Influence of Aviary Design on Musculoskeletal Health and Keel Bone Damage in Hy-Line Brown Laying Hens

Abstract

Bone fractures and keel bone damage as a result of osteoporotic implications on skeletal health due to high rates of egg production are of significant concern in the egg industry. This study was conducted to evaluate the effects of two aviary housing configurations and associated exercise opportunities on musculoskeletal health in laying hens. Two commercial aviary designs were compared: Big Dutchman NATURA STEP (STEP) and Big Dutchman NATURA 60 (N60). Musculoskeletal assessments were performed at 60 weeks of age (n = 180), where measurements included CT imaging and radiography, muscle dissections, tibial and humeral biomechanical properties, and bone ash percentage. Results indicated that hens in the STEP aviary exhibited higher tibial breaking strength, humeral stiffness, and heavier muscle groups compared to the N60 system. However, rates of new and old fractures, as well as rates of deviation, were more prevalent in STEP hens compared to N60 hens. These results indicate that housing system design influences musculoskeletal health in laying hens.

1. Introduction

Osteoporosis in laying hens is characterized by a progressive reduction in mineralized bone structure, predisposing birds to fragility and fractures [1]. Before sexual maturity, pullets deposit medullary bone within the marrow cavities of long bones (e.g., tibia) as a labile calcium reserve for future eggshell formation. With the onset of lay, hormonally driven medullary bone deposition increases, while structural bone mineralization ceases [1]. As the laying cycle progresses, continuous mobilization of structural bone minerals used for eggshell calcification contributes to osteoporosis and increases the risk of fractures and skeletal injury [1,2]. Beyond these intrinsic physiological processes, skeletal health is also influenced by levels of physical activity and the housing environment. Mechanical loading from movement and exercise stimulates adaptive bone remodeling via osteocyte signaling, promoting bone mineral retention and strength [3,4,5,6,7,8,9,10]. Early-life activity further suppresses osteoclastic resorption at endosteal surfaces while enhancing medullary deposition, collectively supporting long-term skeletal integrity during the egg-laying phase [11]. Although these adaptive responses diminish with age, prior studies have linked early and sustained activity to improved bone quality throughout the production cycle [11].

Consumer and legislative drivers for improved hen welfare are motivating the egg industry’s transition from conventional cages to cage-free housing systems that allow hens to express natural behaviors, such as perching, scratching, dustbathing, jumping, and flying. Such alternative housing systems are associated with increased bone mineral density and bone strength in laying hens, largely due to the activity facilitated by providing highly motivating resources and additional space compared with caged systems [7,12]. Specific opportunities for flight or flight-assisted jumping among vertically available resources in aviary-type housing models allow for increased development of the sternal and forelimb musculature through repeated engagement of these muscle groups [11,13,14]. With such increases in muscle mass and mechanical stress on skeletal structures, concurrent increases in humeral strength and maintenance of bone mineral density are expected. Additionally, jumping and perching provide conditions for strengthening the hindlimb muscles. These muscles, which are critical for stabilization and mobility, facilitate increases in mechanical stress on the tibiotarsus and femoral regions [11,13,14]. Enhancing vertical and horizontal activity, in combination with osteoclastic suppression, may help mitigate osteoporosis by generating repeated, progressive mechanical stress on bone structures.

Contrarily, the freedom of movement in extensive housing designs comes with certain repercussions. Increases in skeletal abnormalities, such as fractures or keel bone damage (KBD), have been associated with greater horizontal and vertical space allowances. Old and new fractures—defined by marginal bone impacts or callus formation—can result from collisions, falls, or piling incidents [5,11,15,16]. Keel bone deviation, which reflects angulation rather than fracture, has been linked to perching opportunities where localized pressure from the hens’ body weight induces curvature during bone remodeling [5,17,18,19]. Compared with traditional caged systems, alternative housing designs increase the risk of fractures and deviations; however, their overall impact on hen welfare remains a subject of debate [5,11,15,16]. When evaluating hen welfare, researchers have found that the severe behavioral restriction imposed by conventional cages contributes to high rates of osteoporosis, muscle weakness, and metabolic disorders, as well as frustration from the inhibition of highly motivated behaviors [20,21,22,23,24]. In contrast, housing systems that provide greater space and environmental complexity enhance bone mineral content and muscle deposition through load-driven adaptation without adversely affecting stress responses [25,26]. Mechanical loading is known to stimulate mineral deposition within the skeleton through mechanotransductive pathways [27]. Osteocytes—mature bone cells embedded within the mineralized matrix—serve as mechanosensors that detect strain and trigger remodeling responses to strengthen bone tissue [27]. This load-driven adaptation promotes bone turnover, resulting in greater mineral content and improved structural integrity [28]. In avian species, as in mammals, mechanical stimuli help maintain bone mass and prevent osteoclastic overactivity, thereby supporting bone mineral retention [11]. When muscle development occurs in parallel with adequate nutrition, increased muscle contraction further augments skeletal loading, contributing to higher mineral density and bone strength [28].

Additional influences such as flock synchrony, social facilitation, and genetic strain may also affect how hens use available resources, ultimately shaping exercise-related improvements in musculoskeletal health [29,30,31,32,33,34]. For the present study, the focus lies on the interaction between individual motivation for activity and physical access to resources within multi-tier aviary environments, where internal structural barriers may constrain movement and limit full use of the system [34,35].

