Fishmeal substitutions and their implications for aquatic animal immune and gut function: A review

Highlights

• •Fishmeal is used extensively in fish feed, but sustainability concerns limits its use.
• •Plant and animal-based protein alternatives are researched for its suitability as substitute for fishmeal.
• •The alternative protein lacks suitable nutrient profile, besides containing several toxic elements affecting digestibility.
• •The effect of such substitution on aquatic animal immune and gut function needs careful research.

Abstract

Fishmeal has long been a major ingredient in the aqua feed industry. However, as the aquaculture sector continues to grow rapidly, its heavy reliance on fishmeal is constrained by resource limitations and sustainability goals. Consequently, research has increasingly focused on alternative protein sources for feed preparation. This shift has gained momentum and success through the identification of suitable alternatives that do not compromise the growth of fish and shellfish. While alternative proteins can replace fishmeal to varying degrees, their effects are species-specific and dose-dependent. Unfortunately, many alternatives lack proper micronutrient composition and may contain toxic elements that could harm animal welfare. Plant-based proteins may change the gut flora and induce modest intestinal inflammation, but they may also moderately boost immune responses. Antioxidant capability and disease resistance can be enhanced by microbial and animal-based protein sources. The limited research reported on aquatic animals remains fragmented and requires systematic evaluation to focus on the immune and gut impairments caused by such substitutions. This review examines the impact of fishmeal replacement with alternative protein sources on the immunological function and gut health of aquatic animals in aquaculture. It synthesizes research on plant-based, animal-based, and microbial protein substitutes, evaluating their effects on the host microbiome, immune capacity, disease resistance, and intestinal histo-morphology. Finally, it stresses the need for species-specific and ingredient-specific evaluations to optimize aqua feeds and identifies knowledge gaps in long-term effects and underlying mechanisms.

Introduction

The aquaculture sector has rapidly expanded in recent years, fuelled by a rising need for seafood and a decrease in wild fish populations [[1], [2], [3]]. Nevertheless, the ecological impact of aquaculture methods is being subjected to greater examination as a result of environmental apprehensions, specifically pertaining to the utilization of fishmeal in aquafeeds [4]. Fishmeal, typically obtained from marine fish, is commonly used as a main protein source in aquafeeds because of its high nutritional value and appeal to various aquatic species [5]. Nevertheless, the limited availability and increasing expense of fishmeal, together with the difficulties in maintaining sustainability, have prompted endeavours to discover substitute protein sources for aquafeeds [6]. The replacement of fishmeal with alternative protein sources in aquafeeds has attracted considerable attention as a method to improve the sustainability of aquaculture operations [7]. Researchers have investigated numerous kinds of proteins derived from plants, microorganisms, and insects as possible alternatives to fishmeal in aquafeeds [8]. These alternatives have the potential to provide advantages such as lower dependence on wild fish populations, less environmental impact, and greater cost-efficiency [9]. Nevertheless, the impact of replacing fishmeal on the immunological function and gut health of aquatic animals continues to be a topic of discussion and research [10]. Comprehending the effects of replacing fishmeal on the immunological and digestive function is essential for ensuring the well-being and care of farmed aquatic animals [11]. The immune system and gastrointestinal tract are crucial in safeguarding aquatic species against infections, maintaining physiological balance, and maximizing nutrition use [12]. Modifications in the sources of dietary protein can impact immunological responses, the makeup of gut microbiota, the structure of the intestines, and the integrity of the mucosal lining [13]. These changes have the potential to affect susceptibility to diseases, growth performance, and general health [14].

Although fish meal is one of the most popular and effective sources of protein for use in animal feed, it also has certain drawbacks, the most significant of which is its expensive cost, which leads farmers and other stakeholders to look for other solutions to the aforementioned problem [15]. Consequently, plant-based components, insect meals, bacterium meals, etc., may be effectively used as replacements for fish meals that are also potentially less expensive [16]. Nonetheless, every substance and source have some drawbacks that must be accepted and handled appropriately. For instance, when plant-based components are employed in fish diets, intestinal inflammatory illness is a significant concern [17]. Since the intestinal epithelium serves as the first line of defense against invasive pathogens, any disruption or damage to this barrier may, in addition to morphological changes in the intestine, result in extensive activation of the epithelial immune system, inflammatory responses, decreased barrier functions, and eventually increased pathogen translocation and increased disease susceptibility [18]. Furthermore, enteritis-inducing substances that are common in soybean meal might cause intestinal irritation. The intestine is especially susceptible to harmful substances because it is the first tissue to be exposed to ingested feed and has a variety of effects on the body [19]. As a result, any substance consumed that affects a vital intestinal function poses a risk to fish health [20].

Hence, this review attempts to thoroughly assess the present state of knowledge regarding the impact of replacing fishmeal on the immunological and gastrointestinal function of aquatic animals. This review aims to clarify the mechanisms by which dietary protein sources interact with host physiology in aquaculture systems by combining existing research and identifying areas where understanding is lacking. The knowledge acquired from this review will guide future research endeavours, facilitate the creation of environmentally friendly aquafeed compositions, and promote the progress of ecologically conscious aquaculture methods.

