Chlorogenic acid mitigates high stocking density-induced breast muscle quality decline in broilers via modulating mitochondrial redox homeostasis and glycolytic metabolism

Chlorogenic acid mitigates high stocking density-induced breast muscle quality decline in broilers via modulating mitochondrial redox homeostasis and glycolytic metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chlorogenic acid mitigates high stocking density-induced breast muscle quality decline in broilers via modulating mitochondrial redox homeostasis and glycolytic metabolism Yi Zhang, Dongying Bai, Caifang Guo, Penghui Ma, Ziwei Wang, Xiaodie Zhao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6862999/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract High stocking density ( HD ) in intensive poultry farming induces oxidative stress and mitochondrial dysfunction, leading to compromising meat quality. This study explored the potential role of chlorogenic acid (CGA) as a protective dietary supplement for improving breast muscle quality under HD conditions. In total, 510 broilers were reared under either normal density (14 birds/m², ND) or HD (22 birds/m²), with 0.0, 0.5, 1.0 or 1.5 g/kg CGA added to the HD group. After 42 days, exposure to HD led to a substantial 12% ( P < 0.05) decrease in body weight, a significant ( P < 0.05) rise in feed conversion ratio ( FCR ), and deterioration in meat quality as evidenced by a marked decrease in pH, a rise in cooking and dripping losses, and unfavorable alterations in meat color ( L* , a* , b* ) ( P < 0.05). Supplementation with 0.1% CGA significantly alleviated HD-induced growth suppression and enhanced antioxidant defenses by reducing the levels of malondialdehyde ( MDA ) and reactive oxygen species ( ROS ) ( P < 0.05), while boosting glutathione peroxidase ( GSH-Px ) and superoxide dismutase ( SOD ) activities ( P < 0.05).. Mechanistically, CGA alleviated mitochondrial dysfunction by downregulating heat shock proteins ( HSP60 , HSP70 ) and mitochondrial proteases ( CLPP , CLPX ) ( P < 0.05), restored tricarboxylic acid ( TCA ) cycle activity (via increased SDH and MDH ), and shifted glycolytic flux toward aerobic metabolism. These findings highlight CGA as a promising feed additive for improving meat quality under HD stress through mitochondrial protection and metabolic reprogramming. Biological sciences/Biochemistry Scientific community and society/Agriculture broilers CGA high stocking density meat quality mitochondrial redox homeostasis glycolytic metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The global broiler industry has experienced a consistent growth trajectory over an extended period. In 2024, the global production volume reached an impressive 104.13 million tons (Livestock and Poultry, 2024). However, the escalating costs of feed and energy present significant challenges to the industry's continued profitability. To improve economic returns and production performance, breeders often adopt intensive rearing techniques, such as increasing stocking density to maximize yield (Pesti and Choct, 2023). As a critical parameter in poultry rearing, stocking density is usually represented by the live weight per unit area (kg/m²) or the number of birds (broilers/m²) (Dawkins et al., 2004; El Sabry MI et al., 2022). Based on modern feeding guidelines, a broiler density of 39 kg/m 2 or 16 broilers/m 2 is appropriate, taking into account growth performance, animal welfare, as well as other factors (Cengiz et al., 2015; Son et al., 2022). Nevertheless, to maximize profits, high stocking density (HD) is often chosen. Although HD can lower production costs and enhance profitability, it can also disrupt the physiology, metabolism, and behavior of broiler animals, leading to oxidative stress reactions (Jones et al., 2005; Nasr et al., 2021; Evans et al., 2023). Moreover, as we indicated in our previous study, HD stress-induced oxidative damage can disturb gut microbiota balance (Liu et al., 2023), exacerbate intestinal damage (Li et al., 2023; Zhang et al., 2024), particularly in the ileum and jejunum, and alter the levels of immunobiochemicals (Campbell et al., 2023) in serum and hypothalamic physiological metabolism. Despite the well-documented link between HD stress and oxidative damage, the molecular mechanisms underlying the connection between mitochondrial dysfunction and meat quality deterioration, especially through the dysregulation of glycolytic and TCA cycle pathways, remain poorly understood. Thus, effective interventions targeting mitochondrial homeostasis under HD conditions are urgently needed. In the poultry industry, muscle growth, development and meat quality are key components that are intimately related to the health and productivity of broilers (Mir et al., 2017). Effective husbandry and nutritional strategies can promote muscle development in broiler chickens, thereby optimizing meat quality and enhancing both animal health and meat production (Choi et al., 2023). Nevertheless, numerous studies have shown that elevated ambient temperatures, such as those causing heat stress, can decrease broiler growth performance along with meat quality (Liu et al., 2022; Liu et al., 2023). As mentioned earlier, heat stress also induces oxidative stress, which is mainly attributed to mitochondrial stress. Therefore, it is reasonable to suggest that oxidative stress due to heat stress may be the underlying cause of mitochondrial dysfunction or mammary muscle damage. ROS generation rises as a result of mitochondrial malfunction. This process not only results in low-quality meat but also reduces the pH and antioxidant capacity of broiler meat (Gray et al., 1996; Akbarian et al., 2016; Jing et al., 2024a). Excessive ROS generation and insufficient antioxidant capacity has been identified as a principal cause of oxidative stress (Ahmad et al., 2012; Lu et al., 2017). ROS in excess can alter protein structure, leading to protein polymerization or cross-linking, which can affect major meat qualities for instance juiciness and tenderness (Lu et al., 2017; Jing et al., 2024a). Additionally, further evidence indicates that oxidative stress induces mitochondrial dysfunction, which in turn causes mitochondrial damage. This disorder affects the broiler liver's ability to aerobically metabolize pyruvate due to damage to the mitochondrial tricarboxylic acid ( TCA ) cycle (Jing et al., 2023). Therefore, alleviating