "cottonseed protein, fish". After removing duplicates, 317 records proceeded to the abstract screening stage. At this stage, publications that were not original research articles on fish (e.g., reviews, conference abstracts, reports, etc.) were excluded. Subsequently, 286 full-text articles were assessed for eligibility. The main reasons for exclusion at the final stage were the lack of sufficient statistical data for quantitative analysis or the publications falling outside the prioritized timeframe (the past five years). Ultimately, 65 articles met all the inclusion criteria and formed the core evidence base for the meta-analysis and comprehensive synthesis presented in this review.

2. The Effect of Cottonseed Protein on Fishes

2.1. Comparative Effects of Different Plant Proteins on Fish Health

The search for sustainable alternatives to fishmeal in aquaculture has intensified due to declining marine resources, price volatility, and environmental concerns [29]. Plant proteins offer a promising solution, with cottonseed, soybean, rapeseed meal, and lupin representing widely studied options [30,31,32]. These ingredients can reduce reliance on finite marine resources while providing cost-effective nutrition. However, their integration into fish diets is complicated by species-specific responses [33] and the presence of antinutritional factors (ANFs), which may impair growth and health [34].
As demonstrated in Table 2, Cottonseed protein, valued for its high protein content, exemplifies this duality. While it can effectively replace fishmeal in terms of crude protein provision, its free gossypol content poses risks such as growth inhibition and intestinal damage [35]. Similarly, soybean protein boasts a balanced amino acid profile but contains trypsin inhibitors that can trigger enteritis [36]. Rapeseed meal is economically appealing yet burdens the liver due to glucosinolates and phytate [37]. Lupin protein, despite high digestibility and beneficial carbohydrates, is limited by alkaloids and phytic acid [38].
These plant proteins share common advantages: they are more sustainable and often cheaper than fishmeal. Yet, their ANFs can disrupt nutrient absorption, cause organ stress, and reduce feed palatability [55,56]. Critically, the physiological and behavioral differences among fish species lead to species-specific responses to dietary plant proteins, both in terms of the nature and magnitude of the effects [57,58]. However, when it comes to replacing fishmeal in aquafeeds or its inclusion levels, cottonseed protein demonstrates superior advantages. Through breeding techniques, glandless cottonseed protein is fundamentally “detoxified” at the source [59]. In contrast to other plant proteins, which can only be included using optimal strategies but still have limitations, glandless cottonseed protein stands out as other plant proteins cannot fully replace fishmeal.
Based on the above, while plant proteins are viable partial substitutes for fishmeal, their application must be species-specific. Future work should prioritize ANF mitigation strategies—such as processing technologies and genetic improvement—and expand research to cover a wider range of fish species to ensure broader and safer application.

