Experimental Corticosterone Administration Induces Fluctuating Asymmetry and Bursal Atrophy in Broiler Chickens

Abstract

Corticosterone (CORT) is the primary avian stress hormone and regulates key physiological processes, but chronically elevated levels can be detrimental. This study simulated chronic stress by exogenously increasing CORT to assess effects on gait score, bone morphometry, immune indicators, and fluctuating asymmetry (FA) in broiler chickens. A total of 60 one-day-old male Ross 308 broiler chickens were allocated to six treatment groups (T0–T5; n = 10 per group). T0 received a placebo and served as the control group, while T1–T5 were gavaged daily with increasing corticosterone doses (1–5 mg/kg body weight). Treatments were administered from day 1 to day 42. CORT did not affect gait score on day 11 (p = 0.232) or day 42 (p = 0.112), but gait score was significantly correlated with body weight (day 11: p < 0.001; day 42: p = 0.004). Elevated CORT reduced bursa diameter (p < 0.001) and increased bursa follicle scores (p < 0.001), while heterophil-to-lymphocyte ratios remained unaffected (p = 0.349). Carcass and bone dimensions decreased consistently with higher doses (p < 0.001), and body weight correlated positively with all bone traits (p < 0.001). Length-to-width ratios increased with CORT dose (p < 0.001). Composite carcass FA showed dose-related variation in carcass asymmetry, with highest in T4, intermediate in T2, T3, and T5, and lowest in T0–T1 (p = 0.026). Trait-specific FA effects were detected for metatarsus length (p = 0.005) and wattle (p = 0.018), while bone FA remained unaffected (p = 0.272). Overall, exogenously increasing CORT impaired growth, suppressed immune function, and increased fluctuating asymmetry, indicating reduced developmental stability.

1. Introduction

The poultry industry has grown significantly in recent years, becoming the most widely produced meat worldwide since 2020 [1]. Global poultry meat production reached approximately 135 million tons in 2021, with an increasing trend expected to continue due to poultry’s relatively low production cost, high feed efficiency, and wide consumer acceptance [1]. However, this increased demand has led to the expansion of intensive production systems, which has raised concerns about animal welfare [2] and health issues in broilers [3]. These aspects are intrinsically linked, since poor welfare conditions can trigger chronic stress responses that compromise immune function and overall health.

In this context, understanding the consequences of chronic stress exposure becomes crucial. Baseline stress hormones play a critical role in maintaining normal physiological functions by mobilizing energy for vital processes and supporting the animal’s adaptive responses [4]. However, prolonged elevation of these hormones may compromise immune function and skeletal development [5,6]. As stress and immunosuppression are closely linked [7], several physiological indicators have been explored to evaluate chronic stress responses in broilers. Among these indicators, corticosterone (CORT) is the principal glucocorticoid hormone secreted by birds under stress, analogous to cortisol in mammals [8]. CORT is released by the adrenal glands in response to activation of the hypothalamic–pituitary–adrenal (HPA) axis. It plays a key role in maintaining homeostasis during stress by regulating energy metabolism, immune function, and behavior [4,9]. However, when chronically elevated, CORT can negatively affect growth, bone development, and immune responses [10]. Since skeletal integrity and locomotion are directly related to bone strength, gait scoring is widely used as a practical indicator of leg health and welfare in broilers [11]. Impaired gait can result from reduced bone strength [12] and may be influenced by chronic stress or altered endocrine balance. Oral administration of CORT has been used to pharmacologically elevate circulating levels and mimic the endocrine state associated with chronic stress, allowing researchers to evaluate its effects on welfare and physiological development [9,13].

One of the primary immune effects of sustained high CORT levels is the alteration of leukocyte profiles: under normal conditions, lymphocytes predominate in avian blood, but chronic CORT elevation can suppress lymphocytes and increase heterophils, resulting in a higher heterophil-to-lymphocyte (H/L) ratio [14,15,16]. In parallel, chronic stress can also affect central immune organs. The bursa of Fabricius, a primary lymphoid organ responsible for B cell development, is particularly sensitive to prolonged CORT exposure and undergoes atrophy, especially during early development, reflecting compromised humoral immunity [17].

Beyond immunological alterations, chronic stress may also impair developmental homeostasis. A promising approach to assess developmental instability (DI) is the evaluation of fluctuating asymmetry (FA), which represents small, random deviations from bilateral symmetry in traits that are genetically and developmentally programmed to be symmetrical [18,19]. Because bilateral symmetry is the expected developmental outcome in paired anatomical structures, an increase in FA indicates reduced developmental stability, defined as the organism’s ability to buffer environmental or physiological stress during growth [18]. In poultry, FA has been associated with various stressors, including poor housing conditions [20], high stocking density [21], suboptimal incubation environments [22], dietary imbalances [23], antibiotic use [24], and disruptive lighting conditions [25], making it a quantifiable indicator of welfare [26]. Thus, FA provides a direct morphological link between stress physiology and anatomical outcomes and is relevant for evaluating how chronic corticosterone exposure may disrupt biological symmetry and developmental homeostasis.

Therefore, the objective of this study was to evaluate whether long-term exogenous CORT elevation influences skeletal integrity through gait score, bone development and FA in broiler chickens. Specifically, this study tested whether pharmacologically elevated CORT impairs skeletal growth and increases developmental instability, with FA as an indicator. In parallel, we assessed H/L ratios and bursa of Fabricius diameter and follicle as markers of immune competence.

