Post-biotics as emerging next-generation feed additives in poultry nutrition: A review of gut health, immunity, and production performance

Introduction

Poultry has become one of the fastest-growing livestock industries worldwide because of its ability to produce high-quality meat and eggs. Genetic improvement, balanced nutrition, and management have enabled modern strains of poultry to grow rapidly, improve feed efficiency, and productivity. Such improvement has also improved the bird's ability to use energy, protein, vitamins, and minerals effectively. Despite these advances, poultry are still vulnerable to diseases, environmental stresses, poor management, and nutritional deficiencies. Changing diet, heat stress, high stocking density, pathogen exposure, and intestinal disorders can impact gut function and bird performance. Maintaining intestinal health has become an important concern of modern poultry nutrition. The gastrointestinal tract is not only crucial for the digestion and absorption of nutrients but is also a key defense against pathogens and regulates the function of the immune system. The gut microbiota is instrumental in the metabolism of nutrients, the development of intestinal epithelium after

What are post-biotics and how are they defined?

The International Scientific Association of Probiotics and Prebiotics (ISAPP) defines post-biotics as "prepared of inanimate microorganisms and/or parts that confer a health benefit on host" (Salminen et al., 2021) [74] . Of these three points, the microorganisms must be well-characterized, the inactivation process must be intentional, and described, and the final preparation must have measurable health benefits. Although purified microbial metabolites may exhibit biological activity, they are not considered post-biotics unless they are present as part of the final inactivated microbial preparation. Post-biotics is distinct from other microbial products and is not a generic term for organic acids, antimicrobial peptides, cell-free supernatants, or other fermentation products that do not meet ISAPP criteria (Vinderola et al., 2022; Scott et al., 2022; Ma et al., 2023) [86, 76, 54] . It is essential to use the correct language in scientific communication and product development. There are a number of similar terms commonly used in poultry nutrition. Probiotics are live organisms that are beneficial in sufficient amounts when administered as well as prebiotics, which are substances that provide specific nutrients to the beneficial bacteria in their host, either to grow or to function. Synbiotics are those with live probiotics in combination with prebiotic substrates for added bioavailability. Paraprobiotics are non-live microbial cells or fragments that might retain biological activity (No international standard exists for the term yet) (Swanson et al., 2020; Siciliano et al., 2021; Nataraj et al., 2020; Zókiewicz et al., 2020) [82,78,57,100] . Fermented feed products often contain complex mixtures of viable microorganisms, inactivated cells, microbial metabolites, residual fermentation substrates, and cell-wall components. So, not every fermentation product should be considered a postbiotic. This term should only be used on those preparations that meet the ISAPP definition, that are properly characterised and that have a demonstrated health benefit. Consensus documents have stressed the need to clearly describe viability, composition of the product, manufacturing techniques, and identity of the final product for consistent terminology and easier comparisons between studies.Accurate nomenclature is particularly important for the commercial development of poultry feed additives. Heat-inactivated bacterial biomass, clarified fermentation supernatants, yeast fermentation products, and spray-dried fermentates differ substantially in their chemical composition, stability, mechanisms of action, and regulatory status. Consequently, products marketed as post-biotics cannot be assumed to produce similar biological responses simply because they share the same general classification. Their efficacy depends on several factors, including the microbial strain, fermentation substrate, culture conditions, inactivation method, processing technology, and the composition of the final preparation. Comprehensive characterization of these factors is therefore essential for ensuring product identity, reproducibility, and reliable evaluation of biological efficacy.

