Vitamin D₃ as an antimicrobial and antiviral immunonutrient in aquaculture

Abstract

Abstract

Bacterial and viral diseases continue to remain major problem in aquaculture, and the growing limitations on the use of antibiotics have intensified a growing focus on host-directed nutrition-based disease management strategies. While vitamin D₃ (VD₃) has been recognized for its role in calcium–phosphorus metabolism, it is now increasingly recognized to exert an immunonutrient role in fish, which includes modulation of antimicrobial peptide production, inflammatory regulation, epithelial barrier integrity, oxidative homeostasis, microbiota-associated immune interactions, and antiviral signaling. This review critically synthesizes current knowledge on VD₃ metabolism and vitamin D receptor (VDR)-mediated signaling in relation to disease resilience in cultured aquatic species, with particular emphasis on antibacterial, antiviral, and functional-feed applications. Across experimental studies, VD₃ supplementation has frequently been associated with enhanced phagocytic activity, lysozyme and complement function, antimicrobial peptide induction, cytokine modulation, and improved mucosal stability. Emerging evidence further implicates VD₃ in type I interferon and JAK–STAT-associated antiviral pathways. However, these responses remain highly variable across species, supplementation doses, developmental stages, and environmental conditions, reflecting substantial biological and methodological heterogeneity. Importantly, the current evidence base is still frequently composed of transcriptomic and short-term laboratory studies with comparatively few studies demonstrating functional validity in form of pathogen-load reduction, histopathological protection, improvement of survival, or production level. The translation value for VD₃ is still not fully understood, although it holds promise for use in integrated functional feed systems. Overall, effective application will depend on species-specific dose optimization, long-term safety assessment, and stronger field-scale validation across diverse aquaculture systems.

Key points

• Vitamin D₃ modulate antimicrobial peptides and innate and antibacterial immunity in fish.

• VD₃ activates interferon pathways and strengthens antiviral defenses.

• Optimal VD₃ dosing improves resilience; field validation is needed in aquaculture.

Graphical abstract

Vitamin D₃ supports host-directed antimicrobial and antiviral immunity in fish via VDR-mediated immune modulation rather than direct pathogen targeting. Its application as a functional feed additive may contribute to antibiotic-reduced and sustainable aquaculture in a species-dependent manner.

Introduction

Aquaculture is the most rapidly expanding animal food production sector in the world that accounts for more than half of the world’s seafood consumption today (FAO 2024; Maezono et al. 2025). Increased production intensity, however, has led to greater vulnerability and risks of infectious disease outbreaks in freshwater and marine aquacultures, especially from bacterial and viral diseases, which has affected their productivity, animal welfare, and economic viability (Haenen et al. 2023; Maezono et al. 2025). However, despite improvements in husbandry, vaccination, and disease surveillance, pathogens such as Aeromonas hydrophila, Vibrio anguillarum, Edwardsiella tarda, tilapia lake virus (TiLV), and viral hemorrhagic septicemia virus (VHSV) continue to be significant factors in production losses (Hadfield 2021; Haenen et al. 2023). Traditionally, chemotherapeutics and antibiotics have been widely used for disease control, but their widespread application has exaggerated concerns regarding antimicrobial resistance (AMR), antimicrobial residues, ecological disruption of aquatic microbial communities, and overall environmental contamination (Cabello et al. 2016; Mondal and Thomas 2022; Hossain et al. 2022). Therefore, increasing regulatory and scientific attention has been shifted toward disease-management strategies capable of strengthening host resilience while reducing antibiotic dependence. In this context, immunonutrition has emerged as a promising host-directed approach that aims to improve disease resistance by dietary modulating immune, metabolism, antioxidants, and barrier function, rather than by directly killing pathogens (Preena et al. 2020; Baker et al. 2022; Singh et al. 2024). Vitamin D₃ (VD₃; cholecalciferol) is one of the micronutrients which has received special attention because of its biological actions that are far from just maintaining calcium–phosphorus homeostasis (Cao et al. 2024; Voiculescu et al. 2025). In mammalian systems, VD₃ functions as a potent immunomodulatory hormone involved in antimicrobial peptide production, epithelial barrier maintenance, oxidative homeostasis, cytokine regulation, and antiviral defense (Greiller and Martineau 2015; Ismailova and White 2022). Evolving evidence suggests that some of these functions are also conserved in teleost fish, though the strength of evidence remains highly uneven. This discrepancy is important because in different species, developmental stages, or under varying farming conditions molecular activation does not necessarily translate into uniform protection from disease.

VDR is the primary mediator of the biological activity of VD₃, a ligand-activated nuclear receptor that regulates the transcriptional network involved in mineral metabolism, inflammatory signaling, oxidative balance, and immune homeostasis (Meyer et al. 2022). Some fish species have been found to express VDR, including grass carp (Ctenopharyngodon idella), zebrafish (Danio rerio), turbot (Scophthalmus maximus), and salmonids, which are known to express VDR in immunologically relevant tissues including the intestine, spleen, head kidney, liver, and gills (Kollitz et al. 2014; Liao et al. 2023; Song et al. 2023). Through VDR-mediated signaling, VD₃ has been associated with modulation of antimicrobial peptides including β-defensins, hepcidins, and cathelicidins, as well as inflammatory and antiviral pathways involving JAK–STAT, NF-κB, and interferon-associated signaling cascades (Sun et al. 2022, 2023; Zhang et al. 2021; White 2022). In studies involving grass carp, Nile tilapia (Oreochromis niloticus), yellow catfish (Pelteobagrus fulvidraco), and zebrafish, followed by controlled infection, these responses have been linked with enhanced immune indices including increased lysozyme activity, complement activation, phagocytic responses, and interferon-stimulated gene expression during bacterial or viral challenge (Cheng et al. 2020a, b; Liu et al. 2021b, a; Wang et al. 2023; Wang et al. 2024a, b, c).

However, there is an important distinction exists between molecular responsiveness and validated biological outcome. Although a large amount of current fish literature deals with transcriptomic or short-term experimental tests, there are comparatively few studies that offer functional evidence, including pathogen reduction, histopathologic protection, improved survival, or performance in production-scale tests. This evidence hierarchy is critical because across multiple species, developmental stages, or farming environments transcriptional activation does not consistently translate into reproducible disease resistance. Moreover, depending on supplementation dose, pathogen system, and environmental conditions, several studies report neutral or inconsistent responses. Emerging studies additionally report associations between VD₃ supplementation and altered tight-junction regulation, antioxidant defense, and microbiota composition that may influence host resilience under infection or environmental stress (Liao et al. 2023; Sun et al. 2024). Nevertheless, such mechanisms are often studied independently and as such integration between immune, metabolic, epithelial, and microbial systems is not explored. Also, the current research remains strongly biased toward a limited number of freshwater teleost models, whereas marine species, shellfish, crustaceans, and early developmental stages remain comparatively underrepresented (Shi et al. 2025). The broad extrapolation across aquaculture systems gets restricted by such taxonomic biasness. Likewise, whether reported effects reflect direct immunomodulatory, indirect nutritional consequences, or context-dependent host–microbiome interactions of VD₃ remains insufficiently resolved. These uncertainties mirror an important gap between emerging mechanistic hypotheses and experimentally proven functional outcomes.

Previous reviews have primarily addressed VD₃ physiology in relation to mineral metabolism, skeletal biology, and general nutrition in fish (Lock et al. 2007, 2010; Cheng et al. 2023). In contrast, the present review provides a focused and integrative overview of VD₃ as an antimicrobial and antiviral immunonutrient in aquaculture. Attention is given to VDR-mediated immune signaling, antibacterial and antiviral defense, mucosal and microbiota-associated defense, functional-feed integration, and translational challenges in antibody reduced production systems. Importantly, this review critically evaluates the relative strength of molecular versus functional evidence, identifies recurring inconsistencies across studies, and highlights key sources of variability including species differences, dosage effects, and environmental context. This work outlines major research priorities necessary for validating VD₃-based strategies in sustainable aquaculture health management by integrating mechanistic insight with translational relevance. Overall, this synthesis provides a comprehensive overview of VD₃’s role in innate and adaptive immune modulation and evaluates its potential to ensure robust, evidence-based application in aquaculture practice.

Mechanistic basis of vitamin D₃ immunomodulation in aquatic species

Vitamin D₃ (VD₃) functions as a key regulatory molecule that links endocrine signaling, immune modulation, metabolic regulation, and oxidative homeostasis in vertebrates, including teleost fish. Increasing evidence suggests that VD₃-associated signaling regulates both antibacterial and antiviral responses by regulating innate immunity, cytokine networks, epithelial barrier integrity, and redox balance (White 2022; Cao et al. 2024). Mechanism, however, is significantly less well understood in fish than in mammals and often highly variable between species, environment, and context (Kollitz et al. 2014; Song et al. 2023). The available evidence is largely from short-term nutritional or infection trials and from experiments based on transcriptional changes, while experiments validating functionality in terms of pathogen clearance, survival, or production scale performance are not as abundant. This disparity in evidence necessitates a hierarchical interpretation framework in which functional outcomes are more valid than responses at the molecular level. A major complexity in aquatic species rises from pronounced interspecific inconsistency in VD₃ metabolism, vitamin D receptor (VDR) paralog structure, nutritional ecology, and environmental physiology. Factors such as salinity, temperature, photoperiod, developmental stage, dietary composition, and pathogen exposure can significantly vary VD₃ bioavailability and downstream signaling efficiency (Kollitz et al. 2014; Song et al. 2023). Consequently, conclusions derived from freshwater teleost models such as tilapia, carp, and zebrafish cannot be extrapolated directly to marine fish, shellfish, or early developmental stages without careful physiological consideration. Accordingly, given section synthesizes current knowledge on VD₃ metabolism, receptor biology, and immune regulation while critically evaluating major mechanistic uncertainties and translational limitations.

Metabolism and activation of vitamin D₃ in fish

Vitamin D₃ (VD₃; cholecalciferol) is a lipid-soluble secosteroid that is traditionally known for its role in calcium and phosphorus metabolism in vertebrates but is now being investigated for its broader immunophysiological actions (Lock et al. 2010). In mammals, VD₃ is obtained through dietary intake and ultraviolet-B (UV-B)-mediated photochemical conversion of 7-dehydrocholesterol in the skin (Dominguez et al. 2021; Voiculescu et al. 2025). However, the contribution of endogenous photochemical synthesis is not clearly established in fish and is heavily influenced by the ecological niche, habitat depth, and exposure to solar radiation. Dietary vitamin D is the main and most reliable source of VD₃ in aquaculture systems, though it has been demonstrated in some surface-dwelling fish species, such as salmonids, under experimental conditions and UV-B-dependent VD₃ synthesis (Lock et al. 2010; Fossen et al. 2025). Vitamin D₂ (VD₂) or VD₃ can be used by fish from natural feeds as well as formulated feeds, but VD₃ is more biologically active and nutritionally relevant form in teleost physiology.

Following intestinal absorption, vitamin D-binding protein (VDBP) transports VD₃ to metabolically active tissues, where it undergoes sequential hydroxylation producing biologically active metabolites. The first hydroxylation step occurs mainly in the liver via cytochrome P450 enzymes such as CYP2R1-like isoforms, generating 25-hydroxyvitamin D₃ [25(OH)D₃], the major circulating indicator of VD₃ status in fish. In classical vertebrate models, the second activation step is primarily renal, mediated by CYP27B1, producing the hormonally active form 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (calcitriol) (Meyer et al. 2022). In teleost fish, however, VD₃ activation shows a more distributed and flexible enzymatic architecture. Evidence indicates that conversion of 25(OH)D₃ to active metabolites, including 1,25(OH)₂D₃ and 24,25(OH)₂D₃, occurs not only in kidney but also in liver and potentially other tissues, supported by the presence of tissue-specific 1α-hydroxylase activity (Henry and Norman 1975; Takeuchi et al. 1991; Sundell et al. 1992). In species such as European eel, extrahepatic activation has also been reported where intestine, muscle, and gills may contribute to local VD metabolism (Du Bois et al. 1988). Activated VD₃ primarily acts through the VDR, which heterodimerizes with retinoid X receptor (RXR) and binds vitamin D response elements (VDREs), regulating mineral balance, epithelial integrity, oxidative homeostasis, immunity, and stress adaptation (Fig. 1). Functional VDR homologs in fish suggest conserved roles, although species-specific receptor sensitivity and signaling efficiency limit broad extrapolation (Kollitz et al. 2014, 2015; Meyer et al. 2022; White 2022; Liao et al. 2023; Song et al. 2023).

Fig. 1.

Metabolic pathway of VD₃ from synthesis/acquisition through successive hydroxylation to the active form, enabling VDR-mediated gene regulation

Taken together, these findings indicate that fish have a species- and tissue-specific activation system that is quite different from the mammalian kidney-centered model (White 2022). Importantly, the liver seems to be more central to the teleost VD₃ metabolism than previously assumed, potentially involved in both hydroxylation steps in certain species (Takeuchi et al. 1991; Lock et al. 2010; Newmark et al. 2017). It is important to note that VD₃ metabolism in fishes is a relatively complex physiological system (Fig. 1). This metabolic plasticity is biologically significant because it implies that VD₃ bioactivation efficiency varies across taxa and is influenced by physiological and environmental context. Such variation is further reinforced by varying environmental and nutritional factors, including temperature, salinity, photoperiod, oxygen availability, dietary lipid composition, and pathogen exposure, all of which can modulate enzymatic activity, metabolite turnover, and tissue distribution of overall vitamin D derivatives (Dioguardi et al. 2017; Wang et al. 2024a, b, c). Subsequently, VD₃ status and responsiveness fluctuate markedly between water teleosts such as tilapia and grass carp that often show stronger transcriptional responses to dietary VD₃ than salmonids, although variances in diet and experimental conditions confound comparisons, leading to inconsistencies in physiological and immunological outcomes across studies (Shao et al. 2022; Zhang et al. 2023a, b). Once activated, VD₃ exerts biological effects through binding to the VDR, initiating transcriptional regulation of genes involved in immune modulation, epithelial integrity, oxidative balance, and metabolic regulation. Therefore, downstream signaling intensity and biological responsiveness get directly influenced by variability in metabolic activation.