2. Materials and Methods

This research received approval from the Ethics Committee of Poulpharm bvba (Approval Number LA 1400564) on 17 November 2022.

2.1. Birds and Facilities

A total of 60 one-day-old male Ross 308 broilers were allocated to six floor pens (2 m²) in an incomplete block design, with 10 broilers per pen. The group size (10 birds per pen) and moderate stocking density were chosen to avoid social stress and prevent confounding effects on physiological responses, following established corticosterone-administration models in broilers [9,27]. The pens had concrete floors covered with wood shavings (2.5 kg/m²). At setup, all birds were individually neck-tagged for identification. CORT was administered daily at doses ranging from 0 to 5 mg/kg body weight (BW) to pharmacologically elevate circulating CORT levels (Table 1). Because CORT was administered individually based on each bird’s BW, the individual bird was considered the experimental unit. Each pen contained birds from two adjacent dose levels to minimize behavioral disruption associated with large treatment contrasts while also reducing the number of animals required.

Table 1. Experimental design showing the distribution of birds across pens and corticosterone treatments.

Pen	Number of Birds	Treatment 1
1	10	0
		1
2	10	1
		2
3	10	2
		3
4	10	3
		4
5	10	4
		5
6	10	5
		0

¹ Corticosterone, mg/bw.

In line with commercial practices and breed recommendations, birds had ad libitum access to feed and water provided through feed and water towers. All birds were fed the same commercial diet according to breed-specific guidelines: starter feed from days 0 to 14, grower feed from days 15 to 34, and finisher feed from days 35 to 42. The lighting schedule was 23 h of light and 1 h of darkness for the first 7 days, then from day 7 to day 41 chickens received 18 h of darkness and 6 h of light. During the first week, the temperature was maintained at 36 °C, followed by a gradual reduction of about 4 degrees per week until reaching 22 °C by the third week, which was then maintained for the remainder of the six-week period. The chickens were inspected daily by a veterinarian to ensure their health.

Every day at 9 AM, from day 1 to day 42, each bird was individually weighed to determine the CORT dosage according to its BW for each treatment. The gavage solution contained 10 mg of CORT mixed with 1 mL of ethanol and diluted 1:1 with water (v/v; 5 mg/mL). Fresh batches of the solution were prepared weekly. Each bird was gently handled, and either CORT or placebo was administered orally using a soft rubber tube, which was carefully inserted into the esophagus to ensure full dose delivery. On day 42, chickens were euthanized using sodium pentobarbital (Euthanimal 20%, sodium pentobarbital, Alfasan, Woerden, The Netherlands), with the dose adjusted based on BW. Each chicken was gently and individually restrained, and an experienced veterinarian administered the injection into the brachial vein. The procedure was completed within a maximum of two minutes to minimize stress and discomfort.

2.2. Gait Score

Gait score was performed for all birds on days 11 and 42 following the Welfare Quality^® assessment protocol [28]. Scores ranged from 0 to 5, where 0 indicated normal walking with no visible abnormalities and 5 represented severe lameness or inability to walk. Each bird was gently encouraged to walk approximately one meter within the pen, and its locomotion was evaluated based on walking ability.

2.3. Immunological Indicators

To evaluate stress-related immune responses, blood smears were prepared immediately after collection using fresh whole blood obtained from the wing vein. The smears were air-dried, fixed in methanol, and stained with May–Grünwald–Giemsa stain. Differential leukocyte counts were performed under a light microscope (1000× magnification with oil immersion). For each slide, a minimum of 100 leukocytes were counted and the H/L ratio was calculated by dividing the number of heterophils by the number of lymphocytes. All counts were performed by a single trained observer, blinded to the treatment, to reduce bias.

Bursa of Fabricius samples were collected from all carcasses, and then excised and immediately fixed in 10% neutral buffered formalin (4% formaldehyde) for two days. Sections (5 µm) were stained with hematoxylin and eosin for morphological evaluation. Bursa diameter was measured using a calibrated light microscope (Leica DM2000; Leica Microsystems, Wetzlar, Germany). For each sample, 20 measurements per bird were taken across different histological sections, and the mean value was used to obtain an accurate estimate of the widest transverse diameter. All measurements were performed by the same observer. Bursa follicle scores were assessed microscopically and assigned a score from 0 to 5, where 0 indicated a normal bursa, 1 corresponded to <25% lymphoid depletion, 2 to 25–50%, 3 to 50–70%, 4 to 70–90%, and 5 to >90% depletion, following the criteria described by Muskett et al. [29].

2.4. Carcass and Bone Measurements

On day 42, immediately after culling, bilateral (right and left) morphometric measurements were taken twice from the carcasses of euthanized birds. The traits measured included wattle width, eye length, beak length, nostril length, radius length, metatarsus length and width, as well as the lengths of digits II, III, and IV. All measurements were taken using a digital caliper (Model CD-20APX, Mitutoyo, Kawasaki, Japan; 0.01 mm accuracy). After measurements were completed, all carcasses were stored at −15 °C for seven days for subsequent bone processing and morphometric analyses.