Effect on host-microbiome: plant-based meal substitution

The effects of increasing the percentage of fish meal replacement with ultra-micro ground mixed plant proteins (uPP) were studied by Xie et al. [21]. They discovered that inclusions of 5 % uPP up-regulated the intestinal expression of genes related to inflammation (genes such as NF-B p65, TNF-, IL-1, IL10, and TGF-) and decreased HIF-1 expression. In a study conducted by Hao et al [22], the partial replacement of fish meal by Saccharomyces cerevisiae culture was examined in channel catfish (Ictalurus punctatus). Two equal nitrogen and energy diets were prepared: a basal diet that contained 10 % fish meal (the Control), and an experimental diet that substituted 20 % of the basal diet's fish meal with yeast culture (the RFM). The diets were fed to channel catfish for a period of 12 weeks. While the expressions of NF-κB in the intestine and liver were dramatically reduced in the RFM group, the expression of the intestinal HIF1 gene was significantly enhanced. The study also revealed that as compared to HFM-0, the LFM-0 diet increased LPW (Lamina propria width) and SMW (submucosa width) in the anterior and mid intestine, but the impact was more pronounced in HFM diet.

Chlorella vulgaris (ChM), Clostridium autoethanogenum (CAP), and cottonseed protein concentrate (CSM) were completely substituted for fish meal in the diet of largemouth bass (Micropterus salmoides), with a significant reduction in villi height or width but no discernible changes in the villi of the algal protein [23]. Through modulating pancreatic secretion, protein digestion, and absorption, hepatopancreas transcriptome profiling revealed that excessive inclusion of CAP had a deleterious impact on protein synthesis and the use of nutrients. High-throughput sequencing of the intestinal microbiota in CSM demonstrated an increase in the number of harmful bacteria species (Proteobacteria, Acinetobacter). Contrarily, in CAP and ChM, there were more beneficial bacteria species (Firmicutes, Bacteroidetes and Cetobacterium) [24].

In their study, Willora et al. [25] examined the effects of plant protein concentrates, specifically soy protein concentrate (SPC) and pea protein concentrate (PPC) at 1:1 ratio, provided in the form of three test meals at varying levels of fish meal substitution, including 25 % (PP25), 50 % (PP50), and 75 % (PP75). Fish-fed test diets showed many differences from controls, including increased width of lamina propria (WLP) in the distal intestine (DI), more goblet cells (GCs), and shorter mucosal fold height (MFH) in the anterior intestine (AI). Compared to controls, fish given the PP50 diet showed increased leucine aminopeptidase (LAP) activity in the pyloric caeca and decreased chymotrypsin (CHY) activity in the middle intestine (MI).

Effect on host-microbiome: animal-based feed substitution

Fish fed a low fish meal (LFM) diet exhibited evidence of intestinal inflammation, according to an examination of the intestinal histo-morphometric measures. Commercial feed additives LUMANCE® (0.2 % & 0.5 %) and NOVIGEST® (0.4 %) showed some modulatory effects when added, most notably increased intestinal villi length and lamina propria width in the mid-intestine. In the LFM-0.6 and LFM-0.9 groups, higher intraepithelial cell numbers, increased mucus production, and decreased hepatic vacuolation were also seen, though not statistically significant [26]. Digestive enzymes, which are regarded as indications of digestive capacity, play a major role in fish's capability to absorb nutrients [27]. Additionally, because they are directly related to feed digestibility, digestive enzyme activity is also very important in this assessment [28]. Trypsin is regarded as a crucial enzyme in protein digestion since, among the digestive proteinases, it causes the activation of other enzymes that were first produced as inactive zymogens, such as chymotrypsin or carbo-peptidases [29].

Effect on host-microbiome: microbial meal-based substitution

Yu et al. [30] conducted a study to assess the effects of replacing fish meal (FM) with methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®, FK), in which six isonitrogenous diets were formulated with FK to replace FM (control group, FM content: 35 %). The analysis of gut flora revealed that the relative abundance of Lactobacillus in the treatments with FK concentrations of 7 % and 10.5 % groups Bacteroidetes, Firmicutes, and Proteobacteria made up about 70 % of the entire gut microbiota at the phylum level. When compared to the FM group, the groups given FK meals at 7 % and 10.5 % showed higher relative abundances of Lactobacillus.

Additionally, the effects of an FM-free diet (NoPAP SANA) were evaluated based on plant ingredients, aquaculture by-products, algae oil, insect meal, and bacterial fermentation biomasses as the main dietary oil and protein sources. Results showed no significant differences in diversity or at the phylum level of the intestinal bacterial composition [31]. When fish were fed a diet that substituted yeast culture for 20 % of the fish meal in the base diet, the relative abundance of Firmicutes and Turicibacter both tended to rise [22].