Overall, VD₃ metabolism should be viewed as a heterogeneous, multi-organ, and environmentally responsive system rather than a simplified analogue of mammalian endocrine regulation in fish. This difference is important for interpreting experimental findings and for designing species-specific nutritional strategies in aquaculture systems. Future research focusing comparative physiology, metabolomics, transcriptomics, proteomics, and functional genomics will be critical to resolve these species-specific metabolic pathways and improve evidence-based VD₃ supplementation strategies across aquaculture taxa.

Distribution of VDR and expression dynamics

The vitamin D receptor (VDR) is a ligand-activated nuclear receptor that governs the majority of the biological effects of VD₃, such as the control of mineral metabolism, epithelial integrity, oxidative homeostasis, and immune signaling (Song et al. 2023). In teleosts, VDR expression has been detected in various metabolically and immunologically active tissues, such as the intestine, liver, spleen, gills, skin, and head kidney (Kollitz et al. 2014; Liao et al. 2023; Song et al. 2023). Overall tissue distribution indicates that VD₃ signaling could have a systemic and mucosal effect. In direct comparison with other aquaculture species, the number of receptors, their sensitivity to ligands, and the downstream transcriptional response are quite different, however, and so it would be inappropriate to extrapolate from one species to another in an aquaculture setting. Distinct from mammals, many teleosts have duplicated VDR paralogs (VDRa and VDRb) resulting from teleost-specific genome duplication events (Howarth et al. 2008; Kollitz et al. 2014; Kwon 2016; Aggeletopoulou et al. 2022). These paralogs show differences in tissue localization, ligand binding affinity, and transcriptional activity, indicating functional discrepancy rather than simple redundancy. In Japanese flounder (Paralichthys olivaceus), the earliest identification of fish VDR sequences was reported, providing foundational evidence for the evolutionary diversification of VD₃ signaling in bony fish (Suzuki et al. 2000). Successive studies suggest partial sub-functionalization in grass crap, where VDRa is more strongly associated with osmoregulatory tissues such as gills and kidney, whereas VDRb is comparatively enriched in metabolically active tissues such as intestine and liver (Song et al. 2023). Similar patterns have been proposed in zebrafish and salmonids; however, comparative mechanistic evidence remains limited and not fully consistent across species (Kollitz et al. 2014).

Functional assays also indicate differences in the response to ligands of the two isoforms of VDR. In zebrafish, 1,25(OH)₂D₃ has a higher binding affinity to VDRa than to VDRb, with VDRa mainly involved in calcium–phosphorus regulation and VDRb involved in developmental processes (Lin et al. 2012; Kwon 2016). These results suggest that species-specific differences in VD₃ responsiveness exist, while the functional role of the different VD₃ receptor isoforms in most aquaculture species is not fully understood. VDR activation has been linked to regulation of antimicrobial, inflammatory, and oxidative pathways in fish at the functional level (Nadh et al. 2026). Altered expression of antimicrobial peptides, lysozyme activity, complement components, antioxidant enzymes, and cytokine-related signaling across multiple teleost models has been associated with dietary VD₃ supplementation (Liu et al. 2021b, a; Zhang et al. 2023a, b; Wang et al. 2024a, b, c). Mechanistically, VDR-mediated signaling interacts with NF-κB, MAPK, and interferon-associated pathways, influencing cytokine balance, epithelial barrier integrity, oxidative stress adaptation, and antimicrobial defense (White 2022; Sun et al. 2023; Huang and Huang 2023). Most of available evidence is derived from transcriptional profiling rather than direct measurement of receptor binding dynamics, protein translation, or functional disease resistance, requiring cautious interpretation of biological significance. Experimental perturbation studies provide additional support for receptor-dependent regulation. In zebrafish, VDR knockdown disrupts developmental and immune-associated signaling networks, confirming its functional relevance in vertebrate physiology (Kwon 2024). Additionally, dietary VD₃ has been shown to upregulate VDR transcripts, relating with improved antimicrobial peptide expression, lysozyme activity, and antioxidant responses (Bass et al. 2020), supporting its role in frontline immune defense.

Overall, although VDR signaling is strongly implicated in fish immune regulation, its extrapolative value for functional disease resistance remains inconsistent across species. This variability is driven by environmental factors mainly temperature, salinity, stocking density, nutritional status, and pathogen exposure, all of which influence receptor expression and signaling efficiency (Dioguardi et al. 2017; Wang et al. 2024a, b, c). Existing literature is also heavily biased toward a limited number of freshwater models (tilapia, carp, zebrafish, grass carp), while marine fish, shellfish, and early developmental stages remain underrepresented. Upcoming clarification will require integrative approaches including comparative genomics, single-cell transcriptomics, proteomics, chromatin-level analysis, and functional gene-editing to resolve species-specific receptor dynamics and their relevance to aquaculture disease resilience.

Vitamin D₃ and innate immune modulation

Innate immunity represents the primary defense system in fish and plays a pivotal role in determining disease resistance under aquaculture conditions (Özcan, and Arserim 2026). Research evidence suggests that VD₃ contributes to regulation of multiple innate immune components, including pathogen recognition, antimicrobial peptide (AMP) production, inflammatory signaling, oxidative balance, and mucosal barrier integrity (Liu et al. 2021b, a; Cheng et al. 2020a, b; Wang et al. 2024a, b, c). However, the current available knowledge is largely based on transcriptional profiling and acute short-term supplementation trials, while measurements of functional outcomes such as pathogen clearance, survival improvement, or production-level performance remain comparatively underrepresented. This creates a persistent imbalance between molecular signals and biological validation which limits definitive interpretation about the VD₃ efficacy. Experimental studies indicate that VD₃ supplementation promotes innate immune parameters including lysozyme activity, complement activation, phagocytic capacity, respiratory burst response, and AMP expression (Liu et al. 2021b, a; Cheng et al. 2020a, b; Wang et al. 2024a, b, c). Increased transcription of β-defensins, hepcidins, and cathelicidins has been observed in grass carp and Nile tilapia (Sun et al. 2022; Zhang et al. 2023a, b), implying reinforcement of mucosal and systemic antimicrobial defense. However, these responses are frequently inferred from gene expression patterns, and direct evidence linking these molecular changes to microbial killing efficiency or pathogen load reduction remains limited.

Mechanistically, VD₃ is proposed to regulate innate immunity through pattern recognition receptor signaling, particularly toll-like receptors (TLRs), which activate downstream inflammatory cascades. These include NF-κB, MAPK, and JAK–STAT pathways, which collectively modulate cytokine production, leukocyte activity, and inflammatory resolution (White 2022; Sun et al. 2023; Chen et al. 2025). VD₃ supplementation has been associated with reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and increased anti-inflammatory mediators such as IL-10 (Liao et al. 2023; Wang et al. 2024a, b, c), suggesting tighter inflammatory control during infection. This phased cytokine modulation may optimize immune defense while limiting hyperinflammation, although its effectiveness appears to be species- and context-dependent (Dong et al. 2022; Rao et al. 2023; Rider et al. 2023). This modulation may prevent immunopathology while maintaining antimicrobial capacity.

A key functional aspect of VD₃-mediated innate immunity involves oxidative balance. Controlled ROS production is essential for pathogen killing; however, excessive ROS causes tissue damage and immune dysfunction. VD₃ enhances antioxidant defenses including SOD, CAT, GPx, and Nrf2-associated pathways (Sun et al. 2024; Zhang et al. 2024a, b). In black carp and largemouth bass (Micropterus salmoides) A. hydrophila infection, VD₃ upregulated hepatic lipid peroxide 5 (lpo5) expression, improved antioxidant enzyme activity and infection resistance, and reduced oxidative stress markers (Wu et al. 2020; Li et al. 2021). Nevertheless, causal linkage between oxidative regulation and consistent disease outcomes remains insufficiently validated across species. VD₃ also supports mucosal immunity by maintaining epithelial barrier integrity in intestine, gills, and skin. Reported from tilapia, zebrafish, and yellow catfish, effects include modulation of tight junction proteins, mucin production, and epithelial differentiation (Liao et al. 2023; Shao et al. 2022), potentially limiting pathogen entry and secondary infection. However, functional evidence directly connecting barrier modulation to improved survival or production outcomes remains limited. Overall, innate immune regulation by VD₃ (Fig. 2) is strongly context-dependent, shaped by species, dosage, environmental stress, and pathogen type. Current literature is heavily biased toward freshwater model species, while marine fish, shellfish, and early developmental stages remain underrepresented.

Fig. 2.

Immunomodulatory functions of VD₃ in fish, connecting VDR activation with antimicrobial peptide induction, cytokine regulation, and innate effector responses

Vitamin D₃ in adaptive and antiviral immunity

Compared to innate immune regulation, the effect of VD₃ on adaptive and antiviral immunity in fish remains less extensively characterized, although emerging evidence indicates potential involvement of VD₃ as a modulatory factor for lymphocyte function, antigen presentation, and interferon-mediated antiviral resistance. These effects are mainly exerted by the activation of VDR dependent signaling, establishing a bridge between endocrine regulation and cellular immune activity (Zhang et al. 2025a, b). A role for VD₃ in adaptive immune regulation is supported by VDR expression in other immune-associated organs including the head kidney, spleen, thymus, and intestines (Kollitz et al. 2014; Song et al. 2023). Experimental evidence from zebrafish, tilapia, and carp indicates the impact of VD₃ supplementation on lymphocyte proliferation, immunoglobulin production, and cytokine balance during pathogen exposure (Wang et al. 2023; Zhang et al. 2024a, b). In Cyprinus carpio, the enhancement of antigen presentation capacity by 1,25(OH)₂D₃ has been reported to increase MHC II, CD4, and CD8 transcript expression, suggesting enhanced antigen presentation capacity (Sun et al. 2022). Similarly, VD₃ supplementation enhanced serum IgM and IgD levels post-vaccination in grass carp as measure of humoral immune priming (Mu et al. 2021). Recently, the effects of active VD₃ in inducing FOXP3-like expression in zebrafish larvae where immunoregulatory signaling with regulated, controlled immune resolution were found to occur (Martens et al. 2020; Srichomchey et al. 2023). Taken together, the results indicate an immunomodulatory function of VD₃ in the coordination of adaptive immunity; however, most evidence remains indirect and based on gene expression rather than functional immune protection. VD₃ has attracted attention for its potential to regulate interferon-mediated signaling pathways in antiviral immunity. The ongoing threat posed by viral pathogens, such as tilapia lake virus (TiLV), viral hemorrhagic septicemia virus (VHSV) and largemouth bass ranavirus (LMBV), continues to cause substantial aquaculture loss, necessitating the exploration of host-directed immunomodulatory approaches. Transcriptomic studies indicate that exposure to VD₃ could regulate type I interferon responses, ISGs, and RIG-I-like receptor pathway and JAK–STAT signaling cascades (Chen et al. 2024; White 2022; Wang et al. 2023). VD₃ supplementation has been correlated with increased expression of antiviral effectors such as Mx proteins, ISG15, and IFN-related genes in zebrafish and tilapia models following the viral challenge (Wang et al. 2023; Zhang et al. 2024a, b). In specific experiments,VD₃ treatment has additionally been implicated for enhanced vaccine responses in different studies, where administration occurs only after bacterial or viral challenge (Sazu et al. 2026). These observations imply that VD₃ may have a role as an ancillary basal antiviral weapon at the molecular level. Beyond direct antiviral signaling, VD₃ may contribute in controlling infection by regulating inflammatory homeostasis and maintaining epithelial integrity. While controlling the pathogen, exaggerated inflammatory responses induced by viral infection can also damage tissues and lead to immunopathology reducing host survival. VD₃-mediated regulation of NF-κB signaling and cytokine production could be an approach for counterbalancing antiviral activity with appropriate immune responses (Sun et al. 2023; Liao et al. 2023). This dual regulatory mechanism indicates that VD₃ does not play a direct role as an antiviral effector, but rather as a modulator of immune equilibrium during infection.

Overall, adaptive and antiviral effects of VD₃ in fish appear biologically plausible but remain inconsistently validated across species and experimental systems. The current literature is dominated by transcriptional evidence and short-term challenge models, while functional endpoints such as viral clearance, survival advantage, and long-term immune memory remain underexplored. The extent and consistency of the protection need further in-depth investigations in terms of specific viral challenge systems and multi-species comparisons in aquaculture research. Importantly, the magnitude and direction of VD₃ effects vary depending on species, developmental stage, viral strain, and environmental conditions, indicating strong context dependency rather than conserved antiviral action. From a mechanistic perspective, VD₃ signaling in adaptive and antiviral immunity should therefore be interpreted as a modulatory layer influencing immune responsiveness rather than a primary effector pathway. Its functional relevance is likely determined by interaction with baseline immune status, pathogen pressure, and environmental stressors rather than acting independently as a deterministic antiviral factor.

