Comparative study on encapsulation and sustained release of anti-Campylobacter jejuni Immunoglobulin Y Using Various pH-Responsive Beads

Abstract

To protect egg yolk immunoglobulin (IgY) from degradation by pepsin and acidic conditions during oral administration, Campylobacter jejuni (C. jejuni)-specific IgY was encapsulated in polypeptide beads using various methodologies. This study compared different pH-sensitive bead formulations to optimize IgY release at the target site, enhancing C. jejuni control and improving broiler chicken performance. Among the tested formulations, methacrylic acid copolymer (Mac) and β-cyclodextrin (β-CyD) beads demonstrated high encapsulation efficiency, with Mac beads achieving the highest (94.16 %). The inclusion of β-CyD further improved encapsulation efficiency by 3.5 % compared to alginate (Alg) alone. In vitro and in vivo gastrointestinal digestion models, and enzyme-linked immunosorbent assay (ELISA), were used to assess IgY stability and release. β-CyD beads exhibited the highest stability, followed by Alg and Mac, particularly in the gastric phase. Additionally, in vivo studies confirmed that encapsulated IgY, particularly Mac beads, maintained its biological activity and effectively neutralized C. jejuni post-encapsulation. This approach presents a promising strategy for protecting IgY during digestion and ensuring its targeted release in the intestine, thereby improving intestinal health and broiler performance. Consequently, IgY encapsulation may be an efficient feed additive for mitigating C. jejuni infections in poultry.

Graphical abstract

Introduction

Poultry production is the world leader among all avian species. Producers encounter numerous challenges during the rearing process (Wlaźlak et al., 2023). A significant challenge in the husbandry of domestic poultry species, including chickens, turkeys, and ducks, is their susceptibility to infectious diseases caused by bacteria, viruses, fungi, and parasites (Mehrzad et al., 2024). Such infections may lead to immunosuppression and immunoevasion (Schat and Skinner, 2022). Consequently, the presence of a fully functional immune system is essential, although it may be threatened by various stressors and other detrimental environmental factors (Sarrigeorgiou et al., 2023). Avian species are equipped with sophisticated, diverse, and highly integrated immunological strategies that encompass various organs, cells, and biomolecules to counteract pathogenic microbes effectively (Mehrzad et al., 2024). The distinctive avian immune system has evolved in response to the environmental pressures in all aspects of innate and adaptive immune responses, including localized and circulating lymphocytes, the variability of immunoglobulin (Ig) repertoire, and a variety of cytokines and chemokines (Lu et al., 2023).

Antibodies are important players in both innate and adaptive immune responses, and act as a first-line immune defense against pathogens while playing a significant role in the processes of clearance and immune homeostasis (Sarrigeorgiou et al., 2023). Immunoglobulins (Igs), commonly referred to as antibodies, exhibit diverse structural configurations optimized for their functional roles in recognizing antigens. In avian species, three distinct classes of immunoglobulins (IgY, IgA, and IgM) have been identified (Kaiser and Balic, 2015).

Antibodies are important players in both innate and adaptive immune responses, and act as a first-line immune defense against pathogens while playing a significant role in the processes of clearance and immune homeostasis (Sarrigeorgiou et al., 2023). Immunoglobulins (Igs), commonly referred to as antibodies, exhibit diverse structural configurations optimized for their functional roles in recognizing antigens. In avian species, three distinct classes of immunoglobulins (IgY, IgA, and IgM) have been identified (Kaiser and Balic, 2015).

In chickens, immunoglobulins contribute to health maintenance and pathogen defense through complementary mechanisms. Immunoglobulin M (IgM), the first isotype expressed during embryonic development, provides rapid but short-term protection against novel pathogens (Kianpoor et al., 2025). In the intestinal mucosa of broiler chickens, secretory immunoglobulin A (sIgA) is the predominant isotype. It plays a central role in mucosal immunity by blocking microbial adherence, neutralizing antigens, and maintaining microbial balance, which helps prevent pathogen invasion (Bi et al., 2020).

Egg yolk immunoglobulin Y (IgY) constitutes approximately 75 % of all chicken immunoglobulins (Müller et al., 2015). IgY antibodies are derived from serum antibodies synthesized in the bloodstream and subsequently transferred to the egg yolk. Due to their safety, efficacy, stability, pathogen resistance, lack of toxic residues, and low production cost, IgY antibodies have gained considerable attention as preventive and therapeutic agents for various diseases (Radwan et al., 2024). Early oral administration of IgY can compensate for the deficiency of other immunoglobulins in the digestive tract and reduce mucosal IgA production by lowering the intestinal pathogen load (Mahdavi et al., 2010). Since IgA production is age-dependent, with early-life pathogen exposure influencing its levels (Ricci et al., 2021), and because IgY concentrations are inversely correlated with IgA secretion (Lyte et al., 2025), specific IgY has become a promising candidate for passive immunization strategies (Gadde et al., 2015).

Numerous studies have shown that specific IgY has positive effects in the prophylactic and therapeutic modality against enteric infections in broilers, including Salmonella, Campylobacter jejuni (C. jejuni), Escherichia coli (E. coli), and Eimeria acervulina (E. acervulina) (Hatamzade Isfahani et al., 2020; Hermans et al., 2014; Karthikeyan et al., 2022; Lee et al., 2009).

In our previous study, we demonstrated that anti-C. jejuni IgY powder (specific IgY) significantly reduced C. jejuni counts in the liver and cecal contents and mitigated its adverse effects on intestinal health indices in poultry (Soltani et al., 2025). However, the efficacy of orally administered IgY is often compromised under gastric conditions due to pepsin's low pH (≈2) and high proteolytic activity. These factors contribute to the structural degradation of IgY through the enzymatic hydrolysis of its polypeptide chains, resulting in reduced biological activity (Gu et al., 2021; Li et al., 2007; Wu et al., 2014). As the primary target site of orally administered IgY is the small intestine, protecting the antibody during gastrointestinal transit is essential for preserving its therapeutic efficacy (Li et al., 2007). While unencapsulated IgY powder may exert beneficial effects against intestinal pathogens, encapsulation has emerged as a promising strategy for protecting IgY from gastric degradation, thereby enhancing its bio-efficacy and facilitating targeted, sustained release within the intestine (Lu et al., 2025). Various techniques have been explored to achieve this, including IgY chitosan–alginate microcapsules for inhibition Edwardsiella tarda in turbots (Xu et al., 2020), IgY sodium alginate–chitosan–sodium alginate systems for inhibition E. coli and Salmonella in broilers (Jin et al., 2023), and delivery systems incorporating alginate combined with shellac, lecithin, or chitosan-liposomes in vitro (Dong et al., 2022; Li et al., 2022; Lu et al., 2025).