No differences between the HFM-0 diet and the LFM-0 diets were found in the anterior and mid intestines of juvenile gilthead sea bream (Sparus aurata) in the study by Mallioris et al. [26] that examined the modulatory effect of two commercial feed additives, Lumance® (0.2 % and 0.5 %) and Novigest® (0.4 %) when added to high (HFM-0) and low fish meal (LFM-0) diets. The GC index in the anterior intestine did, however, slightly rise when 0.5 % Lumance® was added to the HFM diet. Similar to this, a modest rise in the index in the mid intestine was seen after the two additions were added to the LFM-0 diet. Trypsin and lipase activity in fish-fed diets with Methanotroph Bacteria Meal (MBM) tend to be reduced, but pepsin and chymotrypsin activity showed a significantly increasing linear pattern with the addition of dietary MBM, according to an analysis of the effects of replacing dietary fish meal (FM) with MBM on the digestive enzyme activity [28]. Methanotroph (Methylococcus capsulatus, Bath) bacterium meal (FeedKind®, FK) was used in place of fish meal (FM) in a study by Yu et al. [30], the treatment with FK inclusion of 3.5 % demonstrated considerably greater trypsin and lipase activities, whereas the other groups had values that were comparable to the FM group.

Effect of fishmeal substitution on immune capacity and disease resistance: plant-based meal substitution

According to the study conducted by Lee et al. [32], where black rockfish (Sebastes schlegelii) juveniles were given various vegetable and fruit juice by-product feed additives, such as garlic juice processing by-product (GLJB), yacon juice processing by-product (YCJB), and blueberry juice processing by-product (BBJB), to assess their lysozyme activity. It was discovered that these diets significantly increased lysozyme activity compared to controls. Additionally, compared to fish fed the control diet, all diets enriched with by-products of plant juice processing increased fish survival rates following the V. harveyi challenge. Chlorella vulgaris (ChM), Clostridium autoethanogenum (CAP), and cottonseed protein concentrate (CSM), as prospective protein sources to replace fish meal, were tested in a study by Li et al. [23] to see how well they worked in largemouth bass (Micropterus salmoides). According to the findings, intestinal malondialdehyde levels were substantially higher in CSM than in ChM and CSM had significantly lower superoxide dismutase activity than in CAP. Additionally, CSM dramatically reduced the expression of the anti-inflammatory cytokines il-10 and tgf-β and increased the expression of the pro-inflammatory cytokines il-8, il-1β, and tnf-α. Comparatively, CAP and ChM reduced intestinal inflammation and improved the integrity of the intestinal barrier as evidenced by the elevated expression of marker genes. Compared to the control group, replacement of FM with RPC up to 25 % resulted in greater mRNA levels of the intestinal cytokines IL-1β, IL-10β, TGF-β, and TNF-α, indicating an unspecific immune system stimulation. The RPC75 group had the largest significant cumulative mortality among fish infected with Aeromonas veronii, followed by the RPC50, RPC25, and control groups. As a result, it was determined that RPC incorporation levels of up to 25 % are advised for O. niloticus to have improved antioxidant capacity, immunocompetence, and disease resistance [33] (Table 1).

Table 1.

Different alternatives of fish meal reported studies.

Alternatives	Effects	References
Methanotroph Bacteria Meal (MBM)	✓Reduced trypsin and lipase activity ✓Increased pepsin and chymotrypsin activity ✓diverse effects on several antioxidant systems ✓reduced CAT activity. ✓SOD activity and GSH content significantly decreased when the replacement level reached 80 % MBM	[28]
Methanotroph (Methylococcus capsulatus, Bath) bacterium meal (FeedKind®, FK)	✓higher trypsin and lipase activities ✓greater proportions of Lactobacillus ✓activities of ACP and AKP significantly decreased ✓replacing FM with FK at a percentage of 10–20 % increases lysozyme and C4 activity	[30]
Ultra-micro ground mixed plant proteins (uPP)	✓lowered HIF-1 expression	[21]
Saccharomyces cerevisiae culture	✓expression of intestinal HIF1 gene greatly increased ✓NF-κB drastically decreased in gut and liver. ✓survival rate of channel catfish significantly increased in the RFM group. ✓increased disease resistance to both Aeromonas veronii Hm091 and Aeromonas hydrophila NJ-1.	[22]
LUMANCE® (0.2 % & 0.5 %) and NOVIGEST® (0.4 %), commercial feed additives	✓increase of intestinal villi length and lamina propria width in the mid-intestine	[26]
Chlorella vulgaris (ChM), Clostridium autoethanogenum (CAP), and cottonseed protein concentrate (CSM)	✓increase in the species of beneficial bacterial species in CAP and ChM. ✓intestine malondialdehyde levels were significantly greater in CSM ✓superoxide dismutase activity significantly reduced in CSM ✓expression of the pro-inflammatory cytokines il-8, il-1β, and tnf- α elevated by CSM ✓CAP and ChM decreased intestinal inflammation and improved the integrity of the intestinal barrier	[23]
Clostridium aethanogenum protein (CAP)	✓higher serum aspartate aminotransferase and phenol oxidase activities ✓CAP-45 and CAP-70 groups had increased lysozyme activity.	[24]
Soy protein concentrate (SPC) and pea protein concentrate (PPC)	✓increased lamina propria width in the distal intestine ✓increase in goblet cells ✓decrease in mucosal fold height in the anterior intestine	[25]
Rice protein concentrate (RPC)	✓Greater mRNA levels of the intestinal cytokines IL-1β, IL-10β, TGF-β, and TNF-α observed following FM substitution with RPC up to 25 % compared to the control group	[33]
Defatted black soldier larvae meal (PD-BSLM)	✓increase in lysozymes in serum, IgM, nitric oxide, and expression of anti-inflammatory cytokines IL-10 and TGF. ✓100 % inclusion level of PD-BSLM, pro-inflammatory cytokine genes (IL-1 and TNF-) were down-regulated	[35]
Tilapia protein hydrolysate	✓Increase disease resistance of silver pompano fingerlings against V. anguillarum ✓Increased lysozyme activity.	[34]