Integration of oxidative and immune homeostasis

Oxidative stress and immune regulation in fish physiology are tightly interconnected processes that jointly determine disease susceptibility and physiological stability, particularly under intensive aquaculture conditions where environmental and biological stressors are persistent (Liu et al. 2026). In the context of this framework, VD₃ is increasingly recognized as a corresponding modulator integrating redox balance and immune reactivity to aid in the preservation of cellular homeostasis during pathogen challenge. Controlled reactive oxygen species (ROS) production at the cellular level is required for pathogen elimination and intracellular signaling in innate immune responses (Ma et al. 2025). But high or long-lasting ROS levels disrupt membrane integrity and enzyme functionality and increase the damage caused by inflammation that leads to a loss of host resistance (Dioguardi et al. 2017). VD₃ appears to contribute to the regulation of this balance by influencing antioxidant defense systems and stress-response signaling pathways, although the strength and consistency of these effects vary across species and experimental conditions.

In teleost fish, VD₃ supplementation has been linked with an increased bioactivity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and enhanced the regulation of glutathione-dependent redox buffering systems (Malekmakan et al. 2020; Sun et al. 2024; Wang et al. 2024a, b, c; Zhang et al. 2024a, b). Such enzymatic responses occur alongside reductions in lipid peroxidation and other markers of oxidative stress, helping to protect cells during inflammatory or infectious stress. However, under most available evidence, traces of biochemical endpoints indicate that a more than causative mechanistic connection between redox modulation is linked to resistance of pathogen. Increasing evidence also indicates that VD₃ may have interactions with transcriptional regulators associated with the adaption pathway to oxidative stress like nuclear factor erythroid 2–related factor 2 (Nrf2), which regulates detoxification and antioxidant gene expression in a cellular context (Li et al. 2015; White 2022; Sun et al. 2023). Moreover, the modulation of inflammatory signaling networks indirectly affects redox homeostasis by preventing chronic oxidative bursts triggered by excessive production of pro-inflammatory cytokines. Nonetheless, such regulatory interactions are still poorly characterized in fish and so far mostly derived from gene expression and enzyme activity profiles rather than a causal functional validation.

Additional studies suggest potential cross-talk between VD₃ signaling and iron metabolism and ferroptosis-associated pathways by modulating hepcidin and lipid peroxidation dynamics (Cai and Zhou 2025). These links rather indicate a wider function for VD₃ in the modulation of immune defense, coordinating with metabolism and redox master regulators, although mechanistic resolution is incompletely established and can vary within species (Ghosh and Chattopadhyay et al. 2026). These new mechanisms are particularly exciting, because they could provide a mechanistic link among antimicrobial defense, nutrient metabolism, and oxidative injury control. Notably, oxidative–immune interaction is highly influenced by environmental variability such as temperature variation, hypoxia, salinity stress, stocking density, and nutritional status (Kumar et al. 2025). These factors affect immune activation and redox balance, which lead to high heterogeneity in VD₃ responsivity between aquaculture systems and induce the lack of direct-extrapolation from controlled laboratory findings to field conditions. Thus, integrative frameworks integrating redox biology with proteomics, metabolomics, and microbiota analysis alongside well‐controlled infection models are warranted to determine if VD₃‐mediated modulation of oxidative–immune homeostasis can truly improve disease resilience and production stability. From a systems perspective, VD₃ need to be considered a modulator of the oxidative–immune regulatory network instead of an direct antioxidant effector from systems perspective, with its efficacy limited by species-specific metabolic capacity and physiological context.

Antibacterial effects and functional evidence in fish

Bacterial diseases still account for one of the most persistent constraints in global aquaculture which lead to significant economic losses due to impaired growth and decreased feed efficiency as well as instability. High stocking densities, dynamic environmental conditions, unsuitable water quality, and chronic physiological stress aggravate these threats by predisposing the host to opportunistic and pathogenic bacteria (Haenen et al. 2023; Maezono et al. 2025). Many bacterial pathogens, such as Aeromonas hydrophila, Vibrio anguillarum, Edwardsiella tarda, Flavobacterium columnare, Streptococcus iniae, Yersinia ruckeri, and Photobacterium damselae have been widely reported to cause different clinical syndromes from hemorrhagic septicemia to ulcerative lesions or systemic infections in both freshwater and marine aquaculture systems (Semwal et al. 2023; Manchanayake et al. 2022; Choi and et al. 2022; Janda and Duman 2024; Zhu et al. 2021; Juárez-Cortés et al. 2024; Attia et al. 2022; Shrivastava et al. 2025; Delghandi et al. 2020; Zhou et al. 2025a, b). The pathogen diversity summarized (Table 1) highlights the multifactorial nature of bacterial disease burden (Maldonado-Miranda et al. 2022), requiring host-resilience strategies that enhance broad-spectrum disease resistance rather than targeting single pathogens.

Table 1.

Representative bacterial pathogens of cultured fish and key symptoms

Pathogen	Disease	Symptoms	References
Aeromonas hydrophila	Motile Aeromonas septicemia	Hemorrhagic lesions, ulcers, abdominal swelling	Semwal et al. 2023
Vibrio anguillarum	Vibriosis	Skin ulcerations, hemorrhaging, organ damage	Manchanayake et al. 2022
Edwardsiella tarda	Edwardsiellosis	Gastroenteritis, systemic infections, skin lesions	Janda and Duman 2024
Flavobacterium columnare	Columnaris disease	Gills and skin infections, yellowish lesions	Zhu et al. 2021
Streptococcus iniae	Streptococcosis	Meningitis, exophthalmia, erratic swimming	Juárez-Cortés et al. 2024
Pseudomonas fluorescens	Bacterial hemorrhagic septicemia	Hemorrhagic skin lesions, internal organ damage	Attia et al. 2022
Yersinia ruckeri	Enteric redmouth disease	Hemorrhaging of the mouth, gills, and fins	Shrivastava et al. 2025
Renibacterium salmoninarum	Bacterial kidney disease (BKD)	White nodules in kidneys, splenomegaly, internal bleeding	Delghandi et al. 2020
Photobacterium damselae	Photobacteriosis	Skin ulcerations, necrosis of fins and tissues	Zhou et al. 2025a, b

Antibiotics and chemotherapeutics have been extensively used in conventional disease control, but the increasing concern about AMR, environmental pollution, microbial dysbiosis, and residue accumulation has stimulated interest in host-directed nutritional strategies (Cabello et al. 2016; Hossain et al. 2022). An interesting potential immunonutritional regulator is associated with modulation of innate immunity, epithelial barrier integrity, inflammatory balance, oxidative stability, and host–microbiome interactions. Studies for the effect of VD₃ supplementation on components of antibacterial defense, especially antimicrobial peptide induction, phagocytic activity, lysozyme production, and even cytokine regulation during a bacterial challenge have been conducted using Nile tilapia (Oreochromis niloticus), grass carp (Ctenopharyngodon idella), zebrafish (Danio rerio), yellow catfish (Pelteobagrus fulvidraco), and turbot (Scophthalmus maximus(Cheng et al. 2020a, b; Liu et al. 2021b, a; Zhang et al. 2023a, b; Wang et al. 2024a, b, c).

Importantly, VD₃ does not act as a direct antimicrobial compound but rather modulates endogenous host-defense systems that shape susceptibility and disease outcome. This distinction is critical because it positions VD₃ not as an antibiotic substitute, but as a component of integrated immunonutritional disease-management strategies whose effectiveness likely depends on species-specific physiology, supplementation dose, environmental conditions, and complementary husbandry practices (Abdul Kari 2025). However, while molecular and immunological evidence is increasingly strong, comparatively fewer studies provide functional validation through pathogen clearance, survival improvement, or production-level outcomes under commercial aquaculture conditions.

Integrated antibacterial activity of VD₃ through four complementary mechanisms

Current evidence suggests that the antibacterial effects attributed to VD₃ signaling in fish are best understood as the outcome of multiple interactive biological pathways rather than as a single dominating mechanism (Huang et al. 2025). In contrast to traditional antimicrobials that exert direct effects on bacterial viability, VD₃ achieves its primary antimicrobial effect through host-regulatory pathways regulating endogenous defenses. Based on literature to date, four principal and overlapping antibacterial mechanisms have been hypothesized: (i) induction of AMPs, (ii) reinforcement of mucosal and epithelial barrier defense, (iii) regulation of inflammatory and cytokine-associated signaling networks, and (iv) modification of intestinal microbial ecology, as well as microbiota-associated immune interactions. Collectively, these mechanisms provide an integrated host-defense framework that modulates bacterial virulence, tissue condition, and overall disease severity in aquaculture.

At the molecular level, biologically active VD₃ interacts with the VDR–retinoid X receptor (RXR) signaling complex to regulate transcriptional programs linked with antimicrobial defense (Sun et al. 2022; Zhang et al. 2023a, b), inflammatory control, oxidative balance, epithelial maintenance, and immune-cell activation (Fig. 3). In this context, VD₃ can be viewed as a nutritional regulator that may coordinate overlapping levels of defense simultaneously. Nevertheless, evidence supporting each pathway is variable in strength. Whereas fish-specific experimental evidence supports stronger endorsement of antimicrobial peptide induction and cytokine-associated regulation, modulation by microbiota and biofilm-linked interactions remains more correlative and mechanistically unclarified. The most direct antibacterial pathway is the regulation of antimicrobial peptides. VD₃ supplementation regulated the expression of cathelicidins, β-defensins, and hepcidins, which contribute to bacterial membrane disruption, iron sequestration, immune-cell attraction, and inhibition of bacterial colonization (Sun et al. 2022; Zhang et al. 2023a, b). As rapid-response innate molecules, they could be among the first in an early protective front line that precedes systemic immune responses. But AMP induction is markedly different in varying studies which suggest a strong species- and dose-dependent responsiveness. VD₃ signaling has also been associated with the regulation of PRR pathways, phagocytic activity, complement-associated responses, and cytokine regulation, thereby influencing both pathogen sensing and inflammatory resolution (Guo et al. 2025). This aspect of inflammation is particularly relevant because robust antibacterial immunity relies on killing the pathogen without causing excessive tissue damage. Experimental observations suggest that VD₃ behaves as an immunomodulator rather than a simple immune activator, fine-tuning the balance between inflammatory activation and resolution. This likely explains why some studies report robust cytokine-associated responses while other investigators see more modest effects under similar challenge conditions. In addition to immune-cell modulation, VD₃ may play a role in the maintenance of mucosal barrier integrity in the intestinal epithelium as well as epithelial cell function and antimicrobial defense (gills and skin), which are among the major pathways through which aquatic pathogens interface within the host (Zhang et al. 2023a, b). Both experimental studies and bioinformatic analysis indicate that VD₃ supplementation may influence tight-junction integrity, epithelial differentiation, mucin-associated pathways, and barrier-associated immune signaling, potentially limiting bacterial invasion and subsequent inflammatory damage (Liao et al. 2023; Shao et al. 2022). In orange-spotted grouper (Epinephelus coioides), VD₃ improved mucosal barrier function and oxidative stress tolerance, contributing to enhanced resistance against Vibrio spp. infections (Yang et al. 2021).

Fig. 3.

Antimicrobial potential of VD₃ for effective reduction of bacterial load

Emerging evidence further indicates that VD₃ may shape gastrointestinal microbial ecology through effects on microbial diversity, short-chain fatty acid–associated metabolism, and inflammatory homeostasis. These microbiota-associated changes may indirectly contribute to antibacterial resilience by supporting epithelial stability and reducing dysbiosis-associated pathogen expansion. However, causal relationships between VD₃-mediated microbiota modulation and measurable disease resistance remain insufficiently resolved, particularly under commercial production conditions. Importantly, these four antibacterial mechanisms should not be interpreted as isolated biological processes. AMP induction, epithelial integrity, inflammatory regulation, and microbiota stability interact extensively within a broader defense network. This systems-level integration provides a conceptual basis for understanding why VD₃ responses frequently exhibit species-, dose-, and context-dependent variability across aquaculture studies. It also establishes an evidence-based framework for the following subsections, where each mechanism is examined according to its functional strength, experimental support, and translational relevance.

VD₃-induced antimicrobial peptides (AMPs)

Antimicrobial peptides (AMPs) are conserved effector molecules of innate immunity in fish that contribute to antibacterial defense by disrupting membranes, forming pores, sequestering iron, recruiting immune-cells, and modulating local inflammatory responses (Akhavan-Bahabadi et al. 2024). Current knowledge indicates that VD₃ may modulate AMP-associated pathways in teleosts based on the VDR-mediated transcriptional control, but the strength of the evidence is predominantly species and AMP class-dependent. Among the best-characterized VD₃-responsive AMPs are β-defensins, cathelicidins, and hepcidins. Experimental studies in grass carp, Nile tilapia, zebrafish, and yellow catfish report increased β-defensin transcription following dietary VD₃ supplementation or bacterial challenge (Sun et al. 2022; Zhang et al. 2023a, b). β-defensins act on bacterial membrane disruption and leukocyte recruitment such as IL-10 increase and direct activation of dendritic cells (Estévez et al. 2018; Fu et al. 2023). Cathelicidins participate in membrane destabilization, chemotaxis, and mucosal defense. Hepcidins are a class of AMPs that functionally link antimicrobial activity with nutritional immunity by restricting iron availability (ferroptosis) to invading pathogens (Chung et al. 2020; Liu et al. 2021b, a; White 2022; Sun et al. 2024).

Mechanistically, VD₃-dependent AMP induction is mediated primarily via VDR–RXR association with VDREs, with additional interactions involving NF-κB, MAPK, and iron-regulatory pathways (Sun et al. 2023; Wang et al. 2024a, b, c). Nevertheless, AMP induction represents one of the most mechanistically plausible antibacterial pathways linked to VD₃; however, substantial interspecies variation in peptide repertoire, VDR sensitivity, and pathogen-specific responsiveness suggests that its protective value is unlikely to be uniformly conserved across aquaculture taxa.