Given the variation in aviary interior design and the resulting differences in movement opportunities for hens, a better understanding of how these housing configurations influence musculoskeletal health is needed. The current study, therefore, aimed to compare the musculoskeletal characteristics of laying hens housed in two commercial aviary systems that differ in vertical access and internal layout. One aviary utilizes an open design with unlimited tier access and passage from side to side (Big Dutchman NATURA Step (STEP)), and one utilizes a closed design with tier access only at the bottom level (Big Dutchman NATURA 60 (N60)). Evaluations focused on bone strength, mineral content, muscle development, and keel bone integrity at 60 weeks of age to identify design-related effects on skeletal welfare. The findings are intended to inform recommendations for housing and management strategies that optimize both musculoskeletal condition and welfare outcomes in commercial production. We hypothesized that hens housed in the STEP system, which offers greater vertical accessibility and activity opportunities, would exhibit superior musculoskeletal qualities but a higher prevalence of keel bone damage compared with hens housed in the N60 system.

2. Materials and Methods

2.1. Ethics

All procedures in this study were reviewed and approved by the Institutional Animal Care and Use Committees of Michigan State University (PROTO202200159—03/25/2023) and Clemson University (AUP2022-0341—04/12/2023).

2.2. Animal Husbandry

The study was conducted at the Michigan State University Poultry Teaching and Research Center (East Lansing, MI, USA). A total of 3696 Hy-Line Brown laying hens were obtained from a commercial hatchery, Hy-Line North America (Mansfield, GA 30055, USA). Chicks were hatched and reared on floor pens in accordance with Hy-Line Brown management guidelines prior to transfer. At 16 weeks of age, pullets were randomly assigned to one of two commercial aviary configurations for the duration of the experiment [36]. Birds were housed in enclosed, climate-controlled rooms. All chicks had been beak-trimmed at one day of age using infrared treatment and were reared in floor pens bedded with wood shavings (5 cm depth). Hens were provided vertical perches prior to transfer.

Throughout the study, dietary formulations and photoperiod schedules were managed according to the breeder’s guidelines [36]. All hens received the same commercially formulated diet, delivered three times daily through automated feed lines. Two additional 10 s feed-line stimulations were provided between feedings to encourage feed intake. Birds were fed a pre-lay ration from 16 to 18 weeks of age (WOA), a pre-peak ration from 18 to 23 WOA, and a peak-lay ration from 23 to 60 WOA. Water was available ad libitum via nipple drinkers.

Lighting schedules gradually increased from 12L:12D to 16L:8D over the 12-week acclimation period following placement. Each day began with a 5 min pre-illumination dim phase and ended with a 45 min dimming sequence, consisting of 30 min of overhead light reduction, followed by 15 min of dimming the rope lights within the systems. Light intensity at bird height across all tiers and litter areas was maintained at approximately 32 lux, in accordance with breeder recommendations.

Environmental conditions were controlled through mechanical ventilation and automated heating to maintain temperatures between 17.8 °C and 25.0 °C and relative humidity between 40% and 60%. Litter areas contain wood shavings at a 5 cm depth, and manure belts beneath the systems were operated twice weekly for waste removal. Bird health, environmental parameters, and system function were monitored daily by trained farm personnel.

Hens exhibiting severe injuries, substantial weight loss, gait abnormalities, or inability to access resources were humanely euthanized to maintain welfare standards. For additional details regarding housing design, management routines, and environmental control, see Baugh et al. [37].

2.3. Housing Design

Two commercially available Big Dutchman (Holland, MI, USA) aviary models were used in this study: the NATURA Step (STEP) and NATURA 60 (N60). A total of six rooms were equipped with aviaries—three rooms with the STEP design and three rooms with the N60 design. Each room was further divided into four sections using wire-mesh partitions that permitted passage for farm personnel and researchers.

Initial stocking densities included a total of 1968 birds in the STEP (n = 164 birds per section) and 1728 in the N60 (n = 144 birds per section). Initial stocking densities followed the recommendations of the United Egg Producers (UEP) guidelines [38], which specify a minimum total floor area of 929 cm² per bird. The STEP system provided 1056 cm² per bird, whereas the N60 provided 943 cm² per bird. UEP guidelines also recommend a minimum of 84 cm² of nest space per bird; the STEP system provided 161 cm² per bird, and the N60 provided 88 cm² per bird. Perch space recommendations of at least 15 cm per bird were also met in both aviaries. All other recommendations for feeder and drinker space were likewise satisfied. For additional schematics and detailed descriptions of aviary construction, internal configuration, and management protocols, see Baugh et al. [37].

In each STEP section (Figure 1), hens were provided three vertical tiers with perches located at each level. This design allowed access to the bottom, middle, and top tiers from the litter area on both sides of the system through its double-sided, open-air structure. Feed troughs were positioned on the top and lower tiers, and nipple drinkers were located on the middle and lower tiers. A centrally located colony nest on the middle tier contained an egg belt beneath and was subdivided into two compartments per side (four total). Perches were present at all tier levels; however, due to room constraints, the lower tier was modified to include ramps for easier access. Birds could move laterally between both sides of the aviary via the bottom and top tiers. Access beneath the system was restricted using wire mesh. In each N60 section (Figure 2), hens were likewise provided with three vertical tiers and perches at each level, but this design permitted litter-area entry only through an opening on the bottom tier. Movement to the upper tiers required internal navigation of the structure. Feed troughs were placed on the middle and lower tiers, and nipple drinkers were positioned on the top and lower tiers. Colony nests were located along the top-tier edge with an externally accessible egg belt and were subdivided into two compartments per section. Internal metal ledges enabled vertical passage among tiers. As in the STEP system, access beneath the structure was blocked with wire mesh, but no ramps were provided for the lower tier. A maintenance aisle was situated along the rear of the aviary to allow personnel access to the egg belt and the lower two tiers. For additional schematics and detailed descriptions of aviary construction, internal configuration, and management protocols, see Baugh et al. [37].

Figure 1. Big Dutchman NATURA STEP Configuration.

Figure 2. Big Dutchman NATURA60 Configuration.

