All experimental procedures involving animals were conducted in strict accordance with national and international guidelines for the care and use of animals in research. The study complied with the principles of the Animal Research: Reporting of In Vivo Experiments 2.0 guidelines, the World Organization for Animal Health Terrestrial Animal Health Code, and applicable Indonesian animal welfare regulations.

The experimental protocol was reviewed and approved by the Animal Ethics Committee of the School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia (Approval No. 209/KEH/SKE/ IV/2024). Ethical review covered all aspects of animal use, including housing conditions, rearing systems, feeding management, handling procedures, sampling strategy, and slaughter methods.

Throughout the study period, birds were monitored daily for health status, behavior, and signs of distress. Stocking density, ventilation, access to feed and water, and environmental enrichment were maintained to minimize stress and ensure animal welfare under both intensive and free-range systems. No invasive procedures were performed prior to slaughter, and no mortality related to experimental handling was observed.

Slaughtering was carried out at 24 weeks of age following a manual halal procedure, after a 24-h fasting period, in accordance with accepted humane slaughter practices to minimize pain and distress. Tissue sampling for histological analysis was performed only postmortem. All efforts were made to reduce the number of animals used while ensuring sufficient statistical power and scientific validity.

The experiment was conducted from January to November 2024. The IPB-D3 chickens were housed at the Field Laboratory of Animal Breeding and Genetics, Faculty of Animal Science, IPB University, Dramaga, Bogor, located at approximately 250 m above sea level. Histological analyses, including hematoxylin-eosin (HE), picrosirius red (PSR), and immunohistochemical (IHC) staining of Pec and Fem muscles, were performed in the Veterinary and Zoology Laboratory, Anatomy and Histology Section, National Research and Innovation Agency (BRIN), Indonesia.

This preliminary study was conducted as part of the research series by Kuswandi et al. [31]. A total of 90 IPB-D3 chickens (12 weeks old) were used. All chickens underwent a 7-day acclimatization period before the experiment, during which their health status was monitored daily. Birds were randomly allocated into two rearing systems, intensive and free-range, each consisting of 45 chickens (21 males and 24 females), maintaining an approximately 1:1 sex ratio. The rearing period lasted 12 weeks. The IPB-D3 chicken is a fast-growing composite line derived from a cross between Pelung × Sentul (male) and Kampung × Broiler Cobb (female), each contributing 25% of the genetic composition.

At 24 weeks of age, chickens were slaughtered following a manual halal procedure after a 24-h fasting period. From each rearing system, five chickens (n = 10 total; 2 males and 3 females per group) were randomly selected for histological analysis. Following slaughter, carcasses were scalded in 70°C water for 10–20 s to remove feathers [33]. Internal organs were excised, and carcass weights (without giblets) were recorded. Samples of Pec and Fem muscles were collected, and 2 × 2 cm sections were fixed in 10% neutral-buffered formalin (Merck, Darmstadt, Germany) for 48 h, then transferred to 70% ethanol before standard paraffin (Merck) embedding. Tissues were sectioned serially at 5 µm thickness (three sections per muscle).

The growth performance of IPB-D3 chickens did not differ significantly (p > 0.05) between the two rearing systems (Table 1). Both intensive and free-range chickens exhibited comparable body and carcass weights, indicating similar growth efficiency under different management conditions.

Microscopic examination revealed that the myofiber cross-sectional area of the Fem muscle was significantly larger in chickens reared under the free-range system compared with those reared intensively (Hedges’ g = 1.088; 95% confidence interval [CI] [0.175, 2.296]; p < 0.05) (Table 2). Likewise, the proportion of high-intensity type IIX myofibers was greater in the Fem muscle of free-range chickens (Hedges’ g = 11.657; 95% CI [−0.170, 2.610]; p < 0.05) (Table 3). In contrast, the relative percentage of intramuscular collagen was higher in the Fem muscle of intensively reared chickens (Hedges’ g = 1.329; 95% CI [−2.668, −0.058]; p < 0.05) (Table 2).

Overall, no distinct morphological abnormalities or fiber arrangement differences were observed between the two rearing systems. The muscle microstructure of IPB-D3 chickens consisted of fasciculi bound by collagenous perimysium, with individual myofibers enclosed by endomysium (EM). Most myofibers displayed a polygonal shape, although some appeared round (Figures 1 and 2). Under polarized light, thick collagen fibers were predominantly localized in the perimysium, while thin fibers were observed mainly in the EM (Figure 3).

Although overall morphology appeared similar, two histomorphometric parameters differed significantly (p < 0.05) between rearing systems. The Pec muscle contained more myofibers per mm² than the Fem muscle, corresponding to its smaller fiber size. Conversely, the Fem muscle in free-range chickens exhibited larger fasciculus and myofibril areas, reflecting muscle hypertrophy associated with higher physical activity. In both rearing systems, the fasciculus area was significantly greater (p < 0.05) in Pec than in Fem muscles. Within the intensive system, the relative intramuscular collagen percentage was significantly higher in Fem than in Pec muscles (Table 2).

IHC analysis revealed clear variations in staining intensity among muscle fibers, representing differential antibody binding to type IIX MyHC. The distribution patterns of type IIX fibers varied between Fem and Pec muscles (Figure 4) but remained generally consistent across rearing systems (Table 3).

High-intensity myofibers, corresponding to type IIX fibers, were more abundant in the Fem muscle and particularly prevalent in free-range chickens. Moderate-intensity fibers were more frequently observed in the Pec muscle, whereas low-intensity fibers showed high variability with no significant differences between muscle types or rearing systems (Table 3). Morphologically, low-intensity fibers exhibited a round shape, while high-intensity fibers appeared polygonal (Figure 5).