Enhancement of mucosal barrier function and epithelial defense

Mucosal surfaces such as intestine, skin, and gills are important physical and immunological interface for fish toward aquatic environment and prevent bacterial adhesion and invasion (Mistri et al. 2025). Characteristically, such barriers are much more susceptible in intensive aquaculture conditions where environmental fluctuations can cause a decrease in epithelial integrity and increased susceptibility to infection due to handling stress and sub-optimal water quality (Dobre et al. 2026). Evidence from multiple teleost models suggests that VD₃ promotes mucosal barrier homeostasis by regulating both epithelial structure and gene expression of barrier-associated genes. Reported responses include modulation of tight-junction proteins, mucins, and epithelial differentiation markers that collectively regulate paracellular permeability and microbial translocation (Liao et al. 2023; Shao et al. 2022). For example, VD₃ increased tight-junction components (claudins, occludin, and ZO-1) associated with decreased epithelial permeability and higher resistance to Aeromonas hydrophila infection in Ctenopharyngodon idella and Pelteobagrus fulvidraco (Liu et al. 2021b, a; Liu et al. 2023a, b; Zhang et al. 2023a, b). Activation of the VDR increases the expression of E-cadherin and claudins, which reduce paracellular pathogen passage, thus strengthening the intestinal barrier (Sun and Zhang 2022). In Danio rerio, similar modulation of mucin-related pathways and expression of junctional proteins such as claudin-10 and TJP-1b have been observed, suggesting partially conserved but context-dependent epithelial responses across teleosts.

However, the functional significance of these molecular changes remains unevenly validated as comparatively fewer assess direct functional endpoints such as bacterial translocation, barrier permeability assays, or infection progression under controlled challenge conditions. This leads to a crucial gap between molecular markers of barrier activation and disease protection validated in large studies. From an evidence hierarchy perspective, epithelial and mucosal responses to VD₃ are therefore best classified as moderately supported at the molecular level, but weakly validated at the functional level. While gene-level regulation of tight-junction proteins suggests biological plausibility, confirmation through survival outcomes, pathogen burden reduction, or production-level performance remains limited. Mechanistically, VD₃-mediated epithelial regulation should be viewed as part of an integrated mucosal defense network in which barrier integrity interacts with immune signaling and environmental stress responses rather than acting as an isolated pathway. The variability observed across studies likely reflects differences in species physiology, developmental stage, and environmental context rather than inconsistent VD₃ functionality. Generally, VD₃-associated modulation of mucosal barrier function represents a promising but incompletely validated component of antibacterial immunity in fish, requiring stronger functional confirmation under production-relevant aquaculture conditions.

Cytokine modulation and regulation of inflammatory networks

Inflammatory regulation is a central determinant of fish antibacterial immunity as it is crucial for pathogen eradication depends on coordinated immune activation while limiting excessive tissue destruction (Buchmann 2022). More recently, a number of studies focused on investigating the role of VD₃ as an immunomodulatory hormone through specific cytokine-associated signaling networks, but the magnitude, direction, and consistency of VD₃ effects are highly variable depending on species and experimental conditions. Several studies report VD₃-induced upregulations and downregulations of pro- and anti-inflammatory cytokines such as TNF-α, interleukin (IL)−1β, IL-6, IL-8, IFN-γ, and IL10 during bacterial challenges or environmental stress (Liao et al. 2023; Wang et al. 2024a, b, c; Sun et al. 2023). Through VDR-associated regulation of pathways such as NF-κB, MAPK, and JAK–STAT signaling, VD₃ appears capable of influencing leukocyte recruitment, inflammatory amplification, and immune resolution. For instance, in yellow catfish (Pelteobagrus fulvidraco), transcriptomic analysis showed that VD₃ administration modulate classical and alternative NF-κB signaling pathways during Edwardsiella ictaluri infection (Cheng et al. 2020a, b). Activation of classical NF-κB results in the expression of inflammatory factors, such as TNF-α and IL-1β, which further stimulate the alternative pathway (Woronicz et al. 1997). Similarly, in abalone (Haliotis discus hannai), VD₃/VDR signaling suppressed NF-κB activation through inhibition of IκB ubiquitination and degradation and influenced autophagy. Importantly, cytokine regulation is one of the more well-validated mechanisms by which VD₃ has been connected to antibacterial actions, supporting that the VD₃-dependent antibacterial protection is due to integrated immune regulation rather than activation of any pathway in isolation. Nonetheless, process oriented experimentation over these interconnected responses is relatively rare. From a functional perspective, cytokine modulation is best interpreted as a balancing mechanism that shapes inflammatory efficiency rather than as a direct predictor of pathogen resistance. This distinction may explain why similar cytokine profiles across studies do not always produce equivalent survival or pathogen-clearance outcomes. Together, VD₃ appears to function less as a simple immune stimulant and more as a regulator of inflammatory homeostasis, where antibacterial relevance depends on balancing immune activation with tissue protection under species- and context-specific conditions.

Microbiota-mediated antibacterial actions

The intestinal microbiota is crucial to fish health, as it modulates nutrient metabolism, epithelial integrity, immune development, and colonization resistance against pathogens (Medina‐Félix et al. 2023). Emerging evidence from turbot (Scophthalmus maximus L.) and grass carp (Ctenopharyngodon idella) indicate that VD₃ may contribute to antibacterial defense partly through modulation of host–microbiome interactions within an integrated nutritional–immunological framework rather than as an isolated immune mechanism (Shao et al. 2022; Arciuch-Rutkowska et al. 2024; Sun et al. 2024). Experimental studies associate VD₃ supplementation with altered microbial diversity, composition of the bacterial communities, and microbial metabolites involved in intestinal homeostasis and immune regulation in experimental studies (Liao et al. 2023; Sun et al. 2024). Metagenomic analyses have shown enrichment of beneficial microbial taxa as well as a decrease in opportunistic pathogens, often correlating with improved intestinal morphology, increased villus height, and enhanced nutrient absorption efficiency (Wu et al. 2024). Moreover, VD₃ was associated with augmented short-chain fatty acid (SCFA) synthesis, bile-acid metabolism status, and intestinal pH regulation; these changes collectively supported the stability of commensal, though restrained, pathogen expansion (Zhao et al. 2022; Zhang et al. 2025a, b).

The current evidence hierarchy remains relatively weak, as most studies are correlative rather than causative relationships. Evidence for causative relationship between VD₃-driven gut microbiome shifts and disease resistance is still correlational, and causal validation through longitudinal, metabolomic, and microbiota-transfer studies remains essential (Yang et al. 2021). Direct demonstration that VD₃-induced microbiota shifts lead to improved pathogen resistance remains limited, and separating primary microbiome effects from secondary consequences of immune modulation or dietary change remains difficult. Species-specific microbiota composition, developmental stage, feeding strategy, salinity, and husbandry practices further complicate generalization across aquaculture systems. Thus, microbiota-associated antibacterial effects of VD₃ are best interpreted as an important integrative layer of host defense (Shao et al. 2022), but one whose direct functional contribution remains less resolved than AMP induction, epithelial protection, or cytokine regulation.

Experimental evidence across fish models

Experimental studies investigating the effect of VD₃ supplementation in fish demonstrate a wide range of immunological, physiological, and disease-associated responses among teleost models but strength and consistency of evidence vary widely within species, pathogens, dietary formulation, and experimental design. Commonly studied species include Nile tilapia (Oreochromis niloticus), grass carp (Ctenopharyngodon idella), zebrafish (Danio rerio), yellow catfish (Pelteobagrus fulvidraco), turbot (Scophthalmus maximus), bluntnose black bream (Megalobrama amblycephala), and black carp (Mylopharyngodon piceus) challenged mostly with bacterium Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, and Streptococcus spp. The comparative data summarized (Table 2) demonstrates not only an increasing interest in VD₃-mediated pathogen resistance but also an inconsistency of functional validation between fish–pathogen systems (Cao et al. 2024). As observed in mammals, VD₃ is known to modulate histone acetylation and innate immune memory (Singh and Paramanik 2025), indicating that similar conserved epigenetic processes may be involved. The comparative evidence summarized (Table 2) highlights that functional validation is available for several fish–pathogen systems although the trend appears uneven across the wider literature. Whereas some studies include controlled infection challenges with quantifiable endpoints such as mortality reduction, histopathological protection, or bacterial suppression, many others remain limited to transcriptional profiling or short-term immune-marker responses assessment.

Table 2.

Immuno-priming and novel VD₃-responsive pathways in fish

Aspect	Findings/mechanisms	Species/model	Interpretation	References
Cytokine modulation	Associated with increased IL-10, TGF-β1/2 and reduced IL-1β, TNF-α; increased AMP transcription (LEAP-2A/2B, β-defensin-1)	Yellow catfish, grass carp	VD3 may contribute to inflammatory regulation and AMP-associated defense, although responses appear species-, tissue-, and dose-dependent and are supported mainly by transcriptional evidence	Cheng et al. 2020a, b; Jiang et al. 2022; Zhang et al. 2024a, b
Pathogen resistance	Associated with reduced tissue injury and partial improvement in pathogen clearance during F. columnare and A. hydrophila challenge	Grass carp, yellow catfish	Supports a potential protective role of VD3 during bacterial challenge; however, evidence remains limited to a small number of freshwater species and controlled laboratory models	Jiang et al. 2022; Zhang et al. 2024a, b
NETs and ROS production	Induction of NET-associated responses, ROS production via NADPH oxidase, cytoskeletal remodeling, and phagosome maturation	In vitro/comparative vertebrate models	VD3 may influence neutrophil-associated antimicrobial responses, although direct mechanistic validation in fish remains limited	Kim et al. 2020
Phagocytosis and respiratory burst	Increased phagocytic activity and respiratory burst responses	Yellow catfish (P. fulvidraco)	Indicates enhancement of innate immune-cell activity following VD3 supplementation, although production-scale validation under aquaculture conditions is lacking	Cheng et al. 2020a, b
Gut microbiota modulation	Altered microbial composition including increased beneficial taxa, AMP expression, mucin-associated genes, and improved villi morphology	Turbot, grass carp, yellow catfish	VD3 may support intestinal homeostasis and barrier-associated immunity; however, long-term microbiome stability and causal links to disease resistance remain unresolved	Jiang et al. 2022; Shao et al. 2022; Zhang et al. 2024a, b
Gut barrier integrity	Improved villi morphology, enterocyte organization, and reduced intestinal injury during A. hydrophila exposure	Grass carp, yellow catfish	Supports a possible protective role of VD3 in mucosal defense, although mechanistic links between epithelial regulation and functional pathogen resistance require further study	Jiang et al. 2022; Zhang et al. 2024a, b
Biphasic cytokine response	Transient increase in IL-1β, IL-8, TNF-α followed by increased IL-10 and TGF-β expression	In vitro/comparative models	VD3 may contribute to balanced inflammatory regulation by coordinating early defense and later inflammatory resolution; in vivo dynamics in fish remain poorly characterized	Ismailova and White 2022
Antibacterial resistance/immune signaling	Increased cGAS–STING–TBK1–IRF3–IFNα signaling, enhanced autophagolysosome fusion, reduced liver necrosis	Largemouth bass (M. salmoides)	Highlights emerging links between VD3 signaling, autophagy, and immune regulation; however, evidence remains limited and mechanistic translation across aquaculture species is uncertain	Cao et al. 2024

Across these models, VD₃ supplementation is often linked with significant increases in indicators of innate immunity such as antimicrobial peptide production, complement activation, lysozyme production, phagocytic activity, cytokine regulation, and mucosal protection (Cao et al. 2024), which reflect integrated antibacterial mechanisms discussed in previous sections. In bluntnose black bream, VD₃ supplementation elevated serum C4 levels while VD₃ doses also increased C3 in fish challenged with Aeromonas hydrophila (Li et al. 2015). Similarly, juvenile black carp fed with VD₃ exhibit increased serum C3 and C4 levels along with upregulated C3 and C9 expression in hemocytes and liver (Wu et al. 2020). In Asian swamp eel, VD₃ reduced mortality, increased lysozyme activity and lymphocyte populations, and expanded the neutrophil indicating broader immune activation. In selected infection studies, these molecular and immunological changes have correlated with improved survival, reduced tissue injury, enhanced vaccine response, or lower pathogen burden after bacterial challenge in selected infection studies (Cheng et al. 2020a, b; Liu et al. 2021b, a; Wang et al. 2024a, b, c). A clear evidence hierarchy emerges across these studies. While many report strong transcriptional or biochemical immune responses, comparatively fewer demonstrate stronger functional outcomes such as pathogen suppression, histopathological protection, or survival improvement. This distinction is important because molecular immune activation does not consistently translate into equivalent disease resistance. Differences in dosage, developmental stage, pathogen virulence, and environmental conditions likely explain much of this variability, reinforcing that VD₃-associated antibacterial protection remains biologically plausible but strongly context-dependent across aquaculture systems (Fernández et al. 2018).

Implications for antibiotic-free aquaculture

The increasing restrictions on antibiotic use in aquaculture, driven by antimicrobial resistance (AMR), environmental contamination, and food-safety concerns, have intensified focus on sustainable host-directed alternatives (Milijasevic et al. 2024). In this context, VD₃ is recognized as a functional immunonutritional element that enhances host defense largely through endogenous immune regulation rather than direct antibacterial activity. As discussed across the preceding mechanisms, VD₃ supplementation is involved in antimicrobial peptide induction, mucosal barrier stabilization, inflammatory regulation, and microbiota-related immune balance. Collectively, these mechanisms support its potential as a modulator of baseline pathogen resistance and reduced susceptibility to infection in aquaculture systems. VD₃ should be regarded as an adjunctive immuno-nutritional strategy in combination with vaccination, husbandry, and biosecurity instead of replacing therapeutics (Varikuti et al. 2018). Its translational application in antibiotic-sparing aquaculture holds promise but underscores the necessity for standardized validation across multiple fish species, long time-frames, and production scale evaluations to realize practical implementation.