Sources, Components, and Production of Post-biotics

Post-biotics are produced from a variety of microorganisms, but most widely from lactic acid bacteria (LAB). The most common genera of LAB include Lactiplantibacillus, Lacticaseibacillus, Limosilactobacillus, Lactobacillus, Bifidobacterium, Bacillus, and Saccharomyces. The organisms produce a range of bioactive compounds, including organic acids, short-chain fatty acids, bacteriocins, enzymes, bioactive peptides, exopolysaccharides, vitamins, and other secondary metabolites. The yeast and fungal fermentations also produce -glucans, mannans, nucleotides, peptides, and antioxidants. The composition of the postbiotic is heavily dependent on the microbial strain, fermentation substrate, culture conditions, and growth phase. Even closely related microorganisms can differ significantly in their antimicrobial, antioxidant, and immunomodulatory activities (Chang et al., 2020 [13,45,79,90] . The production of post-biotics is initiated by controlled microbial fermentation and the bacteria are then intentionally killed, while still maintaining their biological activity. Different inactivation methods have been employed, including heat treatment, high-pressure processing, ultrasonication, enzymatic lysis, irradiation, and chemical methods. Other processing steps may include centrifugation, filtration, freeze-drying, spray-drying, microencapsulation, and absorption onto carrier materials, to maintain product stability, store product, or separate microbial biomass from cell-free supernatant. Inactivation is not only an important preservation step, but also an important factor in product composition and function. Processing may remove heat-sensitive bacteriocins and enzymes as well as peptidoglycan, lipoteichoic acid, teichoic acids, nucleic acids, and intracellular components, while at the same time expose other proteins such as peptidoglycan, lipoteichoic acid, teichoic acids, nucleic acids, and intracellular metabolites. The method used to produce post-biotics may influence the receptor, biological activity, particle size, solubility, and shelf stability (Peluzio et al., 2021; Park et al., 2022; Pimentel et al., 2023) [60,58,61] . Analyzing postbiotic products is critical to ensure product integrity and reproducibility. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis have been used to identify bioactive metabolites, along with highthroughput screening, metabolomics, and computational analyses, allowing the identification of chemical signatures that are associated with antimicrobial, antioxidant, immunomodulatory, and other biological activities (Chan et al., 2021; Bauermeister et al., 2022; Ye et al., 2022; Ayon, 2023) [12,6,91,4] . Post-biotics are composed of more than one active ingredient: organic acids, short-chain fatty acids, bacteriocins, exopolysaccharides, lipoteichoic acid, peptidoglycan fragments, phenyllactic acid, cell-free supernatant constituents, enzymes, peptides, nucleotides,glucans, mannans, and other microbial metabolites (Giordani et al., 2023; Shin et al., 2023; Vitale et al., 2023) [ 29, 77, 87] . The relative concentration of these compounds varies depending on the strain and manufacturing process, highlighting the need to characterize chemical composition prior to measuring effectiveness. [13,56] Bacillus spp. [18,84]

Mechanisms of action of post-biotics in poultry health and production

The biological effects of post-biotics are mediated by several mechanisms, which act both at their site of action in the gastrointestinal tract and systemically by influencing host physiological and immune responses. Such complementary actions result in better intestinal health, increased immune function, nutrient utilization and overall poultry performance. The organic acid generation is one of the main antimicrobial activity mechanisms that help reduce the pH of the gut and thus make it less favorable for the growth of acid-sensitive pathogenic bacteria. Moreover, bacteriocins and antimicrobial peptides cause disruptions in the cell membrane of bacteria and halt the growth of harmful microorganisms. Other components secreted by microbes such as biosurfactants, and EVs (extracellular vesicles), modulate pathogen adhesion and biofilm formation to limit their interactions with the intestinal epithelium. [43,25,87,29] .

Along with the benefits of improving epithelial integrity, promoting mucin production, and interacting with the intestinal microbiota, post-biotics also have many other functions to strengthen the function of intestinal barrier. [35,5,63] . The immunological activity, however, depends on the amount of the post-biotics, and an overstimulation of the receptors leading to immune activity might result in undesirable immune response. Thus the dosage and physiological status of host should be taken into consideration while assessing the efficacy of post-biotics.

Another mechanism that plays a key role is modulating gut flora. Post-biotics are not introduced directly into the gastrointestinal tract, but rather work as modulators of the micro-ecology that lead to the inhibition of pathogenic microorganisms, favouring friendly bacteria cross-feeding, changing intestinal redox conditions, supplying metabolites of bacterial fermentation entailed to maintain the microecological homeostasis of the gastrointestinal tract. [96,71,36,67] . However, future research is needed to further identify causal links between changes in the microbiome and health and production benefits in poultry, using combined microbiome, metabolomics and transcriptomics. [14,56] . But these responses differ with strain of the microbe, formulation, dose and bird genotype as well as under challenge conditions. There is a good correlation between the improvement of intestinal function and the improvement of villus architecture, villus height to crypt depth ratio and maintenance of the TJP (e.g. occludin, claudins and zonula occludens), mucus secretion and decreased intestinal permeability. The changes observed enhance the intestinal barrier, the nutrient absorption and the lower potential translocation of pathogenic microorganisms. Another study also found that fermented fungus extract and cell wall extract from yeast enhanced the numbers of goblet cells and improved intestinal morphology as a supportive agent for the protection of mucous membranes (Lin and Lee, 2021; Pascual et al., 2020) [52,59] . Favorable intestinal morphology is far from a sure sign of increased body weight, or feed efficiency, however pointing to other factors besides gut morphology.

