The poultry industry faces increasing pressure to reduce antibiotic use while maintaining optimal growth performance and disease resistance. Medium-chain fatty acids, particularly lauric acid, have emerged as promising alternatives to conventional growth promoters. This study investigated the dose-dependent effects of dietary lauric acid supplementation on immune cell populations and tissue distribution in broiler chickens. A total of five experimental groups were established, including a control group receiving standard diet and four treatment groups supplemented with lauric acid at 375, 500, 750, and 1000 g/MT feed. Flow cytometric analysis revealed significant dose-dependent increases in B lymphocyte populations (Bu1+ cells), with the 750 g/MT group showing maximum enhancement (28.82 ± 1.03%) compared to control (20.73 ± 0.52%). Pharmacokinetic analysis demonstrated sustained plasma lauric acid concentrations over 48 hours, with dose-proportional increases across treatment groups. Tissue distribution studies showed preferential accumulation in respiratory tissues, with lung and trachea concentrations reaching 8.98 ± 0.21 μg/ml and 6.45 ± 0.25 μg/ml respectively in the highest dose group. These findings suggest that dietary lauric acid supplementation at 750 g/MT provides optimal immunomodulatory effects in broiler chickens, supporting its potential as a natural feed additive for enhancing immune function and respiratory health.
The global poultry industry continues to face substantial challenges related to disease control, antibiotic resistance, and food safety concerns. The extensive use of antibiotic growth promoters (AGPs) in commercial poultry production has led to regulatory restrictions in many countries, necessitating the development of alternative strategies to maintain productivity while ensuring animal health and welfare [1]. Among the various alternatives being investigated, medium-chain fatty acids (MCFAs) have garnered considerable attention due to their multifaceted biological activities, including antimicrobial, immunomodulatory, and metabolic effects [2][3].
Lauric acid (C12:0), a saturated MCFA predominantly found in coconut oil and palm kernel oil, has demonstrated potent antimicrobial properties against various bacterial and viral pathogens [4]. The antimicrobial mechanism of lauric acid involves disruption of lipid membranes of enveloped viruses and gram-positive bacteria, making it particularly effective against common poultry pathogens [5]. Beyond its direct antimicrobial effects, lauric acid has been shown to modulate host immune responses, influence gut microbiota composition, and enhance nutrient metabolism in poultry [6][7].
The avian immune system comprises both innate and adaptive components, with B and T lymphocytes playing critical roles in humoral and cell-mediated immunity respectively [8]. B lymphocytes, characterized by the Bu1+ surface marker in chickens, develop primarily in the bursa of Fabricius and are responsible for antibody production [36]. T helper cells (CD4+) orchestrate immune responses by regulating both cellular and humoral immunity through cytokine production and direct cell-cell interactions [10]. Previous studies have demonstrated that dietary interventions can significantly influence lymphocyte populations and functional immune responses in broiler chickens [11][12].
Recent investigations have revealed that lauric acid supplementation can enhance growth performance, modulate serum metabolome, and alter gut microbiota composition in broilers [6]. Furthermore, lauric acid has been shown to improve intestinal morphology, reduce inflammatory responses, and enhance antioxidant capacity under challenge conditions [14][15]. However, comprehensive dose-response relationships and tissue distribution patterns of lauric acid in broiler chickens remain insufficiently characterized. Understanding the pharmacokinetics and tissue-specific accumulation of lauric acid is essential for optimizing supplementation strategies and elucidating mechanisms of action.
The respiratory tract represents a critical target tissue for disease prevention in poultry, as respiratory pathogens cause substantial economic losses in commercial production [16]. The potential for lauric acid to accumulate in respiratory tissues and exert local antimicrobial and immunomodulatory effects has not been extensively investigated. Additionally, the dose-dependent effects of lauric acid on specific immune cell populations require detailed characterization to establish optimal supplementation levels for practical application.