As the current evidence base does not support VD₃ as a standalone replacement for antibiotics under commercial farming conditions, its strongest practical value lies within integrated disease-management frameworks where nutritional modulation is combined with vaccination, biosecurity, water-quality enhancement, and functional-feed strategies. Such translational positioning is relevant, as it is likely that no single measure will be sufficient to reduce antibiotic use in aquaculture in a meaningful manner. From an applied perspective, VD₃ should therefore be regarded as a supportive component of preventive health-management systems, improving host robustness and larger-scale disease-control strategies rather than standing alone as a complete therapeutic alternative.

Antiviral properties and translational potential

Viral diseases remain one of the major challenges in aquaculture because of their fast spread, high mortality, and limited therapeutic control options, resulting in significant economic losses and production uncertainties across both freshwater and marine systems (Mugimba et al. 2021). Unlike bacterial infections, where chemotherapeutic interventions remain available, antiviral management in fish relies predominantly on preventive strategies such as vaccination, biosecurity, and environmental control, all of which are constrained by viral mutation, host specificity, and variable farming conditions (Hadfield 2021; Mancheva et al. 2021). Major viral pathogens affecting cultured fish, including tilapia lake virus (TiLV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), nervous necrosis virus (NNV), spring viremia of carp virus (SVCV), koi herpesvirus (KHV), and largemouth bass ranavirus (LMBV), collectively illustrate the diversity and clinical complexity of antiviral challenges in aquaculture systems (Table 3). Although antiviral research in fish remains substantially less developed than antibacterial investigations, emerging findings suggest that VD₃ signaling may influence several pathways relevant to antiviral defense in teleosts. In this regard, VD₃ is increasingly regarded as a host-directed immunonutritional factor with potential relevance for antiviral defense. VD₃ does not directly act against viral particles; rather, it influences antiviral competence through the regulation of interferon signaling, inflammatory balance, epithelial protection, and cellular stress resilience (Fig. 4). Currently, VD₃ is best viewed as a promising immunonutritional adjuvant component within the overall integrated antiviral health-management frameworks rather than a broadly validated significant antiviral intervention (Rathor and Swain 2024). Emerging studies in teleost models indicate biologically plausible antiviral roles, though existing evidence remains heterogeneous and follows a clear hierarchy, ranging from transcriptomic and biochemical observations to fewer studies demonstrating viral-load reduction, tissue protection, survival improvement, or production-level outcomes. This distinction is important because mechanistic activation in vitro does not always correlate to validated antiviral protection under aquaculture settings. These observations taken together make position VD₃ as a promising integrated antiviral health-management frameworks (Rathor and Swain 2024), but still not adequately translated as an integral component of strategies against viral infections.

Table 3.

Representative viral pathogens of aquaculture species, associated diseases, and major host species

Virus	Disease caused	Major species affected	Clinical signs	References
Viral hemorrhagic septicemia virus (VHSV, novirhabdovirus)	Viral hemorrhagic septicaemia (VHS)	Salmonids, pike, turbot, and > 80 aquatic species	Systemic hemorrhage, exophthalmos, darkened body coloration, erratic swimming; temperature-sensitive virulence (8–12 °C)	Mohammadisefat et al. 2023
Infectious haematopoietic necrosis virus (IHNV, Novirhabdovirus)	Infectious haematopoietic necrosis (IHN)	Pacific salmonids, especially sockeye salmon (Oncorhynchus nerka)	Necrosis of haematopoietic tissues, pale gills, abdominal distension, elevated juvenile mortality	Balakhnina and Melnikov 2024; Shan et al. 2025
Infectious pancreatic necrosis virus (IPNV, Birnavirus)	Infectious pancreatic necrosis (IPN)	Trout, salmon, and other freshwater species	Abdominal swelling, corkscrew swimming, pancreatic necrosis, growth impairment	Tapia et al. 2022
Koi herpesvirus (KHV, cyprinid herpesvirus-3)	Koi herpesvirus disease (KHVD)	Common carp (Cyprinus carpio), koi carp	Gill necrosis, skin lesions, neurological signs; latent persistence and stress-associated reactivation	Zrnčić et al. 2020
Tilapia lake virus (TiLV, Orthomyxo-like virus)	Tilapia lake virus disease (TiLVD)	Nile and other tilapia species	Lethargy, exophthalmia, skin erosion, liver necrosis, cumulative mortality	Rabeh 2022
Nervous necrosis virus (NNV, betanodavirus)	Viral nervous necrosis (VNN)	Groupers, seabass, marine finfish larvae	Brain and retinal vacuolation, spiral swimming, severe larval mortality; enhanced virulence in susceptible stages	Padrós et al. 2022

Fig. 4.

Antiviral potential of VD₃ in fish. 1,25(OH)₂D₃–VDR/RXR signaling regulates VDRE-linked transcription to strengthen host antiviral readiness and viral clearance

Activation of type I interferon and JAK–STAT pathways

Type I interferon (IFN) signaling represents one of the most conserved antiviral defense systems in vertebrates, including teleost fish (Zhang and Gui 2012). Following viral recognition by pattern-recognition receptors (PRRs), particularly retinoic-acid-inducible gene-I (RIG-I)-like receptors and melanoma differentiation-associated protein 5 (MDA5), downstream signaling involves activation of mitochondrial antiviral signaling protein (MAVS) and interferon regulatory factors (IRF3/IRF7) (Greiller and Martineau 2015; Cheng et al. 2020a, b; Soto-Dávila et al. 2020; White 2022; Wang et al. 2023). Subsequent induction of type I IFNs activates the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway, driving the expression of interferon-stimulated genes (ISGs) that restrict viral replication and support cellular protection mechanisms, including tissue integrity and barrier stabilization in species such as tilapia, seabass, and salmonids (Pereiro et al. 2025; Cheng et al. 2020a, b; Wang et al. 2024a, b, c). Current evidence indicates that vitamin D₃ (VD₃) may modulate multiple components of this antiviral cascade (Table 4). In several teleost models—including Atlantic salmon, Nile tilapia, zebrafish, rainbow trout, and largemouth bass—VD₃ supplementation has been associated with elevated expression of IFNs, JAK1, STAT1, and ISGs such as Mx, IFI56, and ISG15 during viral challenge (Wang et al. 2021, 2023; Shao et al. 2022; Cao et al. 2024; Zhang et al. 2024a, b). In largemouth bass, VD₃ has additionally been linked with the activation of the cGAS–STING–TBK1–IRF3 axis and reduced hepatic damage during viral infection (Cao et al. 2024). These findings suggest that VD₃ can influence upstream antiviral sensing and downstream interferon signaling pathways, although responses are not uniform across studies.

Table 4.

Integrated summary of VD₃-mediated antiviral, anti-inflammatory, and systemic immunoregulatory mechanisms in fish against viruses

Component/pathway/aspect	VD3 effect/mechanism	Species/model	Interpretation	References
Pattern Recognition & Viral Sensing (RIG-I, MDA5)	Associated with increased sensitivity of MAVS-dependent antiviral signaling pathways	Comparative vertebrate evidence	VD3 may prime viral sensing pathways and enhance early pathogen recognition; however, signaling kinetics in fish PRR systems remain poorly quantified	Greiller and Martineau 2015
Type I interferons (IFN-α, IFN-β)	Associated with increased IFN expression via IRF3/IRF7 activation through RIG-I/MDA5–MAVS pathways	Atlantic salmon, yellow catfish	Activation of IFN-associated antiviral signaling; additional validation is required in tropical and eurythermal aquaculture species	Cheng et al. 2020a, b; Soto-Dávila et al. 2020; Wang et al. 2021
Interferon regulatory Factors (IRF3, IRF7)	Increased phosphorylation and nuclear translocation associated with IFN transcription	Salmon macrophages, yellow catfish	Supports transcriptional regulation of interferon signaling by VD3; chromatin-level regulation in fish remains poorly characterized	Cheng et al. 2020a, b; Soto-Dávila et al. 2020
Interferon-stimulated genes (ISGs)	Increased IFI56, IFP35, Mx1, and ISG15 expression associated with antiviral immune activation	Turbot, largemouth bass, yellow catfish	Demonstrates downstream amplification of IFN-associated responses; however, direct linkage to reduced infection severity remains limited in most models	Shao et al. 2022; Wang et al. 2023
ChIP-seq/genomic targets	Putative VDREs identified within promoters of irf3, stat1, and ifi56	Comparative molecular studies	Provides supportive genomic evidence for VDR-associated antiviral regulation; functional in vivo validation remains limited in fish	Zhou et al. 2019; Teymoori-Rad et al. 2019
Hepcidin isoforms (TH1–5)	Hepcidin isoforms associated with interaction against viral proteins or membrane-associated structures	European sea bass, Tilapia	Extends hepcidin-associated defense beyond antibacterial immunity; differential isoform activity may represent promising biomarker targets	Lenin and Antony 2023; Anooja et al. 2024; Cervera et al. 2024
JAK–STAT signaling	Associated with increased STAT1 and JAK1 activation alongside enhanced ISG expression	Rainbow trout, yellow catfish	Strengthening of canonical antiviral signaling pathways; however, pathway cross-talk with inflammatory regulation complicates mechanistic interpretation	Cheng et al. 2020a, b; Cheng et al. 2021; Van Harten et al. 2018
NF-κB/inflammasome modulation	Reduced NF-κB and inflammasome-associated inflammatory activity	Turbot	VD3 may help limit hyperinflammation and preserve tissue integrity during viral stress; interactions with oxidative pathways remain incompletely resolved	Chen et al. 2022
Anti-inflammatory cytokine profile	Reduced IL-1β, TNF-α, IL-6 alongside increased IL-10 and TGF-β expression via VDR-associated signaling	General fish models	VD3 contributes to balancing immune activation and inflammatory resolution, potentially limiting immunopathology in high-density aquaculture systems	Fenercioglu 2024
Autophagy/apoptosis regulation	Associated with modulation of caspase activity, mitochondrial stability, and autophagy-associated pathways	Comparative vertebrate and fish studies	VD3 may contribute to selective autophagy and cellular protection during viral challenge; fish-specific mechanistic evidence remains limited	Chen et al. 2022
Antioxidant and systemic defense	Increased TAOC, TSOD, serum proteins, and oxidative-stress protection	Largemouth bass (ranavirus challenge)	Broader systemic cytoprotective functions beyond interferon signaling, potentially contributing to stress resilience during infection	Wang et al. 2023
Neuro-immuno protection	Associated with restoration of BDNF, serotonin-associated signaling, and microbiota balance under stress conditions	Zebrafish	Broader interactions between stress physiology, microbiota-associated pathways, and immune regulation relevant to disease susceptibility	Mukherjee et al. 2025

Despite consistent molecular activation, functional validation remains limited. Most studies report transcript-level changes, whereas reductions in viral load, transmission, or mortality are less consistently demonstrated. This indicates that the activation of IFN–JAK–STAT signaling does not necessarily translate into proportional antiviral protection under all conditions. Additional reports in zebrafish and largemouth bass suggest that VD₃ may also influence antiviral resilience indirectly through the modulation of oxidative balance, microbiota-associated signaling, and neuro-immune pathways (Mukherjee et al. 2025; Wang et al. 2023), although these associations remain largely correlative. Mechanistically, VD₃ may regulate interferon-related genes via vitamin D response element (VDRE)-mediated transcriptional control, with broader interactions involving autophagy, inflammasome signaling, and mitochondrial antiviral responses (Bikle 2022; Zhou et al. 2019; Teymoori-Rad et al. 2019). However, the strength of evidence varies considerably across species and experimental systems, and direct validation at the level of viral clearance remains scarce. Overall, the interferon–JAK–STAT axis represents one of the most mechanistically supported links between VD₃ and antiviral immunity in fish. However, its functional relevance is strongly context-dependent, and current evidence is largely concentrated at the molecular level, limiting definitive conclusions regarding consistent antiviral efficacy across aquaculture systems.

Modulation of cytokine networks and inflammation control

Effective antiviral defense in fish depends not only on pathogen elimination but also on tightly regulated inflammatory control, as excessive or prolonged immune activation can amplify tissue injury and compromise host resilience (Sullivan et al. 2021). Although cytokine activation is essential for immune responsiveness, prolonged or dysregulated inflammation may promote epithelial damage, oxidative stress, mitochondrial dysfunction, metabolic disruption, and increased disease susceptibility (Dai et al. 2023). Increasing evidence suggests that (VD₃) promotes antiviral immune homeostasis through modulation of cytokine networks and inflammatory-resolution pathways (Siddiqui et al. 2020). Several teleost studies (Table 4) report associations between VD₃ supplementation and altered cytokine profiles during viral challenge or immune stimulation. In particular, VD₃ has frequently been linked with the downregulation of pro-inflammatory mediators including IL-1β, TNF-α, and IL-6 along with the upregulation of anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β (TGF-β) (Fenercioglu 2024; Wang et al. 2023). These patterns suggest that VD₃ may support antiviral defense by preserving inflammatory equilibrium rather than simply amplifying immune activation. Mechanistically, this immunoregulatory effect (Fig. 4) appears associated with interactions among NF-κB, JAK–STAT, IRF-dependent pathways, and VDR-mediated transcriptional regulation (Zheng et al. 2023). Experimental observations in turbot (Scophthalmus maximus), yellow catfish (Pelteobagrus fulvidraco), and largemouth bass (Micropterus salmoides) have suggested that VD₃ supplementation may counteract excessive inflammatory signaling while preserving antiviral competence and epithelial protection (Chen et al. 2022; Wang et al. 2023).VD₃ may influence antiviral resilience through gut–brain–immune signaling, oxidative regulation, and metabolic stabilization in fish. However, current evidence remains largely associative and species-specific, limiting mechanistic resolution and broader translational application across diverse aquaculture systems (Mukherjee et al. 2025; Wang et al. 2023).