Effect of fishmeal substitution on immune capacity and disease resistance with animal based meal substitution

Additionally, Tejpal et al. [34] observed that adding tilapia protein hydrolysate (TPH) to the food increased the disease resistance of silver pompano fingerlings against Vibrio anguillarum and increased lysozyme activity compared to the control group. In a 12-week study, Kishawi et al. [35] examined the effects of replacing all or a portion of the protein in fish meals with defatted black soldier larvae meal (PD-BSLM). Their findings suggested that groups given higher levels of PD-BSLM had a rise in non-specific immunological markers like serum lysozymes, IgM, nitric oxide, and the expression of the anti-inflammatory cytokines IL-10 and TGF-. Pro-inflammatory cytokine genes (IL-1 and TNF-) were down-regulated at a 100 % inclusion level of PD-BSLM, but dietary interventions had no effect on serum C-reactive protein.

Effect of fishmeal substitution on immune capacity and disease resistance: microbial-based meal substitution

Jiang et al. [24] investigated the dietary effects of replacing fish meal with Clostridium autoethanogenum protein (CAP), and it was discovered that all fish meal-substituted groups had increased serum aspartate aminotransferase and phenol oxidase activities, and the CAP-45 and CAP-70 groups had increased lysozyme activity. Serum antioxidant markers, including T-AOC, SOD, CAT, GSH, and MDA, can be used to gauge an individual's antioxidant capability. It appeared that dietary MBM had varying impacts on different antioxidant systems in a study where dietary fish meal (FM) was replaced with MBM (Methanotroph Bacteria Meal) at different amounts. CAT activity was considerably decreased in all the replacement groups. When the replacement level reached 80 % MBM, By replacing fish meal (FM) with methanotroph (Methylococcus capsulatus) bacterium meal (FeedKind®, FK), it was found that the activities of ACP and AKP significantly decreased with increasing amounts of FK in the diet [30]. The fact that the activity of both phosphatases has decreased in comparison to the control suggests that FK does not cause an inflammatory or stress response in fish-fed FK. Both phosphatases are involved in anti-inflammatory and immunological responses; moreover, lysozyme and C4 activities are boosted when 10–20 % FM is substituted with FK, indicating that FK may have an immunostimulatory impact at lower inclusion rates. However, the SOD activity and GSH content considerably decreased [28]. After being challenged with Aeromonas veronii Hm091 and Aeromonas hydrophila NJ-1, the survival rate of channel catfish dramatically increased in the RFM group, according to Hao et al. [22]. Disease resistance to Aeromonas veronii Hm091 and Aeromonas hydrophila NJ-1 was greatly boosted by the intestinal microbiota produced by the RFM diet.

Response of fish meal replacement in crustaceans' immune & digestibility system

Crustaceans, like invertebrates, rely primarily on their inherent immune response to defend and protect themselves against diseases [36]. The innate immune system consists of cellular and humoral immunological responses [37]. The cellular immune response occurs mainly in haemocytes, where a variety of pattern recognition receptors (PRR) on cell membranes identify and remove pathogens through phagocytosis, apoptosis, nodule formation, and encapsulation [38]. In contrast, the humoral immune response is mostly dependent on immunological components contained in the haemolymph, such as prophenoloxidase (proPO), lectins, antimicrobial peptides (AMP), and so on [36]. It has been said that feed elements affect the shrimp's immune system, which the protein level can influence in the diet [39]. For example, using soybean meal as a protein source lowers non-specific immune responses however fermenting the soybean meal with Lactobacillus spp. can mitigate the detrimental effects [40]. In a research found that adding 100 g kg⁻¹ of lupin kernel meal to the formulated diet improved the immunological status of shrimp by increasing phenol oxidase activity [39]. Another study demonstrated that a 25 % substitution level of Arthrospira (Spirulina platensis) improved immunological markers, including an increase in granular haemocyte percentage [41].