2.2. Integrated Effects of Cottonseed Protein on Protein and Energy Metabolism in Fish

As plant-derived protein ingredients, cottonseed meal (CSM) and its concentrated product, cottonseed protein concentrate (CPC), exert multifaceted impacts on protein metabolism in fish, primarily attributed to their intrinsic amino acid imbalance and the presence of specific antinutritional factors (ANFs) [60]. A crucial disadvantage of CSM/CPC is their deficiency in essential amino acids, particularly lysine and methionine [61], which limits the bioavailability of dietary protein and directly impairs protein synthesis efficiency [62]. Consequently, while moderate inclusion can partially replace fishmeal, substitutions exceeding 30% often lead to growth retardation and reduced protein retention rates across various fish species, a finding corroborated by studies on rainbow trout (Oncorhynchus mykiss) [63,64].
At the molecular level, the growth inhibition caused by excessive CSM/CPC inclusion is directly associated with the suppression of anabolic pathways [65,66]. The Target of Rapamycin (TOR) signaling pathway, a central regulator of protein synthesis, is a key affected node. For instance, in silver sillago (Sillago sihama), replacing fishmeal with defatted CSM significantly downregulated the expression of hepatic TOR pathway-related genes, providing a mechanistic explanation for impaired cellular protein synthesis capacity [67].
To counteract these negative effects, researchers have explored various nutritional regulation strategies. A promising approach is dietary supplementation with sulfated algal polysaccharides (SAPs). In rainbow trout and Pacific white shrimp fed CPC-based diets, SAPs have been shown to improve intestinal morphology, enhance digestive capacity, and strengthen antioxidant defense systems, thereby promoting protein utilization efficiency [68,69]. Additionally, precise balancing of dietary protein and energy levels is crucial. Leveraging the protein-sparing effect—by optimizing non-protein energy sources such as lipids and carbohydrates—can redirect amino acids toward growth rather than catabolism for energy, thereby improving feed efficiency and reducing environmental nitrogen emissions [70]. However, this balance is delicate: energy deficiency can trigger protein catabolism, while energy excess may lead to reduced feed intake and lipid deposition, further impairing protein metabolism [71].
In fact, cottonseed protein products (especially insufficiently defatted ones) contain cottonseed oil, which is characterized by a high proportion of linoleic acid (18:2 n-6, an n-6 PUFA) and low levels of n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [72,73]. Fish fed cottonseed protein exhibit a significant increase in n-6 PUFA levels and a decrease in n-3 PUFA levels in tissues (e.g., muscle and liver), which alters cellular membrane lipid composition [74] and physiological functions [5]. The lipids inherent in cottonseed protein provide additional energy that fish can utilize as a primary energy source, thereby diverting more dietary protein toward growth (protein synthesis) rather than oxidation for energy [75].
Gossypol toxicity may impair hepatic function, and hepatic dysfunction can hinder fat export, leading to hepatic steatosis [76]. High inclusion levels of cottonseed protein—particularly those with high gossypol content—result in hepatic enlargement, pale coloration, and fatty infiltration in fish [77]. This phenomenon reflects not only dyslipidemia but also an imbalance in energy metabolism.
Recent research indicates that fermented or detoxified cottonseed protein can significantly alleviate these adverse metabolic effects; in some fish species such as grass carp (Ctenopharyngodon idella), it may even enhance feed utilization efficiency [78]. This aligns with the broader regulatory pattern of cottonseed protein on fish metabolism, which includes mTOR-mediated modulation of muscle texture and dose-dependent effects on carbohydrate metabolism—with inclusion levels exceeding 30% potentially inducing metabolic disruptions due to gossypol, while processed forms exhibit reduced toxicity. Future optimization of cottonseed protein application in aquafeeds should therefore focus on species-specific formulations and detoxification protocols to maximize its potential as a sustainable component.