The frozen carcasses were then thawed at 5 °C for 72 h. Wing (radius, humerus, and ulna) and leg (metatarsus, tibia, and femur) bones were dissected, and soft tissue was removed [30,31]. Bones were immersed in 47–50% sodium hypochlorite for one hour, followed by manual cleaning, rinsing, and bleaching in 27.5% hydrogen peroxide for 24 h. After a final rinse, bones were air-dried at room temperature.

Morphometric measurements of the bones were taken using a digital caliper (Model CD-20APX, Japan) by the same researcher. Each bone (left and right) was measured twice. Assessed traits included: humerus (length, width and radius joint), radius and ulna (length, width), femur and tibia (length, width), and metatarsus (length, width, metatarsophalangeal joint and intertarsal joint widths).

2.5. Statistical Analysis

Statistical analyses were conducted in RStudio (v1.3.1093). Gait score data were analyzed using a regression model, including CORT treatment as a fixed factor with six levels (T0–T5) and BW as a covariate. The significance of treatment and BW effects was evaluated using likelihood ratio tests. Estimated marginal means and pairwise comparisons were adjusted using the Bonferroni correction.

For morphometric analysis, repeated left and right carcass and bone measurements were averaged per side for each bird. Trait size was calculated as the mean of both sides [(L + R)/2], and treatment effects on carcass and bone traits ratios were evaluated using Kruskal–Wallis tests. When significant (p < 0.05), pairwise comparisons were conducted using Dunn’s test with Bonferroni correction. Additionally, we examined whether carcass and bone morphometrics were associated with BW.

FA was calculated for each bilateral trait using the relative asymmetry formula:

$$\mathrm{F}\mathrm{A}={\displaystyle \frac{\mid \mathrm{L}-\mathrm{R}\mid }{\mathrm{L}+\mathrm{R}}}$$

FA was analyzed using a linear mixed-effects model in which treatment was included as a fixed effect, and bird and side were included as random effects to account for repeated bilateral measurements. Directional asymmetry (DA), defined as a consistent bias favoring one side [32], was tested within the same mixed model, and unbiased FA estimates were obtained by subtracting mean DA [33]. The presence of FA was confirmed using likelihood ratio tests (LRTs) comparing models with and without the (1|chicken:side) variance term. To control for intra-observer measurement error, each bilateral trait was measured twice per side, and measurement repeatability was quantified using the signal-to-noise ratio (SNR = FA variance/measurement error), where SNR > 1 indicates reliable estimation [19]. Additionally, traits were not excluded based on the statistical significance of FA itself, since low p-values are not required for FA to be biologically relevant; instead, reliability was determined by SNR values, as recommended by Knierim et al. [18]. Kurtosis of the signed side differences (L − R) was also calculated to assess the potential presence of antisymmetry (AS), which is typically indicated by negative kurtosis values [33,34]. Assumptions of normality and homogeneity of residuals in mixed models were evaluated using Shapiro–Wilk and Levene’s tests, respectively.

The composite FA index was calculated by standardizing relative FA values into z-scores (mean = 0, SD = 1) for all eligible traits within each category (carcass or bones). The z-scores were averaged per animal to create a composite score representing organism-wide asymmetry. Treatment effects on the carcass and bone composite indices were evaluated using Kruskal–Wallis tests followed by Dunn–Bonferroni post hoc comparisons. Additionally, treatment effects on each trait-specific FA z-score (excluding traits with DA) were analyzed separately using the same non-parametric procedures. No traits exhibited AS or had a SNR < 1, so no further exclusions were necessary.

For immunological parameters (bursa of Fabricius diameter, follicle score, and H/L ratio), normality was assessed using the Shapiro–Wilk test. As the data were not normally distributed, treatment comparisons were performed using Kruskal–Wallis tests followed by Dunn–Bonferroni post hoc corrections. The significance level for all analyses was set at α = 0.05.

3. Results

3.1. Gait Score

There was no significant effect of CORT treatment on gait score at either day 11 or day 42 (Table 2). In contrast, BW showed a significant positive association with gait score on both days (day 11: p < 0.001; day 42: p = 0.004).

Table 2. Gait score (mean ± SEM) by corticosterone treatment (T0–T5) on days 11 and 42.

Gait Score	T0	T1	T2	T3	T4	T5	p-Value 1
Day 11	0.05 ± 0.10	0.05 ± 0.05	0.12 ± 0.14	0.41 ± 0.16	0.20 ± 0.02	0.32 ± 0.16	0.232
Day 42	2.53 ± 0.36	2.47 ± 0.27	2.46 ± 0.21	2.72 ± 0.19	2.89 ± 0.17	2.99 ± 0.07	0.112

¹ Reported p-values indicate the treatment effect, with values < 0.05 considered statistically significant.

3.2. Immunological Indicators

On day 42, the H/L ratio did not differ significantly between treatment groups (Table 3).

Table 3. Immune indicators (mean ± SEM) by corticosterone treatment (T0–T5) on day 42.