mitochondrial stress may effectively improve redox homeostasis, promote pyruvate aerobic metabolism, and preserve meat quality of broilers under HD stress. Antibiotic substitutes, including probiotics, antibacterial peptides, and traditional Chinese herbal formulations, have been utilized to promote broiler growth and enhance antioxidant capacity under HD conditions (Jiang and Xiong, 2016; Yue et al., 2024). As a key bioactive polyphenol in herbal extracts, CGA is generated from caffeic and quinic acids derived from green coffee bean extract by esterification (Liang and Kitts, 2015; Tajik et al., 2017). Various studies have confirmed the remarkable pharmacological properties and potent antioxidant capacity of CGA (Naveed et al., 2018; Lu et al., 2020; Xie et al., 2023). According to animal studies, CGA exerts multiple pharmacological effects, such as improving gut microbiota, enhancing immunity, and promoting antioxidant (Liang and Kitts, 2015), lipid metabolism-regulating activities (Huang et al., 2015), antiviral (Gamaleldin Elsadig Karar et al., 2016), anti-inflammatory (Wang et al., 2020) and antibacterial (Zhang et al., 2020). Therefore, CGA has become a prospective feed additive that enhances the quality of meat products and strengthens animal health. In particular, CGA-enriched diet facilitates muscle production in porcine longissimus dorsi muscle and makes its muscle fibers more susceptible to oxidation, thereby enhancing meat quality (Wang et al., 2021). Additionally, CGA may be beneficial in reducing the severity of colitis and controlling inflammation by inhibiting pro-inflammatory and apoptotic pathways, particularly those mediated by mitochondrial mechanisms (Hu et al., 2022; Qu et al., 2024). When coping with HD stress, the use of CGA may be a feasible therapeutic strategy to enhance meat quality and mitigate the damage from oxidative stress and mitochondrial dysfunction. To this end, a HD stress broiler model was established to assess the efficacy of CGA in improving breast muscle quality, mitigating mitochondrial stress while enhancing glycolytic activity, and identifying the optimal CGA concentration for preventing mitochondrial damage caused by HD. 2. Materials and methods 2.1. Animal ethics statement All of the experimental operations and animal handling followed the Guidelines for Experimental Animals issued by the Ministry of Science and Technology, China (Beijing), and authorized by the Experimental Animal Care and Utilization Committee of Henan University of Science and Technology (AW20602202-1-2). 2.2. Animals, diets and experimental design In total, 510 male Arbor Acres broiler chicks, each weighing around 42 ± 3 g at one day of age were supplied by a commercial hatchery (Henan Quanda Poultry Breeding Co., Ltd., Hebi, China). All trials were implemented at the Animal Research Center of Henan University of Science and Technology. On day seven, broilers were divided into five treatment groups at random after being stratified by body weight: (1) ND (14 birds/m², basal diet), (2) HD (22 birds/m², basal diet), (3) HD + 0.5 g/kg CGA, (4) HD + 1.0 g/kg CGA, and (5) HD + 1.5 g/kg CGA. Each group included 10 replicates with 7 (ND) or 11 (HD) birds in each replicate. The standard basal diet for the ND was based on the NRC (NRC, 1994), as listed in Table S1 . 0.0, 0.5, 1.0 and 1.5 g/kg CGA (98% purity, obtained from Changsha Staerb Natural Ingredients Ltd (Changsha, China) was added to HD group the basal diet and directly to the basic diet for 42 days of pretreatment. Prior to the study, all chicken houses and feeding equipment were thoroughly sanitized following standard biosecurity procedures. The broilers were reared in a ventilation-assisted facility using a three-tier stacked cage system with dimensions of 70 cm × 70 cm × 50 cm (L × W × H) per cage. During the entire duration of the experiment, they had free access to water and feed. The feeding program contains two stages: days 1 to 21 and days 22 to 42, with formulation details illustrated in Table 1 . For the first five days, the temperature was maintained at 32–34°C, then dropped by 2°C per week before reaching 22–24°C. The relative humidity was kept constant at 40–60%. For the first three days, the lighting was constant. After that, the photoperiod was 23 hrs of light and 1 hr of darkness. All birds were provided with adequate nutritional support and routine immunizations, while housing hygiene was maintained through regular cleaning. Their health status and growth metrics were monitored carefully and documented in detail throughout the experiment. 2.3. Growth performance assessment On days 21, 28, 35 and 42, feed intake and body weight of broilers were recorded after 8 hours of fasting during the experiment. Based on these measurements, key growth performance indicators, including ADG, ADFI, as well as the FCR were computed with the formulas : ADG = total body gain (g) / test days / number of broilers in each replicate ADFI = total feed intake (g) / test days / number of broilers in each replicate FCR = ADFI / ADG Throughout the experiment, the broiler's daily growth performance was monitored and recorded closely. In cases of mortality, relevant data including the time of death, leftover feed quantity, and the bird’s final body weight were systematically documented for subsequent analysis.. Any resulting deviations due to mortality were subsequently analyzed, and data from deceased animals were excluded during the final data processing to ensure accuracy and consistency of growth performance evaluation. 2.4. Muscle weight and sample collection Six broilers were chosen from each treatment group at the conclusion of the 42-day period, and they fasted overnight. All of the broilers in each group weighed almost the same on average. The broilers were weighed prior to slaughter, and the weight was recorded as the live weight. Following that, the complete pectoral muscle of each broiler was removed, weighed, and its weight was noted, which was calculated using the formula as below: Pectoral muscle percentage (%) = (breast muscle weight / live weight) × 100 Additionally, a small sample of right pectoral muscle tissue was gathered in a sterile test tube and kept immediately at -80°C for follow-up analysis. 