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. 2026 Feb 11;50(2):155. doi: 10.1007/s11259-026-11108-4

Butyric glycerides enhance resistance of chicken enterocytes to pathogen adhesion and cytotoxicity in vitro

Amine Mellouk 1, Marc Maresca 2, Laurence Canaple 3, Virginie Michel 1, Olga Lemâle 1, Tim Goossens 1, Jessika Consuegra 1,✉
PMCID: PMC12894191  PMID: 41670853

Abstract

Butyrate is a short-chain fatty acid naturally produced by microbiota in the lower gut of chicken. It enhances gut integrity and maturity and acts as a primary energy source for enterocytes. To extend its benefits in chickens’ digestive tract, protected butyrate sources are supplemented in the feed. The current study aims to characterize the effects of a mixture of mono-, di-, and triglycerides of butyrate on avian enterocyte resistance to pathogen colonization. This in vitro study shows that the butyric-glycerides (BG) mixture directly enhances the 8E11 chicken enterocytes’ resistance to microbial pathogens. Unlike sodium butyrate (SB), treatments with butyric glycerides at 2 mM notably reduced the adhesion levels of Campylobacter jejuni and Salmonella Typhimurium to enterocytes by 84 and 60%, respectively (P < 0.001). Butyric glycerides also significantly reduced Clostridium perfringens α toxin-induced cytotoxicity, which reached 83% in untreated control group while only 28% of cytotoxicity is induced in presence of BG (P < 0.001). Secondly, only the butyrate released through lipolysis, rather than butyric glycerides themselves, was able to enhance the basal and maximal the oxygen consumption rate in chicken enterocytes by ~ 40 and 56% (P < 0.001). This mitochondrial metabolic response may lead to increased CO2 levels in gut lumen in vivo, thereby favouring the development of anaerobic commensal bacteria and inhibiting the colonization of aerobic and facultative anaerobes, including potential pathogens. In summary, our results suggest that including butyric glycerides in poultry feed is a promising strategy to enhance disease resistance not only by the described antimicrobial properties of α-monoglycerides, but also via the enhancement of enterocytes’ resistance against pathogen adhesion and toxicity. Collectively, these effects may promote chicken resilience and enhance their resistance to bacterial challenges.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11259-026-11108-4.

Keywords: Butyrate glycerides, Antimicrobial, Enterocytes metabolism, Mitochondrial oxygen consumption, 8E11 cells

Introduction

Butyrate is a major short chain fatty acid (SCFA) generated, by the natural activity of gut bacteria during the fermentation of carbohydrates and fibers (Rombeau et al. 1990). It plays a critical role in maintaining gut integrity and promoting maturation while fortifying the intestinal barrier. Studies on human enterocyte cell line Caco-2 cells have shown that butyrate treatments increase the transepithelial electrical resistance (TEER) by modulating the assembly of tight junction proteins (Peng et al. 2009; Beaumont et al. 2020). Furthermore, at the level of gut epithelium, butyrate promotes the cellular proliferation and differentiation while reducing apoptosis. This support a faster and more efficient maturation of gut structures, characterized by increased villi height and crypts depth (Górka et al. 2014). These parameters enable the animals to achieve greater efficiency and capacity for nutrients’ absorption. Colonocytes primarily depend on butyrate as their main source of energy, thereby reducing the use of endogenous energy sources by 82% (Roediger 1982). In mammals, the utilization of butyrate as an energy source enhances mitochndrial respiration and the Oxygen Consumption Rate (OCR) through diverse mechanisms, including the direct oxidation of butyrate or its involvement in pyruvate flux into mitochondria. These processes exert distinct effects on the glycolysis, either stimulating or inhibiting it, respectively (Bekebrede et al. 2021; Park et al. 2022). Such variations in the metabolic processing of butyrate are intricately linked to the balance of pyruvate kinase isoforms M1 and M2 (PKM1/2), which is known to be dysregulated in cancerous colonocytes (Park et al. 2022). Notably, the increase in OCR and the subsequent release of CO2 by colonocytes in the presence of butyrate contribute to creating an anoxic environment within the gut lumen. This shift in intestinal ecosystem fosters anaerobic beneficial bacteria while inhibiting the growth of aerobic and facultative anaerobic pathogens such as Salmonella (Rivera-Chávez et al. 2016). Additionally, butyrate directly suppresses the production of proinflammatory cytokines and enhances the secretion of antimicrobial peptides by enterocyte and/or resident macrophages in the gut (Schulthess et al. 2019; Gupta et al. 2020).