At 60 weeks of age, 180 hens [~5% of the total population; STEP = 96 (8 birds per section); N60 = 84 (7 birds per section)] were humanely euthanized via cervical dislocation and assigned individual identification numbers using wing tags. Identification numbers were linked to room and section designations. Carcasses were immediately frozen and transported to Clemson University for subsequent analyses.

Diagram of the STEP aviary demonstrating allocated resource locations. Each tier was symmetrically accessible from each litter area. Feed opportunities were provided on the bottom and top tiers, while water opportunities were provided on the bottom and middle tiers. Perch space was provided on each tier, with additional perching opportunities found on each cross beam. Nesting space was provided on the middle tier.

Diagram of the N60 aviary demonstrating allocated resource locations. Only the bottom tier was accessible from each litter area. Other tiers and resources were to be navigated internally after entering the bottom tier. Feed opportunities were provided on the bottom and middle tiers, while water opportunities were provided on the bottom and top tiers. Perch space was provided on each tier, with additional perching opportunities found on each metal ledge. Nesting space was provided on the top tier.

2.4. Ex Vivo Computed Tomography (CT) Image Acquisition and Analysis

After transport to Clemson University, frozen hens were moved to the Godley-Snell Research Center for image procurement (60 WOA; n = 180). For each scan, three birds were placed in a single-file line in right lateral recumbency above a hydroxyapatite calibration phantom (QRM Quality Assurance in Radiology and Medicine, Möhrendorf, Germany). The procedures performed ensured no skeletal superimposition and identical alignment of each bird during the scanning process. Individual identification numbers were recorded in the scan description to ensure accurate identification during acquisition and analysis. Computed tomography (CT) images were obtained using a helical acquisition with a bone-sharp protocol for subjects weighing 0–11 kg, a slice thickness of 0.5 mm, and a 200 mA “FC30” algorithm. Image reconstruction was performed post-acquisition using a 0.5 mm “standard” algorithm. All scans were conducted using a Toshiba Aquilion TSX-101A 16-slice CT scanner (Toshiba America Medical Systems, Inc., Tustin, CA, USA). Following scan reconstruction, measurements were recorded using 3D Slicer.

The image analysis protocol was developed by an image analysis expert with 5 years of experience (CH) and in consultation with an American College of Veterinary Radiology (ACVR)-certified veterinary radiologist and a veterinarian specializing in laying hen behavior and welfare (AA). All CT image analyses were performed by a graduate student (AM), trained by an image analysis expert, using the open-source image analysis software 3D Slicer (Slicer 5.0.3). The observer underwent calibration and repeatability training prior to data collection to ensure consistency in image segmentation and measurement procedures.

The standardized protocol for recording CT measures of tibiotarsal bone quality included the (1) segmentation of tibiotarsal cortical and medullary bone to isolate voxels of desired bone material, (2) the use of software tools to identify a standardized region of the tibiotarsal diaphysis for analysis, (3) the use of software tools to exclude the diaphyseal regions of the tibiotarsus and fibula, (4) segmentation of the hydroxyapatite (HA) phantom for converting density values to bone mineral density (BMD), and (5) the use of software functions to calculate the density of the segmented bone and phantom.

Using the bone window level/window width (WL/WW) combination of 300/1500, the tibiotarsus was identified in the transverse, sagittal, and dorsal views. Within the sagittal view, the length of the tibiotarsus was measured from the proximal to distal epiphyseal head, and a line half the length of the total bone was placed on the surface of the bone, with the midpoint of the line overlying the midpoint of the tibiotarsus, effectively selecting a region of bone that could be standardized between birds to account for different tibiotarsal lengths. Next, separate masks were applied to the CT image to select and segment the voxels within Hounsfield unit (HU) ranges corresponding to the densities of cortical and medullary bone. For the current study, minimum threshold values for cortical bone were set to 700, while maximum threshold values for medullary bone were set to 300; however, this is dependent on scan geometry and reconstruction. These relevant sections of the segmented diaphyseal regions were then excised using the scissor tool. The island tool was then used to remove the fibula from the segmentations. Additionally, a similar segmentation technique was used to isolate the three known concentrations within the HA phantom. Densities of the cortical and medullary bone and the phantom were calculated using 3D Slicer software functions (Slicer 5.0.3, Brigham and Women’s Hospital, Boston, MA, USA) and recorded in spreadsheet software (Excel, Microsoft Corp., Redmond, WA, USA) Using the density measures of the three known HA concentrations (0, 100, and 200 mg/cm³), a calibration curve was generated, and the HU values were converted to bone mineral density (BMD), as was performed by Harrison et al. (2023) [39]. A more detailed description of the image analysis protocol is provided in Supplementary Materials (Figure S1).

2.5. Radiograph Acquisition and Analysis

Following CT acquisition, birds (60 WOA; n = 180) were thawed completely prior to radiograph acquisition. The skin and feathers were removed over the sternal and pelvic regions to allow full leg extension and to eliminate superimposition during imaging; however, the remainder of the skin and feathers were retained to preserve muscle and bone integrity for further analysis.

At the time of imaging, hens were laid in right lateral recumbency and individual images of the entire sternal region were acquired. Individual identification numbers were used as image labels to ensure correct identification during analysis. Radiographs were obtained utilizing a mobile radiograph unit [POSKOM VET-20BT (POSKOM Co., Ltd., GOYANG, Republic of Korea; radiograph tube C.E.I. OX-70 with maximum acceleration voltage of 90 kVp (Costruzioni Elettroniche Industriali, Arezzo, Italy); radiograph plate VARIAN PAXSCAN2530W (Varex Imaging, Salt Lake City, UT, USA); software SmartDR 3.9.2.8888.2281 (Sound Technologies, Inc., Carlsbad, CA, USA)] at a distance of 80 cm and average voltage of 76 kV and analyzed using a publicly accessible online software (Horos 3.3.6; distributed under the LGPL license at Horosproject.org and sponsored by Nimble Co LLC d/b/a Purview in Annapolis, MD, USA).