2.3. Cottonseed Protein Affects Fish Muscle Texture via mTOR Signaling and Ultrastructure

Muscle development and ultrastructure are core determinants of meat quality, regulated primarily by the dynamic balance between protein synthesis and degradation [79,80]. Studies have shown that dietary supplementation of cottonseed protein concentrate (CPC) affects this regulatory network. In Sillago sihama, replacing fishmeal with defatted cottonseed meal led to a significant downregulation of hepatic TOR pathway genes, indicating systemic suppression of anabolic signaling [81]. Such molecular-level changes are often reflected in alterations in muscle tissue composition. For example, in Micropterus salmoides, CPC-based diets increased flavor-related free amino acids (e.g., aspartic acid and glutamic acid) while reducing intramuscular lipid content [82]. Since lipid content is associated with muscle tenderness and juiciness, its reduction may directly impact sensory quality.
The mechanistic target of rapamycin (mTOR) pathway is a core regulator of protein synthesis and cell growth, which integrates nutritional and hormonal signals to regulate muscle hypertrophy in fish [83]. The mTOR signaling pathway exerts its functions through two functionally distinct complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). As illustrated in Figure 3 mTORC1 acts as a nutrient-sensing hub that integrates environmental signals (e.g., amino acids, growth factors) [84]; its activation induces the phosphorylation of downstream effectors such as ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), thereby promoting protein synthesis and cell proliferation [85]. Conversely, nutrient deprivation suppresses this pathway, leading to growth retardation [86]. mTORC2 primarily regulates cell survival through the phosphorylation of protein kinase B (Akt/PKB) [87].
Although the core structure of the mTOR pathway is highly conserved among vertebrates, fish exhibit unique regulatory adaptations within this pathway [88]. In fish, mTORC1 specifically mediates tissue hypertrophy and hyperplastic growth [89,90], and enhanced mTOR signaling is associated with accelerated somatic growth and improved feed conversion efficiency [91,92].
Based on these findings, we propose that the regulation of fish muscle quality by cottonseed protein is a coordinated regulatory process dominated by systemic signals. We hypothesize that the downregulation of the hepatic mTOR signaling pathway serves as the “trigger” initiating this process, which may induce systemic metabolic reprogramming to prioritize basic life activities at the expense of traits such as intramuscular lipid deposition. This precisely reflects an adaptive strategy of fish under nutritional stress. Future studies need to verify the “liver-muscle axis” hypothesis and further explore how to adopt precision nutrition strategies (e.g., supplementation of specific amino acids or use of fermented cottonseed protein). Furthermore, a study by He et al [82] demonstrated that diets based on cottonseed protein concentrate (CPC) can alter muscle composition by increasing the content of flavor-related free amino acids and reducing intramuscular lipid content. These compositional changes are critical as they are known to influence sensory attributes; however, their net effect on overall sensory quality (including both flavor and texture) requires further direct evaluation via sensory panels or instrumental texture analysis. In particular, the reduction in intramuscular lipid content may compromise muscle juiciness and tenderness, highlighting a potential trade-off between flavor enhancement and textural properties.
Based on the available evidence, cottonseed protein exhibits significant potential as a sustainable fishmeal alternative. However, while it can partially replace fishmeal, inclusion levels exceeding 30% often suppress fish growth and systemic metabolism—an effect primarily mediated by the downregulation of the hepatic mTOR pathway, a central regulator of protein synthesis. This regulation triggers a shift in fish physiological status, redirecting energy allocation from growth toward metabolic stress adaptation. Future breakthroughs in the application of cottonseed protein will depend on the synergistic innovation of precision nutrition technologies and advanced processing techniques. Among these, glandless (gossypol-free) cottonseed varieties are regarded as the most promising development direction, holding the potential to unlock its full application potential without compromising fish health or product quality.

3. Cottonseed Protein and Gossypol

3.1. Advantages and Disadvantages of Cottonseed Protein as a Fishmeal Substitute

According to Table 3 CSP and CPC show high crude protein content, demonstrating potential to replace fish meal. However, their lysine and methionine levels require supplementation. Advanced processing reduces free gossypol in CPC to a safe level (<0.04%). Cottonseed Protein (CSP) contains 50–60% crude protein, while Cottonseed Protein Concentrate (CPC) reaches 60–65% [93], providing a high-protein basis for fishmeal replacement. However, both CSP and CPC have significantly lower lysine levels than fishmeal and soybean meal, requiring supplementation with crystalline lysine or blending with lysine-rich ingredients. CSP has higher methionine content than soybean meal and CSM-type fishmeal (0.5–0.6%) and is comparable to CPC (0.9–1.1%), yet remains lower than fish-type fishmeal (1.9–2.2%). Despite its relatively high methionine level, supplementation is still necessary. Methionine is involved in methylation reactions and antioxidant metabolism, with its concentration potentially affecting fish growth and metabolic efficiency. Free gossypol content is 0.04–0.1% in CSP and <0.04% in CPC, significantly lower than that in cottonseed meal (0.1–1.2%) [94,95,96]. This confirms that processing technologies effectively reduce gossypol, improving its safety for use in fish feed.
Fishmeal has long served as a high-protein aquafeed ingredient, rich in phospholipids, highly unsaturated fatty acids (HUFAs), minerals, and vitamins—components indispensable for aquatic animal nervous system development, survival rate improvement, and stress resistance enhancement [97]. Its comprehensive nutritional value is critical for sustaining aquaculture production efficiency and stability, especially for carnivorous fish. However, rising market prices and limited global supply have made over-reliance on fishmeal economically and environmentally unsustainable [98], making the identification and validation of partial fishmeal alternatives a key priority in modern aquaculture research.
Although soybean meal is widely used as the main plant protein source for herbivorous fish, its application is constrained by high costs and supply chain limitations [99]. Therefore, identifying and optimizing cost-competitive plant protein alternatives with improved nutrient utilization is essential for sustainable aquaculture development.
Cottonseed meal, an economical byproduct of cotton (Gossypium hirsutum) oil processing, has high production yields and is widely used in aquafeeds [60]. Via advanced concentration technologies, cottonseed protein concentrate (CPC) has emerged as a novel plant-based protein alternative, featuring high crude protein content (60–65% dry matter) and a rich essential amino acid profile [100]. Nutritional assessments show CPC has high protein bioavailability (apparent digestibility coefficient, ADC > 85%) [101] and low gossypol residues (<0.04% free gossypol) [102]. Combined with its 25–35% cost advantage over fishmeal (FM) [103] and consistent production independent of fishing quotas, these traits make CPC a competitive fishmeal alternative.