The dietary composition of protein resources significantly impacts immunological markers, antioxidative reactions, and intestinal health, influencing overall health performance and resistance to infections in crustaceans [42]. In addition, a tight relationship between the immunological and metabolic systems has been discovered. When the immune system is active, energy and metabolic substrate demand rises dramatically. The immune system obtains these metabolic substrates mostly from foods, which supply energy and serve as precursors for the creation of new cells, effectors (e.g., antibodies, cytokines, and acute phase proteins), and protective molecules (e.g., glutathione) [43]. Crustaceans rely heavily on amino acids and/or their metabolites to regulate their immune systems. For example, a study revealed that adding Arg to the diet at 2.7–3.7 % increased immunity and disease resistance in juvenile Chinese mitten crab (Eriocheir sinensis) [44]. In a similar study, Zhao et al. (2012) investigated the effects of histamine on the survival and immunological parameters of the Chinese Mitten Crab. They found that histamine increased PO and SOD activity while decreasing THC, ACP, and AKP [45].

Digestive enzyme activity indicates the most fundamental physiological aspects of an animal's digestion and ability to absorb feed nutrients [46]. Wang et al. (2022) examined the effects of cottonseed protein concentrate (CPC) in place of fishmeal on the growth performance, immune response, digestive ability, and intestinal microbiota of Litopenaeus vannamei, concluding that CPC increased the activities of digestive enzymes in the intestine [47]. Lin and Chen, (2022) investigated the effects of fermented soybean meal (FSBM) substituted for fish meal and concluded that a suitable replacement amount of 75 % is recommended to improve nutritional digestibility for white shrimp [48]. Lin et al. (2022) found that replacing 10 % of the fishmeal with rice protein meal considerably enhances digestibility, protein synthesis, antioxidant capacity, and disease resistance [49].

In evaluating the effects of plant-based meal substitution in crustacean shrimp diets, Fig. 1 illustrates the significant impact on the host-microbiome interaction. The shift from traditional fish meal to plant-based alternatives results in notable changes in gut microbiota composition, which in turn influences both immune response and digestibility. As observed in Fig. 1, these microbial shifts may either enhance or hinder the shrimp's ability to efficiently digest plant-based proteins, highlighting the importance of optimizing such dietary changes to maintain immune health and digestive function.

Fig. 1.

Effect on host-microbiome with plant-based meal substitution in crustacean shrimp.

Response of fish meal replacement in omnivore's immune & digestibility systems

Hossain et al. (2019) conducted a study to assess how yellow catfish's growth, digestive system, and immune systems were affected when soybean peptide was used instead of fish meal particles. Results of creating diets by substituting 0 %, 20 %, 35 %, and 50 % of fish meal revealed that the dietary 50 % group also saw a substantial rise in final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR). Serum globulin concentration (GLB), alkaline phosphatase activity (ALP), and total nitric oxide synthase activity (tNOS) were all greater in the 50 % group than in the control group [50].

In investigating the potential of replacing FM with black soldier fly (Hermetia illucens) meal (HIM) in aquafeeds, using zebrafish, Esmaeili et al. (2017) found that their initial body weights increased, no negative effect on the intestine histology or the main productive performances of zebrafish, such as mortality, feed intake, body weight gain, and feed conversion rate. Instead, some enzyme activities were reduced when the FM replacement rate was increased [51]. Magalhães et al. (2017) conducted to investigate the effects of replacing fish meal with cottonseed protein concentrate (CPC) (free gossypol < 7.9 mg/kg) in the diets of juvenile golden pompano (Trachinotus ovatus). Which contained 340 g/kg fishmeal and five CPC diets each with differing CPC concentrations and found that weight gain rate (WGR) and specific growth rate (SGR) showed no significant difference among groups [52].

Amer et al. (2020) demonstrated that increasing the substitution percentage of fish meal by methylated soy protein isolates (MSPI) reduced the growth performance of Nile tilapia (Oreochromis niloticus) fingerlings. It has also been established that MSPI is an effective immune-modulating agent that may enhance the immunological responses of fish challenged with A. hydrophila as well as the general health of the gut [53]. In Fig. 2, the effects of fishmeal substitution with animal-based meals on the immune capacity and disease resistance in omnivorous fish are demonstrated. The figure highlights how these dietary changes influence the fish's immune response, potentially enhancing disease resistance while maintaining overall health.

Fig. 2.

Effect of fishmeal substitution on immune capacity and disease resistance with animal-based meal substitution in the omnivore fish.

Tacon (1995) was carried out to evaluate the effect of processed canola meal (PCM) as a fishmeal (FM) substitute in the juvenile Nile tilapia diet. No significant changes were observed for ADC of protein amongst various experimental groups. Digestibility of lipids was not affected by the replacement of FM by PCM up to 25 %. However, it was dramatically decreased as dietary PCM level increased in a way that the lowest value belonged to those fish that received the highest dietary PCM. Various digestive enzyme activities of juvenile Nile tilapia, including lipase, alkaline protease, and amylase, were unaffected by dietary PCM content. Results also revealed no significant differences amongst various experimental diets regarding mucosal lysozyme, alkaline protease and alkaline phosphatase activity and total immunoglobulin content (p > 0.05). Although values of alkaline phosphatase activity, alkaline protease activity and total immunoglobulin content decreased by increasing dietary PCM content, values of lysozyme activity increased [54].