Immune Indicators	T0	T1	T2	T3	T4	T5	p-Value 1
Bursa (µm)	931.22 a ± 57.03	638.54 ab ± 15.86	521.67 bc ± 20.24	416.98 c ± 23.02	357.62 c ± 15.39	395.69 c ± 31.24	0.001
Bursa follicle score	0.00 a ± 0.00	2.0 ab ± 0.26	2.60 abc ± 0.16	3.30 bcd ± 0.21	4.00 cd ± 0.15	4.30 d ± 0.21	0.001
H/L ratio 2	95.60 ± 23.37	153.25 ± 33.03	142.75 ± 32.55	95.75 ± 20.33	151.33 ± 12.35	166.00 ± 30.14	0.349

¹ Values within a row lacking a common superscript differ significantly; reported p-values indicate the treatment effect, with values < 0.05 considered statistically significant; ² H/L = heterophil-to-lymphocyte ratio.

T0 exhibited the largest average bursa diameter, significantly larger than all other treatments. T1 and T2 showed intermediate values, with T1 differing significantly from T3–T5 but not from T0 or T2. The smallest diameters were observed in the higher-dose groups T3, T4, and T5, which did not differ significantly from each other (Figure 1). Bursa follicle scores differed significantly among treatments. Scores increased progressively with higher CORT levels, with T0 showing significantly lower values than all other groups, whereas T4 and T5 presented the highest scores, differing from most lower-dose treatments.

Figure 1. Representative histological sections of the bursa of Fabricius from T0 ((A), control) and T5 ((B), highest CORT dose). Images are shown to illustrate structural differences observed between treatments, complementing the quantitative diameter measurements. In T0, follicles appear larger and more organized, whereas in T5, the bursa shows signs of atrophy and reduced follicular structure.

3.3. Carcasses and Bone Measurements

CORT treatments significantly affected all carcass and bone morphometric traits (Table 4). Both carcass and bone dimensions (length and width) showed a consistent dose-dependent reduction (p < 0.001 for all traits). Additionally, the length-to-width ratio of all examined bones increased progressively with higher CORT administration (p < 0.001). Final BW was positively correlated with all bone traits (p < 0.001).

Table 4. Effects ¹ of different corticosterone doses (T0–T5) on broiler chickens’ bone morphometric traits (mean ± SEM).

Trait (mm)	T0	T1	T2	T3	T4	T5
Carcasses
Eye L 2	12.90 a ± 0.26 3	13.2 a ± 0.12	11.76 b ± 0.16	11.45 bc ± 0.21	10.14 c ± 0.36	12.15 bc ± 0.80
Nostril L	7.42 a ± 0.18	7.59 a ± 0.15	6.28 b ± 0.01	5.97 bc ± 0.13	6.38 bc ± 0.23	5.68 c ± 0.08
Wattle L	24.48 a ± 0.52	25.35 a ± 0.45	18.38 b ± 0.52	17.1 bc ± 0.53	16.24 bc ± 0.43	12.29 c ± 0.35
Beak L	32.51 a ± 0.24	30.49 b ± 0.16	29.7 b ± 0.19	27.62 c ± 0.16	27.59 c ± 0.34	26.3 c ± 0.22
Radius	70.52 a ± 1.17	66.88 ab ± 0.53	63.65 b ± 0.62	58.7 c ± 0.74	55.89 c ± 0.60	55.15 c ± 0.65