While these methods show promise, they often have limitations such as formulation complexity, high production costs, or lack of in vivo validation. Alginate-based hydrogels remain attractive for oral delivery due to their low cost, biocompatibility, biodegradability, and non-toxicity (Gu et al., 2021; Lima et al., 2017). Their pH-responsive behavior further supports their utility in gastrointestinal delivery. In acidic environments, protonation of carboxylic acid groups leads to polymer compaction and enhanced protection of encapsulated agents. Conversely, at neutral pH—typical of the intestinal tract—ionization increases electrostatic repulsion, promoting swelling and controlled release of encapsulated compounds such as IgY (Gu et al., 2021).

Nonetheless, the dense network structure of alginate can limit drug loading and release rates (Omtvedt et al., 2019). To overcome this, combining alginate with other natural polymers has been proposed as a cost-effective means of improving its performance (Xie et al., 2024). Cyclodextrins (CyDs), due to their ability to form inclusion complexes, enhance solubility and improve bioavailability, are particularly suitable candidates. Grafting CyDs onto alginate may yield a composite system that integrates alginate's protective and responsive properties with the complexation capabilities of CyDs (Omtvedt et al., 2019), thereby enhancing IgY encapsulation efficiency and controlled intestinal release. In addition to the above, among the evaluated polymeric systems, pH-responsive poly (methacrylic acid-co-methyl acrylate) copolymers (Eudragit®) demonstrated notable efficacy in IgY encapsulation and targeted intestinal delivery. These anionic polymers exhibited controlled release profiles under simulated intestinal conditions, likely due to their pH-dependent solubility properties that enable protection in gastric environments and dissolution in alkaline intestinal media (Hatamzade Isfahani et al., 2020).

This study presents a comparative analysis of various capsule formulations designed for the encapsulation and oral delivery of specific IgY. The primary objective is to identify an economically viable carrier system capable of protecting IgY through the gastric environment while enabling its sustained release in the intestinal tract, thereby inhibiting Campylobacter replication. The research evaluates encapsulation efficiency, release kinetics, and the functional activity of specific IgY following encapsulation, in intestinal health in broiler chickens.

Materials and methods

Experimental design

An available egg yolk powder containing anti-C. jejuni IgY antibodies by Soltani et al. (2025) were used in this study. The IgY antibodies were obtained by the water-soluble fraction (WSF) method, and then lyophilized by a freeze dryer. The primary objective of the study was to assess encapsulation in vitro. Subsequently, experimental trials were conducted on broiler chickens to identify the most effective treatment for promoting gut health. The animal management and experimental procedures (sampling and euthanasia methods) in this study were approved by the Animal Ethical and Welfare Committee of Tarbiat Modares University (ethical review acceptance number: REC.1400.134).

Capsulation of immunoglobulin Y

Preparation of IgY-loaded Alginate beads

Initially, the optimal conditions for preparing alginate beads (Alg) were determined based on the findings of a previous study (Li et al., 2007), with slight modifications. In summary, a solution containing 0.5 % (w/w) IgY and 2 % (w/w) sodium alginate was injected into a hardening solution composed of 0.5 % calcium chloride (CaCl₂) (pH 4) and left to harden for 30 minutes. The resulting calcium alginate beads were collected and freeze-dried using a freeze dryer (Martin-Christ, Osterode am Harz, Germany) for three hours (h). Then, they were stored in sealed plastic bags at room temperature until further analysis was performed. In this study, 2.2 g of encapsulated IgY with Alg was equivalent to 1 g of non-encapsulated IgY.

Preparation of IgY-loaded Alginate/β-cyclodextrin beads

The preparation of alginate/β-cyclodextrin (β-CyD) beads was conducted as follows: Sodium alginate and β-CyD were separately dissolved in distilled water and stirred at 60°C for 15 minutes to ensure complete polysaccharide dissolution. After cooling to room temperature, powdered IgY was dissolved in the β-CyD solution to achieve a final concentration of 0.5 % (w/w). The resulting mixture was then combined with a sodium alginate solution at varying ratios, forming mixed systems containing 2 % (w/w) sodium alginate and 0.5 % (w/w) β-CyD. Next, 10 mL of the mixed polysaccharide solution was extruded through a 0.7 mm needle into 200 mL CaCl₂ solution (0.5 % w/v, pH 4.0) under continuous stirring at 200 rpm and room temperature. The needle was positioned 8 cm above the surface of the CaCl₂ solution, and the solution was dispensed at a rate of one drop per second. The formed beads were incubated in the CaCl₂ solution for 30 minutes to ensure hardening. Subsequently, the beads were collected, rinsed with distilled water to remove residual CaCl₂, and freeze-dried (Moses et al., 2000). The dried beads were then stored in sealed plastic bags at room temperature until further analysis. In this study, 2.5 g of encapsulated IgY with ßCyD-Alg was equivalent to 1 g of non-encapsulated IgY.

Preparation of IgY-Loaded Methacrylic Acid Copolymer Beads

The emulsion, containing 40 % (w/w) IgY, was prepared by dissolving IgY in distilled water and stirring the mixture at 250 rpm for 30 minutes. Subsequently, the prepared IgY emulsion was added to sucrose cores (mesh size 20-25, IPS, Co., Italy) using a peristaltic pump (Masterflex, Cole-Parmer Instrument Company, USA). The emulsion was dispensed at a rate of 0.1 mL/s throughout 4 h in a platen machine, maintained at a temperature of 57°C. Following this, the sucrose core beads loaded with IgY were coated using a peristaltic pump with a solution containing 15 % methacrylic acid copolymer (Mac) (Eudragit® L 100-55, Evonik Röhm GmbH, Darmstadt, Germany) and 10 % polyethylene glycol (PEG) 6000. In this study, 2 g of encapsulated Mac was calculated to be equivalent to 1 g of non-encapsulated IgY.

Morphology examination of microcapsules

The morphology of the IgY-encapsulated dried beads (Alg and β-CyD) was investigated using scanning electron microscopy (SEM, Quanta 450, FEI, USA). The samples were coated with a layer of gold (with a thickness of 4 nm) using a sputtering machine. Imaging was performed under high-vacuum mode at a working distance of 14 mm, with an accelerating voltage of 15 kV.