This trial followed the standardized protocol developed and published under Harrison et al. (2023) [40] for quantifying KBD through radiographic imaging. Within this protocol, the percentages of complete and incomplete cranial and caudal portions of KBD were calculated along with the proportion of deviation (POD), and calluses located in cranial and caudal regions of the keel bone. A full description of the protocol can be found in Harrison et al. (2023) [40]; however, a summation of the protocol is as follows:

To quantify the number of fractures and calluses, each keel bone was divided into cranial and caudal halves using the length tool in the imaging software. A reference line was drawn along the visceral surface of the keel, extending from the carinal apex to the caudal tip. The midpoint of this line was then used to create an additional perpendicular line dividing the keel into equal halves. Within each region (cranial and caudal), the frequency of radiolucent lines—classified as complete or incomplete fractures—was recorded. A fracture was categorized as complete when involving both bone margins, and as incomplete when affecting one or fewer margins. A callus was defined as a localized area of increased opacity with irregular or disrupted bone margins.

To determine the proportion of deviation (POD), the length tool was utilized to create an imitated ventral margin by placing lines at the opposing ends of the deviation and along the ventral margin of the keel. The total keel bone area was measured using the pencil tool within the software by tracing the bone, including the imitated ventral margin, and stopping at the craniodorsal border where the sternum fuses with the keel bone. The proportion of deviation was calculated as:

$$POD={\displaystyle \frac{VentralDeviationArea}{TotalKeelBoneArea}}\times 100$$

POD, therefore, represents a continuous measurement of the proportion of the keel bone affected by deviation for each individual bird. The prevalence of keel bone fractures and calluses—defined as the percentage of sampled birds per unit exhibiting at least one occurrence of either condition—was calculated at the unit level, with individual birds treated as subsamples. To assess the prevalence of keel bone deviations, a binary threshold of ≥10° deviation angle was applied at the bird level. The number of birds exceeding this threshold was divided by the total number sampled within each unit to determine the unit-level prevalence of deviations. For each unit (84–96 sampled birds out of approximately 144–164 housed), the number of affected birds was divided by the total number sampled.

2.6. Musculoskeletal Dissection Protocol

To compare muscle and skeletal anatomy across aviary systems, selected muscles and bones were dissected and prepared for subsequent analyses following CT and X-ray acquisition. Birds were positioned in dorsal recumbency, and any remaining skin and feathers covering the thoracic cavity and limbs were removed. Individual muscles were identified by tracing the natural separation planes at the epimysial fascia and carefully isolated from adjacent tissues using blunt dissection.

The M. biceps brachii and M. triceps brachii of the left forelimb were identified along their epimysial planes and excised by severing at their origins on the brachial heads and their tendinous insertions near the ulna [41]. The M. pectoralis thoracicus (pectoralis major) was then separated bilaterally from the surrounding connective tissue of the coelomic cavity and removed from its origin on the sternal body and insertion on the pectoral crest of the humerus. Following its removal, the M. supracoracoideus (pectoralis minor) was identified bilaterally and excised from its origin on the sternal body and insertion at the dorsal tubercle of the humerus [41,42].

For the hindlimb, the left femoral head was disarticulated from the acetabulum at the articulatio coxae, and the limb was removed intact to preserve muscle integrity. After removal of skin and feathers, the M. fibularis longus [Peroneus longus (PL)] was identified laterally and excised from its origin at the proximal tibiotarsus and patellar region to its insertion on the posterolateral aspect of the medial cuneiform and the lateral side of the first metatarsal base [41,43]. The M. gastrocnemius pars externa (GPE) was subsequently identified and removed from its origin on the lateral femoral condyle to its insertion on the calcaneus [41,44]. Once these individual muscles were isolated, the remaining hindlimb muscles were excised collectively. The PL and GPE were weighed individually (g), followed by the determination of the total hindlimb muscle group mass for each bird.

Following muscle dissections, bones of interest were collected for biomechanical analysis. In the left forelimb, the humerus was disarticulated from the scapula at the articulatio humeri (shoulder joint) and from the radius and ulna at the articulatio cubiti (elbow joint) [41]. In the left hindlimb, the tibiotarsus was disarticulated proximally from the femur at the articulatio genus (knee joint) and distally from the tarsometatarsus at the articulatio intertarsalis (hock joint) [41]. After removal of the tibiotarsus, the fibula was isolated and excised. Left humeri and tibiotarsi were immediately placed in saline solution for transport to biomechanical testing. The right forelimbs and hindlimbs were disarticulated at their respective joints, excised in full, and frozen for subsequent analyses.

2.7. Tibiotarsi and Humeri Biomechanical Testing

Following dissection, the mechanical properties of the left tibiotarsi (n = 180) and humeri (n = 180) were evaluated using a three-point bending test in accordance with the standards established by the American National Standards Institute (ANSI). Testing was conducted using an Instron Dynamic and Static Material Test System (Model 5944; Instron Corp., Canton, MA, USA) equipped with a 500 N load cell and controlled through the Automated Material Test System software (8800 MT Controller; Instron Corp., Canton, MA, USA). The testing protocol for tibiotarsal biomechanics followed previously published methods [39,45], whereas the humeral protocol was adapted from ANSI guidelines for three-point bending of animal bones [46].

Each humerus was positioned with the medial aspect facing upward and centered across the fulcrum, with the midpoint placed equidistant (2 cm) from each support. Load and displacement data were continuously recorded during testing and subsequently used to calculate bone-breaking strength (N) and stiffness (N/mm).

