Hypoxia-induced apoptotic pathways and their contribution to fibrosis progression in wooden breast myopathy of fast-growing broilers.

Fibrotic breast myopathy, including the Wooden Breast (WB) condition, represents one of the most economically and welfare-significant challenges confronting modern commercial broiler production. Despite growing recognition of the syndrome, the cellular and molecular mechanisms linking chronic myofibre hypoxia to the progressive fibrotic pathology remain incompletely defined. This study employed a four-group experimental model using 192 male Ross 308 broilers subjected to chronic hypobaric hypoxia (simulated altitude 3,000 m; 13.0% O₂) with and without pharmacological pan-caspase inhibition via Z-VAD-FMK (2 mg/kg, three times weekly) over a 42-day production period, to characterise the hypoxia-apoptosis-fibrosis axis in the pectoralis major muscle. Chronic hypoxia induced progressive HIF-1α protein accumulation (4.8-fold by Day 42; p < 0.001), accompanied by marked elevation in apoptotic index (14.3% TUNEL-positive nuclei; p < 0.001), a strongly pro-apoptotic Bax/Bcl-2 ratio shift (8.2 vs. 1.0 in controls), and downstream activation of the intrinsic caspase cascade (cleaved caspase-9, caspase-3, and PARP). Concomitantly, hypoxic birds demonstrated severe oxidative stress (4.4-fold MDA elevation; depleted SOD, CAT, GPx, and GSH), robust NF-κB-mediated inflammation (IL-1β, IL-6, IL-8, TNF-α all elevated > 8-fold), and extensive extracellular matrix remodelling (hydroxyproline content 7.8 µg/mg; fibrotic area 31.4%; upregulation of COL1A1, COL3A1, TGF-β1, MMP-2, and MMP-9). The blockade of pan-caspases markedly decreased the severity of WB lesions, fibrosis (19.8%), apoptosis (6.1%), pro-inflammatory cytokines, and ECM turnover without affecting the upstream accumulation of HIF-1α, indicating that the executioner caspase activation was one of the key nodes of signal amplification during the development of the disease. A highly significant positive correlation between the expressions of HIF-1α, apoptotic index, hydroxyproline level, and NF-κB (r = 0.78-0.87; all P < 0.001) provided evidence for a mechanistic involvement of hypoxia-induced apoptosis in the pathogenesis of the disorder.