Effects of post-biotics on intestinal health and gut barrier function

However, recent studies have indicated that the efficacy of post-biotics is also affected by the composition of the food that they are fed. In broiler production research, fiber modification, protein digestibility, buffering capacity, and antinutrient factor caused bird reactions to pro-or postbiotics to vary with changes in the fiber content of the diet. While alterations of gut microbial community was consistently detected, improvements of immune responses were less apparent (Jansseune et al., 2024a; Jansseune et al., 2024b) [37,38] . Based on these results, it seems that the composition of the diet may be important in assessing the efficacy of the postbiotic in commercial production.

The health-promoting properties of post-biotics on the intestinal barrier function, inflammatory responses and microbial balance are also validated in studies conducted on pigs, ruminants, fish and laboratory animals (Feng et [26, 50, 97, 40] . These results support its biological justification for post-biotics supplementation though more poultry targeted studies are needed. Future research needs to include additional outcomes to assess the mechanisms by which post-biotics enhance intestinal health in poultry, such as assess intestinal permeability along with expression of the tight junction proteins, production of short-chain fatty acids, function of gut microbiota, digestibility of nutrients, and lesion score.

Role of post-biotics in pathogen control and disease resistance

In poultry, post-biotics are used to prevent disease through various mechanisms such as lowering the population of pathogenic microorganisms, supporting the intestinal barrier, and regulating host immune response. The mechanisms of action of post-biotics are complementary to antibiotics, such as the production of antimicrobial metabolites, blocking the colonization of pathogens, use of antiadhesive properties, and strengthening the intestinal microbiota, and strengthening the intestinal barriers. These synergistic interactions favor intestinal homeostasis and prevent intestinal infections.

There are several studies that have proved the efficacy of post-biotics against the economically important poultry pathogens of the world. A layer pullet trial using a Saccharomyces cerevisiae-founded publish biarithmetic system lowered enteric Salmonella colonization of the cecal tissue which suggests it might be used as a preharvest intervention to enhance flock health and food safety (Gingerich et al., 2021) [28] . The same for Lactobacillus helveticus postbiotic in commercial layer chicks which inhibited the presence of Salmonella Gallinarum in the guts (Ribeiro et al., 2023) [68] . Control of these pathogens in the intestine and/or in the faeces may help limit the spread of these pathogens in flocks and the risk of faecal contamination of poultry products in the food chain. Additionally, intestinal health and the prevention of pathogen-induced damage to intestinal tissues may help protect against other enteric pathogens, as the case of postbiotics. In broilers challenged with enteric pathogens, beneficial effects on intestinal morphology, gut permeability, microbial composition of the cecum, and biochemical responses have been reported. Many studies that currently exist, however, have compared the effects of post-biotics with other feed supplements, together with prebiotics or probiotics and it is challenging to obtain information on the effect of post-biotics alone. Therefore, more controlled experiments from various programs are required to identify their independent effects in commercial poultry settings.

Results for control of Clostridium perfringens and Campylobacter spp. and pathogens responsible for coccidiosis are promising but are limited. There are various reports in the literature for reduced lesions, integrity of the intestine and microbial balance, but these responses are varying depending on the type of microbial strain and composition of the post-biotics, the disease challenge and the strain of diet (Ramlucken et al., 2020) [66] . Standardised methods for investigating the efficacy of post-biotics against these pathogens need to be developed.

In vitro studies have also shown that post-biotics exhibit antimicrobial properties against S. aureus, as well as against fungi producers of A. flavus, microbial biofilms, and some models of viral infections (Ebrahimi et [25,87,43,46] . The results are significant in terms of the mechanisms and need to confirm through well-designed poultry challenge studies prior to advice being given in practice. The use of standard pathogen challenge models using more than one outcome measure, such as pathogen shedding, tissue invasion, lesion severity, mortality, AMR, persistence during poultry processing, and overall production performance, should be followed in future investigations. Erica's work through such in-depth studies will give better proof of post-biotics disease control and food safety.