2.8. Tibiotarsi and Humeri Ash Percentage

Whole legs previously stored frozen were thawed at refrigeration temperature (~4 °C) for 24 h. Soft tissues surrounding the right tibiotarsi and humeri were carefully removed, and the fibulae were separated from the tibiotarsi. The dry weights of empty ceramic crucibles were recorded prior to sample preparation.

The right tibiotarsi (n = 180) and humeri (n = 180) were fragmented into small pieces, placed into pre-weighed crucibles, and weighed. Samples were air-dried at 100 °C for 1 h, transferred to a desiccator for 1 h, and reweighed. Bones were then ashed at 600 °C for 6 h in a Thermolyne 30400 oven (Barnstead International, Dubuque, IA, USA). After ashing, crucibles were placed in a desiccator for 1 h before final weighing.

The percentage of bone ash was calculated using the following equation:

$$\mathrm{Bone}\mathrm{Ash}(\%)={\displaystyle \frac{\mathrm{Weight}\mathrm{after}\mathrm{ashing}\left(\mathrm{g}\right)}{\mathrm{Dry}\mathrm{weight}\mathrm{before}\mathrm{ashing}\left(\mathrm{g}\right)}}\times 100$$

2.9. Statistical Analysis

All analyses were conducted in R (R Core Team, Version 3.3.1, 2023; Vienna, Austria [47]). Data were analyzed using mixed-effects models implemented in the glmmTMB and lme4 packages [48]. Prevalence was modeled at the section level using a binomial distribution and a logit link, whereas continuous measures (e.g., CT-derived bone mineral density, bone strength, muscle mass, and ash content) were analyzed using linear mixed-effects models with individual birds as observational units. Housing system (N60 vs. STEP) was included as a fixed effect, and section nested within room was included as a random effect to account for clustering. Model assumptions and overdispersion were evaluated using residual diagnostics (DHARMa), and a beta-binomial structure was applied when appropriate. Least-squares means (LSMeans), and their standard errors (SE), were obtained with emmeans [49], and pairwise comparisons were adjusted using Tukey’s method. Results are reported as LSMeans ± SE on the response scale, with significance declared at p < 0.05 and trends at 0.05 ≤ p ≤ 0.10.

3. Results

3.1. Bone Mineral Density Results

Computed tomography analysis revealed significant effects of aviary design on bone mineral density (BMD) in both cortical and medullary regions of the tibiotarsus (Table 1). Hens housed in the STEP aviary exhibited higher cortical and medullary BMD values than those housed in the N60 system (p = 0.029 and 0.036, respectively). These results indicate that the more open STEP configuration, which offers greater vertical access and activity opportunities, supported enhanced skeletal mineralization compared with the N60 system.

Table 1. CT Scan Analysis Results.

	Bone Mineral Density (Tibiotarsus; mg/cm3)
SYS	Cortical Bone	Medullary Bone
STEP	1368.85 ± 35.16 a	76.58 ± 6.25 a
N60	1086.25 ± 21.36 b	51.26 ± 7.35 b
p-value	0.029	0.036

Bone mineral density values across systems (SYS). Data are displayed as LSMeans ± SE, where superscripts of varying letters indicate significant differences (p < 0.05).

3.2. Keel Bone Damage Results

Radiographic evaluation of keel bone damage (KBD) demonstrated significant effects of aviary design on several damage categories (Table 2). Hens housed in the STEP system exhibited a higher prevalence of incomplete cranial fractures (p = 0.036), complete and incomplete caudal fractures (p = 0.042 and 0.029, respectively), and greater frequencies of cranial and caudal calluses (p = 0.039 and 0.026, respectively). Conversely, hens in the N60 system displayed a higher proportion of deviations (POD; p = 0.031). These findings indicate that hens housed in the STEP aviary experienced a greater overall incidence of fracture-related keel damage, whereas deviation-type lesions were more common in hens housed in the N60 configuration.

Table 2. Keel Bone Damage Results.

Percentage of Keel Bone Fractures
SYS	Cranial (Complete)	Cranial (Incomplete)	Caudal (Complete)	Caudal (Incomplete)	Prevalence of Deviations (≥10°)	Cranial Calluses	Caudal Calluses
STEP	5.63 ± 1.36 a	16.52 ± 3.36 a	12.25 ± 2.52 a	31.25 ± 3.66 a	65.63 ± 6.69 a	12.25 ± 1.03 a	66.58 ± 3.66 a
N60	2.36 ± 2.63 a	7.85 ± 2.58 b	6.25 ± 1.03 b	19.52 ± 3.58 b	83.58 ± 7.35 b	7.85 ± 0.99 b	43.85 ± 4.52 b
p-value	0.096	0.036	0.042	0.029	0.031	0.039	0.026

Keel bone damage values across housing systems (SYS). Data are presented as estimated marginal means (LSMeans) ± SE, where different superscript letters indicate significant differences between housing systems (p < 0.05). The prevalence of keel bone fractures and calluses was defined as the percentage of sampled birds per unit with ≥1 occurrence of the condition. Deviation prevalence was defined as the percentage of sampled birds per unit exhibiting keel bone deviation angles ≥ 10°.

3.3. Musculoskeletal Dissection Results

Aviary design significantly influenced muscle development in multiple anatomical regions (Table 3). Hens housed in the STEP system exhibited greater muscle mass in the M. pectoralis thoracicus (p = 0.041), M. supracoracoideus (p = 0.037), M. biceps brachii (p = 0.045), and M. triceps brachii (p = 0.048) compared with hens housed in the N60 system. Similarly, STEP hens displayed heavier hindlimb muscles, including the M. fibularis longus (PL; p = 0.032) and M. gastrocnemius pars externa (GPE; p = 0.039), as well as greater total hindlimb muscle mass (p = 0.028). These results indicate that the increased vertical accessibility and spatial layout of the STEP aviary promoted enhanced forelimb and hindlimb muscle development relative to the N60 configuration.