IgY encapsulation efficiency percentage

The encapsulation efficiency of beads was determined using the method outlined by Li et al. (2007). To measure the IgY content integrated into the capsules, 10 mg of dried IgY beads were dissolved in 5 mL of IgY release media. This media consisted of a mixture of 0.2 M sodium bicarbonate (NaHCO₃) and 0.06 M sodium citrate dehydrate (Na₃C₆H₅O₇•2H₂O), adjusted to a pH of 8.0. The solution was then subjected to ultracentrifugation at 10,000 rpm and 4°C for 30 minutes. After centrifugation, the supernatant was collected for total protein content analysis, which was carried out using a bicinchoninic acid (BCA) protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, USA) following the manufacturer's recommended protocol. IgY encapsulation efficiency (EE%) was calculated using Equation (1), where CP represents the mass of the released protein, and WTP denotes the total mass of the protein used in the capsule preparation process. The experiment was conducted thrice.

$$\text{EE}\%=\frac{\text{CP}}{\text{WTP}}\times 100\%$$ (1)

In vitro IgY release from the capsules

The in vitro release profiles of IgY were assessed at a constant temperature using a shaking incubator with simulated gastric fluid (SGF). Specifically, 50 mg of IgY-loaded capsules were re-dispersed in 50 mL of SGF, consisting of 0.03 M sodium chloride (NaCl) at pH 2, and incubated at 150 rpm and 37°C. After 2 h, the SGF was filtered and replaced with 50 mL of simulated intestinal fluid (SIF), prepared with 0.05 M monopotassium phosphate (KH₂PO₄) at pH 6.8, and incubated under the same conditions for 3 h. At 30-minute intervals, 200 µL of the supernatant was extracted from the SIF and replaced with an equal volume of fresh medium. Then, protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce Inc., New York, NY) on 96-well ELISA plates. The cumulative release percentage (Q%) was determined using Equation (2).

$$Q\%=\text{(Cn}\text{×}\text{V+Vi}\sum _{i=0}^{n-1}Ci)/\left(W\times IgYloading\%\right)\times 100\%$$ (2)

In this calculation, Cn represents the sample concentration at time Tn, V denotes the total volume of the release medium, Vi is the sample volume at time Ti, and Ci indicates the sample concentration at time Ti. Additionally, both V₀ and C₀ were set to zero. The parameter W corresponds to the weight of capsules used for the in vitro IgY release study, as described by Li et al. (2007). The experiment was conducted thrice.

In vitro stability of IgY to gastrointestinal conditions

The stability of IgY under simulated avian gizzard digestive juice (SGDJ) conditions was evaluated using SGDJ, which consisted of 1 M NaCl and 10 g/L pepsin (Merck) at pH 1.5. The treatments included encapsulated and non-encapsulated specific IgY, with the respective amounts of Mac (2 g), Alg (2.2 g), β-CyD (2.5 g), and IgY (1 g). Antibody activity was measured after incubation in SGDJ at 42°C with shaking at 350 rpm. Sampling occurred at 0.5, 1, and 2-hour intervals. Following this, an equal volume of 2x simulated intestinal digestive juice (SIDJ)—containing 0.05 mol/L KH₂PO₄, 3.5 % bile extract, and 0.35 % porcine pancreatin diluted to effective concentrations of 0.35 % and 0.035 %, respectively, at pH 7.4—was added to the samples. The solutions were then slowly shaken for 2 h at 42°C. Sampling was repeated at the same intervals (0.5, 1, and 2 hours) with the samples placed on ice (Hatamzade Isfahani et al., 2020). Antibody activity was defined as the binding ability of anti-Campylobacter IgY to Campylobacter and was determined using the enzyme-linked immunosorbent assay (ELISA) protocol described by Soltani et al. (2025). Each treatment was replicated five times.

In vivo gastrointestinal stability of IgY

The in vivo stability of IgY was assessed as described previously (Hatamzade Isfahani et al., 2020). A total of 100 broiler chicks (ROSS 308), obtained from a commercial hatchery (Kosar Company, Karaj, Alborz Province, Iran), at 28 days of age, were randomly allocated to four treatment groups, with five birds per group, for each designated sampling time point. The treatment groups consisted of Mac (1 g/kg), Alg (1.1 g/kg), β-CyD (1.25 g/kg), and IgY (0.5 g/kg). The different encapsulated IgY and IgY powders were mixed manually through the feed (wt/wt). Throughout the five-hour experimental period, feed and water were provided ad libitum. At predetermined intervals of 0.5, 1, 2, 3, and 4 hours, the birds were humanely euthanized via CO₂ asphyxiation, and necropsies were performed to collect the contents of the proventriculus, gizzard, jejunum, and ileum. The entire contents of this gastrointestinal section were vortexed separately, then mixed with three volumes of phosphate-buffered saline (PBS) containing a protease inhibitor (Merck) and maintained on ice. The samples were homogenized and centrifuged (7500 rpm for 10 min at 4°C). The resulting supernatants were ultra-filtered using a cellulose membrane with a molecular weight cut-off (MWCO) of 50 kDa, reducing their volume to 1/5 of the original. The filtered liquid was subjected to serial dilution in PBS and subsequently utilized via ELISA to quantify residual IgY activity in the samples. Briefly, the contents of the specified regions of the digestive tract containing anti-Campylobacter IgY were assayed for reactivity against C. jejuni. Microtiter plates were coated with C. jejuni bacterin (100 µL) as the primary antigen. After washing and blocking, goat anti-chicken IgY conjugated to horseradish peroxidase (Sigma-Aldrich®, USA) was applied as the secondary antibody (100 µL). Anti-Campylobacter antibody titers were determined at a working dilution of 1:10 for the filtered sample supernatants, following a previously described protocol with minor modifications.

Animals and management

A total of 450 one-day-old male broiler chicks (Ross 308) were obtained from a commercial hatchery (Kosar Company, Karaj, Alborz Province, Iran). The chicks were distributed based on body weight and randomly assigned to one of six treatment groups. All broilers received spray vaccinations for infectious bronchitis and Newcastle disease on days 0 and 21, along with a Gumboro vaccination on day 13. Upon arrival, the broilers were confirmed to be free of Campylobacter by collecting cloacal swabs, which were streaked onto modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid, UK) and incubated for 48 h under microaerobic conditions at 42°C. No Campylobacter were detected in swab samples taken from day-old birds and before infection at 7 days of age. Each treatment group consisted of 15 broilers housed in five floor pens (2 × 1 × 1.2 m) containing wood shavings as litter in a temperature-controlled environment from day 0 to day 28 post-hatch. The temperature started at 32°C and was gradually reduced by 1°C every two days, reaching 24°C. Environmental conditions were maintained using thermostatically controlled heaters, evaporative cooling plates, and exhaust fans to ensure optimal comfort. The broilers were initially kept under a 23L: 1D cycle during the first week, then transformed to a consistent 16L: 8D schedule for the remainder of the study. Relative humidity was maintained at approximately 70 % during the first three days and adjusted to between 55 % and 65 % for the remainder of the study. The broilers had ad libitum access to water and mash diets. The negative control group was housed in a separate isolated room to prevent cross-contamination.