Response of fish meal replacement in carnivore's immune & digestibility systems

An experiment by Shen et al. (2020)[65] using garlic (Allium sativum) powder in place of fishmeal to replace it with meat and bone meal in juvenile rainbow trout showed that an increased dietary content of meat and bone meal hampered the fish's ability to grow and produce as well as their body composition, tissue fatty acid profile, digestive enzyme activity, and overall digestibility [55]. On the other hand, adding garlic powder managed to fix everything. Dossou et al. (2018) evaluated the effects on red sea bream, Pagrus major, of substituting soy protein concentrate (SPC) for fishmeal (FM) and supplementing it with inosine monophosphate (IMP) [56]. The findings showed that the fish with the highest final weight, weight gain, specific growth rate, and feed consumption were those fed a 50 % and 75 % replacement diet. The 50 % substituted diet group had considerably greater levels of ADCDM and ADC Protein. Dietary interventions considerably impacted PA and NBT, two of the several evaluated nonspecific immunological markers. When compared to the control, all FM replacements with IMP-supplemented diet groups showed improved innate immune responses.

In another replacement of fish meal with yeast fermented rapeseed meal (FRM) on juvenile, Pagrus major by Amoah et al. (2022) indicated no differences in final body weight, weight gain, specific growth rate and feed intake among all groups when compared to fish fed the control diet. Feed conversion ratio, protein efficiency ratio and survival were not affected by the test diets and Other blood parameters and innate immune responses parameters were not altered by diet [57]. Fronte et al. (2021) investigate the effects of replacing fishmeal (FM) with castormeal (CM) in juvenile hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂). The results showed that the final weight, weight gain rate, and specific growth rate were high. The serum total protein content first increased compared to the control group, a significant increase (P < 0.05) in the activities of serum and liver immunoglobulin-M, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and complement-3 (except serum activity for CM12 group); liver lysozyme; intestinal amylase, and lipase, was witnessed [58].

Amer et al. (2020) conducted a feeding trial to assess the effect of partially replacing fish meal (FM) by Black soldier fly prepupae meal (HM) in diets for European seabass (Dicentrarchus labrax) juveniles and results demonstrated that growth performance, feed intake, and feed efficiency were not affected by diet composition, The apparent digestibility coefficients (ADC) of dryandorganic matter, protein, lipids and energy were high and unaffected by diet composition. The ADC of arginine, histidine, and valine were higher in HM diets when compared to the control. Amylase and protease activities were not affected by dietary HM, while lipase activity was lower in HM 6.5 diets than in the control and HM 19.5 diets [53].

Dawood et al. (2015) conducted supplementation of fish meal diets with heat-killed Lactobacillus plantarum (HKLP) with graded levels of soybean meal (SBM) in Seriola dumerili, formulation contain 0 %, 15 %, 30 %, and 45 % SBM, and each SBM level was supplemented with HK-LP at 0.0 and 0.1 %. Results demonstrated that Serum lysozyme activity was significantly $(P<0.05)$ increased in the SBM15(0.1) group when compared with other groups. Fish fed SBM30 diet supplemented with 1 g kg−1 HK-LP showed significantly higher serum bactericidal activity than the other groups and serum peroxidase activity recorded the highest significant values $(P<0.05)$ in the SBM15 and SBM30(0.1) groups. The apparent digestibility coefficient (ADC) of protein was significantly $(P<0.05)$ higher in fish fed SBM0, SBM15, SBM15 (0.1), SBM30, and SBM30 (0.1) groups than SBM45 and SBM45 (0.1) groups. ADC of lipid was found to be significantly different $(P<0.05)$ with being higher in the SBM15 and SBM30 (0.1) groups than the other experimental groups [59].

In Fig. 3, the effects of fishmeal substitution with microbial-based meals on the immune capacity and disease resistance of carnivorous fish are detailed. The figure demonstrates that microbial-based meals can significantly alter immune function, potentially enhancing the fish's ability to resist diseases. It also highlights that while these substitutions can maintain or even improve immune performance, the specific response depends on the type and composition of the microbial meal used. This suggests that microbial-based diets offer a promising alternative for sustaining health and resilience in carnivorous species when fishmeal is replaced.

Fig. 3.

Effect of fishmeal substitution on immune capacity and disease resistance with microbial-based meal substitution in the carnivore fish.

Response of fish meal replacement in herbivore's immune & digestibility systems

Herbivorous fish are less dependent on fish meal than carnivorous fish. As a result, herbivorous fish offspring require less fish meal than carnivorous fish [60]. Herbivorous fish can transform plant protein into animal protein with high nutritional value, which is critical to the long-term development of the human food supply [61]. Wu et al. (2015) investigated the effects of fish meal replacement by stickwater meal on the growth and health of grass carp (Ctenopharyngodon idellus) and determined that there are no negative effects on grass carp health when stickwater meal is used to replace all fish meal in a 6 % fish meal diet, and when 2 % stickwater meal is used to replace an equal amount of fish meal, grass carp have higher feed utilization and a faster growth rate, and the effect is superior to fish meal [62].