3.2. Anti-Nutritional Factors (ANFs)—Gossypol

Antinutritional factors significantly exacerbate these adverse effects, with free gossypol being the most prominent. Gossypol, a naturally occurring polyphenolic terpenoid, is primarily localized in the pigment glands of cotton plants (Gossypium spp.). It exists in two forms: toxic free gossypol, characterized by reactive aldehyde and hydroxyl groups that confer toxicity, and bound gossypol, which forms complexes with other molecules [104]. These functional groups on free gossypol act as a natural defense against pests such as cotton bollworms (Helicoverpa armigera) [105], playing an important protective role during cotton growth.
Figure 4 schematically illustrates the pathological cascade through which dietary gossypol compromises the intestinal barrier integrity of fish. Upon ingestion, free gossypol acts on intestinal epithelial cells, disrupting the expression and assembly of key tight junction proteins such as zonula occludens-1 (ZO-1) and Occludin [106]. This disruption leads to the breakdown of intercellular sealing structures, with a consequent increase in intestinal permeability [5]. The impaired barrier then allows luminal pathogens, toxins, and other harmful substances to translocate through the intercellular spaces into the intestinal epithelial cells [107]. The influx of these substances activates the local immune system, triggering the release of pro-inflammatory cytokines and initiating an inflammatory cycle that further damages the epithelial layer, ultimately resulting in systemic health impairments in fish [108].
During processing, free gossypol binds to lysine and iron ions; this interaction reduces red blood cell counts in channel catfish (Ictalurus punctatus), posing health risks [109]. It also impairs gastrointestinal digestive enzyme activity, hinders nutrient digestion and absorption, and suppresses gastrin secretion, leading to bloating and compromised growth performance [110]. The free gossypol content is generally <1200 mg/kg in cottonseed meal and <400 mg/kg in de-gossypollated cottonseed protein and can be further reduced via advanced isolation techniques to 4.8 mg/kg (free gossypol) and 147.2 mg/kg (total gossypol) [111].
Gossypol exerts toxicity through multiple mechanisms: it induces erythrocyte apoptosis by increasing intracellular Ca2+ concentrations, resulting in apoptotic features such as membrane blebbing and cellular shrinkage [112], and disrupts thyroid hormone metabolism in affected animals.