Experiment design

The experiment consisted of six treatment groups, each with five replicates of 15 birds per replicate. The treatment groups were as follows (Table 1):

Table 1.

Experimental design for the in vivo experiment.

Group	Treatment Description	Dose of IgY treatment
NC	Negative control (No IgY treatment, No C. jejuni inoculation)	—
PC	Positive control (C. jejuni inoculation, No IgY treatment)	—
IgY	IgY-treated (C. jejuni inoculation + IgY powder)	0.50 g/kg
Mac	Mac-treated (C. jejuni inoculation + IgY encapsulated)	1.00 g/kg
Alg	Alg-treated (C. jejuni inoculation + IgY encapsulated)	1.10 g/kg
β-CyD	β-CyD-treated (C. jejuni inoculation + IgY encapsulated)	1.25 g/kg

Treatments were manually incorporated into the feed (wt/wt). At days 7 and 8, all broilers, except those in the negative control group, were orally gavaged with approximately 1 × 10⁸ CFU/mL of C. jejuni. C. jejuni ATCC 29428 was provided by Razi Vaccine and Serum Production Research Institute (Karaj, Iran). One bird per replicate was humanely euthanized at days 14, 21, and 28. Growth performance parameters, including average daily weight gain (ADW), daily feed intake (DFI), and feed conversion ratio (FCR), were assessed at days 7, 14, 21, and 28.

Histomorphological analysis of chicken ileum

Sample collection and preparation

Histomorphological assessments of the chicken ileum were conducted in a histological laboratory following the paraffin technique described by Bogucka et al. (2016). After euthanizing the birds, approximately 1 cm of tissue samples were taken from 2 cm before the ileo-cecal transition and immediately immersed in a 10 % formaldehyde solution for preservation. The samples were then dehydrated, cleared, and embedded in paraffin. Thin sections (5–7 µm) were prepared using a rotary microtome. For the evaluation of general histological architecture, sections were stained with haematoxylin and eosin (H&E) following standard protocols. For histochemical analysis, the alcian blue/periodic acid–Schiff (AB–PAS) method was employed to differentiate acidic mucins (stained blue) and neutral mucins (stained magenta/purple) within goblet cells, to enable their enumeration under light microscopy.

Histomorphological measurements

For ileal measurements, five sections were prepared from each tissue sample per bird, and five images of each section were randomly captured at 400 × magnification using an Olympus light microscope. Subsequently, 10 well-oriented villi per bird were analyzed. The following parameters were assessed:

Villus Height (VH), measured from the tip of the villus to its base at the crypt-villus junction (100 × magnification). Villus Width, measured at the midpoint of the villus length (100 × magnification). Crypt Depth (CD), Defined as the invagination depth between adjacent villi (100 × magnification). Subsequently, Villus-to-Crypt Ratio (VH/CD) is calculated to determine the structural relationship between villus height and crypt depth. Goblet Cell Count, Quantified as the number of goblet cells per 100 µm of villus height (400 × magnification).

Liver and Cecal campylobacter jejuni enumeration

To determine the number of C. jejuni in the cecum and liver quantitatively, the number of colony-forming units (CFU) was determined by a direct-plating method. One gram of the samples was vortexed in 9 mL of sterile phosphate-buffered saline for 1 min, and serial dilutions were plated. Aliquots of 100 µL of each dilution were plated on mCCDA (Oxoid, UK) and incubated for 48 h at 42°C under microaerophilic conditions consisting of 5 % oxygen (O₂), 7.5 % carbon dioxide (CO₂), 2.5 % hydrogen (H₂), and 85 % nitrogen (N₂).

Data Analysis

The data were analyzed for variance using a one-way ANOVA, employing the generalized linear model (GLM) procedure in SAS software version 9.4. Mean comparisons were conducted using Duncan’s multiple range test at a significance level of 5 % (P < 0.05) (SAS Institute Inc., 2014).

Results and discussion

Oral passive immunotherapy using hyperimmunized egg yolk antibodies has been investigated by several researchers as a method for controlling enteric infections in broiler chickens (Abadeen et al., 2025; Hajiyev et al., 2024; Vandeputte et al., 2020). However, this approach may not be economically viable, as achieving therapeutic efficacy necessitates substantial quantities of egg yolk powder containing specific IgY antibodies. This requirement arises due to significant degradation of IgY during its passage through the proventriculus and upper small intestine. Moreover, as IgY traverses the gastrointestinal tract, its antigen-binding activity is markedly reduced or even entirely lost due to proteolytic degradation, primarily by gastric acid and pepsin, severely diminishing its biological activity. Additionally, gastrointestinal barriers further exacerbate the reduction or complete loss of IgY antibody bioavailability (Lia et al., 2015; Wang et al., 2024; Xu et al., 2011). While the susceptibility of IgY to enzymatic degradation presents a challenge for its practical application, encapsulation techniques offer a promising solution (Li et al., 2007). In this study, the morphological characteristics of the Alge and β-CyD models were analyzed following the application of various capsule formulations.

Morphology observation of microcapsules

The SEM results show that, in addition to grain size, the freeze-drying technique significantly influences the morphological characteristics of the beads. Fig. 1 (A and B) illustrates the structural properties of dried Alg (A) and β-CyD (B) beads. Both types of beads had a near-spherical shape with a porous, sponge-like microstructure, and an average diameter of approximately 1 mm. These features highlight the distinct textural and morphological attributes resulting from the freeze-drying process. Specifically, the morphological analysis of them revealed a transformation in their structural appearance following the drying process (Fig. 1). Initially exhibiting a spherical shape when wet, the beads underwent notable alterations due to volumetric expansion associated with the phase transition of water into ice (Shi et al., 2012). As drying progressed, the beads experienced shrinkage and surface wrinkling, shown by the SEM images. Therefore, increased size and rough surfaces of the beads enhanced their integration with the feed, thereby improving their practical applicability in formulation processes. The observed changes in the formulation process, including morphological modifications, may influence the encapsulation efficiency of IgY.