This study was designed to evaluate the dose-dependent effects of dietary lauric acid supplementation on immune cell profiles, plasma pharmacokinetics, and tissue distribution in broiler chickens. Specific objectives included: (1) quantification of B and T lymphocyte populations using flow cytometry; (2) characterization of plasma lauric acid concentrations over time following dietary supplementation; and (3) determination of lauric acid accumulation in respiratory tissues. The findings from this investigation provide critical insights into the immunomodulatory mechanisms of lauric acid and establish evidence-based recommendations for its application in commercial broiler production.
Experimental Design and Animal Management
The experimental protocol was designed to evaluate dose-response relationships of dietary lauric acid supplementation in broiler chickens. Five experimental groups were established with appropriate sample sizes for statistical analysis. Group 1 (G1) served as the control group receiving standard commercial broiler diet without lauric acid supplementation. Treatment groups received the basal diet supplemented with lauric acid at four different concentrations: Group 2 (G2) at 375 g/MT, Group 3 (G3) at 500 g/MT, Group 4 (G4) at 750 g/MT, and Group 5 (G5) at 1000 g/MT feed.
All experimental procedures were conducted in accordance with standard guidelines for the care and use of agricultural animals in research. Birds were housed in conventional open pen sytem with ad libitum access to feed and water throughout the experimental period. Temperature, humidity, and lighting conditions were maintained according to commercial broiler production standards to ensure optimal growth and welfare.
The test compound, designated as FineX 3060, containing lauric acid as the active ingredient, was incorporated into the basal diet at the specified concentrations. The basal diet was formulated to meet or exceed the nutritional requirements for broiler chickens as recommended by standard poultry nutrition guidelines. Feed was manufactured using standard mixing procedures to ensure homogeneous distribution of the lauric acid supplement throughout the diet. Dietary treatments were maintained consistently throughout the experimental period.
Flow Cytometric Analysis of Lymphocyte Populations
Peripheral blood samples were collected for immunophenotyping of B and T lymphocyte subsets. Blood samples were processed for flow cytometric analysis using standard protocols for avian lymphocyte identification. B lymphocytes were identified using anti-Bu1 monoclonal antibody conjugated to appropriate fluorochrome, as Bu1 is a well-established surface marker for chicken B cells [17]. T helper cells were identified using anti-CD4 monoclonal antibody, which specifically recognizes the CD4 surface marker on helper T lymphocytes in chickens [18].
Flow cytometric acquisition was performed using a calibrated flow cytometer with appropriate optical configuration for multi-parameter analysis. Forward scatter (FSC) and side scatter (SSC) parameters were used to identify lymphocyte populations based on size and granularity characteristics. A minimum of 10,000 events were acquired per sample to ensure statistical reliability. Data analysis was performed using specialized flow cytometry software, with gating strategies designed to exclude debris, doublets, and non-lymphocyte populations. Results were expressed as percentage of positive cells within the lymphocyte gate.
Blood samples were collected at predetermined time points to characterize the pharmacokinetic profile of lauric acid following dietary supplementation. Sampling time points were established at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours to capture both the absorption phase and steady-state concentrations. Plasma was separated by centrifugation and stored at appropriate temperature until analysis.
Lauric acid concentrations in plasma were quantified using validated analytical methodology. Sample preparation involved lipid extraction followed by derivatization procedures suitable for chromatographic analysis. Quantification was performed using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) with appropriate internal standards for accurate measurement. Calibration standards and quality control samples were included in each analytical batch to ensure accuracy and precision of measurements.
At the terminal sampling point, birds were humanely euthanized, and tissue samples were collected for lauric acid distribution analysis. Lung and trachea tissues were collected, weighed, and processed for lauric acid quantification. Three animals per group (designated A1, A2, and A3) were utilized for tissue distribution studies to provide biological replication.
Tissue samples were homogenized using appropriate mechanical disruption methods, followed by lipid extraction procedures optimized for fatty acid recovery. Lauric acid concentrations in tissue homogenates were quantified using the same analytical methodology employed for plasma samples, with appropriate matrix-matched calibration standards. Results were expressed as micrograms of lauric acid per milliliter of tissue homogenate (μg/ml). Tissue concentrations were compared across treatment groups to assess dose-dependent accumulation patterns.