Metatarsus L	108.83 a ± 1.25	101.21 a ± 0.58	93.51 b ± 0.35	89.13 bc ± 0.79	86.16 cd ± 0.70	82.4 d ± 0.45
Metatarsus W 4	13.87 a ± 0.21	12.13 ab ± 0.09	11.57 bc ± 0.01	11.98 cd ± 0.63	10.66 d ± 0.15	9.62 e ± 0.01
Metatarsus L/W 5	7.89 a ± 0.08	8.36 bc ± 0.08	8.1 ab ± 0.06	7.90 bc ± 0.23	8.11 ab ± 0.11	8.59 c ± 0.08
Middle toe L	27.6 a ± 0.32	25.86 ab ± 0.22	23.88 b ± 0.57	22.13 c ± 0.44	21.9 cd ± 0.19	20.68 d ± 0.11
Outer toe L	16.42 a ± 0.26	15.6 a ± 0.09	14.46 b ± 0.11	13.96 bc ± 0.52	12.74 cd ± 0.16	12.07 d ± 0.11
Back toe L	25.52 a ± 0.34	23.79 a ± 0.16	22.32 b ± 0.08	21.05 c ± 0.20	20.23 cd ± 0.19	18.91 d ± 0.11
Bones
Radius L	63.23 a ± 0.89 3	61.56 a ± 0.20	58.10 b ± 0.33	56.50 b ± 0.32	55.16 bc ± 0.61	52.42 c ± 0.45
Radius W	6.61 a ± 0.20	5.85 ab ± 0.04	5.40 b ± 0.06	5.07 b ± 0.04	5.13 bc ± 0.07	4.51 c ± 0.07
Radius L/W	9.43 a ± 0.18	10.54 b ± 0.08	10.83 bc ± 0.10	11.16 cd ± 0.08	10.76 bc ± 0.03	11.64 d ± 0.08
Ulna L	60.88 a ± 0.92	59.11 a ± 0.24	54.19 b ± 0.33	53.46 bc ± 0.35	52.78 bc ± 0.88	50.35 c ± 0.53
Ulna W	3.23 a ± 0.09	2.88 ab ± 0.02	2.74 bc ± 0.03	2.45 d ± 0.03	2.51 cd ± 0.05	2.22 d ± 0.03
Ulna L/W	19.09 a ± 0.34	20.67 b ± 0.12	20.16 ab ± 0.19	21.96 c ± 0.18	21.23 bc ± 0.20	22.67 c ± 0.16
Humerus L	65.78 a ± 0.97	64.27 a ± 1.44	60.88 b ± 0.29	58.82 bc ± 0.27	58.41 bc ± 0.66	54.49 c ± 0.45
Humerus W	8.41 a ± 0.19	7.46 a ± 0.05	6.97 b ± 0.08	6.59 bc ± 0.06	6.82 b ± 0.08	6.05 c ± 0.05
Humerus L/W	7.87 a ± 0.09	8.63 b ± 0.05	8.76 b ± 0.09	8.93 bc ± 0.05	8.57 ab ± 0.03	9.06 c ± 0.03
Humerus W (joint radius)	15.80 a ± 0.25	15.11 a ± 0.07	14.51 b ± 0.04	14.10 b ± 0.17	13.76 bc ± 0.17	12.93 c ± 0.05
Femur L	71.11 a ± 1.03	69.58 a ± 0.30	65.48 b ± 0.34	64.03 b ± 0.36	62.72 bc ± 0.36	59.15 c ± 0.34
Femur W	9.36 a ± 0.21	8.09 ab ± 0.06	7.80 bc ± 0.08	7.42 cd ± 0.08	7.60 bcd ± 0.16	7.09 d ± 0.09
Femur L/W	7.65 a ± 0.09	8.61 b ± 0.06	8.42 b ± 0.08	8.68 b ± 0.11	8.28 b ± 0.09	8.36 b ± 0.08
Tibia L	94.80 a ± 1.59	91.65 a ± 0.47	86.48 b ± 0.37	83.46 b ± 0.49	81.95 bc ± 0.97	77.95 c ± 0.44
Tibia W	9.23 a ± 0.27	7.90 a ± 0.05	7.25 b ± 0.05	6.55 c ± 0.08	6.83 bc ± 0.13	6.27 c ± 0.06
Tibia L/W	10.47 a ± 0.23	11.62 ab ± 0.09	11.95 b ± 0.07	12.78 c ± 0.14	12.02 bc ± 0.09	12.44 c ± 0.09
Metatarsus L	70.77 a ± 1.22	67.54 a ± 0.27	63.46 b ± 0.27	61.09 bc ± 0.36	58.57 cd ± 0.51	56.32 d ± 0.57
Metatarsus W	11.48 a ± 0.30	9.77 ab ± 0.04	9.25 b ± 0.07	8.41 c ± 0.09	8.30 c ± 0.14	7.76 c ± 0.11
Metatarsus L/W	6.22 a ± 0.08	6.91 b ± 0.04	6.86 b ± 0.04	7.28 c ± 0.06	7.07 bc ± 0.07	7.26 c ± 0.06
Metatarsophalangeal joint W	21.54 a ± 0.30	20.37 a ± 0.08	19.23 b ± 0.08	18.55 b ± 0.25	18.05 bc ± 0.17	17.09 c ± 0.15
Metatarsus Intertarsal joint W	19.13 a ± 0.30	18.07 a ± 0.08	17.40 b ± 0.07	16.53 bc ± 0.25	16.12 c ± 0.21	15.96 c ± 0.22