In Fig. 4, the effects of fishmeal substitution with plant-based meals on immune capacity and disease resistance in herbivorous fish are illustrated. The figure shows how replacing fishmeal with plant-based alternatives can influence the immune system, potentially reducing disease susceptibility. However, the response varies depending on the type of plant-based meal used, emphasizing the importance of selecting appropriate plant sources to maintain optimal immune function and overall health in herbivorous fish.

Fig. 4.

Effect of fishmeal substitution on immune capacity and disease resistance with plant-based meal substitution in the herbivore fish.

Liu et al. (2019) evaluated the effect of replacing dietary fishmeal with cottonseed meal (CSM) on the growth and health of juvenile grass carp. The ideal dietary CSM level was 175.9 g/kg, with a maximum addition of 351.8 g/kg. High levels of dietary CSM may have an impact on nutritional digestion, metabolism, and humoral and cellular immunity [63]. In a study conducted by Mohammadi et al. (2020), various growth metrics, nutrient digestibility, physiological indices, intestine and liver histopathology, as well as gut micromorphology, clearly revealed that the inclusion of processed canola meal (PCM) in the juvenile Nile tilapia diet was limited, with a safe level of dietary PCM of 25 % [64]. On the contrary, Monzer et al. (2017) indicated in their study on the substitution of fish meal by soybean meal in Siganus rivulatus that total replacement of FM by SBM without additional supplements is not advised [65].

Antioxidant capacity effects

Novel protein diets demonstrated superior growth performance in the latter experimental phase despite varying effects on antioxidant capacity. The C. vulgaris diet enhanced antioxidant activity, while the cottonseed protein concentrate diet diminished it. These findings emerged from recent aquaculture research aimed at reducing fishmeal dependency by evaluating alternative protein sources such as Tenebrio molitor, cottonseed protein concentrate, Clostridium autoethanogenum and Chlorella vulgaris [66]. A parallel study assessed fermented rapeseed meal (RM-Koji) in red sea bream diets for the first time. Moderate inclusion levels (25 % and 50 %) of RM-Koji yielded multifaceted benefits: supporting growth, improving nutrient utilization, stimulating immune responses, and bolstering antioxidant defenses. Substituting up to 50 % of fishmeal with RM maintained growth rates and feed efficiency while significantly enhancing immunological parameters and antioxidative status. These results underscore the potential of novel protein sources in sustainable aquaculture development [56].

Alternative protein sources in aquaculture diets have demonstrated varied impacts on fish antioxidant capacity across species. Hydrolyzed soybean meal (HSM) has successfully replaced up to 20 % of fishmeal in silver catfish diets, enhancing growth and antioxidant activity [67]. Likewise, substituting 3–6 % fishmeal with Spirulina platensis and 0.3 % of its polysaccharide extract improved largemouth bass antioxidant defense's antioxidant capacity [68]. In Nile tilapia (Oreochromis niloticus), 5 % and 7.5 % dried bovine hemoglobin (DBH) supplementation enhanced hepatic antioxidant parameters, upregulated related gene expression in the spleen, and reduced malondialdehyde levels [69]. Goldfish (Carassius auratus) exhibited significant improvements in serum total antioxidant capacity, peroxidase activity, and immunoglobulin M levels when 40 % of fishmeal was replaced with fermented soybean meal (FSM) [70]. Contrastingly, increased cottonseed protein concentrate (CPC) proportions in juvenile hybrid culter diets diminished total superoxide dismutase (T-SOD) and catalase (CAT) activities [71]. However, rainbow trout (Oncorhynchus mykiss) showed modest antioxidant and immune function improvements with 50 % insect-based protein substitution, evidenced by decreased malondialdehyde and acid phosphatase levels [72]. These findings underscore the necessity for species-specific and ingredient-specific evaluations to optimize aquafeed formulations, considering the variable effects of alternative protein sources on fish antioxidant systems.

Enhanced gut barrier function

The complex interplay between dietary components and immune function underscores the need for continued research, particularly in elucidating the direct impact of prebiotics on immune responses. These functional molecules have garnered attention for their potential to enhance epithelial barrier integrity, foster beneficial gut microbiota, and augment the production of intermediate metabolites, such as short-chain fatty acids (SCFAs), which play a crucial role in maintaining immune system homeostasis [73]. Probiotics, in turn, have been shown to modulate inflammatory responses through multifaceted interactions with the intestinal milieu. They engage with the intestinal epithelium, M-cells in Peyer's patches, and innate immune components, including dendritic cells and other antigen-presenting cells. Moreover, probiotics reinforce mucosal barrier function by stimulating mucus secretion and antibody production [74].