Immunomodulatory effects of post-biotics in poultry

The avian immune system is central to combating infectiousdiseases in birds and is essential for tolerance to gut-not-damaging microbes and antigens in food. The intestinal immune system plays a critical and always active role in interacting with the gut microbiota and sustaining intestinal homeostasis as an important part of gut-associated lymphoid tissue (GALT [5,18,1] . The goal of postbiotic supplementation should be maintaining immune homeostasis rather than activating immune system. A well-regulated immune response will allow birds to mount an appropriate immune response against pathogenic microorganisms and not overreact and respond to create excessive inflammation that may lead to poor growth and nutrient absorption. In situations of low inflammation, nutrients are free to promote growth and production instead of stimulating the immune system. For instance, some postbiotic products developed from yeast have been reported to improve goblet cell function and modulate inflammatory responses, but not necessarily all the improvements in terms of productive performance have been consistent (Pascual et al., 2020) [59] . Likewise, it was reported that a significant modulation was possible by improving broiler performance (Ward et al., 2020), although immune parameters have been changed only slightly.

Another recent study with broilers demonstrated that while the gut microbiota was significantly affected, immune responses were measured with only minor changes (Jansseune et al., 2024b) [38] . [17,95,26,40] . Increases in individual cytokines, especially without an increase in resistance to the infection and disease or improvements in production, should not be taken as a sign of improved immunity. Immune responses should be assessed in the future in the context of a full complement of immune system biomarkers including cytokine profiles, secretory immunoglobulin A (sIgA), systemic antibody responses, and responses to vaccination and/or pathogen challenge. Furthermore, the effects of age, health status, diet composition, postbiotic dose, and economics need to be taken into account for assessing the potential of post-biotics in commodity poultry feed.

Antioxidant effects of post-biotics in poultry

Oxidative stress is a significant problem in today's poultry industry, and frequently is caused by heat stress, high stocking density, mycotoxin exposure, pathogenic infection, etc. Oxidative stress is a big problem in the modern day with poultry production and is often triggered by heat stress, overcrowding, exposure to mycotoxins, pathogenic infections and other environmental stressors. These factors generate greater amounts of Reactive Oxygen species (ROS) which leads to elevated levels of lipid peroxidation, resulting in damage to the cells, poor immunity and diminished growth. Post-biotics have been shown to exert direct antioxidant activity, as well as influence the body's own endogenous antioxidant system, which further indicates their role in reducing oxidative stress. The products of biotransformation are diverse antioxidants metabolites and bioactive compounds with the ability to neutralize free radicals and reduce oxidative damages as a result of which post biotin products have been prepared. Additionally, postbiotics may act by increasing the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) along with activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. The use of diet supplementation with post-biotics was demonstrated to have beneficial effects on antioxidant enzyme activity, decreased lipid peroxidation, reduced physiological stress, serum lipid profile and meat quality in heat-stressed broilers (Humam et al., 2020) [34] . Post-biotics from Lactobacillus acidophilus and fermented fungal formulations have also been reported to have antioxidant and gut health benefits, respectively (Lin & Lee, 2021; Monika et al., 2024) [52,56] .

The antioxidant activity of a postbiotic varies in intensity depending on the strain of the microorganism, its fermentation substrate, and the fermentation process. [13,45,30] . Hence, the in vitro antioxidant activity is not necessarily correlated with the in vivo biological effect as it is modified by intestinal environment, metabolism and digestion. Other studies in other livestock species also corroborate the antioxidant properties of post-biotics. Lactiplantibacillus preparations have increased the antioxidant capacity and integrity of the intestinal barrier in lambs, and Aspergillus oryzae post-biotics have decreased the effects of heat stress in various animal models (Izuddin et al., 2020; Kaufman et al., 2021) [36,41] . These results do support the biological rationale for the use of post-biotics in poultry nutrition but further research is required to validate their efficacy in commercial poultry production conditions. Future research needs to consider multiple parameters of oxidative stress, such as malondialdehyde (MDA), antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD; and glutathione peroxidase, GPx), inflammatory cytokines, intestinal tissue damage, growth performance, and oxidative stability of poultry product. This integrated approaches will equip a more holistic knowledge of the interactive mechanisms of post-biotics action as antioxidants and its application in the poultry nutrition.