2.5. Meat quality of breast muscle 2.5.1. Meat color and pH A portion of the left breast muscle was taken from each selected broiler in order to assess the meat color and pH. Measurements were conducted with a portable pH meter (pH-Star, Matthäus, Pöttmes, Germany) and a Minolta Chromameter CR-300 (Minolta Camera, Osaka, Japan). Yellowness/blueness ( b* ), redness/greenness ( a* ), and lightness ( L* ) were the parameters utilized to assess the flesh color. First, the samples of breast muscle were cut, left to rest for 45 minutes, and then three separate pH and color measurements were made. After that, the samples were maintained at 4°C in a Ziploc bags. The pH and color measurements were repeated after a 24-hour period. The average of three measurement points per sample was utilized in the analysis. 2.5.2. Cooking loss In brief, from each chosen broiler, 30 g of left-side breast muscle tissue was removed and originally kept at 4°C. Samples were taken out and weighed to determine the initial mass (Wa) following a 24-hour period of refrigeration. For the purpose of thermal processing, 10 samples in all were individually sealed in Ziplock bags and submerged in a water bath set at 90°C. A kerosene thermometer was utilized to monitor the internal temperature of each sample and when the temperature reached 70°C, the samples were immediately removed and cooled naturally at RT. Filter paper was applied to properly remove surface moisture. After that, the post-cooking weight (Wb) was noted. The following formula was utilized to compute cooking loss (%): Cooking loss (%) = (Wa − Wb) / Wa × 100 2.5.3. Drip loss From the right pectoral muscle, a fresh thick piece (5 cm × 5 cm × 1 cm) was taken, weighed, and then designated as W1 according to the initial breast muscle weighing results. Ten of these slices were prepared, sealed in Ziplock bags one at a time, and kept at 4°C. Each sample was reweighed after being carefully blotted dry using filter paper to eliminate surface moisture after 24 and 48 hours. These subsequent weights were recorded as W2 and W3, respectively. The following formulas were applied to calculated drip loss: 24 - hour drip loss (percent) = (W1 − W2) / W1 × 100 48 - hour drip loss (percent) = (W1 − W3) / W1 × 100 2.5.4. Peak shear force Following the 24-hour measurements of pH and meat color, the peak shear force was measured using the remaining muscle samples. Each piece was sealed in a Ziplock bag and thermally processed in a water bath at 90°C. Once removed, the samples were let cool naturally to RT and then placed in a 4°C refrigerator from which the internal temperature reached 70°C. After 24 hours, 5 cores (with a diameter of 1.5 cm) were taken from each sample utilizing the same sampler. Ten of these cores underwent shear testing using a Texture Analyzer (XT2, Stable Micro Systems Ltd., Godalming, Surrey, UK). The findings of the five cores extracted from the same pectoral muscle were averaged to establish the peak shear force value for each sample. 2.6. Assessment of antioxidant capacity and enzymatic activity TBA test was exploited to determine the serum levels of MDA. WST-1 test and Colorimetric analysis were employed to assess the activity of T-SOD and serum GSH-PX, separately. The ABTS test was also applied to measure the serum's T-AOC. All tests were carried out with commercially available kits provided by the Nanjing Jiancheng Bioengineering Institute Co., Ltd. (Nanjing, China). Using certain test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the levels of lactic acid, glucose, glycogen, mitochondrial citric acid, pyruvate, ATP and MDA in breast muscle samples were measured. ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) were applied to measure the levels of oxaloacetate ( OA ) and acetyl-CoA, along with the activity of the enzymes isocitrate dehydrogenase ( ICD ) and citroyl synthetase ( CS ) in breast muscle samples. Via applying an ELISA kit (Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China), the ROS concentration in the identical samples was measured. Furthermore, through the appropriate test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the enzyme activity of MDH, SDH, LDH, T-SOD, T-AOC, and GSH-Px in breast muscle samples were assessed. With a commercial test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the protein content of the sample extract was identified. The instructions included with the kit were followed when performing sample pretreatments. 2.7. RT-PCR analysis of mRNA expression in breast muscle Samples of muscle tissue were thawed on ice as described previously (Zhang et al., 2024), and then ~ 100 mg of tissue was transferred into 1.5 ml sterile test tubes. Extraction of total RNA was carried out from each sample through TRIzol reagent (Invitrogen Inc., Carlsbad, CA), and Nanodrop 2000 spectrophotometry (Thermo Scientific, Ottawa, Canada) was applied to evaluate the RNA purity. Only samples with an A260/A280 ratio > 1.9 were considered suitable for downstream analysis. RNA concentrations were determined by absorbance, afterwards, the RNA samples were aliquoted and preserved at -80°C before use. A reverse transcription kit (Vazyme, Nanjing, China) was exploited to create cDNA from the isolated RNA. The primers (Supplementary Table S2 ) were designed with Primer 3.0 and generated from Shanghai Shenggong Bioengineering Co., Ltd., and the housekeeping gene GAPDH served as an internal reference for normalization. On a CFX Connect Real-Time PCR system, qRT-PCR was implemented with the SYBR Green PCR kit (Vazyme Biotechnology Co., Ltd., Nanjing, China). Thermocycling conditions included initial denaturation at 95°C for 5 min, denaturation at 95°C for 15 s, and annealing/extension at 60°C for 30 s, for a total of 40 cycles. 2 −ΔΔCT method was utilized to determine the levels of relative gene expression. 