Table 3. Muscle Dissection Results (g).

SYS	Bicep Brachii	Triceps Brachii	Pectoralis Major	Pectoralis Minor	PL	GPE	Leg Muscle Group
STEP	3.93 ± 0.04 a	3.43 ± 0.08 a	135.63 ± 1.85 a	55.96 ± 0.51 a	7.26 ± 0.11 a	9.36 ± 0.09 a	139.89 ± 1.36 a
N60	3.53 ± 0.05 b	3.36 ± 0.06 a	116.52 ± 1.31 b	52.06 ± 0.43 b	7.24 ± 0.11 a	9.33 ± 0.08 a	129.26 ± 1.23 b
p-value	0.026	0.065	0.031	0.039	0.203	0.163	0.042

Muscle weight values across systems (SYS). Data are displayed as LSMeans ± SE, where superscripts of varying letters indicate significant differences (p < 0.05).

3.4. Tibiotarsi and Humeral Biomechanical Testing

Aviary design had a significant effect on multiple indicators of bone mechanical integrity and composition (Table 4). Hens housed in the STEP system exhibited higher bone-breaking strength in both the humerus and tibiotarsus (p = 0.033 and 0.041, respectively) and greater humeral stiffness (p = 0.046) compared with those housed in the N60 system. Ash content was also higher in STEP hens for both skeletal elements (p = 0.038 for humerus; p = 0.029 for tibiotarsus), indicating enhanced mineralization. Collectively, these findings demonstrate that hens maintained in the STEP aviary possessed stronger and more mineralized bones, consistent with the increased opportunities for movement and load-bearing activity provided by its vertical design.

Table 4. Bone Biomechanical Testing Results.

	Bone Biomechanical Testing
	Breaking Strength (N)		Stiffness (N/mm)
SYS	Tibia	Humerus	Tibia	Humerus
STEP	159.96 ± 7.58 a	66.96 ± 6.89 a	89.60 ± 5.24 a	26.58 ± 2.12 a
N60	126.52 ± 9.58 b	50.58 ± 5.30 b	83.44 ± 5.35 a	15.58 ± 2.21 b
p-value	0.033	0.048	0.049	0.043

Bone biomechanical testing values across systems (SYS). Data are displayed as LSMeans ± SE, where superscripts of varying letters indicate significant differences (p < 0.05).

3.5. Tibial and Humeral Ash Percentage Results

Ash analysis revealed significant effects of aviary design on bone mineral content (Figure 3). Hens housed in the STEP aviary exhibited higher tibial and humeral ash percentages than those housed in the N60 system (p = 0.032 and 0.039, respectively). These findings indicate that skeletal mineralization was enhanced in hens maintained in the STEP configuration, consistent with greater opportunities for movement and mechanical loading.

Figure 3. Ash percentage of tibial and humeral bones in hens housed in the STEP and N60 systems. Values are presented as LSMeans ± SE where superscripts of varying letters indicate significant differences (p < 0.05).

4. Discussion

Research on the effects of exercise on musculoskeletal health has demonstrated significant biological changes in bone and muscle structure in laying hens associated with increased opportunities for enrichment and movement in alternative housing systems compared to traditional colony cages [5,7,50]. As osteoporosis remains a prominent contributor to reduced hen welfare within the egg industry and continues to pose concerns for both producers and consumers, further investigation into the implications of exercise on skeletal health is clearly warranted.

Recent behavioral research has identified perching, flying, dustbathing, foraging, and nesting as highly motivated and desirable activities for laying hens [24]. These behavioral tendencies, derived from their ancestral jungle fowl, demonstrate that hens utilize elevated perches to avoid predation and show a strong preference for the highest available perch, particularly during nighttime hours [51,52,53,54,55,56]. Similarly, studies examining nest availability have shown that hens are highly motivated to access nest sites and, when deprived of them, exhibit prolonged frustration and restlessness [12,56,57,58]. Consequently, the spatial distribution of essential resources may impact exercise opportunities within extensive housing systems. However, increased locomotive freedom and tiered configurations in such environments also introduce potential risks, with incidents of collisions and falls more likely [5]. Thus, research involving housing configurations that employ these behavioral opportunities is necessary to promote beneficial management practices that refine hen welfare in commercial settings.

4.1. Bone Mineral Density

This study found that hens housed in the N60 aviary—where limited pathways exist for a variety of vertical movement within the system—had lower cortical bone mineral density (BMD) than hens housed in the STEP system, which provided unrestricted choice of vertical movement and access to all levels of the system. Medullary BMD followed a similar pattern, with N60 hens exhibiting lower values than those in the STEP aviary. This pattern suggests that differences in tier accessibility and movement requirements between systems contributed to altered mechanical loading on long bones. Additional research on these behaviors is to be published in a subsequent paper; however, these findings are consistent with previous research demonstrating that increased opportunities for movement and exercise enhance musculoskeletal development, leading to greater muscle mass and bone strength in hens with greater freedom of movement compared with those housed in restricted environments [5,9,13,45,59]. This relationship is likely largely due to mechanical stress on bone during muscle contraction [3,60,61].