Fig. 1.

SEM micrographs of surface structures of freeze-dried (A) Alg and (B) β-CyD beads. Abbreviations: SEM: scanning electron microscope, Alg: alginate beads of IgY powder, β-CyD: alginate/β-cyclodextrin beads of IgY powder.

Encapsulation efficiency percentage

The encapsulation efficiency (EE%) of IgY depends on the method of preparation. Fig. 2 illustrates the effect of different encapsulation techniques on IgY EE%. As shown in Fig. 2, the EE% of Mac, Alg, and β-CyD beads was 94.16, 83.92, and 87.44 %, respectively (P > 0.05). The highest encapsulation efficiency of IgY was observed in Mac, indicating greater stability. However, encapsulation efficiency decreased in other beads. In β-CyD, the encapsulation efficiency was higher than Alg (P > 0.05), possibly due to Alg aggregation induced by interactions with β-CyD fragments.

Fig. 2.

Encapsulation efficiency (EE%) of specific IgY in beads. ^a-c means in a column, without the same letter is significantly different: P < 0.01. Data are mean ± standard error of the mean (SEM) (n = 3 samples/treatment per group). Abbreviations: Mac: methacrylic acid copolymer beads of IgY powder; Alg: alginate beads of IgY powder; β-CyD: alginate/β-cyclodextrin beads of IgY powder.

Encapsulation efficiency may serve as key parameter to select the optimal oral delivery system for IgY by preventing its inactivation in the gastric environment. Higher encapsulation efficiency of Mac and β-CyD beads, compared with Alg (p > 0.05), is due to their multilayered structure (Fig. 2). The enhancement in encapsulation efficiency suggests that these beads exhibit greater stability, with the multilayer coating effectively preventing IgY leakage (Li et al., 2007). Moreover, studies investigating the use of chitosan as a membrane for IgY-liposome (Li et al., 2022) and IgY-alginate (Li et al., 2009) capsules have demonstrated improved encapsulation efficiency, indicating that multilayer encapsulation contributes to bead stability and facilitates controlled release within the digestive system.

In vitro IgY release from the capsules

The in vitro release profiles of IgY from Mac, Alg, and β-CyD beads in SGF and SIF are shown in Fig. 3. As expected, IgY release from all bead formulations remained minimal in the acidic environment. Upon exposure to SIF, IgY release occurred much more quickly, particularly in the Alg beads. In contrast, Mac beads exhibited a 30-minute lag phase before a substantial release of IgY was observed. Meanwhile, β-CyD beads demonstrated a gradual release profile. Under acidic conditions, all capsules remained structurally intact, with IgY release being insignificant (below 10 %). However, IgY release in SIF progressively increased, reaching approximately 93, 88, and 83 % for Alg, Mac, and β-CyD beads, respectively, following a 3-hour incubation period (P < 0.05).

Fig. 3.

The release profile of IgY from different particles in simulated gastric fluid (SGF) for 2 h, and then transferred to simulated intestinal fluid (SIF) for 3 h. The IgY activity was measured by ELISA. ^a-d means in a column, without the same letter is significantly different: P < 0.01. Data are mean ± standard error of the mean (SEM) (n = 3 samples/treatment per group). Abbreviations: Mac: methacrylic acid copolymer beads of IgY powder; Alg: alginate beads of IgY powder; β-CyD: alginate/β-cyclodextrin beads of IgY powder.

Assuming an average retention time of feed in the digestive tract, excluding the ceca, is probably around 3 to 4 h (Svihus, 2011), in vitro release profiles of IgY from Mac, Alg, and β-CyD beads were evaluated around 5 h. The physicochemical properties of beads are a fundamental prerequisite for the sustained release of IgY during digestion. In the SGF medium, the cumulative release rate of IgY from beads remained close to 0 % after 2 h (Fig. 3). However, since IgY must be released and remain functionally active in the intestine, the drug release behavior in the SIF buffer varied among bead formulations. Specifically, Alg beads exhibited a more rapid release of IgY than Mac (Fig. 3). These results align with the established mechanism of drug release from the alginate matrix. The release of IgY exhibited an approximately linear correlation with time in SGF, suggesting that diffusion was a primary mechanism governing the drug release process (Wu et al., 2014). Additionally, the environmental transition from acidic to neutral conditions, resulted to proton hydrolysis at low pH, may have accelerated matrix degradation, leading to enhanced IgY release upon re-equilibration at neutral pH (Goh et al., 2012). The Mac exhibited significant resistance to gastrointestinal conditions, which explains its extensive use in enteric coating and sustained-release formulations. The Mac formulation effectively inhibits protein release in acidic gastric fluid, with IgY being released at pH levels exceeding 5.5 (Kim et al., 2022). This release behavior is likely attributed to the carboxyl protonation of Mac during its passage through the low-pH environment. As the pH increases, ionization of carboxyl groups occurs, leading to bead swelling and disruption of hydrogen bonding, thereby facilitating IgY release (Ullah et al., 2022). In contrast, β-CyD beads demonstrated a more gradual and sustained release of IgY in SIF compared to the other two formulations. Hence, we inferred that capsulations could protect the biological activity of IgY in the low-pH.

In vitro stability of IgY in gastrointestinal conditions

Beads containing IgY exhibited relative stability during in vitro digestion in SGDJ. In contrast, non-encapsulated IgY underwent hydrolysis, leading to a reduction in its activity from 2.44 to 0.73 optical density at 450 nm (OD₄₅₀) after 2 h of incubation in SGDJ (Fig. 4A). This degradation is attributed to the susceptibility of IgY to pepsin and the extremely low pH (1.5) of the SGDJ medium, which led to near-complete hydrolysis. Consequently, upon transfer to SIDJ medium, IgY demonstrated the lowest activity among all treatments (P > 0.05, Fig. 4B). The hydrolysis of Mac and Alg beads commenced after 2 h of incubation in SGDJ medium, with their activity reaching a peak relative to β-CyD and non-encapsulated IgY powder after 30 minutes of incubation in SIDJ medium. Among all formulations, β-CyD beads displayed a gradual hydrolysis process, maintaining a consistently lower IgY activity than the other beads types throughout the experimental period (P > 0.05, Fig. 4B).

Fig. 4.

