The study protocol was reviewed and approved by the Animal Ethics Committee of Kasetsart University, Bangkok, Thailand (Approval No. ACKU66-AGR-018). All procedures involving laying hens were conducted in accordance with the approved institutional protocol and applicable guidelines for the ethical care and use of animals in research. The study was designed to minimize animal use and discomfort while obtaining scientifically valid data, consistent with the principles of replacement, reduction, and refinement.

Only clinically healthy LB and WL laying hens were included. Before enrolment, all birds were visually examined and confirmed to be free from apparent clinical signs of disease. The hens were maintained under uniform housing, feeding, lighting, ventilation, vaccination, and management conditions at the Animal Research Farm, Department of Animal Science, Kasetsart University. Animal health and welfare were monitored throughout the study, and blood sampling was performed by trained personnel using appropriate handling and restraint procedures to minimize stress and discomfort.

A total of 2 mL of blood was collected once from the brachial vein of each hen into an ethylenediaminetetra-acetic acid-coated tube for hematological analysis and peripheral blood mononuclear cell isolation. The sampling volume was limited to that required for the planned analyses, and no experimental infection, surgical intervention, prolonged restraint, or terminal procedure was performed. Following blood collection, the birds remained under routine farm management and observation. The sample size was limited to the minimum number of animals deemed necessary, based on animal availability, ethical considerations, and previous poultry studies evaluating hematological and immune-related gene expression parameters.

The experiment was conducted from September 2023 to September 2025 at the Animal Research Farm, Department of Animal Science, Kasetsart University, Thailand.

A total of 20 clinically healthy laying hens were selected from LB (population size = 1,381) and WL (population size = 79) flocks at 42 and 80 weeks of age. All birds were visually examined and confirmed to be free from clinical signs of disease. Five hens were then randomly chosen from each breed and age group under the same housing and management conditions for blood collection and subsequent analyses. All hens were housed in conventional layer cages under the same environmental and management conditions. The house was equipped with mechanical ventilation and maintained in accordance with standard commercial management practices. A 16-h light program was provided throughout the study. Birds were fed a commercial diet (120 g/day; 17% crude protein and 3,500 kcal ME/kg) with ad libitum access to water. All birds were clinically healthy at the time of sampling and showed no apparent signs of disease. The flock was managed according to the routine vaccination program of the research farm. A formal a priori statistical power analysis was not performed. The sample size was determined based on ethical considerations, animal availability, and previous poultry studies evaluating hematological and immune-related gene expression parameters.

Blood samples (2 mL) were randomly collected from the brachial vein of laying hens (n = 5 per breed and age) into ethylenediaminetetraacetic acid-coated tubes to prevent coagulation. Each sample was divided into two aliquots: 1 mL for hematological analysis and 1 mL for PBMC isolation. Blood samples were transported to the laboratory immediately after collection and processed on the same day. Hematological analyses were performed within a few hours of collection to minimize storage-related alterations in blood cell characteristics. PBMC isolation was performed immediately after blood collection.

Hematological parameters, including red blood cells (RBC), hemoglobin, hematocrit, white blood cells (WBC), lymphocytes, monocytes, basophils, eosinophils, and heterophils, were determined using a Neubauer hemocytometer following dilution (1:200) with Natt and Herrick solution, according to the method described by [9].

PBMCs were chosen as they represent a readily accessible, non-invasive source of immune cells that reflect systemic innate immune status. PBMCs were isolated from whole blood following the method of [10]. Briefly, 1 mL of blood was layered over 2 mL of Lymphoprep™ (Stemcell Technologies, Cologne, Germany) on first use and centrifuged at 277 × g for 30 min. The mononuclear cell layer was collected and stored at −80 °C for further analysis.

Total RNA was extracted from PBMCs using a QIAamp RNA Mini Kit (Qiagen, Courtaboeuf, France) on first use according to the manufacturer’s instructions. RNA purity was assessed using a NanoDrop spectrophotometer (A260/280 ratio of 1.80–2.10). Gene expression analysis was performed using a MyGo Pro® real-time polymerase chain reaction (PCR) system (IT-IS Life Science Ltd., Middlesbrough, UK) on first use with the QuantiNova SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) on first use. This one-step RT-qPCR system combines reverse transcription and quantitative PCR amplification in a single reaction. Reactions were prepared according to the manufacturer’s instructions, and amplification was performed on a real-time PCR instrument under the recommended cycling conditions. Each 20 µL reaction contained 10 µL of 2× master mix, 1 µL each of forward and reverse primers (10 µM), 0.2 µL of RT mix, 5 µL RNA template, and nuclease-free water. Amplification conditions consisted of pre-denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. Melting curve analysis was performed from 60 to 97 °C. Each sample was analyzed in duplicate, and gene expression levels were calculated from the quantification cycle ratio of the target gene to the housekeeping gene β-actin, which is widely used as a stable reference gene in avian gene expression studies [11]. Primer sequences are shown in Table 1.