2.8. Western blot analysis of protein abundance in breast muscle Western blotting was implemented based on the procedures described by Liu et al. (2022). In brief, a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), manufactured by Beo Tianmei in Shanghai, China, was added to pre-chilled RIPA lysis buffer buffer prior to homogenizing the breast muscle samples. After centrifuging the resultant homogenates for 30 minutes at 4°C at 13,000 × g, the supernatants were gathered. A BCA assay kit (catalog no. A045-3 from Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was exploited to measure the total content of protein in each sample after the supernatant was gathered, and the results were then standardized to 6 µg/µL. 400 µL of each protein sample was then combined with 100 µL of 5 × SDS-PAGE sample loading buffer (catalog no. P0015L from Beyotime, Shanghai, China), and the mixture was denaturated in a water bath at 95°C for 10 minutes. Proteins were resolved on SDS-PAGE gels with concentrations ranging from 8–12%, and then electrotransferred onto PVDF membranes (Bio-Rad, CA, USA). For lowering non-specific binding, membranes were blocked for 120 min utilizing 5% non-fat dry milk. This was followed by three TBST buffer washes. The membranes were incubated with primary antibody after blocking (Supplementary Table S3 ). for a whole night at 4°C. HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG, 1:5000 dilution; Proteintech Group, IL, USA) were then added. The bands were visualized via an enhanced chemiluminescence system (Bio-Rad, CA, USA), and the quantification was done through the Image Lab software system (Bio-Rad, CA, USA). 2.9. Statistical analysis Each cage was regarded as an experimental unit in order to assess growth performance. All other parameters were taken as experimental units of a single bird in each replicate. One-way ANOVA with SPSS 20.0 (SPSS Inc., Chicago, Illinois) was employed for the statistical analysis. With Tukey's multiple range test, significant differences across groups were determined; P < 0.05 was deemed statistically significant. GraphPad Prism Version 9 (GraphPad Software Inc., San Diego, CA) was employed for data visualization and result graphing. 3. Results 3.1. Growth performance of broilers The results demonstrate how high-density ( HD ) stress and chlorogenic acid ( CGA ) dietary conditions influenced production performance throughout the trial, as depicted in Table 1 . No significant difference in growth metrics, including FCR, ADG, and ADFI, were observed between the HD and normal-density ( ND ) groups or among the three CGA concentration levels during the initial feeding phase ( days 1–21 ). However, exposure to HD circumstances from day 22 to day 42 remarkably affected the development of broiler chicks versus the ND group. In the HD group, broiler body weight dropped by 12% ( P < 0.05), and broiler FCR increased while ADG and ADFI decreased ( P < 0.05) under HD. Supplementation with dietary CGA, especially at concentrations of 0.1% and 0.15%, partially mitigated the adverse effects of HD, leading to a rise ( P < 0.05) in final weight of body and improvements ( P < 0.05) in ADFI and ADG for broilers. Only a few broilers died in the ND group, while the mortality rate in the HD group was 2.56%. The addition of CGA remarkably raised the broiler mortality rate under HD. 3.2. Breast muscle integrity and meat quality alterations Under HD, broilers reared revealed a substantial ( P < 0.05) decrease in the weight of the breast muscles (Fig. 1 A) together with breast muscle index (Fig. 1 B) in contrast to the ND group. In contrast to ND group, the serum CORT concentration in HD group was considerably higher ( P < 0.05) (Fig. 1 C), indicating a physiological stress response. Additionally, HD conditions adversely affected the meat quality of breast muscle, resulting in a considerable decline ( P < 0.05) in the L* values at both 45 minutes and 24 hours, as well as a rise ( P < 0.05) in the b* values at the same time points. Conversely, the a* value at 45 minutes was remarkably decreased ( P < 0.05) (Fig. 1 D-I). Furthermore, at 24 and 48 hours, under HD, drip loss markedly increased (p < 0.05), as did cooking loss together with peak shear ( P < 0.05). Yet, the pH showed a substantial drop after 24 hours ( P < 0.05) (Fig. 1 J-O). Incorporation of CGA to the diet led to improved broiler meat quality under HD, resulting in a reduction ( P < 0.05) in 24 h L* , both 45 min and 24 h b* as well as the peak shear force, cooking and drip losses. 3.3. Antioxidant status of serum and breast muscle Broilers exposed to HD displayed a considerably lowered ( P < 0.05) amount of MDA in their serum than the ND group, similar differences ( P = 0.06) were noted in the tissue of breast muscle ( Fig. 2A and E ). Additionally, the antioxidant status of breast muscle meat was adversely affected by HD, resulting in a substantial decrease ( P < 0.05) in the blood levels of T-AOC and GSH-Px ( Fig. 2B and D ). At the same time, the amount of T-SOD ( Fig. 2G ) in the breast muscle declined ( P < 0.05). When in contrast to the ND group, the ROS content was considerably raised ( P < 0.05) and the level of ATP in the pectoral muscles was lowered ( P < 0.05) in HD group ( Fig. 2J and K ). As demonstrated by lower ( P < 0.05) levels of MDA and higher ( P < 0.05) serum levels of T-AOC and GSH-Px, together with higher ( P < 0.05) levels of ATP and T-SOD in breast muscle, dietary with CGA enhanced the antioxidant status of broilers under HD ( Fig. 2A, B, D, G, J, and K ). It is worth noting that a 0.1% concentration of CGA boosted ( P < 0.05) ATP and T-SOD levels in breast muscle and considerably raised ( P < 0.05) blood levels of T-AOC and GSH-Px ( Fig. 2B, D, G, and J ). 3.4 Mitochondria homoeostasis in breast muscle To assess mitochondrial stress induced by HD and the potential protective effects of varying doses of CGA, we analyzed several mitochondrial function indicators in broiler breast muscle. The findings suggested that, under HD, the levels of ATP were markedly lower ( P < 0.05), whereas the that of ROS were considerably higher ( P < 0.05), indicating oxidative stress (Fig. 3 A and 3 B). Further analysis showed that HD resulted in a marked upregulation ( P < 0.05) in the mRNA expression of heat shock proteins HSP60 and HSP70, as well as mitochondrial quality control markers CLPP and CLPX (Fig. 3 C). The expression of CLPX and CLPP at the protein level was also notably raised in the HD group ( P < 0.05) (Fig. 3 D), suggesting activation of mitochondrial stress response pathways. Dietary CGA supplementation effectively ameliorated mitochondrial dysfunction, as evidenced by increased ATP concentrations and reduced ROS accumulation (Fig. 3 A and 3 B), highlighting its protective role under HD stress conditions. Furthermore, different concentrations of CGA have demonstrated a reduction or a tendency to decline the mRNA expression of HSP60, HSP70, CLPP, and CLPX (Fig. 3 C). The findings also suggested that CGA supplementation decreased the CLPX and CLPP protein expression (Fig. 3 D). In comparison to the other two concentrations of CGA, broilers administered 0.1% CGA displayed the lowest levels of CLPP in the breast muscle, as displayed by both protein and mRNA expression levels. 