Comparison of the two aviary configurations, supported by previous behavioral observations, indicates that the STEP system likely promoted greater opportunities for exercise than the N60. In the STEP design, hens could move vertically between tiers in a variety of ways, ranging from short flights to long flights, hops, walking on a ramp, or flight-assisted jumps to access feed, perches, or litter areas at the various levels. In contrast, the confined nature of the N60 system restricted hens to a constrained internal pathway between tiers, reducing flight patterns and eliciting jumping responses, whereas hens housed in the STEP aviary could choose their level of exercise. Hens in the N60 are eligible to make almost exclusively small wing hops or wing-assisted jumps from level to level, as they moved between floors, perches, and ledges, with flight only possible to and from the litter and lowest tier. Consequently, hens in the STEP aviary likely experienced greater mechanical stimulation on the appendicular skeleton through more frequent and varied movement patterns due to the aviary configuration. For example, hens occupying the middle tier in the STEP system could make longer jumps (or flights) to reach the litter area, whereas hens in the N60 must perform a series of shorter movements to navigate down to the litter area with the enclosed structure of the system. Thus, these differences in system structure and placement of some resources likely explain the higher cortical and medullary BMD observed in STEP hens through increased mechanical loading.

All pullets were reared under similar conditions prior to placement in the aviary systems, minimizing the confounding effects of early management. Although Hy-Line Brown hens typically begin laying around 16 weeks of age, our hens first began laying at 17–18 weeks of age and reached peak production at 24 weeks of age [37]. Because hens were transferred into the aviaries at 16 weeks, cortical bone deposition likely continued for several weeks until egg production commenced [1]. Increased vertical movement during this period would have further stimulated bone formation, as documented in previous studies linking activity and mineral deposition [9,27,28,59]. Thus, the potential for greater and more varied vertical movement in the STEP system, while pullets continued to deposit bone, could be associated with enhanced cortical and medullary bone development, the latter also supported by estrogen-driven medullary deposition. By contrast, hens in the N60 aviary—restricted to internal vertical pathways—likely underwent normal estrogenic medullary bone formation but experienced reduced cortical mineralization due to limited mechanical stress on the tibiotarsi. Collectively, these results demonstrate that aviary design can directly influence skeletal mineralization. The enhanced vertical mobility and spatial openness of the STEP configuration promoted greater bone mineral density and overall musculoskeletal health compared with the more internally partitioned N60 design.

4.2. Keel Bone Damage

Keel bone damage (KBD) remains one of the most prevalent welfare issues in commercial egg production, with 50–80% of hens reported to exhibit some form of fracture or deformity by the end of lay [16,62,63]. In the present study, radiographic evaluation revealed a higher prevalence of new and old fractures in hens housed in the STEP aviary compared with those in the N60, as evidenced by marginal breaks and callus formation. These findings are consistent with previous reports identifying KBD as a multifactorial condition influenced by genetics, high egg production, calcium depletion, and collisions within housing systems [63,64,65].

The keel bone’s anatomical position and curvature render it particularly susceptible to impact and mechanical strain, especially in the caudal region where muscling is limited [16,62,66]. Similar to earlier studies, this research found that both complete and incomplete caudal fractures, as well as caudal calluses, occurred more frequently than cranial lesions [66]. This pattern may reflect the protective role of the pectoral musculature over the cranial keel, which absorbs impact forces during collisions, while the caudal apex remains comparatively exposed and less cushioned.

System differences observed in the current study also support the role of aviary design in influencing fracture risk. The lower incidence of fractures among hens housed in the N60 could be associated with reduced opportunities for flight and higher-force collisions, due to its enclosed, internally connected structure. In contrast, the open configuration of the STEP system, which allows hens to traverse greater vertical distances through fight-or-flight-assisted jumping, increases the likelihood of accidental impacts and keel fractures [5,7,8,11]. These observations align with previous reports linking elevated collision risk to housing designs that promote high levels of aerial movement [5,11].

Keel deviations also contribute significantly to welfare concerns, as they can cause chronic pain and impaired performance [67]. Deviations typically develop gradually through bone remodeling rather than acute impact, often occurring in response to load-bearing stress during perching [17]. In this study, hens housed in the N60 system exhibited a higher proportion of deviation (POD) than those in the STEP. Previous work has shown that, during roosting, hens bear most of their body weight on the keel bone, generating forces up to five times greater than those placed on a single footpad [18]. Because perches in the N60 were located internally, adjacent to feed and water resources, hens may have perched more frequently or for longer durations while navigating between tiers. The increased perching frequency, combined with sustained load pressure on the keel, could have contributed to the higher deviation rates observed in N60 hens.

Additionally, perch characteristics, including material, shape, and placement, are known to influence both the incidence and severity of KBD [5,18,19]. The STEP system incorporated external beams of varied profiles that hens could also use as perching sites, potentially dispersing body weight across different contact points on the keel. Differences in perch design and orientation between the two systems may therefore have modulated keel loading patterns and contributed to the contrasting lesion profiles observed.

4.3. Musculoskeletal Muscle Dissections

The results of this study indicate that hens housed in the N60 aviary had consistently lower M. biceps brachii, M. triceps brachii, M. pectoralis major, and M. pectoralis minor weights compared with those housed in the STEP system. These muscles are central to flight and flight-assisted locomotion: the forelimb muscles enable bending and extension of the wing, while the pectoral and supracoracoideus muscles power the upward and downward strokes required for vertical movement [41]. The higher muscle weights observed in STEP hens are therefore consistent with the potential increase in opportunity for flight and jumping activity in this system, which would provide greater mechanical loading compared with the more internally restricted design, such as the N60. Similar associations between increased movement and muscle deposition have been documented in laying hens provided with enriched or spacious environments [5,13,50].

Muscle hypertrophy, characterized by an increase in muscle fiber size, is primarily driven by mechanical loading when protein synthesis exceeds degradation [28]. Chronic stimulation through repeated contractions promotes the addition of myonuclei and expansion of muscle fiber cross-sectional area, leading to measurable increases in muscle mass. Conversely, when mechanical activity is limited, atrophy can occur as catabolic processes dominate. The present findings support this concept, as hens housed in the more vertically accessible STEP system could have experienced greater mechanical stress on the flight musculature, resulting in higher muscle mass even after sexual maturity.