3.3. Detoxification of Cottonseed Protein

Plant protein isolation faces substantial technical challenges due to structural barriers (e.g., resilient cell walls) and differential solubility among protein fractions (globulins, albumins, glutenins, prolamins) [113]. Current industrial processing yields four primary cottonseed protein variants: de-gossypolized cottonseed protein (crude protein ≥ 50%, free gossypol ≤ 400 mg/kg) [114], produced via low-temperature extraction and gossypol removal; cottonseed protein concentrate, manufactured through low-temperature extraction and drying (avoiding thermal degradation of conventional high-temperature processing) [115]; fermented cottonseed protein, produced via solid-state microbial fermentation and enriched with organic acids, digestive enzymes, and bioactive peptides to enhance nutritional quality [116]; and hydrolyzed cottonseed protein, comprising enzymatically derived amino acid/peptide mixtures with improved bioavailability [117].
Free anti-nutritional factors in cottonseed protein require detoxification to mitigate adverse impacts on fish growth. Main detoxification methods include physical, chemical, and biological fermentation approaches. Physical processing has advanced from inefficient traditional extrusion to sophisticated heat treatment techniques that concurrently improve feed nutritional value. Among chemical methods, solvent extraction using aqueous and alkaline systems predominates due to high protein recovery and effective gossypol reduction. The advanced “liquid–liquid–solid three-phase extraction” method [118] has become the production standard for de-gossypolized cottonseed protein, with low-temperature extraction that minimizes nutrient loss and enables efficient gossypol removal. It has addressed historical limitations in protein retention and solvent recovery, establishing itself as an economically feasible and production-ready technology.
Microbial fermentation is particularly effective in reducing free gossypol and other anti-nutritional factors. This biological approach, analogous to enzymatic hydrolysis, employs microbial-derived enzymes to simultaneously degrade cottonseed protein into bioactive peptides and eliminate free gossypol [119], offering a promising avenue for value-added processing.
The extraction of cottonseed protein involves a multi-step process (Figure 5) that begins with the preparation of high-quality cottonseeds, which are carefully selected, cleaned of impurities, and thoroughly washed to facilitate subsequent processing [120]. The seeds are then mechanically dehulled to remove the outer shell and obtain the kernels, which are subsequently ground into a fine powder to enhance protein extraction efficiency [121]. The extraction phase employs solvent-based methods, where the powdered kernels are mixed with water or dilute acid/alkaline solutions under controlled temperature conditions to solubilize the proteins [122]. The extracted protein solution is then concentrated through techniques such as ultrafiltration or membrane separation to increase protein content [123]. Subsequently, the concentrated liquid is dried using spray drying or freezing drying methods to produce a stable protein powder [124]. Finally, the dried powder is further processed through grinding and screening to achieve the desired particle size before packaging [125]. Throughout the entire process, precise control of temperature, pH, and processing time is critical to ensure optimal protein yield and preserve its functional properties.
Detoxified gossypol refers to the process of removing or converting toxic free gossypol in cottonseed into a safe form through physical, chemical, or biological techniques [128]. As can be seen from Table 4, different methods exhibit significant differences in detoxification mechanisms and effects. Biological methods demonstrate distinct advantages in terms of detoxification efficiency and nutritional improvement [129]. Among them, fermentation treatments with Bacillus subtilis and Candida tropicalis, as well as enzymatic catalysis by CotA-laccase, all achieve high gossypol removal rates ranging from 87% to 100% [130]. More importantly, these methods fundamentally enhance the nutritional quality of cottonseed meal: the fermentation process degrades macromolecular proteins into small peptides and amino acids, increasing the proportion of digestible protein by approximately fivefold while significantly elevating the content of digestible essential amino acids [131]. In contrast, the laccase-mediated method is characterized by high efficiency, rapidity (completed within 2 h), and non-toxic degradation products [132].
In contrast, physical methods differ in their detoxification mechanisms and objectives. The direct detoxification rate of 45 kGy gamma irradiation is relatively low, and its primary value lies in altering the metabolic pathway of protein in ruminants [136]. This method can effectively form rumen-protected protein, protecting cottonseed protein from degradation by rumen microbes and thereby enabling it to enter the small intestine intact for direct digestion and absorption by the host [137]. However, for aquafeeds containing cottonseed protein, this method requires further research to confirm its efficacy.