The beneficial effects of butyrate are predominantly observed in gut segments with high microbial fermentation activity, such as the colon or caeca (Rombeau et al. 1990), and are largely absent in the upper segments of the gastro-intestinal tract (GIT), including the duodenum and jejunum. In livestock industries, the inclusion of butyrate in animal feed is widely recommended to extend its benefits throughout the entire GIT, thereby improving gut health and the overall performances (Ahsan et al. 2016). However, administrating non-protected forms of butyrate in animal feed results in its rapid absorption in the upper GIT, such as the stomach and duodenum, preventing it from reaching the small intestine (Moquet et al. 2018). To address this limitation, various strategies have been developed, including chemically protected butyrate in the form of mixture of butyric glycerides comprising mono-, di- and tributyrin (Bedford and Gong 2018). Monobutyrin has been demonstrated to possess direct antimicrobial properties, effectively inhibiting the growth and proliferation of Salmonella serotype Typhimurium and Clostridium perfringens (Sun et al. 2003) while reducing their colonization in chicken gut (Namkung et al. 2011). Moreover, dietary supplementation with monobutyrin and tributyrin positively influences the microbial composition and diversity in the chicken caeca, leading to improved microbial activity and SCFA production (Nguyen et al. 2017). It has been demonstrated that butyric glycerides undergo enzymatic cleavage by pancreatic lipase in mammals (Clement et al. 1962; Sampugna et al. 1967). Similar observations have been reported in chicken when these glycerides are included in their diet (Namkung et al. 2011). The efficiency of cleavage and the subsequent release of butyrate depends on factors such as the degree of branching in the glycerides (mono-, di- or tributyrin) and the availability of cleavage sites (Clement et al. 1962; Sampugna et al. 1967).

To address the limited understanding of how butyrate derivatives modulate host–pathogen interactions at the cellular level, we hypothesized that butyric glycerides, compared to free butyrate, exert distinct protective effects on chicken enterocytes through both direct and metabolic mechanisms. To test this, we conducted a series of experiments using the 8E11 chicken enterocyte model, which is highly susceptible to bacterial adhesion and invasion (John et al. 2017; Pascoe et al. 2019; Ali et al. 2020; Kolenda Rafał et al. 2021). First, in vitro assays evaluated whether sodium butyrate and butyric glycerides could prevent adhesion, infection, and cytotoxicity caused by pathogenic bacteria, specifically C. jejuni and S. Typhimurium. Second, we quantified mitochondrial and glycolytic activities in 8E11 enterocytes to determine whether these compounds induce metabolic shifts that could influence cellular resilience.

Materials and methods

8E11 chicken enterocytes

Chicken epithelial intestinal cells (clone 8E11) were provided by Tentamus/Micromolar (Germany). They were first characterized John and colleagues as being highly sensitive to bacterial pathogens adhesion and invasion (John et al. 2017). Subsequently, 8E11 cells have been widely utilised as a model for bacterial (Pascoe et al. 2019; Ali et al. 2020; Kolenda Rafał et al. 2021) and viral (Han et al. 2019) pathogenic infections in avian species.

8E11 cells were routinely cultured, according to the provider’s recommendations in DMEM: F12 Glutamax medium supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin (Thermo Fisher Scientific, MA, US). Cells were used between passages 4 and 10 passages. They were maintained in a humidified incubator at 37° C, 5% CO2 and 95% humidity. Upon reaching 80–90% confluency, the cells were detached using a 0.25% trypsin-EDTA solution (Thermofisher Scientific, MA, US) and either sub-cultured into new flasks or seeded into 96 wells plates for the different assays.

Butyric glycerides

The butyric glycerides mixture used in the in vivo and in vitro experiments consisted of 75% mono-, 23% di- and 2% tri- butyric glycerides. All reported concentrations of butyric glycerides were adjusted to reflect the equivalent final concentration of butyric acid.