Effects of post-biotics on growth performance and production efficiency

One of the essential measures for determining the effectiveness of poultry feeds is their growth performance. Many studies have shown that supplementation of the diet based on post-biotics can increase feed intake, feed conversion ratio (FCR), production efficiency, and improve the body weight gain. These responses are greatly affected, however, by the nutritional status, bird genotype, management conditions and liposome preparation. Lactobacillus-derived post-biotics, stabilized non-viable lactobacilli, microbial product-based fermentation, and yeast-based preparations have yielded promising results in various studies assessing the effects on growth performance, feed efficiency, and physiological health of fish ( [56,1,18,14] . Conversely, certain studies have reported that intestinal tract health and immune system function were enhanced, while body weight and feed conversion ratio were unaffected. Likewise, feed conversion ratio was enhanced by yeast-cell wall related preparation during particular developmental stages of broilers, but these supplements yielded relatively poor results in terms of body weight and carcass traits (Pascual et al., 2020) [59] . These results suggest gut health may not have a direct impact on production performance. The composition of the diet can also influence the postbiotic supplementation. In recent studies on broilers there are large variation among the findings regarding the impact of postbiotics on growth performance, intestinal physiology and metabolism whether broils are fed nutritionally balanced diets or challenged by dietary or environmental stressors (Jansseune et al., 2024a) [37] . Microbial cross-feeding and nutrient utilization are functions of microbial ecosystem, saltability, nutrient density, and nutritive value of dietary fiber, which can change the efficacy of post-biotics. A number of biological and management factors influence the responses evoked by post-biotics, such as: bird age, genetic strain, health status, environmental temperature, stocking density, hygiene, dose, period of the supplementation and type of microbial inactivation as well as the carrier material. Variableness of outcome reported across different studies points to the need for standardized assessment protocols, and these variables account for the different results reported. Overall, available current evidence indicates that postbiotics have a significant potential to enhance nutrient utilization and production efficiency, especially when stressed by environmental or diseases ( 84,72,88,99] . But under all practices there is not any proof to the improvement of growth rate consistently. Thus, assessment of postbiotic supplementation needs to be done beyond feed conversion ratio. This should be complemented by a comprehensive economic evaluation of the product, encompassing feed costs per kg of marketable product, mortality, medication costs, the processing loss, carcass value and overall flock profitability.

Effects of post-biotics on carcass traits and meat quality

Post-biotics may influence carcass characteristics and meat quality by improving nutrient utilization, modulating lipid metabolism, reducing oxidative stress, and enhancing intestinal health. Improved gut function and nutrient absorption can promote lean tissue deposition while reducing fat accumulation, thereby contributing to better carcass composition. In addition, the antimicrobial and antioxidant properties of post-biotics may improve the microbial safety and shelf life of poultry meat. Several studies have reported beneficial effects of postbiotic supplementation on carcass yield, meat quality, and oxidative stability, particularly under conditions of heat stress. Improvements have been observed in lipid peroxidation, antioxidant status, and meat quality parameters following dietary supplementation with postbiotics. Enhanced intestinal health has also been associated with increased breast muscle yield and reduced abdominal fat deposition. However, the effects of post-biotics on carcass traits are generally less consistent than their effects on gut health and feed efficiency. For example, supplementation with a yeast-derived cell wall preparation improved feed conversion and intestinal health but produced inconsistent responses in carcass characteristics (Pascual et al., 2020) [59] . The effects of post-biotics on meat quality should be evaluated using a comprehensive range of carcass and physicochemical parameters, including dressing percentage, breast and thigh yield, abdominal fat percentage, muscle pH, drip loss, cooking loss, water-holding capacity, tenderness, color, lipid oxidation, fatty acid composition, sensory characteristics, and microbial shelf life. Standardized evaluation of these parameters will improve the comparability of studies and help establish the relationship between postbiotic composition and meat quality outcomes (Urban et al., 2024; Waqas et al., 2024) [84,88] . Beyond their effects during poultry production, postbiotic metabolites also show potential for improving food preservation. Studies have demonstrated that antimicrobial compounds produced during microbial fermentation can inhibit spoilage microorganisms and foodborne pathogens in packaging systems and food matrices, suggesting potential applications as natural preservatives in poultry products (Mohammadi et al., 2022; Anvar et al., 2023) [55,3] . Nevertheless, improvements in carcass characteristics resulting from dietary supplementation should not be interpreted as evidence of enhanced post-harvest meat preservation. Separate investigations are required to evaluate the direct effects of postbiotic compounds on meat storage stability, microbial safety, and shelf life.