All data were subjected to appropriate statistical analysis to determine treatment effects and dose-response relationships. Descriptive statistics including mean and standard deviation (SD) were calculated for all measured parameters. Flow cytometry data for B and T lymphocyte populations were analyzed separately for each cell type. Pharmacokinetic data were evaluated at each time point, and area under the curve (AUC) calculations were performed to assess overall systemic exposure.
For tissue distribution data, individual animal values were recorded, and group means with standard deviations were calculated. The limit of detection for lauric acid in control group samples was established, with concentrations below detection limits designated as “ND” (not detected). Statistical comparisons between treatment groups were performed, with significance established at appropriate probability levels. All statistical analyses were conducted using Graphpad prism 5.0.
Effects on B Lymphocyte Populations
Flow cytometric analysis of peripheral blood B lymphocytes revealed substantial dose-dependent effects of dietary lauric acid supplementation (Table 1). The control group (G1) receiving standard diet exhibited a baseline B cell percentage of 20.73 ± 0.52%. Supplementation with lauric acid at 375 g/MT (G2) resulted in an increase to 23.76 ± 0.77%, representing a 14.6% enhancement compared to control.
Table 1: Flow cytometric analysis of B and T lymphocyte populations in peripheral blood. Values represent mean ± SD.
Progressive increases in B cell percentages were observed with increasing lauric acid concentrations. The 500 g/MT group (G3) demonstrated 25.80 ± 0.42% B cells, while the 750 g/MT group (G4) exhibited the maximum B cell percentage at 28.82 ± 1.03%, representing a 39.0% increase relative to control. Interestingly, the highest supplementation level of 1000 g/MT (G5) showed a reduction in B cell percentage to 23.10 ± 0.50%, suggesting a potential plateau or optimal dose effect at intermediate concentrations. The standard deviation values indicated consistent measurements within groups, supporting the reliability of the observed dose-response pattern.
Effects on T Helper Lymphocyte Populations
T helper cell (CD4+) populations exhibited a different response pattern compared to B lymphocytes (Table 1). The control group demonstrated a CD4+ cell percentage of 51.54 ± 1.70%. All lauric acid supplementation groups showed numerical reductions in CD4+ percentages relative to control, though the biological significance of these modest changes requires careful interpretation.
The 375 g/MT group (G2) showed 49.52 ± 0.74% CD4+ cells, while the 500 g/MT group (G3) exhibited 48.89 ± 1.00%. The 750 g/MT group (G4) demonstrated the lowest CD4+ percentage at 46.05 ± 0.80%, representing a 10.7% reduction compared to control. The highest dose group (G5) showed 49.45 ± 1.27% CD4+ cells. The inverse relationship between B and T helper cell percentages suggests potential compensatory mechanisms in lymphocyte homeostasis, as total lymphocyte populations maintain relatively constant proportions within the circulation.
Plasma Pharmacokinetics of Lauric Acid
Plasma lauric acid concentrations demonstrated clear dose-dependent patterns across all treatment groups and time points (Table 2). The control group (G1) exhibited baseline lauric acid concentrations ranging from 1.31 ± 0.10 μg/ml at time zero to 2.59 ± 0.35 μg/ml at 1 hour, likely representing endogenous lauric acid from standard dietary lipids and metabolic processes.
Table 2: Plasma concentrations of lauric acid (μg/ml) over 48 hours following dietary supplementation. Values represent mean ± SD. G1 = control; G2 = 375 g/MT; G3 = 500 g/MT; G4 = 750 g/MT; G5 = 1000 g/MT.
The 375 g/MT supplementation group (G2) demonstrated substantially elevated plasma concentrations, with initial levels of 10.54 ± 0.26 μg/ml at baseline, representing an 8-fold increase compared to control. Plasma concentrations in G2 remained relatively stable throughout the 48-hour sampling period, ranging from 10.04 ± 0.83 to 12.64 ± 1.04 μg/ml, indicating sustained systemic exposure following continuous dietary supplementation.