Bacterial adhesion (CellELISA)

Micro-organism used in the study were obtained from the German Leibniz Institute (DSMZ) and correspond to S. Typhimurium (strain 14028; DSMZ, Germany) and C. jejuni (strain BAA-2151; DSMZ, Germany). Micro-organisms were cultured on Luria Bertani (LB) agar plates in aerobic condition for S. Typhimurium and in microaerobic condition generated using BD GasPak system for C. jejuni. The day prior the assay, S. Typhimurium or C. jejuni was inoculated from a LB agar plate into 3 mL of LB broth using sterile loops. The cultures were incubated overnight at 37 °C without shaking.

8E11 cells were cultured as described earlier then seeded into 96-wells plates at a density of 5 × 104 cells/well. Once the cell reached confluency, they were exposed to sodium butyrate or butyric glycerides at different concentrations (0–10 mM) for 3 h at 37 °C. After this, the cells were infected with S. Typhimurium or C. jejuni at 105 CFU/mL in culture medium without FBS for an additional 3 h at 37 °C.

Post infection, wells were washed six times with PBS and fixed for 20 min at room temperature in 4% paraformaldehyde diluted in PBS with. Following fixation, wells were saturated with PBS containing 5% BSA. Next, 100 µL of primary rabbit antibodies against C. perfringens or S. Typhimurium (Thermo Fisher, MA, US; references PA1-7244 and PA1-7205, respectively) were added at a 1 : 10 000 dilution in PBS supplemented, with 5% BSA, and incubated at room temperature for 60 min. Supernatants were removed, and the wells were washed 3 times with PBS. Secondary goat anti-rabbit IgG antibodies conjugated to HRP (Jackson Immunoresearch, PA, US) were then added at a 1 : 10 000 dilution in PBS supplemented with 5% BSA and incubated at room temperature for 60 min. Wells were aspirated and washed 6 times with PBS. Then, 100 µl of HRP substrate (OPD) were added to each well, and the plates were incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 50 µl of 2 N H2SO4 and the optical densities were measured at 490 nm using a spectrophotometer. The results are presented in percentages reported signal obtained in presence of the bacteria at 1 : 10 000 dilution. The log decrease of the adhesion was calculated according to the adhesion level of the bacteria at different concentrations presented in Supp. Fig.1.

Alpha-toxin cytotoxicity

8E11 cells were seeded into 96-wells plates and allowed to reach confluency. The cells were then treated with the indicated concentrations of butyrate or butyric glycerides (0–10 mM) for 1 h, followed by before exposure to PLC derived from C. perfringens (Sigma Aldrich, MO, US) at varying concentrations (0–1000 mU/mL) for 3 h. After incubation, the wells were aspirated, and cell viability was assessed using resazurin-based assay (TOX8 kit; Sigma Aldrich, MO, US) following the manufacturer’s instructions. The cells were incubated with the assay reagents for 2 h at 37 °C, and the fluorescence was measured at 530 nm excitation and 590 nm emission. Fluorescence values were corrected and normalized against untreated cells, which were set as controls with 100% viability.

Enterocyte’s metabolism seahorse real time metabolic function analysis

The butyrate sources were digested in vitro following Menezes-Blackburn protocol (Menezes-Blackburn et al. 2015), simulating the chicken GIT from crop-gizzard to jejunum phases. Concentrations of free butyrate were controlled by chromatography at the end of digestion. 8E11 chicken enterocytes (3.104 cells/well) were preincubated for 72 h in presence or absence (control condition) of various treatments: 5 mM sodium butyrate (Sigma-Aldrich, MO, US), butyric glycerides equivalent to 5 mM butyric acid (Adisseo, France), 1.66 mM tributyrin at (to achieve 5 mM equivalent butyric acid; Sigma-Aldrich, St Louis, US), or digested butyric glycerides or tributyrin releasing 5 mM butyric acid. Cell viability and morphology were assessed prior to the metabolic analysis using brightfield imaging.