Effects of post-biotics on laying performance and egg quality

Comparatively to broiler chickens, the research on the use of post-biotics in laying hens is less specific, but growing evidence suggests that may help maintain intestinal health, reduce environmental and metabolic stress, and increase longevity throughout the laying cycle. This is important because commercial laying hens can be in a production environment for extended periods of time during which gut health is paramount to egg production and overall well-being. Post-biotics have been evaluated to control enteric pathogens in laying hens. A postbiotic from fermentation of Saccharomyces cerevisiae was used as a preharvest strategy to reduce cecal colonization by Salmonella Enteritidis in layer pullets (Gingerich et al., 2021) [28] . A Lactobacillus helveticus postbiotic reduced intestinal colonization by Salmonella Gallinarum in commercial layer chicks (Ribeiro et al., 2023) [68] . This study suggests that post-biotics may provide improved flock health and decrease the risk of foodborne pathogen transmission through the egg production chain. But, the available evidence does not provide consistent improvement in egg production or laying throughout the production process. Consider evaluating the efficacy of post-biotics in laying hens by assessing production, reproductive ability and egg quality. Egg production on hen day, egg mass, feed ratio per kilogram of egg mass, egg weight, eggshell strength, shell thickness, Haugh unit, albumen height, yolk color, yolk lipid profile, cholesterol concentration, oxidative status, mortality, and bone mineralization are measures that will help determine the beneficial and economic value of postbiotic supplementation. Even breeder chickens have also been sparsely studied. A fungicide derived from yeast Saccharomyces cerevisiae has been reported to enhance reproductive performance without impairing the intestinal barrier (Forder et al., 2024) [27] . However, reproductive performance, intestinal health, and immune responses should be evaluated independently to accurately determine the specific effects of post-biotics. Future studies should focus on long-term experiments involving commercial laying hens and breeder flocks under practical production conditions. Multi-cycle studies that integrate productive performance, egg quality, gut health, immune function, and economic analyses will provide stronger evidence for the successful application of postbiotics in the poultry industry (Roque et [70,84,72] . [34] Heat-stressed broilers

Lactobacillu

Post-biotics as potential alternatives to antibiotic growth promoters

The global ban on antibiotic growth promoters (AGP) has made it all the more urgent to look for safe and effective substitute feed additives that can ensure poultry health and productivity without contributing to antimicrobial resistance (AMR). Due to their greater stability and safety and biological efficacy, post-biotics are regarded as promising feed additives. Post-biotics do not require the living and active microbial cells and do not rely on colonizing the intestine and are therefore more resilient to feed processing, such as pelleting, and to storage and transportation. Additionally, post-biotics do not contain viable microorganisms, which means that there is a lower theoretical risk for microbial translocation or horizontal transfer of antimicrobial resistance genes (Salminen et al., 2021; Vinderola et al., 2022) [74,86] . After their synthesis, post-biotics exert their biological effects via a combination of bioactive compounds such as organic acids, bacteriocins, antimicrobial peptides, enzymes, exopolysaccharides and microbial cell components. Further comparative studies are required to assess the relative contribution of each metabolite of the purified preparations and the full postbiotic preparations and the mechanisms responsible for their beneficial effects. A standardized manufacturing process and comprehensive chemical characterization is crucial to guarantee the uniformity of product quality, reproducibility, and biological efficacy, ( [74,86,57] . Although the potential of post-biotics is great, they cannot be considered as a general or all-purpose replacement for antibiotics in all production scenarios. These are typically multi-factorial, strain specific and depend on diet composition, environment, pathogen challenge, bird genotype, and management practices. Where product stability and safety are well controlled, viable microbial products like Bacillus-based probiotics and synbiotics are still effective alternatives in certain cases (Ramlucken et al., 2020; Slawson et al., 2020) [66] . Thus post-biotics should be viewed as a piece of a puzzle aimed at reducing antibiotics in combination with other measures, not a silver bullet in itself. Achieving successful antibiotic free production must be through a multi-faceted approach that integrates effective biosecurity, vaccination, litter and water management, nutritionally balanced and digestible diets, scientifically proven microbial feed additives (MFAs), and organic acids.