The 500 g/MT group (G3) exhibited baseline concentrations of 15.45 ± 1.56 μg/ml, with peak concentrations reaching 19.25 ± 0.48 μg/ml at 24 hours. The 750 g/MT group (G4) showed initial concentrations of 20.62 ± 0.65 μg/ml, with maximum levels of 25.54 ± 0.65 μg/ml observed at 1-hour post-sampling initiation. The highest supplementation level of 1000 g/MT (G5) produced the most substantial plasma concentrations, with baseline values of 28.57 ± 2.11 μg/ml and peak concentrations of 46.10 ± 1.81 μg/ml at 1 hour.
The sustained plasma concentrations observed across all timepoints reflect the continuous nature of dietary supplementation, where lauric acid intake occurs throughout the day rather than as a single bolus dose. The relatively stable concentrations over 48 hours suggest achievement of steady-state kinetics under continuous feeding conditions. The dose-proportional increases in plasma concentrations indicate predictable pharmacokinetic behavior across the tested dose range.
Tissue Distribution in Respiratory Organs
Analysis of lauric acid distribution in lung and tracheal tissues revealed dose-dependent accumulation patterns with substantial tissue-specific concentrations (Table 3). In the control group (G1), lauric acid was not detected (ND) in either lung or tracheal tissue samples, indicating that tissue concentrations from endogenous sources were below the analytical detection limit.
Table 3: Tissue distribution of lauric acid (μg/ml) in lungs and trachea. Individual bird values (A1, A2, A3) and group means ± SD are presented. ND = not detected.
In lung tissue, the 375 g/MT group (G2) showed mean concentrations of 3.26 ± 0.77 μg/ml, with individual animal values ranging from 2.45 to 3.99 μg/ml. The 500 g/MT group (G3) exhibited lung concentrations of 4.22 ± 0.61 μg/ml, representing a 29.4% increase compared to G2. Progressive dose-dependent increases were observed, with G4 (750 g/MT) achieving 6.29 ± 1.21 μg/ml and G5 (1000 g/MT) reaching the highest lung concentrations of 8.98 ± 0.21 μg/ml.
Tracheal tissue demonstrated similar dose-dependent accumulation patterns, though absolute concentrations were generally lower than corresponding lung values. The 375 g/MT group showed tracheal concentrations of 1.77 ± 0.21 μg/ml, increasing to 3.68 ± 0.52 μg/ml in the 500 g/MT group. The 750 g/MT and 1000 g/MT groups exhibited tracheal concentrations of 5.56 ± 0.55 and 6.45 ± 0.25 μg/ml respectively.
The ratio of lung to tracheal concentrations remained relatively consistent across treatment groups, ranging from 1.15 to 1.84, suggesting similar tissue penetration characteristics despite anatomical and physiological differences between these respiratory structures. Inter-animal variability, as reflected by standard deviation values, remained modest across treatment groups, indicating consistent tissue accumulation patterns within dose groups.
This study provides comprehensive evidence for dose-dependent immunomodulatory effects and tissue distribution patterns of dietary lauric acid supplementation in broiler chickens. The findings demonstrate significant enhancement of B lymphocyte populations, sustained plasma pharmacokinetics, and substantial accumulation in respiratory tissues, supporting the potential application of lauric acid as a natural feed additive for improving immune function in commercial poultry production.
The observed dose-dependent increase in B lymphocyte percentages represents a significant finding with important implications for humoral immunity in broilers. B lymphocytes play essential roles in antibody production and adaptive immune responses against bacterial and viral pathogens [19]. The maximum enhancement of B cells at the 750 g/MT dose level, with a subsequent decrease at 1000 g/MT, suggests an optimal dose range for immunostimulatory effects. This biphasic dose-response pattern has been observed with other immunomodulatory compounds and may reflect complex regulatory mechanisms involving cytokine networks and lymphocyte homeostasis [20].
Previous investigations have demonstrated that lauric acid modulates immune function through multiple mechanisms, including direct effects on immune cell membranes, alteration of signaling pathways, and modification of inflammatory mediator production [13][15]. The enhancement of B cell populations observed in this study aligns with previous reports showing that medium-chain fatty acids can stimulate lymphocyte proliferation and activation [21]. The bursa of Fabricius, the primary site of B cell development in chickens, may be particularly responsive to dietary fatty acid composition, potentially explaining the substantial effects observed on circulating B cell percentages [9].