Metabolic functions were evaluated using the Seahorse XFe96 extracellular flux analyser (Agilent, CA, US). Oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) were measured in XF DMEM medium supplemented with 10 mM glucose, 1 mM Pyruvate and 2 mM Glutamine (Agilent, CA, US). Baseline OCR and ECAR levels were determined through four consecutive measurements (0–20 min). Subsequently, sequential injections of metabolic modulators were performed. At 20 min, the glycolysis inhibitor, 2-deoxiglucose (2DG, Sigma-Aldrich, St Louis MO, US) was injected, at 50 mM, to inhibit glycolysis and force the cells to rely on mitochondrial respiration, allowing the quantification of maximum mitochondrial activity. OCR and ECAR were measured 3 times (20–37 min). At 40 min, a mixture of rotenone and antimycin A, at 1.25 µM and 2.5 µM respectively (Sigma-Aldrich, St Louis MO, US), was injected to completely inhibit the mitochondrial respiratory chain. At the end of the metabolic assay, the number of the cells was determined by automated counting of nuclei stained with NucBlue live cells stain (Life Technologies, CA, US). OCR and ECAR values were normalized to the cell count, and analysed using Wave software (Seahorse Biosciences, MA, US).

Statistical analysis

Cell survival, pathogen adhesion, and metabolic changes were analysed using 1-way-ANOVA and Tukey’s honestly significant difference (HSD). A Student t-tests were performed to evaluate metabolic changes in presence or absence of sodium butyrate. All statistical analysis were performed using JMP statistical software (SAS institute, NC, US). Results were considered as significantly different when obtained P values were inferior to 0.05.

Results

Butyric glycerides decrease the adhesion of S. Typhimurium and C. jejuni to chicken enterocytes more efficiently than sodium butyrate alone

The susceptibility of the 8E11 enterocytes to adhesion by S. Typhimurium and C. jejuni was assessed in the presence of sodium butyrate or butyric glycerides (Fig. 1). For this purpose, 8E11 enterocytes were preincubated for 3 h with various concentrations of sodium butyrate (0–10 mM) or butyric glycerides mixture (equivalent to 0–2 mM of butyrate). Following preincubation, the enterocytes were exposed to S. Typhimurium or C. jejuni at a concentration of 105 CFU/mL, and bacterial adhesion was quantified using the CellELISA method.

Fig. 1.

Fig. 1

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Campylobacter jejuni and Salmonella Typhimurium adhesion ability to 8E11 chicken enterocytes pre-incubated with sodium butyrate or butyric glycerides 8E11 enterocytes were preincubated, or not (CTRL: blue circles), for1 hour with sodium butyrate (SB: red circles) or butyric glycerides (BG: green circles) before being exposed for 3 h to C. jejuni or S. Typhimurium at 105 bacteria/mL (A) Average percentages (± SD) of C. jejuni adhesion quantified by cellELISA and reported to their maximal (100%) ability to adhere on 8E11 enterocytes in absence (blue) or presence and of treatments with sodium butyrate (red) or butyric glycerides (green) at 2 mM. (B) Average percentages (± SD) of S. Typhimurium adhesion quantified by cellELISA and reported to their maximal (100%) ability to adhere on 8E11 enterocytes in absence (blue) or presence of treatments with sodium butyrate (red) or butyric glycerides (green) at 2 mM. Statistical differences were evaluated by 1-way-ANOVA and Tukey’s honestly significant difference (HSD). n = 3

The results showed that preincubation with butyric glycerides at 2 mM provided significant protection to 8E11 enterocytes, reducing the adhesion normalised OD signals of C. jejuni and S. Typhimurium by 84 and 61%, respectively (P < 0.001, Fig. 1A-B). These reductions correspond to decreases in adhesion of approximately > 2 and 0.8 log folds in absolute bacteria adhesion, respectively (Supp. Fig. 1).

In contrast, preincubation with sodium butyrate at the same concentration (2 mM) did not confer protection against C. jejuni (Fig. 1A) and led to only 20% reduction in S. Typhimurium adhesion levels (Fig. 1B, ~ 0.15 log fold decrease in absolute CFU). Increasing the butyrate concentration to 10 mM did not result in a further decrease of S. Typhimurium adhesion (20%; P < 0.001; Supp. Fig. 2B). However, higher concentrations of sodium butyrate provided partial protection against C. jejuni adhesion reducing it by 25% (P = 0.004; Supp. Fig. 2A).