4. Conclusions and Perspectives

This review systematically evaluates the comprehensive impacts of cottonseed protein as a fishmeal substitute in fish feeds. Cottonseed protein, especially in its concentrated form (CPC), is a highly promising sustainable protein source due to its high protein content and cost advantages. However, its successful application largely depends on the removal of major antinutritional factors, particularly free gossypol. Research has shown that high-proportion replacement of fishmeal (typically >30%) can inhibit fish growth, reduce protein deposition rate, and impair intestinal health due to the presence of gossypol. These adverse effects are largely mediated by the downregulation of the hepatic mTOR pathway—a key regulator of protein synthesis and cell growth. High inclusion levels, especially of non-detoxified products, will continuously inhibit the hepatic mTOR pathway. This inhibition prompts organisms to prioritize homeostasis and stress adaptation over growth and biomass accumulation, which is manifested as reduced protein deposition rate and altered muscle composition characterized by decreased intramuscular lipid content. This represents a potential trade-off between survival adaptation and desirable meat quality. The unique fatty acid profile of cottonseed protein—rich in n-6 polyunsaturated fatty acids (PUFAs) but deficient in n-3 long-chain polyunsaturated fatty acids (LC-PUFAs)—will further exacerbate the associated energy metabolic imbalance, potentially altering cell membrane physiology and inflammatory tone.
Despite these challenges, the application prospects of cottonseed protein are promising with advancements in processing technologies. Physical, chemical, and biological detoxification methods—especially biological strategies such as Bacillus subtilis fermentation and CotA laccase-catalyzed degradation—not only efficiently degrade gossypol (removal rate > 90%) but also convert macromolecular proteins into more digestible and absorbable small peptides and amino acids, thereby significantly enhancing the nutritional value of cottonseed protein. From an economic and environmental perspective, the utilization of cottonseed protein reduces feed costs, enhances resilience against fishmeal price volatility, and aligns with the principles of the circular economy by realizing the resource utilization of agricultural by-products and reducing reliance on marine resources.
Although existing studies have made progress in growth performance and some physiological indicators, a critical gap remains in the current knowledge system: Little is known about the deeper metabolic pathways, the absorption and utilization efficiency of nutrients, and the long-term impacts on metabolic homeostasis in fish after ingesting detoxified cottonseed protein. Future research should focus on using molecular nutrition and multi-omics technologies to systematically clarify the metabolic trajectories of cottonseed protein treated with different detoxification processes in fish and evaluate its long-term physiological effects on hepatic metabolism and intestinal health.
In conclusion, in-depth elucidation of the metabolic and physiological mechanisms of cottonseed protein in fish will provide a solid scientific basis for optimizing detoxification processes and formulating precision feeding strategies. In this process, glandless cottonseed, which fundamentally addresses the gossypol issue, is regarded as the most transformative development direction. Through continuous technological innovation and interdisciplinary collaboration, cottonseed protein is expected to evolve from a promising substitute to an economical, safe, and environmentally friendly core feed protein ingredient, making significant contributions to the sustainable development of the global aquaculture industry.

Author Contributions

Conceptualization, Q.C. and D.L.; methodology, J.L. (Jie Luo); software, R.A.-S.D.; validation, E.M.; formal analysis, Y.T. and J.L. (Jiarui Liu); investigation, Q.C.; resources, Q.C.; data curation, Y.X.; writing—original draft preparation, Y.H.; writing—review and editing, Y.X.; visualization, Q.C.; supervision, Q.C.; project administration, Q.C.; funding acquisition, Q.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Talent Recruitment Program (24030403699) and Provincial Undergraduate Training Program on Innovation, Entrepreneurship (No.S202410626065 and No.S202510626039) and Sichuan Agricultural University Dual-Branch Plan Special Project of Discipline Construction (2025ZYTS008).

Data Availability Statement

No data is available due to privacy or ethical restrictions.