The modest reduction in T helper cell percentages accompanying B cell increases likely reflects physiological mechanisms maintaining lymphocyte homeostasis rather than immunosuppressive effects. Total lymphocyte counts typically remain relatively constant, and reciprocal changes in lymphocyte subset proportions are commonly observed [22]. Furthermore, the absolute numbers of CD4+ cells may remain unchanged or even increase despite proportional reductions, emphasizing the importance of considering both relative and absolute cell counts when interpreting immunophenotyping data.
The immunomodulatory effects of lauric acid may involve multiple molecular mechanisms. Lauric acid has been shown to influence lipid raft composition in cell membranes, potentially affecting receptor clustering and signal transduction pathways critical for lymphocyte activation [23]. Additionally, lauric acid and its metabolites can modulate gene expression patterns through effects on transcription factors and epigenetic mechanisms [24]. The sustained plasma concentrations observed in this study provide continuous exposure of immune tissues to lauric acid, potentially explaining the substantial immunological effects observed.
The pharmacokinetic data reveal sustained plasma lauric acid concentrations over the 48-hour sampling period, reflecting the continuous nature of dietary supplementation in commercial poultry production. Unlike single-dose administration studies, continuous feeding results in steady-state kinetics where input from dietary absorption balances elimination through metabolism and incorporation into tissues [25]. The dose-proportional increases in plasma concentrations across treatment groups indicate predictable absorption and distribution characteristics.
Medium-chain fatty acids, including lauric acid, undergo absorption and metabolism through pathways distinct from long-chain fatty acids [26]. Following intestinal absorption, lauric acid is transported via the portal circulation to the liver, where it undergoes β-oxidation or incorporation into lipoproteins for systemic distribution [27]. The relatively rapid achievement of steady-state concentrations observed in this study suggests efficient absorption and distribution kinetics.
The sustained plasma concentrations have important implications for biological activity. Continuous exposure of immune cells to lauric acid in circulation provides ongoing immunomodulatory stimulation rather than transient effects. Additionally, sustained plasma levels support consistent delivery of lauric acid to peripheral tissues, including the respiratory system, where local antimicrobial and anti-inflammatory effects may contribute to disease resistance [28].
Inter-individual variability in plasma concentrations, as indicated by standard deviation values, remained modest across most time points and dose groups. This consistency suggests predictable dose-response relationships suitable for practical application in commercial production settings. However, factors such as individual variation in feed intake, digestive function, and metabolic capacity may influence actual lauric acid exposure in production environments, warranting consideration during implementation.
The substantial accumulation of lauric acid in lung and tracheal tissues represents a particularly significant finding given the importance of respiratory health in poultry production. Respiratory diseases caused by bacterial and viral pathogens impose substantial economic losses through mortality, reduced growth performance, and increased medication costs [35]. The preferential accumulation of lauric acid in respiratory tissues may provide localized antimicrobial and immunomodulatory effects that enhance resistance to respiratory pathogens [29].
The antimicrobial properties of lauric acid against respiratory pathogens have been demonstrated in vitro, with efficacy against gram-positive bacteria and enveloped viruses [30]. The tissue concentrations achieved in this study (up to 8.98 μg/ml in lung tissue) may be sufficient to exert direct antimicrobial effects against colonizing pathogens. Additionally, lauric acid can modulate local inflammatory responses in respiratory tissues, potentially reducing excessive inflammation that contributes to tissue damage during infection [14].
The dose-dependent accumulation pattern observed in both lung and trachea indicates predictable tissue distribution characteristics. The slightly higher concentrations in lungs compared to trachea may reflect differences in blood perfusion, tissue composition, or active transport mechanisms. Both tissues demonstrated clear dose-response relationships, supporting the ability to modulate tissue concentrations through dietary supplementation levels.
The mechanism of lauric acid accumulation in respiratory tissues likely involves incorporation into cellular phospholipids and tissue lipid pools. Medium-chain fatty acids can be incorporated into membrane phospholipids, potentially altering membrane fluidity and affecting cellular functions [31]. This incorporation may provide sustained local effects beyond the duration of plasma exposure, contributing to prolonged biological activity.