3.5. Myofiber types and glycolysis status in breast muscle Glycolytic activity and muscle fiber types in broiler breast muscle were assessed. The HD group considerably increased ( P < 0.05) the protein expression of Fast-MyHC in breast muscle while dramatically reducing ( P < 0.05) that of Slow-MyHC versus the ND group(Fig. 4 A). Regarding glycolysis activity, broilers under HD conditions revealed a remarkable rise ( P < 0.05) in breast muscle concentrations of lactic acid, glucose, and glycogen (Fig. 4 B-D). In addition, the enzymatic activity of lactate dehydrogenase ( LDH ) was evidently raised ( P < 0.05) in the HD group versus ND group (Fig. 4 E). The findings also suggested that the LDH activity was obviously higher ( P < 0.05) in HD broilers versus ND broilers (Fig. 4 F). Furthermore, Supplementation with CGA caused a marked decline ( P < 0.05) in LDH activity, glucose, and glycogen, alongside a marked rise ( P < 0.05) in the content of pyruvic acid. Follow-up analysis exhibited that HD considerably elevated ( P < 0.05) the glycolysis-associated enzyme mRNA expression, encompassing PKM, PFKM, HK1, LDHA and PDK4, (Fig. 4 H). Additionally, dietary CGA supplementation potentially decreases the expression of biomarkers related to glycolysis, including PFKM, PGK, PKM, PDK4, and LDHA (Fig. 4 G). In contrast, birds receiving CGA-supplemented diets demonstrated a downregulation of several glycolytic markers, notably PFKM, PGK, PKM, PDK4, and LDHA (Fig. 4 G). Additionally, in contrast, the mTOR pathway components such as S6K1, 4EBP1, and mTOR were not appreciably altered in the ND group in terms of their expression ( P > 0.05). Notably, CGA supplementation, particularly at a 0.1% inclusion rate, was found to enhance the transcription of mTOR and S6K1, indicating potential involvement in protein synthesis regulation (Fig. 4 G). 3.6. Mitochondria tricarboxylic acid cycle in breast muscle Pyruvate, a byproduct of glycolysis, is used by muscle tissue primarily through the TCA metabolic pathway. In the present investigation, HD substantially decreased the acetyl-CoA concentration ( P < 0.05) by 12.2% (Fig. 5 A and B ) along with the levels of mitochondrial CA ( P < 0.05) and oxaloacetic acid ( P < 0.05) by 17.4% and 35.7%, separately (Fig. 5 A, C, and D ). Follow-up analysis suggested that CS, ICD, SDH, and MDH enzyme activities were decreased by 19.4%, 47.2%, 39.6%, and 13.9%, separately, in the breast muscle of broilers exposed to HD conditions (Fig. 5 A, E–H). The dietary supplementation of CGA exhibited mitigating effects, resulting in a rise in the acetyl-CoA concentration ( P < 0.05) (Fig. 5 B) and a trend towards a raised oxaloacetic acid concentration ( P < 0.05) (Fig. 5 D). Furthermore, it strengthened MDH, SDH and ICD activities (Fig. 5 G and H ). In the meantime, when compared to the three concentrations of CGA, broilers that received 0.1% CGA exhibited higher Acetyl-CoA activity in the breast muscle (Fig. 5 B). Additionally, compared to the 0.05% and 0.15% concentrations, supplementation with 0.1% CGA showed a tendency to increase the MDH and SDH concentrations in HD-stressed broiler breast muscle (Fig. 5 G and H ). 3.7. Correlation analysis For the exploration of the intrinsic mechanism of the CGA's protective effect on the meat quality under HD, we correlated the parameters of meat quality with a range of physiological indices, including antioxidant status, glycometabolic activity, and mitochondrial function. This analysis aimed to identify key factors associated with variations in meat quality in broilers stressed by HD (Fig. 6 ). Regarding the meat color value of breast muscle, glycogen levels displayed a negative correlation with the 24-hour L* value ( P < 0.05), while the glucose and GSH-Px levels were positively correlated with the 24-hour a* value ( P < 0.05). After 24 hours, drip loss in breast muscle had a negative relationship with muscle ROS and lactate levels ( P < 0.05). Likewise, muscle ATP and T-SOD levels showed a negative association with 48 hr values of drip loss ( P < 0.05). Peak shear in breast muscle has a positive association with the levels of glucose in breast muscle ( P < 0.05). Similarly, muscle ROS and GSH-Px levels positively correlated with glucose and lactic acid, respectively ( P < 0.05). 4. Discussion We created a broiler model that was exposed to HD stress and examined the possible protective function of CGA in promoting growth and preserving meat quality. Furthermore, we examined potential differences in the effectiveness of CGA from various sources with varying concentrations. During the initial 21 days of HD exposure, no significant differences in production performance were observed. However, by day 42, broilers subjected to HD conditions exhibited a decrease of 22.0% in BW, 8.2% in ADFI, and 15.5% in ADG, in contrast to the ND group. This discovery is in line with previous investigations (Liu et al., 2023; Li et al., 2023; Zhang et al., 2024), which indicate that the onset of density stress is associated with a phase of accelerated growth. The current study demonstrates that CGA supplementation enhances production performance, as evidenced by increased BW, ADFI, and ADG while reducing mortality rates. Although CGA supplementation at 0.1% and 0.15% did not completely restore performance or quality indicators to levels of the ND group, these concentrations still exerted a significant mitigating effect on the physiological stress induced by HD. Breast muscle is the primary edible portion and a source of high-quality protein in broilers. This research observed that HD can decrease the proportion of pectoral muscles in broilers. In agreement with the previous findings, restricted growth in broilers after exposure to oxidative stress, including heat stress, is generally linked to a reduction in the percentage of breast muscle (Liu et al., 2023; Jing et al., 2024). The findings display that the progressive onset of HD may have triggered oxidative stress responses in broiler chickens, eventually causing a loss of breast muscle mass. Additionally, the findings present that HD-stress considerably elevated the CORT index. The CORT is regarded as a cellular stress sensor and is commonly utilized to evaluate damage induced by oxidative stress (Lee et al., 2018; Ma et al., 2020). This suggests that in broiler chickens, HD