Nonetheless, available data still focuses on cytokines transcriptional profiles and biochemical markers (Cao et al. 2024), whereas comparatively fewer studies directly connect inflammatory modulation with viral-load reduction, histopathological protection, or survival improvement. This distinction is important because cytokine regulation alone does not necessarily indicate effective antiviral resistance. Together, these results position VD₃-mediated cytokine modulation as a context-dependent mechanism of immune homeostasis whose antiviral significance is species and viral-system dependent as well as infection severity.

Antiviral effects in specific fish models

Experimental evidence evaluating VD₃-associated antiviral responses in fish remains comparatively limited but is gradually expanding across several teleost models and viral systems. Studies have investigated species including zebrafish (Danio rerio), Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss), largemouth bass (Micropterus salmoides), Atlantic salmon (Salmo salar), and yellow catfish (Pelteobagrus fulvidraco) challenged with pathogens such as tilapia lake virus (TiLV), infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), nodavirus, ranavirus, and spring viremia of carp virus (SVCV) (Wang et al. 2021; Cervera et al. 2024; Tong et al. 2024; Gao et al. 2025; Rizwan et al. 2025). The summarized comparative findings (Table 5) highlight both increasing interest in VD₃-associated antiviral regulation and substantial heterogeneity across fish–virus systems. Across these models, VD₃ supplementation has frequently been associated with enhanced interferon signaling, increased interferon-stimulated gene (ISG) expression, moderated inflammatory responses, and improved tissue stability during viral challenge (Wang et al. 2023; Cao et al. 2024; Cervera et al. 2024). In largemouth bass, ranavirus challenge was accompanied by the activation of cGAS–STING–TBK1–IRF3 signaling and reduced hepatic necrosis following VD₃ supplementation, whereas rainbow trout exposed to IHNV showed enhanced antiviral signaling with reduced viral burden (Wang et al. 2023; Van Harten et al. 2018). Similar transcriptional activation of antiviral genes has also been reported in Atlantic salmon and zebrafish under experimental viral stimulation.

Table 5.

Comparative evidence of vitamin D₃-mediated antiviral responses in fish

Virus/disease	Species/model	VD3 mechanism/effect	Key molecular markers/pathways	Interpretation	References
Tilapia lake virus (TiLV)	Oreochromis mossambicus (tilapia)	VD3-induced hepcidin isoforms associated with interaction against TiLV envelope glycoproteins, potentially influencing viral assembly and release	Hepcidin (TH1–5) peptides	Demonstrates a plausible indirect antiviral mechanism via peptide–virus interaction; however, in vivo replication and transmission studies remain limited	Wang et al. 2021
Nodavirus (NNV)	Dicentrarchus labrax (European sea bass)	VD3-associated hepcidin activation linked to reduced infection severity and preservation of gill and hepatic integrity	Hepcidin isoforms (1–5)	Supports potential cross-protective hepcidin activity; however, evidence derives primarily from small-scale laboratory trials lacking long-term validation	Cervera et al. 2024
Viral hemorrhagic septicemia virus (VHSV)	Scophthalmus maximus (turbot)	Dietary VD3 supplementation associated with reduced viral load and increased antiviral gene expression	Increased Mx, ISG15, tissue integrity	Consistent with activation of IFN-associated signaling pathways; however, commercial-scale validation in turbot farming systems remains lacking	Tong et al. 2024; Gao et al. 2025; Rizwan et al. 2025
Ranavirus	Micropterus salmoides (largemouth bass)	High dietary VD3 supplementation associated with reduced viral titers and enhanced cytokine responses	Increased IL-1β, IFN-γ, Mx, complement proteins	Broad immunostimulatory effects rather than direct virus-specific inhibition; dosage substantially exceeds typical nutritional ranges and requires safety evaluation	Greiller and Martineau 2015; Wang et al. 2023
Infectious hematopoietic necrosis virus (IHNV)	Oncorhynchus mykiss (rainbow trout)	VD3-associated activation of JAK–STAT signaling and ISG expression linked to lower viral titers	ISGs, JAK–STAT activation	Supports mechanistic linkage between VDR signaling and canonical interferon pathways; however, evidence remains heavily dependent on cell-culture studies	Van Harten et al. 2018
Infectious pancreatic necrosis virus (IPNV)	Oncorhynchus mykiss (rainbow trout)	VD3 supplementation associated with reduced viral replication and improved cytokine balance	Reduced viral load, increased JAK–STAT and ISGs	Regulatory modulation of antiviral immune equilibrium, although protein-level validation and dose–response analyses remain limited	Van Harten et al. 2018
General antiviral regulation	Multiple fish species and cell models	VD3-associated regulation of antiviral gene networks through VDR-binding elements identified by ChIP-seq analyses	JAK–STAT, Mx, ISG15, complement, hepcidin pathways	Provides molecular-level evidence supporting VDR-associated transcriptional regulation; however, relatively few studies link these genomic responses to measurable reductions in infection severity	Teymoori-Rad et al. 2019; multiple sources

Current evidence (Table 5; Fig. 4) supports a plausible role for VD₃ in antiviral immunity, yet most studies emphasize transcriptional responses rather than viral kinetics, survival, or production outcomes. Yet antiviral evidence follows an uneven validation hierarchy, ranging from transcriptomic activation and immune-marker profiling to stronger endpoints such as viral-load reduction, histopathological protection, and survival outcomes. At present, most studies remain concentrated in the lower tiers of this hierarchy, and several report elevated ISG or interferon expression without measurable survival advantage or effective viral suppression. This distinction indicates that molecular responsiveness alone may overestimate functional antiviral protection. Variation in host species, viral tropism, infection severity, supplementation dose, and environmental conditions likely explains much of this inconsistency. Collectively, current evidence supports VD₃ as a biologically plausible but context-dependent antiviral immunonutrient whose functional value remains strongest in selected fish–virus systems rather than as a universal antiviral intervention.

Dietary application and integration in functional feeds

Increasing demand for sustainable aquaculture production, combined with growing restrictions in antibiotic use (e.g., prophylactic), has intensified interest in functional feeds capable to promote host resilience by nutritional immunomodulation (Dawood et al. 2018). Within this framework, VD₃ has gained increasing attention not only for its classical role in calcium–phosphorus metabolism but also for its broader contributions to immune regulation, epithelial maintenance, oxidative balance, and stress. Unlike the therapeutic interventions that are applied in disease outbreak scenarios, dietary supplementation presents a feasible tool for long-term regulation of innate immune factors via routine feeds, making it advantageous in preventive health management strategies within intensive aquaculture systems. Experimental evidence summarized in earlier sections (Tables 2, 4, and 5) suggest that VD₃ supplementation may impact antibacterial and antiviral defenses by facilitating collaborative immune and physiological responses imposed under challenge conditions (Cao et al. 2024; Wang et al. 2024a, b, c). It has been shown the VD₃ and its metabolite 25-hydroxyvitamin D₃ [25(OH)D₃], given at more moderate levels (2000–5000 IU kg⁻¹) provides a cost-effective option to support growth and survival or immune competence during bacterial and viral challenges (Cheng et al. 2020a, b). In practical aquafeed systems, VD₃ has been investigated both as a standalone additive and in combination with probiotics, prebiotics, phytogenics, β-glucans, trace minerals, and organic acids, reflecting its integration into broader multifunctional nutritional strategies (Huang and Huang 2023; Vijayaram et al. 2025). The physiological effects of VD₃ supplementation are highly context-dependent, with effects modified by species, environment, and nutrient interactions. Although molecular immune responses are frequently reported, consistent gains in survival, growth, or disease resistance remain limited under commercial conditions (EFSA 2017). Dose optimization, formulation stability, and applicability to production scale still need standardized long-term trials.

Forms and sources of vitamin D₃ used in aquafeeds

Vitamin D supplementation in aquaculture feeds is provided predominantly as VD₃ (cholecalciferol), but vitamin D₂ (VD₂; ergocalciferol) and hydroxylated metabolites including 25-hydroxyvitamin D₃ [25(OH)D₃] have also been experimentally evaluated (Jenkinson 2019). Among these forms, VD₃ continues to be the principle component of commercial aquafeeds due to its well-established nutritional significance and wide utilization in teleost production systems. Natural dietary sources includes fish oil, fishmeal, and zooplankton and photosynthetic algae-associated trophic pathways; however, modern intensive aquaculture is primarily reliant on synthetic supplementation to maintain formulation consistency (Thazeem et al. 2022). Recently, hydroxylated derivatives such as 25(OH)D₃ have governed growing interest due to their ability to bypass the need for initial hepatic hydroxylation step in the liver and may therefore provide a faster physiological response and potentially greater bioavailability than conventional VD₃ (Lock et al. 2010). Studies in salmonids, carp, and tilapia have linked reported that 25(OH)D₃ supplementation with improved calcium metabolism, growth performance, oxidative stability, and immune-associated signaling processes (Lock et al. 2010; White 2022). Yet, direct comparisons of functional immune outcomes after VD₃ and 25(OH)D₃ supply remain scarce and the better biological activity of hydroxylated metabolites seems to vary with species, feed composition, or production goals rather than any enhanced effect in general. Alternative delivery systems including microencapsulation, nano-emulsions, lipid-coated formulations, and slow-release matrices have also gained attention for improving stability and intestinal bioavailability, particularly under high-temperature extrusion and prolonged storage conditions where oxidative degradation may reduce efficacy (Kumar et al. 2018; Masoomi Dezfooli et al. 2019; Cheng 2024). Nevertheless, most advanced delivery systems remain investigated primarily at experimental scales, while practical feasibility, manufacturing compatibility, and cost-effectiveness under commercial aquafeed production remain incompletely established. Thus, formulation choice in aquafeeds is increasingly becoming a balance between bioavailability, processing stability, and species-specific nutritional demands rather than simply selecting a single vitamin source.

Optimal dosage and nutritional requirements

Determining optimal VD₃ supplementation levels in aquaculture remains challenging because nutritional requirements vary considerably among fish species, developmental stages, environmental conditions, dietary composition, and the physiological endpoints evaluated (Cheng et al. 2023; Flo et al. 2025). Traditionally, dietary VD₃ requirements have been established based on skeletal development, calcium–phosphorus homeostasis, and prevention of deformities related to deficiency. Yet an increasing body of evidence indicates that the immunological and stress-associated roles of VD₃ may function within dietary intake ranges that deviate from these classical minimum nutritional thresholds. Experimental studies report a broad range of dietary inclusion levels depending on species and study design (Wu et al. 2020; Zhang et al. 2023a, b; Wang et al. 2024a, b, c). In salmonids and marine finfish, supplementation often ranges from several hundred to several thousand International Units per kilogram of feed, whereas antibacterial and antiviral studies frequently apply higher doses to evaluate immunomodulatory effects (Lock et al. 2010; Wang et al. 2023). Notably, moderate supplementation levels between 2000 and 5000 IU kg⁻¹ have repeatedly been associated with improved growth stability, enhanced survival, and stronger immune competence under pathogen challenge (Wu et al. 2020; Zhang et al. 2023a, b; Wang et al. 2024a, b, c). These findings indicate that practical immunonutritional benefits may occur above basal nutritional requirements but below pharmacological ranges. The reported optimal doses are likely influenced by differences in species physiology, feed formulation, lipid content, calcium–phosphorus balance, and bioavailability within the context of pathogen severity or environmental stress intensity. Species-specific variability further complicates establishment of standardized recommendations (Taylor et al. 2025). Warm-water freshwater species such as tilapia and grass carp often display stronger VD₃ responsiveness than salmonids or marine species, likely due to differences in metabolic activation, digestive physiology, and growth dynamics (Viegas 2019; Taylor et al. 2025).

In addition, high-dose supplementation could pose physiological risks including hypercalcemia, abnormal mineral deposition, oxidative stress, and pro-inflammatory signaling dysregulation (Miao et al. 2015), particularly during prolonged feeding. Environmental variables and developmental stage substantially influence VD₃ bioavailability and physiological demand, creating marked variability in supplementation outcomes across aquaculture systems (Cheng et al. 2023; Liu et al. 2023a, b; Lee and Lee 2025; Kumar et al. 2026). Consequently, universal immuno-nutritional dosing recommendations remain premature, emphasizing the need for species-specific precision frameworks. Thus, optimal VD₃ supplementation should be viewed less as a universal numerical threshold and more as a species-specific nutritional window shaped by biological demand, environmental stress, and production objectives, aligning more closely with precision aquaculture nutrition than fixed-dose standardization.