Recent studies on aquatic species have illuminated the nuanced impact of alternative protein sources on gut health. In Atlantic salmon, dietary Candida utilis induced aquaporin 8ab downregulation without compromising growth or intestinal health [75]. Similarly, partial fishmeal substitution with insect meal in zebrafish maintained gut morphology and functionality gene expression, suggesting its viability as a sustainable alternative [76].

Investigations into feed additives have yielded varied outcomes. Functional compound additives in soybean meal mitigated enteritis in Japanese seabass without altering gut microflora [77]. Conversely, high inclusion of cottonseed meal significantly reduced microbial diversity in juvenile golden pompano, potentially compromising intestinal barrier function [78].

These findings underscore the complexity of diet-gut-immune interactions across species and emphasize the need for targeted research to optimize dietary formulations that balance growth performance and gut health. As understanding deepens, innovative nutritional strategies in aquaculture and beyond will emerge.

Gut-related immune gene expression & histological effects

"For last, an important up-regulation of the immune-related gene studied on the skin was also detected. In addition, the expression of certain immune-related genes was evaluated in the skin [79]. Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1β gene expression 2 h after stress test, whereas StAR expression was significantly (p < 0.05) up-regulated". "Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1β gene expression 2 h after stress test, whereas StAR expression was significantly (p < 0.05) up-regulated [80].

Gut histological assessment only revealed minor alterations related to an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response. In these fish, gut histological assessment only revealed minor alterations related to an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response [81].

To determine the effect of plant protein feeding on the innate immune response to bacterial pathogens, an ex vivo procedure of intestine explant culture was implemented. Results showed Fish showed less tolerance to dietary plant protein in phase I than in phase II, while the ex vivo assays indicated that the intestine from fish fed at short-term plant diets showed a higher immune response than at long term feeding. Concerning the immune response to bacterial challenge, a significant expression in pro-inflammatory cytokines IL-1β and IL-6 after 6 h of exposure to V. algynoliticus, while COX-2 expression was significantly induced by P. damselae subsp. pisicida, showing a positive high correlation between them. Differential health status was observed depending on the growth stage, being stricter with the plant protein inclusion of the younger fish. Under ex vivo conditions, the bacterial challenge induced inflammatory and immune intestinal response, responding stronger to that intestine of fish fed during a short term with a total substitution of fish meal [82]. The replacement of FM by plant protein in the diets induced nitric oxide (NO) and lysozyme production, while immunoglobulins (Ig), monocytes percentage and gut interleukin 10 (IL10) gene expression were inhibited [83].

Alternative protein sources in aquaculture diets significantly influence the expression of immune-related genes in fish, with varying outcomes across species and ingredients. Cricket meal (Gryllus bimaculatus) substitution up to 75 % in fish diets up-regulated intestinal expression of IL-1β, IL-8, IL-10, and HIF1α genes, while 100 % substitution elevated TNF-α, IL-22, and IFN-γ levels [84]. Conversely, reduced fishmeal diets in juvenile blunt snout bream decreased the expression of IL-8, TNF-α, IL-10, and NF-κB [85].

Histological studies on Atlantic salmon parr revealed that fish meal (FM) and FM with 20 % Candida utilis maintained normal distal intestine (DI) morphology. However, soybean meal (SBM) - based diets induced mild SBM-induced enteritis, characterized by reduced simple fold height and fewer supranuclear vacuoles. Despite promoting growth, C. utilis supplementation failed to mitigate these histological changes [75].

In shrimp aquaculture, low-fishmeal diets supplemented with sulfate-based alginate polysaccharide (SAP) improved antioxidant capacity, immunity, and disease resistance in Litopenaeus vannamei, as confirmed by transcriptomic analysis [86]. Interestingly, in Nile tilapia (Oreochromis niloticus), 10 % inclusion of Aspergillus oryzae-fermented olive cake increased mRNA expression of gut-related inflammatory cytokines TNF-α and IL-1β [87].

These diverse findings underscore the complex interactions between alternative feed ingredients and the fish immune system, necessitating a comprehensive evaluation of each ingredient's impact on specific species under various conditions.

Conclusion

In conclusion, this review provides valuable insights into dietary protein-gut health-immunity interactions in aquaculture. It emphasizes the need for species-specific and ingredient-specific evaluations to optimize aquafeed formulations. Moving forward, research should focus on understanding the long-term consequences of alternative protein usage and developing strategies to mitigate potential adverse effects. By carefully selecting and combining alternative protein sources, it may be possible to formulate functional aquafeeds that support optimal growth and enhance health and welfare while promoting environmental sustainability. This holistic approach will be crucial for the future development of the aquaculture industry.

CRediT authorship contribution statement

Venerability Dhar: Writing – original draft, Methodology, Investigation. Soibam Khogen Singh: Writing – review & editing, Supervision, Resources, Conceptualization. Swapnil Ananda Narsale: Writing – original draft, Data curation. Sourabh Debbarma: Writing – original draft, Visualization, Validation. Pritisha Saikia: Writing – original draft, Investigation, Conceptualization. Yilbong Yirang: Validation, Software.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• No data was used for the research described in the article.