¹ Values within a row lacking a common superscript differ significantly; reported p-values indicate the treatment effect, with values < 0.05 considered statistically significant; ² L: length; ³ Standard error of mean; ⁴ W: width; ⁵ L/W: Length-to-Width ratio.

3.4. Fluctuating Asymmetry

To assess the developmental stability of carcasses and skeletal traits, DA, FA, SNR, and kurtosis were analyzed for 15 bilateral bone traits and 10 carcass traits (Table 5). Five carcass traits showed significant DA: eye, beak, radius, metatarsus, and back toe length. SNR values were high for most carcass traits, and kurtosis ranged from 2.5 to 85.0. Regarding the bones, five traits showed significant DA: radius width, radius length, femur width, metatarsophalangeal joint width, and metatarsus intertarsal. FA was estimated for all traits, with significant evidence of FA detected in each case (p < 0.001). SNR values were uniformly high, and kurtosis ranged from 1.8 to 6.9. As a result, both carcass and bone FA composite indices were calculated using all traits.

Table 5. Significance of directional asymmetry, fluctuating asymmetry, signal-to-noise ratios, and kurtosis for different carcasses and bone traits measured on broiler chickens.

Trait	DA	FA	SNR 2	Kurtosis 3
Carcasses
Eye L	F1,56 = 13.98 (p < 0.001)	LR = 27 (p < 0.001)	11.11	2.6
Nostril L	F1,56 = 1.66 (p = 0.203)	LR = 20 (p < 0.001)	3.93	6.0
Wattle L	F1,42 = 0.28 (p = 0.602)	LR = 112 (p < 0.001)	4.51	4.1
Beak L	F1,56 = 8.64 (p = 0.004)	LR = 25 (p < 0.001)	12.26	2.5
Radius L	F1,50 = 11.22 (p = 0.001)	LR = 60 (p < 0.001)	7.57	3.5
Metatarsus L 4	F1,56 = 14.71 (p < 0.001)	LR = 49 (p < 0.001)	48.41	3.4
Metatarsus W 5	F1,56 = 1.45 (p = 0.234)	LR = 3.13 (p < 0.001)	203.25	6.4
Middle toe L	F1,56 = 0 (p = 0.962)	LR = 0.74 (p = 0.380)	38.68	85.0
Outer toe L	F1,56 = 0.88 (p = 0.352)	LR = 0.05 (p = 0.808)	128.74	61.2
Back toe L	F1,56 = 0.27 (p < 0.001)	LR = 3.01 (p = 0.080)	152.44	4.3
Bones
Radius L	F1,36 = 6.02 (p = 0.019)	LR 1 = 490 (p < 0.001)	272.65	6.9
Radius W	F1,36.1 = 4.29 (p = 0.045)	LR = 257 (p < 0.001)	76.46	3.2
Ulna L	F1,27 = 3.09 (p = 0.090)	LR = 366 (p < 0.001)	720.08	1.8
Ulna W	F1,30.2 = 0.90 (p = 0.351)	LR = 258 (p < 0.001)	52.4	2.1
Humerus L	F1,36 = 0.36 (p = 0.552)	LR = 454 (p < 0.001)	158.87	3.2
Humerus W	F1,37.1 = 0.10 (p = 0.749)	LR = 360 (p < 0.001)	47.96	3.3
Humerus W (joint radius)	F1,34.1 = 1.04 (p = 0.314)	LR = 346 (p < 0.001)	100.14	4.2
Femur L	F1,39 = 2.91 (p = 0.095)	LR = 472 (p < 0.001)	291.21	6.3
Femur W	F1,38.1 = 12.25 (p = 0.001)	LR = 375 (p < 0.001)	49.07	2.3
Tibia L	F1,38 = 0.03 (p = 0.860)	LR = 499 (p < 0.001)	534.35	2.3
Tibia W	F1,39 = 8.14 (p = 0.060)	LR = 421 (p < 0.001)	76.37	4.5
Metatarsus L	F1,33 = 0.79 (p = 0.381)	LR = 548 (p < 0.001)	153.61	3.0
Metatarsus W	F1,34.1 = 0.18 (p = 0.676)	LR = 369 (p < 0.001)	117.61	2.6
Metatarsophalangeal joint W	F1,31.1 = 7.89 (p = 0.008)	LR = 472 (p < 0.001)	33.33	2.4
Metatarsus Intertarsal joint W	F1,33.2 = 9.67 (p = 0.003)	LR = 443 (p < 0.001)	39.36	4.8

¹ LR = likelihood ratio; ² Signal-to-noise ratio: FA/ME (measurement error); ³ Kurtosis of signed side differences (L − R); ⁴ L: Length; ⁵ W: width

The composite FA index of carcass traits (Table 6) differed between treatments (p = 0.026). The highest mean composite FA values were observed in T5, while T2 and T3 showed intermediate values, and the lowest FA values were found in T0 and T1. Regarding the composite FA index of bone traits, no significant differences were detected between treatments.

Table 6. Effects ¹ of different corticosterone doses (T0–T5) on FA in single and composite traits measured on the carcasses and bones.