In vitro stability of encapsulated and non-encapsulated specific IgY to (A) simulated gizzard digestive juice and (B) simulated intestinal digestive juice for 120 minutes. ELISA measured the IgY activity. ^a-c means in a column, without the same letter is significantly different: P < 0.01. Data are mean ± standard error of the mean (SEM) (n = 5 samples/treatment per group). Abbreviations: IgY: immunoglobulin Y powder; Mac: methacrylic acid copolymer beads of IgY powder; Alg: alginate beads of IgY powder; β-CyD: alginate/β-cyclodextrin beads of IgY powder.

The stability of IgY during simulated digestion was further evaluated using ELISA. As shown in Fig. 4, non-encapsulated IgY displayed an increase in OD values after 2 hours in SGDJ, confirming its instability in acidic gastric conditions and substantial degradation in the proventriculus-like environment. This observation aligns with the findings of Wang et al. (2021), who used ELISA, SDS-PAGE, and immunoblotting to demonstrate extensive degradation of IgY in the chicken gizzard. Consequently, the amount of functional IgY reaching the intestine is minimal.

In contrast, encapsulated IgY retained significant activity following transfer to SIDJ medium and continued incubation for 2 hours, indicating that encapsulation effectively limited its release in SGDJ while promoting its release under intestinal conditions. These results are consistent with previous studies (Li et al., 2022; Lu et al., 2025) which reported improved IgY stability through encapsulation. Similarly, Hatamzade Isfahani et al. (2020) demonstrated that encapsulated IgY remained stable after 3 hours in a simulated digestive environment.

The improved performance of β-CyD beads can be attributed to their capacity to form inclusion complexes with IgY, which modulate its solubility and release kinetics, thereby enhancing stability (Moses et al., 2000). Collectively, encapsulation offers a protective barrier against detrimental conditions, enhancing IgY stability throughout digestion (Lu et al., 2025).

It is important to note that digestion is a highly dynamic process involving the transit of food through gastrointestinal compartments at rates influenced by factors such as structural complexity, caloric density, osmolarity, and rheological properties. The physicochemical conditions—pH, ionic strength, and enzyme activity—vary continuously over time. However, static in vitro digestion models fail to capture these temporal and physiological fluctuations, limiting their predictive accuracy. Therefore, to fully characterize the release profile and functional delivery of encapsulated IgY, further in vivo studies are warranted.

In vivo gastrointestinal stability of IgY

Fig. 5 presents the encapsulation stability of IgY during in vivo digestion within the GIT. As illustrated in Fig. 5A, non-encapsulated IgY exhibited a significant increase in activity within the proventriculus environment but underwent substantial degradation, losing nearly all activity after 2 h in the proventriculus and 1 h in the gizzard. Compared to encapsulated IgY powder in various formulations, non-encapsulated IgY powder exhibited a significant initial increase in OD₄₅₀ during digestion in the proventriculus (60–120 minutes) and the gizzard (at 60 minutes). Following this phase, the OD remained relatively stable and low throughout the remainder of the GIT digestion. As expected, bead digestion was initiated by the gizzard's mechanical activity. Among the tested formulations, the Mac bead exhibited the highest IgY release rate in the jejunum (1.14 ± 0.13; P < 0.05, Fig. 5C). In the distal small intestine, Alg and β-CyD beads demonstrated significantly higher optical densities at 3 h (1.76 ± 0.11 and 1.81 ± 0.06, respectively) than other formulations (P < 0.05, Fig. 5D).

Fig. 5.

ELISA evaluation of in vivo stability of encapsulated and non-encapsulated specific IgY in each gastrointestinal tract area: (A) Proventriculus, (B) Gizzard, (C) Jejunum, and (D) Ileum. ^a-c means in a column, without the same letter is significantly different: P < 0.01. Data are mean ± standard error of the mean (SEM) (n = 5 samples/treatment per group). Abbreviations: IgY: non-encapsulated IgY powder; Mac: methacrylic acid copolymer beads of IgY powder; Alg: alginate beads of IgY powder; β-CyD: alginate/β-cyclodextrin beads of IgY powder.

The activity of orally administered IgY may be rapidly diminished or entirely degraded under gastric conditions due to the high acidity and pepsin activity of gastric fluids. Since the primary target site of IgY is the small intestine, a major reservoir of zoonotic bacteria (Cosby et al., 2015), this study evaluated the effectiveness of various encapsulation strategies in protecting IgY throughout different segments of the chicken GIT. Various IgY encapsulation formulations enhanced IgY stability in the gastrointestinal tract, with Mac beads demonstrating the highest IgY activity upon initial entry into the small intestine, followed by Alg and β-CyD. Hatamzade Isfahani et al. (2020) reported that, in vivo, encapsulated IgY (methacrylic acid polymer) exhibited the highest antibody activity in the proximal intestine, followed by the distal intestine, proventriculus, and gizzard in broilers. Among the tested formulations, β-CyD provided effective protection against IgY degradation under gastric conditions and during transit through the proximal small intestine (Fig. 5B, C, D). This protective effect may be attributed to the denser polysaccharide network of composite beads compared to pure alginate beads, which likely hinders the infiltration of pepsin and gastric acids (Gu et al., 2021). Overall, encapsulated IgY retained significantly greater antibody activity in the jejunum and ileum compared to the unencapsulated group (P < 0.05, Fig. 5).

Growth performance

The effects of various treatments on the quantitative performance of broiler chickens—specifically ADW (g), DFI (g), and FCR—are presented in Fig. 6. The experimental results were analyzed: the pre-challenge period (days 1–7) and the post-challenge period (days 8–28).

Fig. 6.

Impact of encapsulated and non-encapsulated specific IgY on broiler chicks' growth performance for 4 weeks, (A) In vivo experiment, (B) Average Daily Weight Gain (g) (ADW), (C) Daily Feed Intake (g) (DFI), (D) Feed Conversion Ratio (FCR). ^a-b Columns with no common letters differ: P < 0.01; P < 0.05. Data are mean ± standard error of the mean (SEM) (n = 5 samples/treatment per group). Abbreviations: NC: Negative Control; PC: Positive Control; IgY: Positive Control + non-encapsulated IgY powder; Mac: Positive Control + methacrylic acid copolymer beads of IgY powder; Alg: Positive Control + alginate beads of IgY powder; β-CyD: Positive Control + alginate/β-cyclodextrin beads of IgY powder.