Data were analyzed using the General Linear Model (GLM) procedure of SAS statistical software version 9.2 (SAS Institute Inc., Cary, NC, USA) in a completely randomized 2 × 2 factorial arrangement, with breed (LB and WL) and age (42 and 80 weeks) as the main factors. Prior to analysis, data were evaluated for normality and homogeneity of variance. When significant effects were detected, least squares means were compared using Tukey’s multiple comparison test. Differences were considered significant at p < 0.05. The results were presented as least squares means with standard errors. The model used in statistics was:

Y_ijk = µ + α_i + β_j + (αβ)_ij + ε_ijk

where Y_ijk = the value of individual samples of the data from each replicate, µ = overall mean, α_i = breed effect (i = LB and WL), β_j = age effect (j = 42 and 80 weeks of age), (αβ)_ij = interaction between breed and age, and ε_ijk = the random residual error.

The hematological parameters were affected by laying hen breeds, including hemoglobin, lymphocyte count, and the heterophil-to-lymphocyte (H:L) ratio (Table 2). The results showed that the hemoglobin concentration in WL hens was significantly higher than in LB hens (15.32 ± 0.55 vs. 12.42 ± 0.59 g/dL) (p < 0.01). LB hens had a significantly higher lymphocyte level compared to WL hens (62.70 ± 2.54 vs. 53.90 ± 2.39%, respectively) (p < 0.05). Additionally, the H:L ratio in WL hens was significantly higher than in LB hens (1.07 ± 0.11 vs. 0.57 ± 0.12, respectively) (p < 0.01). However, there were no significant differences between breeds in basophil, eosinophil, hematocrit, heterophil, monocyte, RBC, and WBC counts (p > 0.05).

The influence of age on hematological parameters was determined. The results revealed that laying hen age affected hematocrit, hemoglobin, heterophil, lymphocyte, H:L ratio, monocyte, and WBC counts (p < 0.05) (Table 2). Laying hens at 80 weeks of age had a significantly higher hematocrit level compared to laying hens at 42 weeks of age (33.00 ± 1.11 vs. 27.40 ± 1.18%, respectively) (p < 0.01). Meanwhile, the hemoglobin concentration in laying hens at 80 weeks of age was significantly higher than in laying hens at 42 weeks of age (16.52 ± 0.55 vs. 11.22 ± 0.59 g/dL) (p < 0.001). The lymphocyte, monocyte, and WBC levels in laying hens at 80 weeks of age were 68.60 ± 2.39%, 2.10 ± 0.29%, and 10.88 ± 0.83 × 10³ cells/mm³, respectively. These values were 48.00 ± 2.54%, 1.10 ± 0.31%, and 6.68 ± 0.88 × 10³ cells/mm³, respectively, in laying hens at 42 weeks of age. However, the heterophil level in laying hens at 42 weeks of age was significantly higher compared to laying hens at 80 weeks of age (48.98 ± 2.90 vs. 27.60 ± 2.73%) (p < 0.001). In addition, the H:L ratio in laying hens at 42 weeks of age was 1.23 ± 0.12, which was significantly higher than in laying hens at 80 weeks of age (0.42 ± 0.11) (p < 0.001). There were no significant differences between ages in basophil, eosinophil, and RBC counts (p > 0.05).

The interactions between breed and age on hematological parameters are presented in Table 3. There were no significant differences in basophil, eosinophil, hematocrit, monocyte, RBC, and WBC counts (p > 0.05). Significant differences were observed in hemoglobin, heterophil, lymphocyte, and the H:L ratio. The hemoglobin concentrations were the lowest in the LB × 42 group (7.68 ± 0.87 g/dL) (p < 0.001). The heterophil level was the highest in the WL × 42 group (61.20 ± 3.87%) (p < 0.001). The lymphocyte level in the WL × 42 group was the lowest (36.00 ± 3.39%) (p < 0.001). The H:L ratio was the highest in the WL × 42 group (1.81 ± 0.16) (p < 0.001).

TLR2 gene expression showed significant differences between breeds (p < 0.001) and age (p < 0.01). WL hens had a higher expression level of the TLR2 gene than LB hens (0.84 ± 0.01 vs. 0.74 ± 0.01, respectively) (Figure 1).

The expression of the TLR2 gene in laying hens at 42 weeks of age was higher than in laying hens at 80 weeks of age (0.82 ± 0.01 vs. 0.76 ± 0.01, respectively) (Figure 1). The interaction between breed and age was significant for TLR2 gene expression (p < 0.001) (Figure 2). Notably, the highest TLR2 expression was observed in WL hens at 42 weeks (0.90 ± 0.02). This represents a novel breed × age interaction, suggesting heightened innate immune vigilance during peak production in this genetic line. The expression of the TLR2 gene in the WL × 80, LB × 42, and LB × 80 groups was 0.78 ± 0.02, 0.73 ± 0.02, and 0.74 ± 0.02, respectively. Pearson correlation analysis demonstrated a significant positive association between the H:L ratio and TLR2 gene expression (r = 0.784, p < 0.001) (Table 4). The pronounced elevation of the H:L ratio combined with high TLR2 expression in the WL × 42 group provides original evidence that genetic background modulates the relationship between production-related stress and innate immune activation.