prevents the growth of skeletal muscle and damages the breast muscle. CGA has been proved to have strong antioxidant performance (Chen et al., 2023). When oxidative stress is present, dietary containing CGA can support the growth and development of skeletal muscle (Zhao et al., 2019). In this work, CGA reduced the concentration of CORT in serum and numerically increased the breast muscle mass in broilers under HD conditions. These findings indicate that CGA may mitigate oxidative stress, thereby safeguarding the quality and quantity of breast muscle against damage induced by HD. HD stress negatively influences broiler meat quality, mainly by lowering the pH and color of the meat, while elevating peak shear force together with drip loss (Choi et al., 2023; Yue et al., 2024). In this research, we examined the influence of HD on meat quality in broilers, as well as the potential protective effects of CGA. As a result, HD elevated the drip loss, b * value, peak shear force and cooking loss of breast muscle while decreasing the pH and both the a* and L* values. The color of meat is a critical quality characteristic because consumers associate its appearance with freshness, which influences purchasing choices (Yue et al., 2024). The L* value indicates paleness, with higher values representing lighter meat (Tong et al., 2015). Our results align with research reported that HD stress leads to paler meat, which is linked to decreased meat quality and lower muscle pH (Pekel et al., 2020; Weng et al., 2022). The percentages of drip and cooking loss in this work is in line with the results of Wu et al. (2020), who reported elevated water loss from thigh and breast muscles of Arbor Acres broilers raised under HD. Furthermore, their findings indicate that in line with the current investigation, final muscle pH dropped under HD circumstances as drip and cooking losses increased. This may be explained by the substantial negative association between pH and drip and cooking loss, which is due to the accelerated deposition of lactate, leading to a subsequent increase in meat toughness (Huff-Lonergan and Lonergan, 2005; Li et al., 2019). The experimentally obtained data, which are logically consistent, further indicate that HD stress significantly compromises chicken breast muscle quality. According to this investigation, adding CGA to the breast meat of broilers under HD stress decreased both cooking and drip losses. This implies that CGA may be useful in improving meat's ability to retain water. The water-holding capacity of broiler breast meat has also been displayed to be improved by dietary supplementation with E. ulmoides extract, which contains 8% CGA, according to several studies (Jiang and Xiong, 2016; Zhao et al., 2019). A number of variables, encompassing final pH and oxidation of lipids and proteins, affect water-holding capacity of meat (Zhao et al., 2019). Previous studies, including our own, have shown that stress related to HD induces oxidative stress, which can disrupt mitochondrial homeostasis (Lu et al., 2017; Zhang et al., 2024). Typical features of this stress are elevated ROS levels and diminished antioxidant capacity (Lu et al., 2017; Jing et al., 2023). The reduced antioxidant capacity in muscle tissue is frequently associated with a decline in meat quality (Jing et al., 2024a). The efficient functioning of the TCA cycle relies on the preservation of mitochondrial homeostasis, which, in turn, affects the antioxidant capacity (Martínez-Reyes and Chandel, 2020). In this study, it was discovered that HD lowers the antioxidant capacity in blood and breast muscle and disturbs mitochondrial homeostasis. As a result, ROS and MDA levels elevated, T-SOD and GSH-Px activity decreased, the concentration of ATP dropped, and HSP60, HSP70, CLPP and CLPX gene and protein expression raised. Through mitochondrial oxidative phosphorylation, ATP and ROS are produced. However, mitochondrial dysfunction results in an elevated production of ROS and a concomitant decrease in ATP synthesis (Murphy, 2013). Hsp70 is essential for proteostasis and alleviates damage from various stressors, including oxidative stress (Zhang et al., 2022). HSP60 and CLPP are key mitochondrial chaperones and proteases, involved in maintaining mitochondrial homeostasis and serving as biomarkers of mitochondrial stress (Venkatesh, S., & Suzuki, 2017; Luo et al., 2021). CLPX is known to interact with the mitochondrial complex II subunit, SDHB. Mitochondrial dysfunction is commonly associated with raised CLPX, CLPP, and HSP60 levels. This research revealed that HD enhanced the abundance of these proteins in breast muscle, suggesting that HD induces mitochondrial dysfunction. Dietary supplementation with CGA restored ROS levels, decreased the HSP70, HSP60, CLPX and CLPP expression, and elevated the levels of ATP, while also elevating T-SOD levels in breast muscle. These findings indicate that CGA enhances mitochondrial stability and boosts antioxidant capacity in broiler skeletal muscle under heat distress.. To investigate the mechanisms by which CGA preserves broiler meat quality under HD conditon, we initially assessed muscle fiber type expression in the breast muscle. From a physiological metabolic standpoint, myofibers with higher fast myosin heavy chain ( MyHC ) expression typically display a lower pH and increased drip loss, whereas those with elevated slow MyHC levels show reduced drip loss and a higher pH (Wang et al., 2021; Chauhan and England, 2018). This study revealed that in the breast muscle, HD boosted fast MyHC protein expression while reducing protein expression of slow MyHC. In broiler breast muscle under HD stress, dietary CGA supplementation maintained a larger proportion of fast MyHC fibers and declined expression of slow MyHC fibers by preventing muscle fiber type conversion. Research indicates that myofibers with higher concentrations of fast MyHC exhibit increased level of glycogen and lactic acid, which reduced the pH and weaken the water retention capacity of the meat (Lu et al., 2017; Huo et al., 2022). Research has discovered that HDL raises the concentration of lactate, glucose, and glycogen in the pectoral muscles while lowering the concentration of pyruvate. Subsequent analysis demonstrated that HD raised mRNA expression of glycolysis-associated enzymes together with LDHA, and strengthened the LDH enzymatic activity. PKM and HK1 are essential rate-limiting enzymes