Butyric glycerides treated enterocytes are more resistant to alpha toxin induced cell death

The preventive effect of sodium butyrate and butyric glycerides against C. perfringens alpha toxin (PLC) were assessed using resazurin assay. Without pretreatments, PLC-induced cytotoxicity in 8E11 enterocytes was dose-dependent, reaching a maximum mortality of 95% at 15.6 mU/mL. The estimated EC50 for PLC was 4 mU/mL (Fig. 2A). However, pretreatment with sodium butyrate (10 mM) or butyric glycerides (2 mM) significantly increased enterocyte resistance to PLC toxicity. Pretreatment shifted the mortality curve, raising the EC50 to 31.2 mU/mL and increasing the maximal effective PLC dose to 125 mU/mL (P < 0.001; Fig. 2A) providing protection against PLC doses approximately 8 times higher than in untreated cells. Notably, sodium butyrate required a concentration five times higher (10 mM) than butyric glycerides (2 mM) to achieve equivalent protective effects. Further, when comparing equal concentrations of sodium butyrate and butyric glycerides (both at 2 mM) in the presence of PLC at 7.8 mU/mL, butyric glycerides demonstrated significantly greater protection. Enterocyte mortality decreased to 28.2% with butyric glycerides compared to 82.8% in the control group (P < 0.001; Fig. 2B). In contrast, sodium butyrate at 2 mM did not improve survival compared to the control (17.2% vs. 22.2% survival, P = 0.138). These results indicate that butyric glycerides are substantially more effective at protecting chicken enterocytes from toxin-induced cell death than free butyrate at equivalent concentrations.

Fig. 2.

Fig. 2

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Protective effects of sodium butyrate and butyric glycerides against C. perfringens alpha toxin 8E11 enterocytes were pre-incubated, or not (CTRL: blue line or circles), for 1 h with sodium butyrate (SB: red line or circles) or butyric glycerides (BG: green line or circles) before being exposed for 3 h to α-toxin (PLC) from C. perfringens at different concentrations (0 to 1000 mU/mL). Cell survival was quantified by Resazurin toxicity assay (A) Values represent the average survival percentage (± SD) of enterocytes after3 hours of exposition to PLC at different concentrations (from 0 to 1000 mU/mL).Before PLC challenge, 8E11 enterocytes were preincubated for 1 h in presence of sodium butyrate at 10 mM (red line) or butyric glycerides at 2 mM (green line). Non treated control group is represented in blue line. (B) The average percentage of enterocytes survival (± SD) after3 hours of exposition to PLC at 7.8 mU/mL. Before PLC challenge, the 8E11 enterocytes were preincubated for 1 h in presence of sodium butyrate (red circles) or butyric glycerides (green circles) at 2 mM. Non treated control group is represented in blue circles. Statistical differences were evaluated by 1-way-ANOVA and Tukey’s honestly significant difference (HSD). n = 3

Butyric acid changes the metabolic profile and enhances the oxygen rate consumption of chicken enterocytes

In mammals, one mechanisms for preventing bacterial pathogens colonization involves the oxidation of butyrate in enterocytes mitochondria, creating an anoxic gut lumen environment unfavourable to aerobic pathogens (Rivera-Chávez et al. 2016). To explore whether a similar mechanism occurs in avian enterocytes, the metabolic effects of butyrate on 8E11 chicken enterocytes were investigated. Specifically, mitochondrial respiration and glycolytic energy production pathways were assessed after 72-hour sodium butyrate treatments using Seahorse® technology.

The basal oxygen consumption rate (OCR), an indicator of mitochondrial respiration, in 8E11 enterocytes treated with sodium butyrate compared to untreated controls during the initial phase (0–20 min, Fig. 3A). Upon glycolysis inhibition with 2-deoxyglucose (2DG), OCR further increased in both treated and control cells (20–35 min, Fig. 3A), indicating enhanced mitochondrial activity as a compensatory response to the glycolytic inhibition. The average maximal OCR was determined by calculating the area under the curve (AUC) from 20 to 35 min before mitochondrial respiration was fully inhibited by rotenone and antimycin A (R/A). Sodium butyrate-treated cells showed significantly higher AUC values than control cells (Fig. 3B), demonstrating improved mitochondrial respiratory capacity due to butyrate treatment.