Integration of the immunological and pharmacokinetic findings suggests that the 750 g/MT supplementation level provides optimal effects for practical application. This dose level produced maximum B cell enhancement, substantial plasma concentrations, and significant respiratory tissue accumulation while avoiding potential adverse effects that might occur at higher doses. The reduction in B cell percentages observed at 1000 g/MT suggests that excessive supplementation may not provide additional benefits and could potentially interfere with normal immune regulation.
Economic considerations are important when determining optimal supplementation levels for commercial applications. While the 1000 g/MT dose produced highest tissue concentrations, the marginal increase compared to 750 g/MT may not justify the additional cost. Feed cost represents a major component of poultry production expenses, and supplementation strategies must balance biological efficacy with economic feasibility [32].
The findings of this study align well with previous investigations examining lauric acid effects in poultry. Studies by Zhang et al. [6] demonstrated that lauric acid supplementation improved growth performance and immune responses in broilers, though their study utilized different dose levels and assessment methods. The tissue distribution data extends previous knowledge by providing quantitative measurements of lauric acid accumulation in respiratory tissues, an aspect not thoroughly characterized in earlier investigations.
Research by Zhao et al. [14] examining lauric acid monoglyceride and cinnamaldehyde combinations reported improvements in intestinal morphology and inflammatory markers, supporting the anti-inflammatory properties of lauric acid observed indirectly through immunological measurements in the current study. The enhancement of B cell populations observed here complements previous findings showing beneficial effects on humoral immune responses.
Studies examining medium-chain fatty acids in other livestock species have reported similar immunomodulatory and antimicrobial effects [33]. This cross-species consistency supports fundamental mechanisms of action that may be broadly applicable across animal production systems. However, species-specific differences in immune system organization, particularly the unique role of the bursa of Fabricius in avian immunity, warrant caution when directly extrapolating findings between species.
The results of this study have direct implications for commercial broiler production. Implementation of lauric acid supplementation at 750 g/MT could enhance immune function, potentially reducing the disease incidence and improving production efficiency. Respiratory tissue accumulation may be particularly beneficial in production systems where respiratory challenges are common, such as high-density housing or environments with suboptimal air quality.
Integration of lauric acid supplementation into comprehensive health management programs could reduce reliance on antibiotic interventions while maintaining or improving flock health. This approach aligns with global initiatives to reduce antibiotic use in animal production and consumer preferences for products raised with minimal pharmaceutical interventions [34]. The natural origin of lauric acid from coconut and palm kernel sources may provide marketing advantages for products from supplemented flocks.
Limitations and Future Directions
Several limitations of this study warrant consideration. The investigation focused on immunological endpoints and tissue distribution but did not include molecular mechanisms underlying the immunomodulatory effects warrant further investigation. Gene expression studies examining effects on immune-related genes, cytokine production, and signaling pathways would elucidate mechanisms of action. Microbiome analyses could reveal effects on gut microbial composition and metabolic activity that may contribute to systemic immune modulation.
This study demonstrates that dietary lauric acid supplementation produces dose-dependent immunomodulatory effects and achieves substantial accumulation in respiratory tissues of broiler chickens. The optimal supplementation level of 750 g/MT enhanced B lymphocyte populations by 39%, maintained sustained plasma concentrations, and achieved lung tissue concentrations of 6.29 μg/ml. These findings support the potential application of lauric acid as a natural feed additive for enhancing immune function and respiratory health in commercial broiler production.
The enhancement of B cell populations suggests improved capacity for humoral immune responses, while respiratory tissue accumulation provides localized antimicrobial and immunomodulatory effects relevant to disease resistance. The dose-response relationships characterized in this investigation establish evidence-based recommendations for practical implementation.
Further research should evaluate protective efficacy against specific pathogens, long-term production performance effects, and molecular mechanisms of immune modulation. The integration of lauric acid supplementation into comprehensive health management programs offers a promising strategy for reducing antibiotic dependence while maintaining optimal flock health and productivity in modern poultry production systems.