in the glycolytic pathway. Specifically, via catalyzing the glucose phosphorylation, HK1 initiates glucose metabolism, while PKM facilitates the final step, converting phosphoenolpyruvate into pyruvate (De Jesus et al., 2022; Horemans et al., 2022). Furthermore, via regulating the activity of the pyruvate dehydrogenase complex, PDK4 regulates the irreversible conversion of pyruvate to acetyl-CoA (Lu et al., 2017; Jing et al., 2024). According to research, pyruvate is rapidly converted to lactic acid in avian breast muscle by LDH (Wilson, Cahn, Kaplan, 1963). The raised PDK4 mRNA, increased LDH activity, and reduced mTOR expression suggest a diminished glucose entry into the tricarboxylic acid cycle, indicating a shift toward anaerobic glycolysis. These findings imply that HD adversely influences meat quality through the enhancement of glycolysis and modulation of muscle fiber type conversion. CGA supplementation lowered lactate and glycogen levels. Furthermore, it was linked to a rise in the expression of protein synthesis-associated genes (S6K1 and mTOR) and a reduction in the activity of glycolysis-associated enzymes (PDK4 and PFKM), which is in line with the report of Wang et al., (2021). Plant extracts rich in specific polyphenols (e.g., CGA) have been reported to have potential anabolic actions that may facilitate muscle production and produce anti-catabolic and protein-sparing effects on muscle tissue. The results indicate that CGA treatment significantly enhanced the breast muscle quality in broilers, although the effect of the CGA concentration gradient was not highly significant. In the expression of genes linked to protein synthesis (S6K1 and mTOR), the 0.01% CGA concentration was most effective, suggesting the need for further clarification of the concentration gradient. As previously mentioned, the efficiency of the mitochondrial TCA cycle relies on the maintenance of mitochondrial homeostasis, and pyruvate catabolism proceeds through two primary metabolic routes: anaerobic metabolism, leading to lactate production, or entry into the mitochondria for aerobic metabolism (Pithukpakorn, 2005; Che et al., 2024). Previous studies, primarily conducted using heat stress broiler models, have shown that HS causes mitochondrial stress, disrupts TCA cycle, and compromises metabolism of aerobic pyruvate (Jing et al., 2024). Furthermore, lactic acid concentration in breast muscle increased by 66.7%, indicating that HD enhances anaerobic pyruvate metabolism in the muscle. As a central pathway for aerobic pyruvate oxidation, the TCA cycle is dependent on enzymes for instance ICD, CS, MDH and SDH (Martínez-Reyes and Chandel, 2020). In this study, HD markedly inhibited the ICD, CS, MDH and SDH activities, and lowered the concentrations of crucial metabolic intermediates like oxaloacetate and CA in the TCA cycle. Based on the previously described findings, HD inhibits the TCA cycle, which prevents pyruvate from being aerobically metabolized. This causes the breast muscle to produce more lactic acid and accumulate glycogen. The results of this experiment align with conclusions observed in heat stress studies, suggesting that there was less glucose entering the tricarboxylic acid cycle, cells became more dependent on anaerobic glycolysis (Lu et al., 2017; Jing et al., 2023). In comparison to the two other concentrations of CGA, supplementation with 0.1% CGA revealed the most protective effects on the mitochondrial TCA cycle in the breast muscle of broilers exposed to HD stress, as indicated by the increased activities of Acetyl-CoA, SDH, and MDH. Previous research on CGA has mainly concentrated on its role in enhancing oxidative stress resistance (Bao et al., 2018; Wang et al., 2021). This study proves for the first time that CGA augments TCA cycling in broiler muscle mitochondria, highlighting its targeted effect on mitochondrial function. This study focused on the correlation analysis, which uncovered complex relationships between meat quality parameters and biochemical factors in broilers under HD conditions. It also assays pectoral muscle meat color, shear and drip loss. The factors taken into account included antioxidant indicators (such as GSH-Px, T-SOD, ROS), glycometabolism-related substances (glycogen, glucose, lactic acid), and mitochondrial products (ATP). Correlations between these factors and meat quality parameters were tested at various time points (24 and 48 hours). These findings highlight the potential mechanisms by which CGA may exert protective effects. Higher glycogen levels are closely associated with darker-colored meat, as manifested by a lower L* value at 24 hours post-mortem. This phenomenon can be primarily ascribed to the significant role of glycogen in post-mortem glycolysis (Petracci and Cavani, 2012). Glycogen-driven glycolysis exerts a profound influence on the decline in pH levels and the stability of myoglobin, which are crucial factors determining meat color (Yue et al., 2024). Additionally, elevated concentrations of the antioxidant enzyme GSH-Px and glucose are positively correlated with an enhancement in meat redness. GSH-Px, with its potent antioxidant capacity, likely mitigates oxidative damage to heme pigments, thereby effectively maintaining the natural red color of the meat (Yue et al., 2024). Meanwhile, glucose may serve to support the energy metabolism process, which in turn stabilizes the color-related chemical compounds within the meat. In contrast, negative correlations are observed between meat quality parameters and lactic acid as well as ROS (Schieber & Chandel, 2014). A greater lactic acid concentration can accelerate the pH drop, which improves the meat's ability to retain water through protein denaturation (Yue et al., 2024). Notably, a decrease in ROS levels is unexpectedly associated with higher drip loss. This finding implies that oxidative stress, despite its common perception of causing damage, may have an unpredictable impact on cell membrane integrity, potentially leading to increased fluid leakage from the muscle tissue. Negative correlations also exist between meat quality and ATP and T-SOD levels. Higher levels of ATP serve a key role in preserving the cellular energy state, which is vital for maintaining the cell membrane integrity (Jing et al., 2024a). The antioxidant activity of T-SOD effectively reduces oxidative damage to proteins, contributing to enhanced water retention over