Functional outcomes of dietary VD₃ supplementation

Dietary VD₃ supplementation in aquaculture has been associated with a broad range of physiological and production-related outcomes across multiple fish species (Cheng et al. 2023). Beyond its established role in calcium–phosphorus metabolism and skeletal development, increasing evidence indicates that VD₃ may influence growth performance, feed efficiency, intestinal condition, survival, and stress resilience under intensive farming conditions. Several studies report positive associations between dietary VD₃ supplementation and improved production parameters, including weight gain, feed conversion ratio (FCR), specific growth rate (SGR), and nutrient-utilization efficiency in Nile tilapia (Oreochromis niloticus), grass carp (Ctenopharyngodon idella), yellow catfish (Pelteobagrus fulvidraco), seabream, and salmonids (Lock et al. 2010; Wang et al. 2023; Sun et al. 2024). These improvements are often accompanied by the enhancement of intestinal villus morphology, enterocyte organization, and tight-junction integrity which indicated better feed utilization and gut performance (Liao et al. 2023; Shao et al. 2022). Dietary VD₃ supplementation is increasingly linked with enhanced antimicrobial peptide production, cytokine modulation, interferon signaling, and oxidative defense, contributing to improved pathogen resilience in fish (Cheng et al. 2020a, b; Wang et al. 2023). Epithelial integrity and microbiota regulation may further strengthen disease resistance under intensive aquaculture conditions (Feng et al. 2026). In selected bacterial and viral systems, especially in European seabass (Dicentrarchus labrax), VD₃ is also associated with improved survival and less tissue damage under pathogen challenge (Dioguardi et al. 2017; Villa et al. 2025). These results provide more robust functional evidence than molecular immune responses by themselves, as they are a direct measure of host performance under biological stress. Beyond infectious disease, aquaculture species frequently encounter handling, thermal fluctuation, salinity shifts, crowding, transport, and hypoxia (Kumar et al. 205). Emerging studies in zebrafish and largemouth bass suggest that VD₃ supplementation may improve resilience under these production-related stressors by modulating oxidative balance, inflammation, mitochondrial stability, and microbiota–gut–brain–immune interactions, although mechanistic validation remains limited (Mukherjee et al. 2025; Wang et al. 2023). However, the functional evidence remains hierarchical and uneven: growth and biochemical improvements are more commonly reported than survival, long-term productivity, or economic performance. This suggests that the practical value of VD₃ in aquaculture lies not in universal performance enhancement but in species- and context-specific improvement of production robustness.

Synergistic integration with other functional feed additives

Growing interest in sustainable nutritional strategies for aquaculture has accelerated the innovative formulation of multi-component functional feeds that pair VD₃ with probiotics, prebiotics, phytogenics, β-glucans, essential oils, organic acids, trace minerals, and other immunomodulatory compounds (Huang and Huang 2023; Buonaiuto et al. 2025). Rather than functioning independently, these additives often interact through overlapping effects on nutrient utilization, intestinal integrity, microbiota stability, inflammatory regulation, and physiological resilience. Representative VD₃ co-supplementation strategies and their reported synergistic effects are summarized (Table 6). These findings support possible synergy between VD₃ signaling and microbiota-mediated epithelial regulation; however, short study duration, probiotic strain specificity, and limited validation under commercial farming conditions constrain broader interpretation. Several studies indicate that probiotic–VD₃ combinations may improve gut-associated immunity, epithelial organization, and feed efficiency (Wellington et al. 2021; Wei et al. 2022). In Atlantic salmon (Salmo salar) and gilthead seabream (Sparus aurata), formulations containing Lactobacillus spp. have been associated with improved feed conversion ratio (FCR), increased villus height, enhanced mucosal markers, and reduced mortality under challenge conditions (Klakegg et al. 2020; Sun et al. 2024; Jiang et al. 2024; Abualreesh 2026). Similarly, phytogenic additives such as carvacrol and thymol in European seabass have been linked with improved specific growth rate (SGR), hepatic stabilization, and reduced lipid peroxidation (Dinardo et al. 2021). Trace elements including selenium and zinc may further reinforce epithelial stability and antioxidant balance, while omega-3 fatty acids contribute to membrane integrity and inflammatory regulation (Gao et al. 2025; Moustafa et al. 2024; Shakeri et al. 2024). In yellow catfish, zinc-associated strategies have additionally been linked with improved tight-junction expression, inflammatory regulation, and growth performance (Liu et al. 2024; Rizwan et al. 2025). Similarly, zebrafish studies involving VD₃ analogs and essential oils suggest the enhancement of glutathione-associated antioxidant activity and reactive oxygen species (ROS) detoxification pathways (Dharshan et al. 2025). In African catfish and rainbow trout, β-glucan co-supplementation has also been associated with enhanced phagocytosis, respiratory burst activity, lysozyme production, and improved survival during pathogen challenge (Rodrigues et al. 2020; Arciuch-Rutkowska et al. 2024; Villa et al. 2025). Zinc-associated strategies in yellow catfish have additionally been linked to improved antioxidant activity, tight-junction expression, inflammatory regulation, and growth performance (Liu et al. 2024; Rizwan et al. 2025).

Table 6.

Synergistic effects of VD₃ with functional additives in aquaculture

Species	Additive type	Synergistic effect	Interpretation	References
Atlantic salmon (Salmo salar)	Probiotics (Lactobacillus spp.)	Enhanced mucosal immunity, reduced mortality, improved FCR	Evidence indicates VD3 + probiotics enhance gut–immune interactions; strain specificity and study duration limit generalization	Klakegg et al. 2020
Gilthead seabream (Sparus aurata)	Probiotics	Improved gut health, increased immune markers, FCR, villi height	Morphofunctional gut improvements observed; absence of pathogen-challenge limits conclusions on disease resistance	Sun et al. 2024
European seabass (Dicentrarchus labrax)	Essential oils (carvacrol, thymol)	Hepatic protection, antioxidant support, reduced lipid peroxidation, increased SGR	Supports antioxidant synergy; composition variability may affect reproducibility	Dinardo et al. 2021
Zebrafish (Danio rerio)	Essential oils + VD3 analogs	Increased glutathione, enhanced antioxidant enzymes, improved ROS detox	Provides mechanistic redox insights; extrapolation to commercial species is limited	Dharshan et al. 2025
African catfish (Clarias gariepinus)	β-glucans	↑ phagocytic activity, lysozyme, respiratory burst, survival	Demonstrates innate immune co-activation; temporal dynamics and long-term persistence remain underexplored	Arciuch-Rutkowska et al. 2024
Rainbow trout (Oncorhynchus mykiss)	β-glucans	↑ antiviral cytokines (IFN-γ), improved FCR	Evidence supports antiviral immune priming; studies limited to lab conditions	Villa et al. 2025
Yellow catfish (Pelteobagrus fulvidraco)	Zinc	↑ growth, antioxidant enzymes, tight-junction proteins, inflammatory regulation	Broader immunometabolic enhancement; optimal dosing and mineral interactions require further study	Liu et al. 2024; Rizwan et al. 2025

Despite these synergistic outcomes, direct evidence demonstrating that VD₃ supplementation reduces the efficacy of other functional feed additives in aquaculture remains limited. However, comparative nutritional studies indicate that alternative vitamin D metabolites such as 25-hydroxyvitamin D₃ [25(OH)D₃] may outperform conventional VD₃ in regulating antioxidant and inflammatory responses under similar dietary conditions, suggesting that the biological effectiveness of vitamin D forms can vary substantially (Dharshan et al. 2025). In addition, mechanistic evidence indicates that excessive calcium in phytase-supplemented diets may reduce phosphorus availability through calcium–phytate complex formation, potentially attenuating both phytase efficacy and VD₃-associated mineral utilization (Selle et al. 2009). These findings indicate that nutrient compatibility and balanced dosage are still key to functional integration.

However, interpretation remains complex because multi-component formulations make it difficult to isolate the specific contribution of VD₃ from concurrent additive effects. Importantly, not all nutrient interactions are beneficial. Excess dietary calcium, phosphorus imbalance, phytate-rich formulations, mineral competition, and oxidative degradation during feed processing may attenuate VD₃ absorption, metabolic activation, or receptor responsiveness. Consequently, the biological value of VD₃ in integrated feed systems depends less on additive diversity itself and more on formulation compatibility, nutrient balance, and species-specific digestive physiology.

Application in early life stages and stress management

During early developmental stages, experimental studies in zebrafish, salmonids, tilapia, and marine larvae correlate VD₃ supplementation with changes in the expression of antimicrobial peptides, cytokines, epithelial-related genes, or developmental markers (Lock et al. 2010; White 2022; Wang et al. 2023; Rizwan et al. 2025). These findings suggest that VD₃ contributes to the developmental programming of skeletal and immune systems potentially influencing later-life health resilience and physiological robustness.

Since early microbial colonization has a profound impact on epithelial maturation and immune programming, nutritional regulation by VD₃ particularly during these stages may indirectly modulate early microbiota assembly and therefore host long-term resilience (Wu et al. 2020).

During juvenile production, fish are also exposed to frequent handling, grading, transport, salinity fluctuation, thermal stress, hypoxia, crowding, and dietary transitions, all of which increase metabolic and physiological instability (Varsamos et al. 2006). Handling, grading, transport, vaccination, salinity fluctuation, thermal stress, hypoxia, crowding, and dietary transition may collectively disrupt oxidative balance, epithelial integrity, microbiota composition, and immune coordination. Several studies suggest that VD₃ supplementation may support stress adaptation by improving epithelial stability, maintaining mineral balance, and reducing physiological disruption under these conditions (Rizwan et al. 2025). VD₃ may shape early-life immune programming and stress resilience through modulation of gut-associated, neuro-immunological, and microbiota-driven pathways, with potential long-term effects on disease susceptibility (Mukherjee et al. 2025). However, marked species-specific developmental demands and risks of over- or under-supplementation highlight the need for precise larval dose optimization and long-term commercial validation (Boison and Turnipseed 2015). Importantly, VD₃ requirements during early life are highly species-specific because of differences in developmental rate, digestive maturation, endocrine regulation, and skeletal growth dynamics. Both deficiency and excessive supplementation may disturb developmental balance. This makes early-life VD₃ application less a question of supplementation intensity and more one of timing, developmental stage, and species-specific nutritional precision.

Research gaps and future perspectives

Despite growing interest in VD₃-mediated immunonutrition in aquaculture, substantial mechanistic, translational, and commercial uncertainties remain unresolved. Current evidence supports potential roles for VD₃ in fish immunity; however, most studies remain based on short-term laboratory experiments emphasizing transcriptomic, enzymatic, or biochemical endpoints rather than stronger functional outcomes such as pathogen reduction, histopathological protection, survival improvement, or production-level performance. Importantly, only a minority of available studies extend beyond molecular or biochemical observations to include pathogen burden, survival analysis, or production-scale validation. The available evidence therefore follows a clear hierarchy, ranging from transcriptomic responses (lowest confidence), biochemical indicators, and controlled challenge models to survival outcomes and production performance as the strongest forms of validation. This imbalance is a sustained bottleneck in the application of mechanistic discoveries into farm-level approaches. A second major limitation lies in limited taxonomic representation. Research is still focused on a few species and taxa, including zebrafish (Danio rerio), Nile tilapia (Oreochromis niloticus), grass carp (Ctenopharyngodon idella), yellow catfish (Pelteobagrus fulvidraco), salmonids, and a small number of teleost models; while marine finfish, shellfish, crustaceans, larval stages, or environmental specialist taxa are largely still understudied. VD₃ responsiveness is additionally influenced by extensive species biological, developmental, environmental, and supplementation dose heterogeneity (Tables 2, 4, and 5), which complicates interpretive cross-study extrapolation. A recurring challenge is the disconnect between molecular responsiveness and validated biological outcomes. Enhanced cytokine expression, interferon signaling, antimicrobial peptide induction, or antioxidant activity do not consistently translate into improved survival, reduced pathogen burden, or enhanced production performance. Not all studies report beneficial outcomes; several show neutral responses at basal supplementation levels, while others report inconsistent functional gains despite clear molecular activation. Advances in CRISPR/Cas editing, ChIP-seq, single-cell transcriptomics, epigenomics, and VDR-target mapping may help resolve these uncertainties and clarify species-specific regulatory networks underlying VD₃ biology in fish (Yu et al. 2022; Friedman et al. 2024; Zhou et al. 2025a, b). Collectively, these limitations indicate that the principal challenge is no longer establishing mechanistic plausibility, but defining when, where, and under what biological conditions VD₃ supplementation produces reproducible functional benefit across aquaculture systems.

Species-specific metabolism and VDR function

The most critical but unresolved dimensions of VD₃ biology in aquaculture concerns species-specific variation in metabolism, receptor signaling, and physiological responsiveness (Lock et al. 2010). While mammalian models often guide interpretation of fish VD₃ physiology, the accumulating evidence shows significant variation in absorption efficiency and hydroxylation pathways among teleosts that ultimately results in different tissue distribution and receptor function with varying downstream immune consequences. Such variability complicates the establishment of universal strategies for supplementation across aquaculture systems. As discussed earlier, many teleost fish have duplicated vitamin D receptor paralogs (VDRa/VDRb) as a consequence of the teleost-specific genome duplications (Kollitz et al. 2014, 2015; Kwon 2019). The observations made in zebrafish (Danio rerio), grass carp (Ctenopharyngodon idella), and salmonids indicate that these paralogs differ in tissue localization, ligand sensitivity, transcriptional behavior, and physiological function (Kollitz et al. 2014; Song et al. 2023). This divergence implies a functional specialization rather than mere redundancy, yet its immunological relevance has remained unresolved for most fish with commercial importance. Life-stage and salinity variation further complicated the system because of their differential dynamics on receptor expression, metabolic demand of different life stages (larvae vs. juveniles), and developmental sensitivity to VD₃ (Fernández et al. 2018). Uncertainty also remains regarding the relative contribution of dietary acquisition versus endogenous synthesis (Rider et al. 2025). Although dietary intake is predominant in most species that are cultured, photochemical synthesis and tissue-specific activation behaviors may differ greatly by habitat (freshwater vs marine), feeding strategy (pelagic fish vs benthic feeders), and developmental stage. Salinity, temperature, and photoperiod as well environmental conditions may also modify VD₃ utilization and physiological requirements. Importantly, these biological differences help explain why comparable dietary doses can produce inconsistent immune or growth outcomes across species. As reflected earlier (Tables 4 and 5), antiviral and immunoregulatory responses are often context-dependent rather than universally conserved. This makes species specificity not a secondary variable, but a central determinant of VD₃ efficacy in aquaculture nutrition.