During the pre-challenge phase, no significant differences in ADW (g) were observed among the treatment groups (P > 0.05). However, in the post-challenge period, a comparison between the negative and positive control groups demonstrated that the challenge, occurring on days 7 and 8, had a significant impact on ADW (g) between days 14 and 21. Notably, the positive control group exhibited the lowest body weight gain (BWG) among all experimental groups (P < 0.05, Fig. 6B). While the administration of both encapsulated and non-encapsulated specific IgY did not yield a statistically significant effect on weight gain, these treatments contributed to an improvement in the average daily body weight gain of broilers compared to the positive control group (P > 0.05, Fig. 6B).

As illustrated in Fig. 6C, neither encapsulated nor non-encapsulated IgY had a significant effect on FDI during the pre-challenge period (P > 0.05). However, following the challenge, encapsulated IgY—particularly Mac and Alg—reduced FDI, resembling the negative control group's response when compared to the positive control group. Notably, Alg and the negative control exhibited a more pronounced reduction in FDI than Mac (P < 0.05). Among the treatment groups, β-CyD and IgY, along with the positive control group, demonstrated the highest feed consumption (P < 0.05, Fig. 6C).

Prior to the challenge, FCR exhibited a significant difference between the positive control and Mac compared to other experimental groups (P < 0.01, Fig. 6D). Following the challenge, Mac, Alg, and the negative control group demonstrated an improving effect on FCR among the experimental groups (P < 0.01, Fig. 6D). Moreover, Mac and Alg consistently improved FCR throughout the entire experimental period when compared to the IgY and β-CyD groups (P < 0.01, Fig. 6D).

Previous assumptions characterized C. jejuni as a harmless commensal inhabitant of the chicken gut, with a tolerogenic immune response that was believed to have no effect on broiler chicken performance (Hermans et al., 2012). However, C. jejuni infection induces a prolonged inflammatory response, challenging its classification as a benign commensal (Humphrey et al., 2014). This inflammatory reaction may contribute to the poor performance observed in the positive control group. In our study, both encapsulated and non-encapsulated specific IgY significantly decreased FDI and improved FCR, without affecting ADW (Fig. 6). Notably, encapsulated IgY, particularly formulations with Mac and Alg, further reduced FDI and enhanced FCR compared to the positive control group (P < 0.05). Our findings are consistent with those of Rehan et al. (2022), who demonstrated that IgY supplementation significantly improves performance. These beneficial effects likely stem from IgY’s capacity to inhibit pathogenic bacterial proliferation and modulate inflammatory responses, ultimately enhancing body weight, feed intake, and immune function (Mahdavi et al., 2010; Rehan et al., 2022).

Histomorphological measurements

Broiler performance is closely linked to the health of intestinal morphology. Hence, the effects of dietary encapsulated and non-encapsulated specific IgY on ileal morphology at 28 days of age are summarized in Table 2. A significant increase (P < 0.01) in villus height, crypt depth, and the villus height-to-crypt depth ratio was observed across the different treatment groups, whereas no statistically significant differences (P > 0.05) were detected in villus width or goblet cell count among the groups. The highest mean villus height was recorded in broilers receiving the Mac, Alg, and NC treatments, measuring 870, 846, and 929 μm, respectively, while the shortest villus height was observed in the positive control group (632 μm, P < 0.01). Furthermore, crypt depth was significantly greater (P < 0.01) in the PC and β-CyD groups compared to all other treatments. Importantly, the NC group, followed by the Mac group, exhibited a significant increase in the villus height-to-crypt depth ratio relative to the positive control (P < 0.01, Table 2).

Table 2.

Effect of encapsulated and non-encapsulated specific IgY on histomorphological parameters of the ileum in broiler (28 d of age).

	Parameters
	Villus height (μm)	Villus width (μm)	Crypt depth (μm)	Villus height to crypt depth	Goblet Cell Number
Treatments
NC	929a	116	146b	6.36a	10.18
PC	632b	75	210a	3.04c	11.28
IgY	846a	98	156b	5.69ab	10.06
Mac	870a	102	150b	5.9a	10.36
Alg	654b	76	138b	4.88abc	11.46
β-CyD	660b	68	180ab	3.69bc	10.6
SEM	39.82	12.80	11.15	0.454	0.598
P-Value	0.0001	0.082	0.0012	0.0001	0.465

^a-c Means within each column with no common superscript differ: P < 0.01; P < 0.05. SEM: standard error of the mean. (n: 5 samples/treatment per group). Abbreviations: NC: Negative Control; PC: Positive Control; IgY: Positive Control + non-encapsulated IgY powder; Mac: Positive Control + methacrylic acid copolymer beads of IgY powder; Alg: Positive Control + alginate beads of IgY powder; β-CyD: Positive Control + alginate/β-cyclodextrin beads of IgY powder.

Intestinal morphology serves as a key indicator for assessing the digestive and absorptive functions of the gut. Campylobacter infection compromises gut barrier integrity, triggering an excessive pro-inflammatory response that leads to structural distortion and atrophy in the ileal villi and crypts (Chick et al., 2025). The results of this study demonstrated, Campylobacter-induced ileal damage was mitigated to varying degrees by oral administration of encapsulated and non-encapsulated specific IgY (Table 2). This protective effect may be attributed to the ability of specific IgY to reduce intestinal permeability and inflammatory responses. These findings suggest that specific IgY effectively alleviates villous atrophy and crypt hyperplasia, thereby improving intestinal tissue morphology in broilers challenged with C. jejuni. Similarly, Han et al. (2021) reported that anti-E. coli IgY alleviated the inflammatory response by reducing the production and expression of proinflammatory cytokines, thereby improving intestinal morphology in mice.

Campylobacter jejuni enumeration

The analysis of C. jejuni counts per gram of cecal content and liver samples collected from broiler chickens, prophylactically fed either non-encapsulated or encapsulated specific IgY powder at 14, 21, and 28 days, is presented in Fig. 7. The total C. jejuni burden in the cecal content and liver tissue of birds receiving encapsulated specific IgY powder was significantly lower at all time points compared to those fed non-encapsulated specific IgY (P < 0.01).

Fig. 7.

Cecal (A) and liver (B) C. jejuni counts in broiler chickens fed encapsulated and non-encapsulated specific IgY, ^a-c Columns with no common letters differ: P < 0.01. Data are mean ± standard error of the mean (SEM) (n = 5 birds/treatment per group). Abbreviations: NC: Negative Control; PC: Positive Control; IgY: Positive Control + non-encapsulated IgY powder; Mac: Positive Control + methacrylic acid copolymer beads of IgY powder; Alg: Positive Control + alginate beads of IgY powder; β-CyD: Positive Control + alginate/β-cyclodextrin beads of IgY powder.