Fig. 3.

Fig. 3

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Modulation of enterocytes metabolism pathways by chronic treatment with sodium butyrate 8E11chicken enterocytes were cultured for 72 h in presence, or not (control: blue line and circles), of sodium butyrate at 5 mM (red) before seahorse measurements: oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) (A) The right panel shows OCR means (± SD) at each time point measurement. Left panel shows the OCR means (± SD) of basal (0–20 min), maximal (20–37 min) and non-mitochondrial OCR (> 37 min) OCR. (B) Area under curve (AUC) of maximal OCR (± SD) from 20 to 37 min, in presence of 2-deoxyglucose (2DG) and absence of rotenone and antimycin (R/A). (C) The right panel shows ECAR means (± SD) at each time point measurement. Left panel shows the ECAR means (± SD) of basal (0–20 min), ECAR after glycolysis pathway (20–37 min) and mitochondrial pathway inhibition (> 37 min). (D) Area under curve (AUC) of basal ECAR (± SD) from 0 to 20 min, in absence of 2DG and R/A. Statistical differences were evaluated by Student t-test. n = 8

The extracellular acidification rate (ECAR), reflecting lactate production during glycolysis, revealed a slight but significant increase in basal glycolysis following sodium butyrate treatment (0–20 min, Fig. 3C-D).

These findings suggest that butyrate enhances both mitochondrial respiration and basal glycolysis in avian enterocytes. This dual effect could contribute to improved cellular energy metabolism and potentially support mechanisms that create an anoxic gut environment, reducing pathogenic bacterial colonization.

Released butyrate from butyric glycerides, but not the intact butyric glycerides, enhanced oxygen rate consumption by chicken enterocytes

The effects of butyric glycerides on metabolic pathways in chicken enterocytes were evaluated using experiments similar to those described previously for sodium butyrate (Fig. 3). Considering the potential hydrolysis of butyric glycerides in the GIT by lipases, which releases free butyrate, glycerol and partially cleaved glycerides (Sampugna et al. 1967; Gu et al. 2022), experiments were conducted with both undigested and the product of in vitro digested butyric glycerides. At jejunum level, the in vitro digestion released 55.1 and 60.7% of initial butyrate contained in buyric glycerides and tributyrin, respectively, confirming the progressive release of free butyrate in the GIT. Controls included pretreatment with glycerol and digestion medium (Fig. 4).

Fig. 4.

Fig. 4

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Effects of butyric glycerides, before and after pancreatin-digestion, on chicken enterocytes mitochondrial and glycolytic activities 8E11chicken enterocytes were cultured for 72 h in presence, or not (control: light blue), of glycerol (dark blue), butyric glycerides (dark green) and digested butyric glycerides (light green) at 5 mM of butyric acid before seahorse measurements: oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) (A) The right panel shows OCR means (± SD) at each time point measurement. Left panel shows the OCR means (± SD) of basal (0–20 min), maximal (20–37 min) and non-mitochondrial OCR (> 37 min) OCR. (B) Area under curve (AUC) of maximal OCR (± SD) from 20 to 37 min, in presence of 2-deoxyglucose (2DG) and absence of rotenone and antimycin (R/A). (C) The right panels show ECAR means (± SD) at each time point measurement. Left panels show the ECAR means (± SD) of basal (0–20 min), ECAR after glycolysis pathway (20–37 min) and mitochondrial pathway inhibition (> 37 min). (D) Area under curve (AUC) of basal ECAR (± SD) from 0 to 20 min, in absence of 2DG and R/A. Statistical differences were evaluated by 1-way-ANOVA and Tukey’s honestly significant difference (HSD). n = 8

Only free butyrate, released following the digestion of butyric glycerides, significantly increased both basal (Fig. 4A, 0–20 min) and maximal (Fig. 4B) OCR. Undigested butyric glycerides did not affect the mitochondrial respiration, as no changes in OCR were observed compared to the glycerol and digestion medium controls (Fig. 4A-B). These findings suggest that chicken enterocytes are unable to directly metabolize intact butyric glycerides for energy production.