This study provides original comparative data demonstrating that both breed and physiological stage significantly modulate hematological traits and TLR2-mediated innate immunity in laying hens, revealing patterns not previously reported in the literature. Both chicken breed and age significantly influenced hematological parameters in laying hens, reflecting differences in physiological status and immune regulation during the laying cycle. Breed significantly affected hemoglobin concentration, lymphocyte levels, and the heterophil-to-lymphocyte (H:L) ratio, whereas age influenced hematocrit, hemoglobin, heterophil, lymphocyte, monocyte, and total WBC counts. In addition, significant breed–age interactions were observed for hemoglobin, heterophils, lymphocytes, and the H:L ratio. Hemoglobin plays a key role in oxygen transport and reflects the oxygen-carrying capacity of blood [12], and lower hemoglobin concentrations may indicate reduced erythropoiesis or changes in metabolic demand [13]. In the present study, hens at 42 weeks of age exhibited lower hemoglobin levels than those at 80 weeks of age, consistent with previous findings indicating that hemoglobin levels decrease during the peak-laying period and increase again during later stages of production [4]. However, all hematological values remained within normal physiological ranges for healthy laying hens [4, 14], indicating that the birds were not anemic [15]. Age-related changes in hemoglobin levels may reflect adjustments in erythropoiesis associated with reproductive activity and metabolic demand during egg production [16, 17].

Differences in leukocyte profiles further indicated variations in immune responses between breeds and ages. LB hens showed higher lymphocyte levels than WL hens, suggesting breed-related differences in immune regulation and adaptability [13, 18]. In contrast, heterophil levels and the H:L ratio were higher in hens at 42 weeks than at 80 weeks, indicating increased physiological stress during the peak-laying period. Heterophils are primary phagocytic cells involved in innate immune defense against pathogens [19] and increases in heterophil counts accompanied by decreased lymphocytes are commonly associated with stress responses in poultry [20]. Accordingly, the H:L ratio is widely used as a physiological indicator of stress in birds [21], with higher values typically observed under stressful conditions [22]. Peak egg production typically exceeds 90% at around 42 weeks of age [23], which may increase metabolic and physiological demands and, consequently, elevate stress indicators [2]. The interaction between breed and age further suggested that WL hens exhibited higher H:L ratios during peak-laying, whereas LB hens showed more stable hematological profiles across ages. These findings indicate that breed-specific physiological responses may contribute to differences in stress resilience and productive performance in laying hens. In addition, environmental and management factors such as nutrition, housing conditions, and environmental stressors may further influence immune responses and health status in poultry [24].

This study provides original comparative data demonstrating that both breed and physiological stage significantly modulate hematological traits and TLR2-mediated innate immunity in laying hens, revealing patterns not previously reported in the literature. The higher TLR2 expression in WL compared to LB hens is a novel finding that may reflect differences in genetic selection pressure. Commercial lines like LB have been intensely selected for egg production, potentially at the cost of certain innate immune traits, whereas purebred WL may retain more robust baseline TLR2 responsiveness. Unlike Tantikositruj et al. [11], who evaluated TLR2 expression in indigenous chickens, the present study focused on commercial and purebred laying hens during different physiological stages, demonstrating that both genetic background and age may contribute to variation in innate immune-related gene expression. TLR2 is an important pattern-recognition receptor that detects PAMPs and initiates innate immune responses. When animals are infected or tissue barriers are compromised, microbial components can bind to TLR2 and activate intracellular signaling pathways, including nuclear factor kappa B (NF-κB), which regulates inflammatory responses and stimulates the production of pro-inflammatory mediators such as cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) [25]. TLR2 is an important pattern-recognition receptor involved in the early detection of microbial components and the initiation of innate immune responses [7, 8, 25–27]. In the present study, TLR2 expression was higher in WL hens than in LB hens and was greater at 42 weeks than at 80 weeks of age, with the highest expression observed in the WL × 42 group. Previous studies have reported that TLR2 expression varies among chicken breeds and may be influenced by age, genetic background, and physiological status [28–31]. The higher TLR2 expression observed in younger laying hens may indicate greater innate immune responsiveness during the peak-laying period. Increased TLR2 expression may also reflect activation of innate immune pathways associated with physiological stress, inflammatory processes, or exposure to subclinical microbial challenges. Notably, hens at 42 weeks of age exhibited higher heterophil percentages and H:L ratios, which are commonly regarded as indicators of physiological stress in poultry. Therefore, the elevated TLR2 expression observed in the WL × 42 group may represent a combination of increased immune responsiveness and stress-related immune activation. However, because only TLR2 gene expression was evaluated in the present study, further investigations involving additional immune-related genes and cytokines are required to clarify the underlying molecular mechanisms. The significant positive correlation between the H:L ratio and TLR2 expression suggests that birds with higher stress-related hematological responses also tended to show increased TLR2 expression. However, further studies including additional inflammatory biomarkers are required to clarify the biological mechanisms underlying this association.