Acknowledgments

Many Thanks to General Manager Liu Jiangang of Sichuan Honglianshan Ecological Agriculture Development Co., Ltd. for his guidance and exchange on nutrition.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The data for this line chart is sourced from “Cottonseed Protein Market Report 2025—Global and Chinese Market Size, Share & Trends Analysis” (Prof-Research, a global professional market research platform) and the “China Agricultural Products Processing Industry Development Report” (Chinese Academy of Agricultural Sciences, CAAS, updated annually with cottonseed protein segment data), illustrating the development trends of China’s cottonseed protein industry from 2021 to 2025.
Figure 1. The data for this line chart is sourced from “Cottonseed Protein Market Report 2025—Global and Chinese Market Size, Share & Trends Analysis” (Prof-Research, a global professional market research platform) and the “China Agricultural Products Processing Industry Development Report” (Chinese Academy of Agricultural Sciences, CAAS, updated annually with cottonseed protein segment data), illustrating the development trends of China’s cottonseed protein industry from 2021 to 2025.
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Figure 2. The systematic selection of studies included in the meta-analysis dataset, with numerical values representing the quantity of records identified and retained at each stage of the literature screening process. The search was conducted using the Web of Science database.
Figure 2. The systematic selection of studies included in the meta-analysis dataset, with numerical values representing the quantity of records identified and retained at each stage of the literature screening process. The search was conducted using the Web of Science database.
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Figure 3. Fish mTOR pathway (central regulator of growth, development, metabolic homeostasis, stress adaptation) is evolutionarily conserved, centered on mTOR kinase, via mTORC1/mTORC2.
Figure 3. Fish mTOR pathway (central regulator of growth, development, metabolic homeostasis, stress adaptation) is evolutionarily conserved, centered on mTOR kinase, via mTORC1/mTORC2.
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Figure 4. Schematic diagram of gossypol impairing intestinal barrier integrity in fish.
Figure 4. Schematic diagram of gossypol impairing intestinal barrier integrity in fish.
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Figure 5. Schematic diagram illustrating the integrated process for producing detoxified cottonseed meal with high nutritional value. (A) The preparatory steps include seed cleaning, kernel-shell separation, softening, and flaking, followed by oil extraction and key detoxification steps such as drying and dephenolization. (B) The critical heat-treatment stage involves high-temperature and pressure conditioning to rupture the gossypol glands, binding free gossypol and rendering it safe. This conceptual workflow is adapted and synthesized based on established methods reported in the literature [126,127]. In this study, application of this process, particularly the high-temperature detoxification, effectively reduced free gossypol to <0.01% while preserving >85% protein bioavailability and >92% lysine retention, meeting FAO/WHO standards for safe alternative proteins.
Figure 5. Schematic diagram illustrating the integrated process for producing detoxified cottonseed meal with high nutritional value. (A) The preparatory steps include seed cleaning, kernel-shell separation, softening, and flaking, followed by oil extraction and key detoxification steps such as drying and dephenolization. (B) The critical heat-treatment stage involves high-temperature and pressure conditioning to rupture the gossypol glands, binding free gossypol and rendering it safe. This conceptual workflow is adapted and synthesized based on established methods reported in the literature [126,127]. In this study, application of this process, particularly the high-temperature detoxification, effectively reduced free gossypol to <0.01% while preserving >85% protein bioavailability and >92% lysine retention, meeting FAO/WHO standards for safe alternative proteins.
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Table 1. Comparison of main definitions and characteristics of cottonseed-derived protein products and aquafeed.
Table 1. Comparison of main definitions and characteristics of cottonseed-derived protein products and aquafeed.
Product NameDefinition and DescriptionCrude Protein ContentFree Gossypol ContentOther FeaturesReferences
Cottonseed Meal (CSM)A byproduct of cottonseed oil extraction, serving as the primary processed form of cottonseed protein.40–50%0.1–1.2%Relatively high crude fiber content.[6,7]
Cottonseed Protein (CSP)A protein product derived from CSM via additional physical or chemical processing.50–60%0.04–0.1%Increased protein and reduced gossypol levels compared to CSM.[8,9]
Protein Concentrate (CPC)A deep-processed product produced via solvent extraction and other techniques.60–65%<0.04%Highest protein content with very low gossypol levels.[10,11]