Trait	T0	T1	T2	T3	T4	T5	p-Value
Carcasses
Composite FA index 2	−0.115 a ± 0.06	−0.180 a ± 0.09	−0.070 ab ± 0.07	0.053 ab ± 0.10	0.316 b ± 0.12	−0.001 ab ± 0.08	0.026
Eye L 3	−0.297 ± 0.21	−0.475± 0.31	0.428 ± 0.32	−0.012 ± 0.21	0.449 ± 0.36	0.109 ± 0.47	0.197
Nostril L	−0.063 ± 0.21	−0.291 ± 0.26	−0.319 ± 0.28	0.606 ± 0.23	0.209 ± 0.77	−0.093 ± 0.12	0.089
Wattle L	−0.340 ± 0.14	−0.499 ± 0.14	0.544 ± 0.31	1.181 ± 0.60	−0.836 ± 0.28	−0.177 ± 0.34	0.018
Beak L	0.172 ± 0.34	−0.401 ± 0.24	0.039 ± 0.31	−0.367 ± 0.29	0.723 ± 0.31	−0.042 ± 0.36	0.265
Radius L	−0.231 ± 0.25	0.196 ± 0.30	0.447 ± 0.54	−0.073 ± 0.28	0.091 ± 0.18	−0.609 ± 0.20	0.448
Metatarsus L	−0.396 a ± 0.25	−0.100 ab ± 0.33	−0.474 a ± 0.21	−0.389 a ± 0.27	1.222 b ± 0.67	0.607 ab ± 0.29	0.005
Metatarsus W 4	−0.172 ± 0.17	−0.159 ± 0.33	−0.213 ± 0.12	−0.012 ± 0.20	0.437 ± 0.38	−0.095 ± 0.41	0.980
Middle toe L	0.026 ± 0.12	−0.151 ± 0.08	0.524 ± 0.73	−0.094 ± 0.09	−0.211 ± 0.05	−0.198 ± 0.04	0.561
Outer toe L	0.009 ± 0.10	−0.182 ± 0.84	−0.182 ± 0.10	0.481 ± 0.72	−0.11 ± 0.11	−0.157 ± 0.12	0.707
Back toe L	0.138 ± 0.39	−0.065 ± 0.36	0.001 ± 0.21	−0.470 ± 0.25	0.147 ± 0.17	0.389 ± 0.39	0.403
Bones
Composite FA index	0.119 ± 0.11	−0.193 ± 0.11	0.069 ± 0.10	0.058 ± 0.13	0.098 ± 0.08	−0.136 a ± 0.08	0.272
Radius L	0.872 ± 0.66	0.052 ± 0.27	−0.053 ± 0.25	−0.381 ± 0.14	−0.459 ± 0.26	−0.377 ± 0.22	0.559
Radius W	−0.078± 0.25	−0.429 ± 0.22	0.244 ± 0.31	0.687 ± 0.42	−0.763 ± 0.12	0.128 ± 65	0.099
Ulna L	−0.077 ± 0.39	−0.200 ± 0.40	0.247 ± 0.24	0.091 ± 0.60	0.321 ± 0.52	−0.199 ± 0.49	0.980
Ulna W	−0.063 ± 0.33	−0.146 ± 0.40	−0.06 ± 0.39	−0.312 ± 0.48	1.045 ± 0.50	0.388 ± 0.59	0.649
Humerus L	0.107 ± 0.14	−0.065 ± 0.38	−0.166 ± 0.24	−0.029 ± 0.30	−0.964 ± 0.25	0.833 ± 0.83	0.298
Humerus W	0.523 ± 0.43	−0.692 ± 0.21	0.465 ± 0.44	−0.224 ± 0.24	−0.357 ± 0.39	0.096 ± 0.38	0.167
Humerus W (joint radius)	−0.537 ± 0.15	−0.193 ± 0.43	0.188 ± 0.25	0.167 ± 0.50	0.842 ± 0.91	0.115 ± 0.43	0.210
Femur L	0.290 ± 0.62	−0.184 ± 0.31	0.051 ± 0.33	0.271 ± 0.33	−0.062 ± 0.31	−0.415 ± 0.23	0.669
Femur W	−0.324 ± 0.21	0.030 ± 0.27	0.034 ± 0.33	0.545 ± 0.59	−0.453 ± 0.70	−0.117 ± 0.74	0.748
Tibia L	0.532 ± 0.47	−0.102 ± 0.34	−0.232 ± 0.28	0.456 ± 0.41	0.531 ± 0.19	−0.867 ± 0.23	0.063
Tibia W	0.312 ± 0.46	0.546 ± 0.45	−0.203 ± 0.24	−0.081 ± 0.32	−0.750 ± 0.19	−0.443 ± 0.16	0.397
Metatarsus L	0.718 ± 0.40	−0.107 ± 0.53	−0.30 ± 0.23	−0.197 ± 0.32	0.551 ± 0.58	−0.633 ± 0.34	0.182
Metatarsus W	−0.213 ± 0.28	−0.663 ± 0.19	0.238 ± 0.27	−0.447 ± 0.18	1.823 ± 0.82	0.306 ± 0.62	0.061
Metatarsophalangeal joint W	−0.313± 0.25	0.399 ± 0.49	0.404 ± 0.56	0.197 ± 0.21	0.217 ± 0.27	0.121 ± 0.29	0.431
Metatarsus Intertarsal joint W	0.159± 0.34	−0.566 ± 0.15	0.029 ± 0.42	0.793 ± 0.47	−0.315 ± 0.32	−0.882 ± 0.31	0.227

¹ Values within a row lacking a common superscript differ significantly; reported p-values indicate the treatment effect, with values < 0.05 considered statistically significant; ² z-score FA: standardized relative fluctuating asymmetry (mean = 0, SD = 1); ³ L: length; ⁴ W: width.

Regarding single carcass traits, a treatment effect was detected for the wattle, although post hoc comparisons did not reveal clearly separated groups; numerically, birds from T3 and T4 showed higher FA values. For metatarsus length, FA differed among treatments, with birds from T5 showing the highest asymmetry, significantly higher than T0, T1, T2, and T3. Other carcass traits did not show significant treatment effects. For bones, traits did not differ significantly among treatments.

4. Discussion

Our results showed that broilers gavaged with increasing CORT doses (T0–T5) exhibited broad effects on immunity and growth. Elevated CORT levels reduced bursal diameter and follicle size and progressively decreased carcass and bone dimensions. Dose-related variation was observed in carcass asymmetry, with FA increasing with higher doses up to T4. Together, these outcomes confirm the immunosuppressive and growth-inhibiting role of CORT, and indicate that lymphoid organ and carcass traits can be used as reliable markers of stress-induced developmental instability.

The absence of CORT effects on gait score may be explained by the fact that locomotion impairments in broilers are primarily driven by mechanical factors such as growth rate [11], rather than physiological stress itself. CORT mainly affects metabolic and immune pathways [10], and its influence on locomotor ability is therefore likely indirect. In this study, BW showed a stronger association with gait score than CORT treatment, suggesting that heavier birds experienced greater mechanical load on their legs, leading to poorer walking ability, as previously reported in other studies [35,36].