Moreover, the colonization of C. jejuni in the cecal content was reduced (P < 0.01) in the Mac (6.43 ± 0.03 CFU/g), Alg (7.4 ± 0.02 CFU/g), and β-CyD (7.4 ± 0.19 CFU/g) groups at 14 days post-inoculation (dpi) relative to the IgY (7.85 ± 0.02 CFU/g) and PC (8.96 ± 0.47 CFU/g) groups. This trend persisted at both 21 and 28 dpi. By day 28, the Mac, Alg, and β-CyD groups exhibited reductions of 6.24, 5.04, and 4.84 log₁₀ CFU of C. jejuni compared to the PC group, respectively (P < 0.01; Fig. 7A).

On day 14, the Mac and Alg groups exhibited the lowest levels of C. jejuni colonization in liver samples, with bacterial counts of 3.4 ± 0.08 and 3.42 ± 0.03 log₁₀ CFU/g, respectively. There was no statistically significant difference in colonization levels between these two groups (P > 0.01). In contrast, the β-CyD and IgY groups displayed higher colonization levels, with reductions of 3.82 ± 0.07 and 4.23 ± 0.15 log₁₀ CFU, respectively. On days 21 and 28, C. jejuni was not isolated from the liver samples of birds receiving either non-encapsulated or encapsulated specific IgY treatment. However, C. jejuni was detected in the liver of birds in the PC group, indicating continued bacterial presence in the absence of IgY intervention (Fig. 7B).

We investigated the effects of IgY treatment on the colonization of C. jejuni in the cecum and liver of birds (Fig. 7). Notably, IgY administration was particularly effective against long-lived pathogens such as the microaerophilic C. jejuni (Czoska et al., 2025). Targeted delivery of IgY to the distal intestine, rather than the upper GIT, was significantly enhanced when IgY was coated with chitosan–alginate (Li et al., 2009) or encapsulated within hydrogel-carbon nanotube composites (Alustiza et al., 2016) in a pig model. The successful release of IgY at the intended site may exert its protective effects by inhibiting bacterial adherence, enhancing opsonization, altering bacterial structure, and neutralizing toxins (Esmaeili et al., 2024). When administered prophylactically, both encapsulated and non-encapsulated specific IgY treatments significantly reduced C. jejuni colony counts, with the Mac and Alg groups exhibiting a marked decrease in colonization rates in the cecum and liver of chickens (Fig. 7). Additionally, a previous passive immunization study demonstrated that dietary supplementation with anti-Salmonella Infantis IgY encapsulated in methacrylic acid resulted in a significant reduction in cecal and liver colonization in broilers, likely due to IgY-mediated protection against premature degradation (Hatamzade Isfahani et al., 2020).

The results of the present study indicate that different encapsulation formulations of egg yolk powder effectively protect against specific IgY during transit through the proventriculus and upper small intestine. In summary, methacrylic acid copolymers are among the most widely used pH-sensitive polymers in the pharmaceutical industry. At pH levels above 5.5, Eudragit L100-55 effectively controlled IgY release (Salawi, 2022). Additionally, under gastric conditions, Alg beads remained compact, whereas in the small intestine, increased electrostatic repulsion led to swelling, thereby expanding pore size and significantly affecting IgY release (Jadach et al., 2022). Moreover, the incorporation of both β-CyD and Alg within the beads resulted in a denser internal microstructure. Consequently, Mac beads demonstrated superior efficacy as a carrier for specific IgY delivery to the cecum in the treatment of C. jejuni infection, outperforming both β-CyD and Alg. This advantage is likely due to the beads’ simpler protective layers, which facilitate the controlled release of the enclosed material.

Overall, the immunoglobulins encapsulated within the beads exhibited enhanced resistance to release in both in vitro and in vivo experiments due to the formation of an inclusion complex. The presence of complex multilayer structures, particularly in β-CyD beads, may enhance targeted release in vitro. However, these structures also pose a risk of complete excretion without releasing the active compound in the gastrointestinal tract. Additionally, the high production costs and the current absence of industrial-scale production of β-CyD and Alg beads present substantial limitations that require further investigation. Achieving large-scale production of beads suitable for industrial applications while ensuring economic feasibility remains a significant challenge, emphasizing the need for further research in this area.

Conclusion

Based on the results of this study, it can be concluded that Mac beads significantly enhanced encapsulation efficiency compared to various IgY-bead formulations. The slower release rate observed in IgY-β-CyD beads in simulated intestinal fluid suggests improved control over IgY delivery. Additionally, all IgY-bead formulations demonstrated the protective effects of wall materials, which enhanced IgY’s resistance to gastric acid degradation. The presence of pepsin, pancreatin, and the mechanical activity of the gizzard markedly impacts IgY's biological function. Comparative analysis between IgY beads and IgY powder, as assessed by ELISA testing, indicates that IgY beads exhibit superior stability and resilience under both in vitro and in vivo conditions. Furthermore, different IgY-bead formulations tailored for cecum-specific delivery against C. jejuni showed ideal in vitro conditions, supporting improved protein stability for effective in vivo application. In broiler chickens challenged with C. jejuni, certain performance indicators—including feed intake and feed conversion efficiency—were positively influenced in groups receiving an IgY-supplemented diet, especially the Mac group, compared to those fed an unsupplemented diet. This improvement may be attributed to the ability of various IgY treatments to reduce C. jejuni populations, leading to significant enhancements in ileum health indices. Consequently, these findings underscore the potential of IgY encapsulated beads to preserve IgY’s biological activity in vitro while facilitating IgY-specific targeted release in the cecum against C. jejuni. This targeted delivery mechanism supports improved digestion, nutrient absorption, and antibacterial efficacy, contributing to enhanced gut health and performance in broiler chickens.

However, further studies are required to elucidate the biologically active substances and physiological mechanisms of IgY in combating pathogenic bacterial species. These investigations should assess the effects of IgY on inflammatory responses, intestinal health, and the growth performance of broiler chickens. Additionally, advancements in large-scale IgY encapsulation techniques are necessary to enhance the cost-effectiveness and feasibility of egg yolk antibody applications in the poultry industry.

CRediT authorship contribution statement

Nazanin Soltani: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft. Shaban Rahimi: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. Ebrahim Vasheghani-Farahani: Investigation, Methodology, Resources, Validation. Bahareh Eskandari: Investigation, Methodology. Jesse Grimes: Writing – review & editing.

Disclosures

The authors declare that they have no conflict of interest in this study.