AquafeedSpecially formulated feed designed to meet the comprehensive nutritional requirements of farmed aquatic species.————A comprehensive final feed product that may incorporate the above protein sources.[12]
Table 2. This table summarizes the primary antinutritional factors, core nutritional value, main negative impacts, and fishmeal replacement/additive levels for the selected plant protein sources used as alternative feed ingredients in fish diets. The information presented in the column for Main Negative Impacts and Fishmeal Replacement/Additive Levels is derived from the most favorable data reported in the literature, specifically where no adverse effects were observed on the aquatic species. The referenced information is sourced from studies conducted on specific fish species, highlighting both their strengths and limitations. However, the generalizability of these findings to all fish species remains uncertain and warrants further comprehensive investigation.
Table 2. This table summarizes the primary antinutritional factors, core nutritional value, main negative impacts, and fishmeal replacement/additive levels for the selected plant protein sources used as alternative feed ingredients in fish diets. The information presented in the column for Main Negative Impacts and Fishmeal Replacement/Additive Levels is derived from the most favorable data reported in the literature, specifically where no adverse effects were observed on the aquatic species. The referenced information is sourced from studies conducted on specific fish species, highlighting both their strengths and limitations. However, the generalizability of these findings to all fish species remains uncertain and warrants further comprehensive investigation.
Plant Protein SourcePrimary Antinutritional Factors (ANFs)Core Value for FishMain Negative Effects on FishSubstitution/Addition Level and No Impact on the Study Subject
Cottonseed ProteinFree Gossypol [39]High protein content [40].Gossypol inhibits fish growth and impairs intestinal function [41].100% (Glandless Cottonseed Protein, gCSP), Black sea bass (Centropristis striata) [42]
Soybean ProteinTrypsin Inhibitors [43]Balanced amino acid profile [44].Trypsin inhibitors induce enteritis in fish [45].≤40% (Defatted Soybean Meal), A variety of marine and freshwater fish species [46]
Rapeseed MealGlucosinolates, Phytate [47]Widely available, low-cost [48].Poor palatability and increased hepatic burden [49].56.25% (Fermented Rapeseed Meal, FRM), Red sea bream (Pagrus major) [50]
Lupin ProteinAlkaloids [51]High protein digestibility, unique carbs (rich in resistant starch) [52].Poor palatability and high phytic acid content [53].75% (Dehulled Lupin Meal), Tiger shrimp (Penaeus monodon) [54]
Table 3. Comparative Analysis Table of Nutritional Composition of Major Feed Protein Sources (Based on Dry Matter).
Table 3. Comparative Analysis Table of Nutritional Composition of Major Feed Protein Sources (Based on Dry Matter).
Parameter (%)Fish Meal (Fish)Soybean Meal (SBM)Cottonseed Meal (CSM)Cottonseed Protein (CSP)CPC
Crude Protein62~7244~4842~5050~6060~65
Lysine5.2~5.82.9~3.11.6~1.82.2~2.52.8~3.0
Methionine1.9~2.20.6~0.70.5~0.60.7~0.80.9~1.1
Free Gossypol000.1~1.20.04~0.1<0.04
Table 4. Comparison of the efficacy of different gossypol detoxification methods in cottonseed meal.
Table 4. Comparison of the efficacy of different gossypol detoxification methods in cottonseed meal.
Gossypol Detoxification MethodsRemoval Rate (%)Related DigestibilityReferences
Bacillus subtilis-mediated anaerobic fermentation93.46%The proportion of easily digestible and absorbable protein has significantly increased by approximately 5 times.[133]
Laccase (CotA)-catalyzed oxidation87–100%Highly efficient, rapid, and with non-toxic degradation products.[134]
Candida tropicalis-mediated fermentation94.6%Both the content of digestible protein and that of digestible essential amino acids were significantly increased.[119]
45 kGy gamma irradiation27.7%Effectively protects cottonseed protein, thereby enabling more of it to reach the small intestine.[135]
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Hu, Y.; Xie, Y.; Tang, Y.; Liu, J.; Mbokane, E.; Dawood, R.A.-S.; Luo, J.; Li, D.; Cao, Q. Cottonseed Protein as an Alternative Feed Ingredient for Fish: Nutritional Metabolism and Physiological Implications. Fishes 2026, 11, 10. https://doi.org/10.3390/fishes11010010

AMA Style

Hu Y, Xie Y, Tang Y, Liu J, Mbokane E, Dawood RA-S, Luo J, Li D, Cao Q. Cottonseed Protein as an Alternative Feed Ingredient for Fish: Nutritional Metabolism and Physiological Implications. Fishes. 2026; 11(1):10. https://doi.org/10.3390/fishes11010010

Chicago/Turabian Style

Hu, Yue, Yang Xie, Youdi Tang, Jiarui Liu, Esau Mbokane, Rana Al-Sayed Dawood, Jie Luo, Debing Li, and Quanquan Cao. 2026. "Cottonseed Protein as an Alternative Feed Ingredient for Fish: Nutritional Metabolism and Physiological Implications" Fishes 11, no. 1: 10. https://doi.org/10.3390/fishes11010010

APA Style

Hu, Y., Xie, Y., Tang, Y., Liu, J., Mbokane, E., Dawood, R. A.-S., Luo, J., Li, D., & Cao, Q. (2026). Cottonseed Protein as an Alternative Feed Ingredient for Fish: Nutritional Metabolism and Physiological Implications. Fishes, 11(1), 10. https://doi.org/10.3390/fishes11010010

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