Released butyrate after digestion also significantly enhanced glycolytic activity, as indicated by increased ECAR, compared to controls (Fig. 4C-D). Conversely, undigested butyric glycerides significantly reduced glycolytic activity in chicken enterocytes (Fig. 4C-D, P < 0.001).

Similar results were observed with cleaved and non-cleaved tributyrin (Supp. Fig. 3). Only free butyric acid released from tributyrin improved the metabolic profile of chicken enterocytes, indicating the importance of butyrate liberation for its metabolic effects. These data highlight that the metabolic benefits of butyric glycerides in chicken enterocytes depend on their digestion and release of free butyrate in the GIT.

Discussion

Our results indicate that supplementing chicken diets with a mixture of butyric glycerides, including mono-, di- and tributyrin, ensures a sustained delivery of butyrate throughout all the gut segments. This provides a continuous source of energy for enterocytes and may promote a beneficial shift in the gut’s bacterial ecology. Additionally, butyric glycerides exhibited a direct protective effect by preventing bacterial adhesion and mitigating toxicity to enterocytes. In summary, butyric glycerides exert both direct and indirect effects on chicken enterocytes, enhancing their resistance to pathogen colonization and supporting greater resilience against infection and colonization by pathogenic bacteria.

The supplementation of chicken feed with butyric glycerides is a common strategy employed to deliver butyrate throughout the gastrointestinal tract, with the aim of improving gut health thanks to its well-known effects on epithelial barrier and anti-inflammatory properties (Ahsan et al. 2016). Moreover, previous studies have highlighted the direct antimicrobial effects of α-monobutyrin against bacterial pathogens such as S. Typhimurium and C. perfringens (Sun et al. 2003; Namkung et al. 2011). In this study seeks to investigate additional potential modes of actions of butyric glycerides mixtures.

To evaluate the effects of butyric glycerides on the adhesion of pathogenic bacteria to chicken enterocyte, experiments were conducted, revealing that butyric glycerides inhibit the adhesion of C. jejuni and S. Typhimurium to chicken enterocytes, thereby enhancing the cellular resistance to bacterial colonization. Furthermore, incubation of chicken enterocytes with butyric glycerides protected them from the cytotoxic effects of α-toxin produced by C. perfringens. While butyrate showed high efficacy in preventing pathogen adhesion, this was observed only at high concentration(10 mM, Supp. Fig. 2), that are rarely achieved physiologically in the small intestine, where concentrations typically range from 0.1 mM to 0.4 mM in the ileum (Józefiak et al. 2013; Ren et al. 2019). This suggest that butyric glycerides directly affect enterocytes, inducing functional changes that enhance their resistance to bacterial adhesion and toxin cytotoxicity.

Although butyrate is well established as a primary energy source for enterocytes (Rombeau et al. 1990), various and often controversial mechanisms have been proposed to explain its effects and influence on other metabolic pathways, including glycolysis. The prevailing consensus is that butyrate is oxidized directly in the mitochondria (Rombeau et al.; Park et al. 2022). However, this oxidation process is significantly impaired in colonic cancer cells (Chapman et al. 1994), necessitating caution when employing immortalized or cancerous in vitro models. The levels of butyrate oxidation in mitochondria, in both healthy and cancer conditions, depends on the balance of pyruvate kinase isoforms M1 and M2 (PKM1/2), which is altered in cancerous colonocytes and indirectly affects glycolytic activity(Park et al. 2022). Other models have demonstrated that butyrate induces pyruvate flux into the mitochondria of enterocytes, reducing glycolytic activity and leading to fatty acids accumulation in the cytosol (Bekebrede et al. 2021). In this study, we show that sodium butyrate enhances both mitochondrial and glycolytic activities (Fig. 3), suggesting that 8E11 chicken enterocytes may exhibit similar profile to healthy mammalian enterocytes with high PKM1 activity unlike cancerous colonocytes (Park et al. 2022). These characteristics make the 8E11 enterocytes a suitable model for studying the metabolism of healthy chicken enterocytes. To the best of our knowledge, these results provide the first evidence that both glycolysis and mitochondrial activity are increased in the presence of butyrate on chicken enterocytes, likely through its direct oxidation (Park et al. 2022). Consequently, butyrate increases cellular energy production, supporting crucial cellular functions such as activation, proliferation, differentiation, biosynthesis and adaptation to microenvironmental changes.