Combination of these complementary approaches offers increased resistance to enteric diseases with production efficiency and food safety. There are still a number of limitations to the use of postbiotics. These involve differences in formulations, lack of full characterization of active ingredients, variation in the manufacturing method, uncertainty over the optimum dose, differences in regulation across countries and lack of validation in commercial production. Standardized manufacturing procedures, product characterization, doseresponse studies and large-scale field trials should be the priorities of future research. The product composition, stability, safety, efficacy and economic performance should be assessed in comparative evaluations with negative controls, conventional AGPs, probiotics and other feed additives. Importantly, health and performance claims, if used, must be based on a solid scientific foundation and be specific to the product of the postbiotic evaluated. [93, 83] . Postbiotic preparations should be subject to quantitative analysis of characteristic bioactive marker compounds, analysis of potential contaminants and/or residual toxins, and product stability during storage and feed processing (including pelleting). New analytical techniques like chromatography, metabolomics, proteomics and compositional fingerprinting can help achieve batch-tobatch consistency and set objective release criteria. The addition of information regarding the microbial strain, fermentation substrate, processing conditions, chemical composition and biological activity will help in the production of standardized and reproducible postbiotic products (Li et al., 2022; Sahayasheela et al., 2022) [49,73] . The biological effectiveness is also affected by product formulation. Encapsulation technologies can enhance storage stability and targeted delivery of bioactive compounds, but the properties of the bioactive compound may be affected by the process of encapsulation and the material used. The use of microencapsulated butyrate provides a proven case in which the site of release in the gastrointestinal tract plays an important role in the biological activity (Lewandowski et al., 2022) [48] . These factors are likely to also be relevant for other formulations of post-biotics. Food, clinical and animal studies have demonstrated that the use of microbial preparations, fermented food and matrices have generally demonstrated favourable safety profiles and are well tolerated (Vandenplas et al., 2020; Béghin et al., 2021; Bellaiche et al., 2021; Bellaiche et al., 2023) [85,11,8,9] . However, other species safety evaluations may not be a substitute for targetspecies safety evaluation in poultry. Evaluation of the final commercial formulations should be done directly, as formulation components, processing techniques and carrier materials can affect biological activity and safety. Heattreated and fermented microbial preparations have been shown to have beneficial effects on the integrity of the intestinal barrier, inflammatory responses, metabolic markers, and overall physiological condition in several studies (Choi et [17,95,7,81] . These effects are product-and formulation-specific, however, and cannot be translated to other products for use as a postbiotic. Regulatory approval for postbiotic feed additives must be based on comprehensive safety data for the target species, data showing that there are no harmful residues and/or toxins in the product, control of product consistency during manufacture, product stability and stability of the product in use, efficacy data under conditions of use, no interactions of the product with other feed additives and unexpected adverse data from post-market monitoring. The regulatory dossier should be well described to cover the microbial source, genomic safety, fermentation substrate, inactivation process, compositional fingerprint, analytical specifications, shelf life, intended conditions of use and efficacy data. The successful commercialization of post-biotics in the poultry sector will require scientific advances as well as the reliability of product performance, cost, application and proof of return on investment in commercial production systems.