Dose–response relationships and long-term safety

Determining optimal VD₃ supplementation strategies in aquaculture remains complicated by incomplete understanding of dose–response relationships, long-term physiological consequences, and species-specific tolerance thresholds (Flo et al. 2025; Chen et al. 2026). Although dietary requirements for skeletal development and calcium–phosphorus homeostasis are relatively established for some species, the dose ranges required for immunological enhancement remain less clearly defined. This distinction is critical because the amounts beneficially affecting immune function do not generally occur at classical nutritional concentrations. Across existing studies, not all supplementation levels produce corresponding outcomes. Basal nutritional doses often show neutral or restricted immune effects, whereas moderate supplementation ranges have more consistently been associated with improved immune competence, survival, and stress resilience. In contrast, in some bacterial and viral challenge studies, higher inclusion levels used frequently produce stronger molecular responses, including antimicrobial peptide induction, cytokine modulation, and interferon-associated signaling, but these benefits are not always accompanied by proportional improvements in growth or survival.

This inconsistency highlights an important uncertainty boundary. High molecular activation should not be presumed as evidence of biological optimization. The excess exposure to VD₃ may lead calcium hypercalcemia, rock mineralization, oxidative stress, and endocrine disruptor activity, as well as epithelial injury or inflammatory signaling dysregulation especially in the context of chronic feeding (Miao et al. 2015). Early developmental stages may be especially sensitive because larvae and juveniles undergo rapid skeletal growth and endocrine programming (Conceição et al. 2010). At the same time, insufficient VD₃ availability may impair immune maturation, epithelial stability, skeletal integrity, and physiological resilience. Interpretation of this biological window is further complicated by species-specific physiology, temperature, salinity, photoperiod, lipid metabolism, calcium–phosphorus balance, microbiota composition, and co-supplemented feed additives (Wu et al. 2020). Collectively, these uncertainties position dose optimization as one of the central precision-nutrition challenges for practical VD₃ application in aquaculture.

Future potential: VD₃ analogs, nano-formulations, and environmental/epigenetic modulation

Recent advances in nutritional immunology, molecular biology, and nanotechnology have extended new interest in VD₃ based interventions for aquaculture. In addition to traditional dietary supplementation, innovative strategies such as synthetic VD₃ analogs or even nano-formulations, environmental-responsive systems, and epigenetic modulation may enhance bioavailability, tissue specificity, immunoregulatory precision (Christensen et al. 2021). Particular attention has been placed on VD₃ analogs, such as calcitriol derivatives, alfacalcidol, paricalcitol, and modified metabolites which maintain some immunoregulatory activity without the associated hypercalcemic side effects (Leyssens et al. 2014; Patel et al. 2025). Comparative studies of different vertebrate groups suggest that some analogs may facilitate antimicrobial peptide induction, regulation of inflammation, epithelial protection, and oxidative stability in addition to relatively attenuating excessive calcium mobilization (White 2022; Teymoori-Rad et al. 2019). However, comparative studies with fish are still few and far between, and their translational value is often limited to specific species. Concurrently, further advances in nanotechnology and microencapsulation will bring new opportunities to increase VD₃ stability during feed processing and mitigate oxidative degradation, thus permitting a greater degree of controlled intestinal delivery. These technologies might be particularly advantageous in species or developmental stages where absorption efficiency is variable. At the same time, environmental modulation of VD₃ signaling is gaining attention. Temperature fluctuation, salinity, hypoxia, photoperiod, pollutant exposure, and pathogen pressure may alter VDR-associated transcription, chromatin accessibility, DNA methylation, and histone regulation (Kollitz et al. 2014; Sigueiro et al. 2022; Hussain et al. 2025), suggesting that VD₃ may function within broader adaptive immunometabolic networks (Kari 2025). Emerging precision-aquaculture tools, including sensor-driven nutritional monitoring and machine-learning-assisted feed modeling, may further refine VD₃ application by predicting species-specific dosage windows under changing environmental conditions (Senthilkumar et al. 2025). Together, these technologies shift VD₃ supplementation from static nutritional inclusion toward adaptive, data-driven immunonutrition strategies with greater biological precision.

Development of novel VD₃ delivery systems

Optimization of VD₃ delivery systems is an increasingly important goal for enhancing nutritional efficacy and practical use options in aquaculture (Cheng 2024). Conventional feed supplementation continues as the primary commercial approach due to its simplicity, scalability, and perpendicular integration into daily routine production systems. Yet, oxidative degradation during feed processing, leaching of nutrients in water, heterogeneous intestinal absorption, and inconsistent uptake when at physiological stress or a diseased state are likely to limit biological efficiency (Zhang et al. 2026). Alternative delivery methods such as immersion and injectables have gained experimental interest, especially during larval and juvenile stages when feed intake can be quite variable (Rivas-Aravena et al. 2013; Lusiastuti et al. 2025). Immersion strategies could be beneficial for early-life supplementation and hatchery applications providing direct exposure to the environment; however, injectable approaches may provide more accurate dosing precision during vaccination or therapeutic intervention. However, they are also limited by the fact that both systems are labor-intensive, can be prone to stress discharge, do not scale easily, and are economically straining under commercial conditions. Importantly, delivery efficiency is unlikely to be uniform across aquaculture species. Bioavailability may vary according to digestive physiology, lipid metabolism, salinity adaptation, microbiota composition, developmental stage, and feed matrix interactions (Annappa et al. 2025). These biological differences mean that effective delivery is not solely a technological issue but also a species-dependent physiological challenge. Accordingly, the future of VD₃ delivery in aquaculture will likely depend on matching delivery routes with species biology, production stage, and management objectives rather than relying on a single universal supplementation system.

Bridging mechanistic insights with field applications

Despite increasing understanding of vitamin D₃ (VD₃)-associated immune regulation at molecular and cellular levels, translating these mechanistic insights into reliable field-scale aquaculture applications remains one of the major unresolved challenges in fish immunonutrition. Current evidence indicates that VD₃ can influence antimicrobial peptides, interferon-associated pathways, epithelial integrity, microbiota interactions, and inflammatory regulation; however, the strength of evidence supporting these effects remains highly uneven. The major limitation across the literature is the imbalance between mechanistic and functional validation. The available evidence follows a clear hierarchy, ranging from transcriptomic responses and biochemical markers to histopathological outcomes, survival improvement, and production performance as the strongest forms of validation. Currently, most studies are still mainly focused in lower tiers of this hierarchy, while only a minority extend to pathogen burden, survival analysis, or field-level production assessment (Natnan et al. 2021a, b). Such an imbalance makes interpretation difficult, since molecular activation does not lead directly to either biological or economic outcome. Commercial aquaculture conditions also add complexity via fluctuating water quality, stocked density, thermal heterogeneity, mixed-pathogen exposure, dietary diversity, and handling stressors all of which may differentially shape VD₃ responsiveness compared to laboratory-controlled studies. As a result, experimentally distinct supplementation effects often do not translate to predict farm-level performance.

Bridging this translational gap will require integrated frameworks linking mechanistic omics with long-duration field trials, standardized challenge systems, and production-scale traits including growth, feed efficiency, mortality, skeletal integrity, and reproductive performance (Tayyab et al. 2025). Emerging precision-aquaculture tools such as environmental biosensors, metabolomics, and machine-learning-assisted nutritional modeling may further refine species-specific supplementation strategies (Xiao et al. 2025; Wang et al. 2026). The precision aquaculture systems use sensor-based platforms to monitor multiple environmental parameters such as temperature, dissolved oxygen concentration, feeding behavior, and stress indicators in real time and dynamically modifies nutritional interventions under different farming conditions. In parallel, machine learning–assisted nutritional modeling can analyze large multidimensional datasets integrating growth, immune responses, environmental parameters, and dietary composition to predict species-specific VD₃ requirements and optimize supplementation windows with greater precision. Together, these technologies may help transform VD₃ supplementation from fixed dietary inclusion toward adaptive, evidence-driven nutritional management. Ultimately, the principal bottleneck is no longer mechanistic plausibility, but reproducible field-level consistency.

Policy, regulation, and sustainable development goals

Regulatory oversight and sustainability considerations are becoming increasingly central to the implementation of functional-feed strategies in aquaculture (Okon et al. 2025). Although vitamin D₃ (VD₃) is widely recognized as an essential nutritional component of aquafeeds, its practical application is governed by varying regulatory approaches based on the permitted inclusion level, approval process as an additive, safety standards for feedstuffs, and labeling requirements among countries. Consequently, the use of VD₃-based nutritional strategies must be evaluated not only for biological efficacy but also for regulatory compliance, food safety, environmental compatibility, and animal welfare (Allegra et al. 2024).

In many jurisdictions, VD₃ supplementation falls within broader feed-additive regulations monitored by authorities such as EFSA, the U.S. FDA, and national feed-control agencies, which evaluate safety thresholds, toxicological risks, environmental discharge, and product consistency (Vlaicu et al. 2023; Okon et al. 2025). Excessive supplementation may raise concerns regarding hypervitaminosis, tissue mineral imbalance, food-safety implications, and environmental accumulation through aquaculture effluents. Likewise, promising technologies such as nano-formulations, synthetic VD₃ analogs, and innovative encapsulation systems may be subjected to greater regulatory scrutiny due to uncertainties with the potential for nanoparticle persistence, bioaccumulation, and ecological interaction (Cheng 2024). From a sustainability perspective, VD₃-associated nutritional strategies align indirectly with several United Nations Sustainable Development Goals (SDGs), particularly SDG 2 (Zero Hunger), SDG 3 (Good Health and Well-Being), SDG 12 (Responsible Consumption and Production), and SDG 14 (Life Below Water) (Yarkina and Logunova 2022; Atanassova and Ktari 2024; Haque and Mahmud 2025; Siwach et al. 2025). By improving disease resilience and potentially reducing antimicrobial dependence, VD₃-based functional feeds may contribute to more sustainable aquaculture systems (Kari 2025; Mahmud et al. 2026). Next-generation precision aquaculture systems may also strentgthen this sustainability framework by utilizing integrated sensor technologies to monitor water quality, feeding behavior, growth performance, and physiological stress in real-time (Xiao et al. 2025). Concurrently, nutritional modelling assisted by machine learning can assimilate growth, immune, dietary, and environmental datasets to estimate species-specific VD₃ requirements and accurately model supplementation under variable farming conditions (Wang et al. 2026). Collectively, these technologies will enhance the accuracy and appropriateness of supplementation while minimizing unnecessary overuse, at the same time helping to maintain regulatory compliance through adaptive evidence-based feeding strategies. However, broader implementation remains constrained by the absence of harmonized supplementation standards, field-scale safety benchmarks, and environmental-risk frameworks, making policy alignment a critical prerequisite for safe and sustainable VD₃ adoption in global aquaculture.

Conclusion

VD₃ has progressed beyond its classical role in calcium–phosphorus metabolism to emerge as a biologically relevant immunonutrient with increasing significance in sustainable aquaculture. VD₃ serves as a regulatory candidate in the majority of experimental fish systems with respect to enhancing antimicrobial peptide production, modulating inflammatory cytokine networks, reinforcing epithelial and mucosal barrier integrity, maintaining oxidative balance, associated microbial regulation (e.g., basic characteristic of gut microbiota), and interferon-mediated antiviral signaling. Together, these interconnected processes position VD₃ as a multifunctional regulator of host defense rather than a single-target nutritional factor. Current evidence supports a plausible role for VD₃ in improving disease resilience under bacterial and viral challenge while contributing to broader antibiotic-reduction and prevention-oriented health-management strategies. Its integration with functional-feed additives, including probiotics, β-glucans, phytogenics, essential oils, and trace minerals, further strengthens its relevance within multi-component nutritional systems designed to improve immune competence, stress tolerance, epithelial stability, and production robustness. However, the strength of available evidence remains uneven and follows a clear hierarchy, with most studies concentrated at transcriptomic and biochemical levels, while comparatively fewer provide stronger functional validation through pathogen-load reduction, histopathological protection, survival improvement, or production-level outcomes. This distinction remains critical because a molecular response does not necessarily translate into a biological response that can be measured, and some studies report neutral or inconsistent outcomes depending on species, dose, and environmental context. Novel innovations in relation to VD₃ analogs as well as nano-formulations, encapsulation technologies, and precision-delivery systems are emerging which may render supplementation more efficacious and physiologically targeting. Collectively, the current evidence proposed uses of VD₃ not as a universal intervention, but a promising component of immunonutritional strategies, the utility of which will ultimately be determined by species-specific optimization and reproducible field-scale performance.

Author contribution

Syeda Maira Hamid: Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization. Hussain Syed Babar: Writing – review & editing, Methodology, Conceptualization. Zheng Xiaoting: Review, editing, Formal analysis, Zhulan Nie: Supervision, Project administration, Conceptualization, Funding acquisition, Review, Editing.

Funding

The authors acknowledge financial support from the following research grants: Key Technology Research and Demonstration Project for Large-Scale Seedling Cultivation of Saline-Alkali Tolerant Fishes (Yili Perch, E’erhe Silver Crucian Carp, Sea Bass, Golden Pompano), funded by the Science and Technology Bureau of Xinjiang Production and Construction Corps (Grant No. 2025YD016); National Natural Science Foundation of China (NSFC) Project “Resolving Species Validity and Cryptic Diversity of Schizothoracine Fish in Xinjiang Using Morphology and DNA Barcoding” (Grant No. 32460920); National Natural Science Foundation of China (NSFC) Project “Study on Alkalinity Reduction Pathways and Effect Mechanisms of Fish Aquaculture in Saline-Alkaline Water of Xinjiang” (Grant No. 32541123).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors. Therefore, ethical approval is not applicable.

Consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work, the author(s) used online tools to improve sentence structure and avoid grammatical mistakes. After using these tools/services, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the published article.