Regarding bursa diameter and follicle size, elevated CORT led to a reduction, confirming its immunosuppressive effects. These findings are consistent with previous reports showing that stress elevates plasma corticosteroids and contributes to the degeneration of immune organs such as the bursa of Fabricius [37]. Zhang et al. further demonstrated that dietary administration of CORT at 30 mg/kg downregulated more than 1200 genes in the bursa, providing mechanistic insight into its broad immunosuppressive impact [38]. CORT-mediated stress has also been shown to disrupt lipid metabolism and alter systemic immune responses, inducing epithelial atrophy and inflammation in the bursa [39,40]. Together, this evidence reinforces the sensitivity of the bursa as a target organ for stress-induced immunosuppression and demonstrates that the birds in the current experiment were immunosuppressed.

However, despite the pronounced effects on the bursa, H/L ratios remained unaffected in our study. Although H/L is often used as a stress marker, its reliability has been questioned. Nwaigwe et al. showed that changes in H/L only appeared after acute stressors, while Thiam et al. reported that low H/L ratios were more related to immune robustness and resistance to Salmonella than to stress itself [16,41]. This suggests that CORT-induced immunosuppression was more clearly detected in lymphoid organs than in peripheral blood leukocyte profiles, showing that organ-level responses, such as bursa atrophy, may represent a more sensitive indicator of immunosuppression. Thus, chronic CORT exposure may suppress immune tissues directly while allowing circulating leukocyte ratios to stabilize over time, resulting in minimal detectable changes in H/L.

Carcasses and bone morphometry in this study also showed clear dose-dependent reductions under CORT exposure, with all measured traits decreasing as the dosage increased. Strong positive correlations were found between BW and bone dimensions, confirming that heavier birds developed proportionally larger bones. The increase in the length-to-width ratio with higher doses further indicates that CORT altered not only absolute growth but also bone proportions.

Our findings are consistent with previous work showing that prolonged CORT exposure compromises bone growth and development. In histological studies, Luo et al. reported that daily injections of CORT at 4 mg/kg BW for one week shortened the proliferative and pre-hypertrophic zones of the growth plate and inhibited chondrocyte proliferation and differentiation, ultimately slowing longitudinal bone growth [27]. Similarly, Zhang et al. demonstrated that CORT had opposite effects on chondrocytes depending on its concentration [6]. Very low levels stimulated cell viability and the expression of differentiation markers, whereas higher levels suppressed these processes, reducing alkaline phosphatase activity, type X collagen, and parathyroid hormone-related protein expression. At a broader level, Lui and Baron [42] highlighted that glucocorticoids directly inhibit bone growth by reducing proliferation, hypertrophy, and matrix synthesis in the growth plate, while also slowing growth plate maturation. Although our study did not assess histological parameters directly, the observed morphological reductions in bone dimensions are consistent with cellular and tissue-level processes described in the literature.

Regarding FA, a CORT dose-related variation was observed in carcass asymmetry, with increasing doses leading to higher FA values up to T4 followed by a reduction at T5. Because FA reflects the organism’s ability to buffer developmental disturbances [18], this increase in FA indicates reduced developmental stability under elevated CORT exposure. The slight reduction in FA at the highest dose may reflect a ceiling effect of stress, where extreme growth suppression reduces overall morphological variation. Within carcass traits, metatarsus length showed the clearest dose-dependent increase in asymmetry, which may reflect this trait’s particular vulnerability because, unlike the tibia and femur that show accelerated growth and mineralization peaks, the metatarsus grows more steadily and has lower mineral content and strength [43]. Moreover, external carcass traits, which integrate soft tissues, joints, and alignment, are more plastic and therefore more responsive to stress. Phenotypic plasticity arises because environmental stimuli can modify developmental processes, leading to differences in adult morphology [44], which may explain why bone FA remained largely unchanged. Importantly, this contrast is unlikely to reflect measurement error since SNRs were high for both carcass and bone traits.

Previous studies support a link between stress and FA, showing that elevated CORT in fertilized chicken eggs increased embryonic mortality and FA in tarsus length [45], and that higher baseline CORT levels in Eurasian treecreeper nestlings were associated with greater FA [46]. However, this association is not universal, and hormone–FA relationships can depend on species, developmental stage, or methodology [47]. While these earlier studies examined FA responses either during embryogenesis or in wild avian species, evidence in broilers under controlled chronic CORT exposure remains scarce. Our findings therefore extend the CORT–FA relationship to fast-growing commercial chickens, demonstrating that developmental instability can be detected in carcass traits.

Finally, the FA results should be interpreted with caution. The composite FA index combines trait-specific z-scores, and some traits showed directional asymmetry or heavy-tailed side differences, which can bias the index even with high measurement precision. To better capture relationships, bursa and H/L ratio should ideally be analyzed at the individual level. In particular, the high SEM observed for the H/L ratio indicates substantial variability, suggesting that larger sample sizes would be needed to obtain more reliable results. Future research should explore longitudinal monitoring of stress biomarkers. Such approaches would provide a more comprehensive understanding of how exogenous CORT affects immune suppression and developmental instability.

5. Conclusions

This study shows that broilers gavaged with exogenously increasing CORT levels exhibited immunosuppression and growth impairment, evidenced by reduced bursa diameter and follicle size, together with progressive decreases in carcass and bone size. Carcass asymmetry showed a CORT dose-related variation, with FA increasing with higher doses up to T4 and with the clearest pattern observed for the metatarsus. However, bone FA remained largely unchanged. Together, these findings support integrating immune, growth, and FA measures to better capture the multifaceted impacts of stress on broiler health and welfare.