In contrast, no significant differences were detected in the M. fibularis longus (PL) or M. gastrocnemius pars externa (GPE) between aviary systems. The PL, located on the anterolateral tibia, primarily contributes to leg stability and foot eversion during walking and perching [41,43], while the GPE, positioned posteriorly, supports posture and limb extension during perching and standing [44]. Because both aviaries provided equal perch space per bird (15 cm), these behaviors—and thus the mechanical demands on the PL and GPE—were likely similar across systems.

However, total hindlimb muscle group mass was greater in STEP hens than in N60 hens. This difference likely reflects additional loading on muscles surrounding the femur and tibiotarsus during jumping and landing behaviors, which could occur more frequently in the open, multi-tiered STEP configuration. Such repeated, load-bearing movements have been shown to stimulate muscle development in both avian and mammalian species through adaptive increases in protein accretion and muscle fiber hypertrophy [3,4,5,6,7,8,9,10]. Collectively, these results suggest that although each aviary shared vertical height and size characteristics, the increased opportunities for varying types and lengths of vertical locomotion provided by the STEP aviary could be associated with the enhanced overall muscle development relative to the N60 design.

4.4. Bone Biomechanical Properties

Biomechanical testing revealed that hens housed in the N60 aviary exhibited significantly lower tibial and humeral breaking strength values, as well as reduced humeral stiffness, compared with hens in the STEP system. Bone biomechanical measures serve as important indicators of skeletal integrity: breaking strength represents the force required to induce structural failure, while stiffness reflects the degree of deformation a bone undergoes before breaking [68,69,70]. Both parameters are closely associated with bone mineralization and architecture, with breaking strength primarily associated with cortical integrity in pneumatic bones such as the humerus [3] and stiffness with cortical thickness and density [68,69]. The lower tibial and humeral strength values observed in N60 hens were consistent with their lower BMD values, whereas STEP hens displayed stronger and stiffer bones. These results align with previous findings that show that bone density and breaking strength are positively correlated and that enhanced exercise opportunities promote superior biomechanical properties [2,5,9,13,45,59].

The improved bone strength observed in STEP hens could reflect greater mechanical loading associated with increased opportunities for external vertical-tier access and flight-assisted activity in this aviary design. Mechanical stress applied to bone stimulates osteogenic responses that promote mineral deposition and structural reinforcement through adaptive remodeling [3,4,5,6,7,8,9,10]. In contrast, the limited variety of exercise types in the N60 aviary may have reduced the frequency and intensity of such loading stimuli, leading to weaker bone tissue. Bone remodeling at the endosteal surface involves medullary deposition often at the expense of cortical mass, and excessive osteoclastic activity in this region can compromise overall bone strength [1,2]. Any enhanced mechanical stress experienced by STEP hens could have mitigated this process, thereby maintaining cortical density and improving biomechanical outcomes.

Previous studies have demonstrated that access to vertical activities, such as perching and jumping, enhances bone strength and helps preserve cortical bone compared with caged environments [3,71]. However, the positive effects of exercise on bone maintenance are known to diminish with age [11]. Consistent with this pattern, tibial stiffness did not differ significantly across aviary systems in the present study, suggesting that while increased activity enhanced overall bone strength, housing configuration had limited long-term effects on bone tissue’s elastic properties.

4.5. Ash Percentage

Bone mineral content was evaluated using an ashing procedure, in which the ash percentage represents the proportion of inorganic minerals within the bone matrix [72]. In the present study, hens housed in the N60 aviary exhibited significantly lower tibial and humeral ash percentages than those housed in the STEP system. Higher ash content generally indicates greater skeletal mineralization and is widely used as a proxy for bone quality and strength [73]. The reduced ash values observed in N60 hens are consistent with their lower bone mineral density and weaker biomechanical performance, reinforcing previous evidence that constrained movement opportunities can negatively influence skeletal mineral retention.

The STEP aviary, which offered opportunities for more varied vertical locomotion and greater physical exertion, enabled increased mechanical loading of the skeleton, stimulating mineral deposition and preserving bone integrity. Conversely, the more enclosed N60 configuration may have restricted such exercise opportunities, contributing to reduced mineral content through diminished mechanical strain and potential muscle atrophy. It should also be noted that the two aviary systems differ in several structural characteristics, including space allocation, nest space availability, and the presence of ramps in the STEP system. Therefore, the observed differences between systems may reflect the combined effects of multiple design features rather than vertical access alone. Collectively, the ash data corroborate the CT and biomechanical findings, underscoring that aviary design is pivotal in supporting mineralized, structurally robust bones in laying hens. This study was conducted using three rooms per aviary configuration within a single commercial flock cycle of Hy-Line Brown hens. Although room- and section-level replication provided structural independence under commercial-scale conditions, replication across additional facilities or flock cycles would further strengthen external validity. Moreover, as this study investigated only a brown-feathered genotype, the results may not directly extrapolate to lighter white-layer strains that differ in body mass and locomotor behavior. Accordingly, findings should be interpreted within the context of the population and production setting evaluated.

5. Conclusions

The findings of this study demonstrate that aviary design exerts a measurable influence on musculoskeletal health in laying hens. Although a limitation of this study was a lack of behavioral data collection, this study showed that hens housed in the STEP system, which provided the potential for greater flight-assisted movement opportunities due to system configuration, exhibited higher bone mineral density, strength, ash content, and greater muscle development, compared with hens in the more enclosed N60 configuration. However, these benefits were accompanied by a higher incidence of keel bone fractures, underscoring the trade-off between increased mobility and collision risk. Collectively, the results support the hypothesis that housing systems providing greater physical activity opportunities enhance skeletal and muscular condition in laying hens. Future work should integrate behavioral monitoring with biomechanical and physiological assessments to better understand how specific movement patterns within aviary systems contribute to long-term bone integrity and welfare outcomes.