Butyric glycerides supplemented in chicken feed are progressively cleaved in the gut lumen, releasing of less-branched butyric glycerides, glycerol, and, most importantly, free butyrate (Moquet et al. 2018). Glycerol and undigested butyric glycerides showed no effect on basal and maximal mitochondrial activity (Fig. 4 and Supp. Fig. 3), indicating that chicken enterocytes are unable to oxidize these forms or use them as energy sources. However, cleaved butyric glycerides (derived from either the glycerides mixture or tributyrin alone) significantly enhanced both basal and maximal mitochondrial and glycolytic activities, as evidenced by OCR and ECAR measurements (Fig. 4 and Supp. Fig. 3). These findings show that, in addition to their direct protective effects against pathogens adhesion and cytotoxicity, butyric glycerides can serve as source of energy for enterocytes, but only after cleavage by lipase in GIT. Like mammals, these results demonstrate, for the first time, the ability of avian enterocytes to utilize butyrate as an energy source, either when supplemented as sodium butyrate or derived from cleaved butyric glycerides. Nonetheless, the in vivo efficacy of butyric glycerides is likely greater due to their capacity to deliver butyrate throughout the digestive tract.

The oxidation of butyrate by enterocytes results in increased CO2 levels the in gut lumen (Rombeau et al. 1990). Prior studies have shown that hypoxic environment created by butyrate oxidation induces an anaerobic shift in microbial ecology in mice, favouring anaerobic bacteria and inhibiting the growth of aerobic pathogens such as S. Typhimurium (Rivera-Chávez et al. 2016). In this context, it has been reported that butyrate treatment stabilizes hypoxia-induced transcription factor (HIF) of colonocytes (Kelly et al. 2015), which plays a crucial role in maintaining the intestinal epithelial barrier (Colgan et al. 2016).

To confirm the effects of butyric glycerides on the gut ecology in chicken, further in vivo studies are needed under both healthy and challenged conditions, including assessments of microbial composition, activity and pathogen colonisation in chickens fed diets supplemented or not with butyric glycerides. Numerous studies have demonstrated that butyrate positively influence gut microbiota in chickens by stimulating beneficial populations and reducing potential pathogens (Hofacre et al. 2020; Deleu et al. 2021). However, the mechanisms behind these effects remain incompletely understood notably for butyric glycerides mixtures.

Taken together, our findings indicate that butyric glycerides exert two complementary mechanisms on chicken’s enterocytes. First, they induce direct functional changes on chicken enterocytes reducing their susceptibility pathogen adhesion and toxicity. These protective effects were more pronounced with butyric glycerides compared to sodium butyrate. Second, enterocytes exhibit indirect metabolic adaptations that may promote a beneficial ecological shift within the gut microbiota which in turn may disfavour aerobic pathogen colonization and infection. This dual action may ultimately enhance animal resilience against microbial challenges.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 396 KB) (395.9KB, docx)

Acknowledgements

We kindly acknowledge Dr. Quentin Mièvre and analytics team of Adisseo’s R&I department for their help and support with chromatography quantification.

Author contributions

A. M., M. M., L. C. and J. C. designed the study. The experiments were performed by M. M., L. C., and V. M.. The results were analysed and interpreted by all authors. Resources were provided by J. C.. The Manuscript was written by A. M., J. C., M. M., and L. C. and revised by all the authors.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary figures. Any other information or data are available from the corresponding authors upon request.

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Not applicable for the current manuscript.

Competing interests

The authors declare no competing financial interests that could have influenced the integrity of the research. While our organization is involved in the development and production of feed additives, including various forms of protected butyrate, this study was conducted with a commitment to scientific rigor and objectivity.

Footnotes

Publisher’s Note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file 1 (DOCX 396 KB) (395.9KB, docx)

Data Availability Statement

All data supporting the findings of this study are available within the paper and its Supplementary figures. Any other information or data are available from the corresponding authors upon request.

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