Notably, IHC for slow-type I myofibers yielded negative results in all samples, indicating a predominance of fast-twitch fiber composition in the muscles of IPB-D3 chickens.

No significant differences were observed in the body growth performance of IPB-D3 chickens between the intensive and free-range systems. This outcome is consistent with the genetic potential of the IPB-D3 line, which is a fast-growing composite breed derived from IPB-D1 chickens registered under the Ministry of Agriculture of the Republic of Indonesia (Decree No. 693/KPTS/PK.230/M/9/2019). The parent line, IPB-D1, is known for its adaptability to both intensive and free-range systems, showing efficient feed conversion ratios of approximately 2.88 (intensive) and 3.44 (free-range) [41]. The comparable performance of IPB-D3 chickens in both rearing systems indicates a stable growth efficiency and adaptability of this improved local line across diverse management conditions.

The significant increase in the myofiber cross-sectional area of the Fem muscle in free-range chickens suggests enhanced muscle activity and hypertrophy. This effect can be attributed to greater locomotor activity and environmental stimulation in the free-range system, which promotes muscle fiber enlargement. Similar findings were reported by Fu et al. [42], where the thigh muscle diameter was significantly higher in free-range chickens than in caged counterparts at 26 weeks of age. Free-range conditions have also been associated with increased thigh muscle development in Lueyang black-bone chickens [43]. However, since the initial myofiber size at the start of this study was not measured, longitudinal evaluation across growth stages is recommended for future work.

The current study provides the first characterization of myofibers in IPB-D3 chickens. The Pec fasciculus area of IPB-D3 (1.83–1.95 × 10⁵ µm²) was slightly smaller than that of Pelung chickens (2.1 × 10⁵ µm²), one of its parental lines. Similarly, the cross-sectional area of breast myofibers in IPB-D3 chickens (≈1210–1300 µm²) was smaller than that of mature Pelung chickens (1937.74 µm² at 40–56 weeks, body weight ≈3 kg) [11]. Kampung chickens at 63 days (924 g) had smaller myofibers (960.56 µm²) [15], while broilers aged 21 days and weighing 48 g showed even smaller fiber areas, ranging from 310.02 to 769.59 µm² [13, 14, 22]. In Sentul chickens (12 weeks old), thigh and breast myofiber diameters were 27.74 µm² and approximately 39 µm², respectively. These comparisons highlight the broad range of myofiber dimensions across chicken breeds, influenced by genotype, age, nutrition, and muscle type [44]. Since meat quality is closely related to histological and biochemical muscle properties [45, 46], understanding fiber size variation is critical. Larger myofibers generally correlate with reduced tenderness due to denser muscle texture [47].

The Pec muscle contained more myofibers per mm² than the Fem muscle, reflecting smaller fiber dimensions. According to Scheuermann et al. [48] and Huo et al. [49], increased fiber number contributes to higher breast meat yield, although fiber hypertrophy remains the dominant determinant of total muscle mass. Other biological factors such as bone growth and fat deposition may also influence body weight, as noted by Lawrence and Fowler [50] and Li et al. [51].

Histological observations revealed discontinuous intramuscular collagen fibers within the perimysium, predominantly composed of thick fibers, while the EM consisted mainly of thin fibers. In the intensive rearing system, the Fem muscle contained a higher proportion of intramuscular collagen than its free-range counterpart. Variability in collagen deposition among samples suggests its contribution to meat texture and tenderness [52]. Thickened perimysial connective tissue is known to increase shear force values in various meats, including chicken, whereas higher intramuscular fat content generally decreases shear force [53–55]. These findings indicate that collagen composition, though important, is only one of several factors determining overall meat tenderness.

High-intensity type IIX myofibers were more abundant in the Fem muscle of free-range chickens, suggesting rearing-related differences in fiber-type distribution. Muscles with a predominance of type IIX fibers tend to exhibit poorer meat quality, characterized by paler color, reduced water-holding capacity, and lower tenderness, primarily due to enhanced glycolytic metabolism and a rapid postmortem pH decline [56–58]. The larger myofiber cross-sectional area observed in the Fem muscle further supports the hypertrophy of fast-twitch fibers under active movement. Du et al. [59] similarly reported that fast-growing chickens possess larger myofiber diameters and a greater proportion of type II fibers than slow-growing breeds. Type IIX fibers, identified as high-intensity white fibers with low myoglobin content, are typically associated with coarser meat texture [60]. Intermediate-intensity fibers may represent mixed fiber types, as seen in Ross 308 broilers and Xueshan chickens [23, 40, 61]. Such heterogeneity indicates the adaptive plasticity of skeletal muscles, where environmental and physiological stimuli can induce fiber-type shifts [62–64]. The higher occurrence of type IIX fibers in the Fem muscle of free-range IPB-D3 chickens may therefore influence the textural properties of thigh meat and warrants further biochemical and sensory evaluation.

The present study also noted that low-intensity stained myofibers were predominantly round, whereas high- and moderate-intensity fibers exhibited polygonal shapes in both Pec and Fem muscles. Rounded fibers with variable diameters are often indicative of regenerating or immature fibers, whereas polygonal fibers represent mature, functionally stable myofibers [65, 66]. Soendenbroe et al. [61] associated round myofibers with type I fibers, consistent with the current observation that round fibers in IPB-D3 chickens showed low IHC intensity for type IIX. However, all samples were negative for slow-type I myofiber staining, despite positive control reactivity in Ross 308 and Xueshan chickens [23, 40]. This suggests that the IPB-D3 line predominantly consists of fast-twitch fibers, though further molecular confirmation is required.