Future perspectives and research priorities

While the research and promising results on post-biotics' application in poultry nutrition extend their use to commercial production, there are still some scientific and practical issues to be addressed for wider adoption. The standardisation of terms, production methods, analytical characterization and experimental procedures are among the most important. Further research is required which should include detailed data on microbial strain accession numbers, culture media, fermentation pH and time, inactivation methods, confirmation of non-viability, biomass/ supernatant ratio, drying process, carrier material, chemically equivalent marker compounds and biologically equivalent doses. This will enhance the reproducibility of studies, make it easier to compare between different studies and aid regulatory approval. In addition, sophisticated analytical methods, such as high-resolution metabolomics, proteomics, lipidomics, extracellular vesicle analysis and functional bioassays, are needed to elucidate the bioactive molecules involved in the beneficial actions of post-biotics and to identify those that survive feed processing and reach their target tissues in the gastrointestinal tract (Qiu et [64,90,79,91] . Large dose-response trials and multi-site trials under both conventional and antibiotic-reduced production systems are needed for efficacy research to go beyond the small, single-site trials. Experimental model should include nutritionally balanced and challenge diets, heat stress, coccidiosis, necrotic enteritis, bacterial infection and various genetic lines of broilers to more closely mimic commercial production conditions. Similarly, long-term trials should be conducted for commercial laying hens and breeder flocks in full production cycles to assess the longterm impacts of post-biotics on productivity, health, and reproductive performance. Multigenomic analysis such as microbiome sequencing should be coupled with metagenomics, metabolomics, transcriptomics, and other multi-omics studies to gain a better overall understanding of host-microbiome interactions. While research on fish, pigs, ruminants, and other species offers important mechanistic insights, they should be used mainly to formulate biological hypotheses to predict the size of the responses in poultry (Quintanilla-Pineda et al [65,50,71,36] . The future challenge is to move away from an empirical formulation and towards mechanism based design for future product development. Novel opportunities for discovery of new bioactive metabolites and post-biotics-producing microbial strains are emerging, such as advances in microbiomics and microbial cultivation of previously unculturable microorganisms, high throughput screening technologies, and next generation microbial therapeutic platforms (Bodor et [10,47,4,21] . These strategies could enable the creation of precision post-biotics solutions to specific production challenges, such as heat stress, necrotic enteritis, controlling Salmonella, preserving laying persistence and enhancing meat oxidative stability. Reliable biomarkers linked to responsive flocks would further aid in targeted nutritional interventions. This is always a crucial balance to achieve safety and efficacy, which will be a key consideration for the success of postbiotics on the market. The biological activity of fermented products, heat treated microorganisms and post-biotics preparations are consistently different from the biological activity of live microbial counterparts. Several factors, such as formulation characteristics, delivery matrix, route of administration, physiological status of the host and production environment, affect these responses (D' Auria et al., 2021; Jeong et al., 2020; Heydari et al., 2023; Chen et al., 2023) [24,39,32,15] . Consequently, mechanistic evidence obtained from in vitro systems or alternative animal models should always be complemented by well-designed targetspecies studies conducted under practical poultry production conditions. Microecological and nutritional research further emphasizes the importance of distinguishing mechanistic plausibility from demonstrated efficacy in poultry, highlighting the need for formulation-specific validation under commercial conditions (Yu et [92, 20, 98, 89] . Therefore, future recommendations and regulatory approval of postbiotic products should be based on robust, reproducible evidence generated for each specific formulation. Emerging research also suggests that traditional fermented food microorganisms, co-culture fermentation systems, and microbiome-informed approaches that consider microbial interactions and metabolic cross-talk represent promising strategies for discovering novel postbiotic candidates (Dobreva et al., 2023; Kaur et al., 2021; Kienesberger et al., 2022; Chung et al., 2022 [18a]) [23, 42, 44, 19] . In addition, increasing evidence indicates that host responses to post-biotics are influenced by formulation characteristics, developmental stage, nutritional status, metabolic context, and the biological compartment being evaluated (Plaza-Díaz et al., 2023; Rodriguez-Herrera et al., 2022; Yu et al., 2023) [62,69,93] . Future research should integrate these factors into precision nutrition strategies to maximize the effectiveness of postbiotic supplementation. Ultimately, the successful adoption of post-biotics in poultry production will depend on formulation-specific, scientifically validated, and reproducible evidence demonstrating consistent improvements in bird health, production efficiency, food safety, and economic return.

Conclusion

Post-biotics have become an exciting new category of feed ingredients that could provide a safe, stable and biologically active means to enhance poultry health and production. However, the current evidence suggests that post-biotics can positively impact gut microbiota composition, integrity of the intestinal barrier, pathogen control, antioxidant defense, immune homeostasis, nutrient utilization, feed efficiency, stress resilience, meat quality and, to a lesser extent, reproductive performance. Post-biotics have a variety of effects, making them potential addition to sustainable poultry production systems and antibiotic-reduction programs. The evidence is strongest for broiler chickens; there are numerous studies showing broiler chickens have improved intestinal health, growth performance, antioxidant activity and production efficiency. Research on laying hens has mainly been on pathogen control, intestinal health and productive performance, whereas there is comparatively less evidence on egg quality, laying persistence, and performance of breeders. Therefore, further research is needed on postbiotic supplementation in commercial laying hens and breeder flocks during entire production cycles to determine the practical effects of this supplementation. Though there are promising results, post-biotics cannot be viewed as a uniform class of feed additives. The biological activity they exert is dependent upon a variety of interacting factors such as microbial strain, fermentation substrate, manufacturing, inactivation methods, separation and drying methods, carrier material, dietary inclusion level, bird genotype, age, nutritional status, environmental conditions and disease challenge. Variability in these areas can add to the findings differences seen from one study to another, and thus underscores the importance of product-specific evaluation. More consistent manufacturing procedures, indepth compositional characterization, validated identity fingerprints, optimized dosage recommendations, targetspecies safety assessments, and multi-site field validation on a large scale in commercial production will be important to future progress. Just as crucial are strong economic assessments which show improvements that can be repeated in terms of production efficiency and profitability. As the microbial biotechnology sector continues to develop, along with the analytical characterization and precision nutrition, post-biotics have a great potential to be a part of the antibiotic-free poultry production process. However, the successful adoption of their use should be based on formulation specific evidence, scientifically validated and reproducible evidence, and not on general claims across all postbiotic products.

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