time. Moreover, higher glucose levels are correlated with increased meat toughness. This is likely due to the accelerated post-mortem glycolysis process triggered by high glucose. The rapid glycolysis leads to a sharp drop in pH, followed by protein denaturation, which ultimately impairs the tenderization process of the meat (Yue et al., 2024). Furthermore, a positive relationship is detected between GSH-Px and glucose levels. This positive correlation suggests that the GSH-Px antioxidant activity may have a regulatory effect on glucose metabolism. Similarly, ROS and lactic acid are positively correlated, indicating that oxidative stress can exacerbate the glycolytic flux. This exacerbation not only affects the pH balance within the muscle tissue but also intensifies the oxidative damage, further influencing the overall quality of the meat. 5. Conclusion This study developed a HD stress model for broilers and examined the effects of CGA supplementation. Results indicate that HD stress lowers the body weight of broilers, improves feed conversion, and negatively affects the mass of breast muscle, including reduced pH, higher drip and cooking losses, and altered color. However, CGA supplementation, especially at 0.1%, alleviates these negative effects by enhancing growth, increasing body weight, feed intake, and daily gain, while lowering mortality. In terms of meat quality, CGA reduces drip and cooking losses, improving water-holding capacity. Mechanistically, CGA alleviates mitochondrial stress, restores redox balance by reducing ROS and MDA, and boosts antioxidant enzyme activity and ATP levels. Additionally, CGA regulates glycolytic metabolism, preventing fiber-type conversion, lowering glycogen and lactic acid levels, and reducing glycolytic enzyme activity. It also enhances mitochondrial TCA cycle activity and metabolite concentrations. In conclusion, although further study is required to adjust its concentration and examine long-term impacts on meat quality and broiler health, CGA exhibits potential as a feed addition to enhance meat quality of broiler under HD stress. Declarations Supplementary Materials: The supporting information is available free of charge. Institutional Review Board Statement: Animal experiments were approved by the Animal Care and Use Committee of Henan University of Science and Technology (DWFL36891-2023) on October 1, 2023. Author Contributions: Investigation, Y.Z. and D.B.; Methodology, Y.Z., D.B., C.G., P.M., Z.W. and X.Z.; Software, Y.Z. and D.B. and C.G.; Validation, X.X. and X.M.; Formal analysis, Y.Z., W.Z. and D.B.; Resources, Y.M., D.B., B.Z., X.X. and X.M.; Data curation, C.G., P.M., Z.W. and X.Z.; Writing the original draft preparation, Y.Z. and D.B.; Reviewing and editing, D.B., K.I., B.Z. and Y.M; Visualization, Y.Z.; Supervision, Y.Z., W.Z. and Y.M.; Project administration, Y.M.; Funding acquisition, Y.M. All authors have read and agreed to the published version of the manuscript. Funding Sources: The research was supported in part by the National Key Research and Development Program of China (Grant Number 2022YFE0111100), the Key Research and Development Program of Henan Province (Grant Number 241111113800), the Program for International S&T Cooperation Projects of Henan (Grant Number 232102521012), the Key Scientific Research Foundation of the Higher Education Institutions of Henan Province (Grant Number 22A230001), the Frontier exploration Projects of Longmen Laboratory (Grant Number LMQYTSKT037)，and the Doctoral Research Grant of Henan University of Science and Technology (13480103). Institutional Review Board Statement: Not applicable. Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the authors on request. Acknowledgments: The authors extend their gratitude to the College of Animal Science and Technology, Henan University of Science and Technology for providing access to experimental facilities. Gratitude is also extended to the Animal Welfare and Health Breeding of Henan Province and the Expat Scientist Studio for Animal Stress and Health Breeding of Henan Province for their valuable academic guidance throughout this research. Conflicts of Interest: The authors declare no competing financial interest. References Ahmad H, Tian J, Wang J, et al. Effects of dietary sodium selenite and selenium yeast on antioxidant enzyme activities and oxidative stability of chicken breast meat. J Agric Food Chem. 2012; 60(29): 7111–7120. https://doi.org/10.1021/jf3017207. Akbarian A, Michiels J, Degroote J, Majdeddin M, Golian A, De Smet S. Association between heat stress and oxidative stress in poultry; mitochondrial dysfunction and dietary interventions with phytochemicals. 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Tables Table 1 Effects of HD stress and CGA supplementation on broilers growth performance. Item 1 ND HD HD + HD + HD + SEM/ᵪ2 P -value 0.05 CGA 0.1 CGA 0.15 CGA 1-21d BW, g/bird 1 d 46.9 47.0 47.1 46.9 47.1 0.020 0.892 21 d 654.7 634.2 644.7 659.8 649.3 0.010 0.437 ADFI, g/bird 44.89 46.02 45.56 44.21 45.73 0.121 0.331 ADG, g/bird 32.34 31.56 30.08 30.77 31.42 0.235 0.773 FCR 1.39 1.46 1.51 1.44 1.46 0.014 0.458 22-42d BW, g/bird 22 d 699.6 680.3 690.3 704.0 695.1 0.230 0.243 42 d 2527.8 a 2225.4 c 2429.7 b 2477.2 a 2459.5 a 31.780 < 0.01 ADFI, g/bird 142.16 a 130.09 c 133.89 b 138.42 a 135.67 a 3.822 < 0.01 ADG, g/bird 87.05 a 73.57 c 82.83 b 84.44 a 84.01 a 2.074 < 0.01 FCR 1.63 b 1.77 a 1.61 b 1.64 b 1.61 b 0.023 0.04 Mortality, % 0.01 2.56 1.78 1.23 1.34 χ 2 = 1.323 0.216 1 BW = body weight; ADFI = average daily feed intake; ADG = average daily gain; FCR = feed conversion rate. ND = normal stocking density fed basal diet group; HD = high stocking density fed basal diet group; HD + 0.05 CGA = high stocking density fed with 0.05% CGA; HD + 0.1 CGA = high stocking density fed with 0.1% CGA; HD + 0.15 CGA = high stocking density fed with 0.15% CGA. Results for BW, ADFI, ADG and FCR were expressed as mean with SEM (n = 10). a-c Different letters indicate significant differences ( P < 0.05). Additional Declarations No competing interests reported. Supplementary Files supplementdata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6862999","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":477088645,"identity":"b4b62220-ae69-4159-95c7-c8f5925d149d","order_by":0,"name":"Yi Zhang","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":477088646,"identity":"5ef7d3dc-bfcb-4e61-a89b-222d022a8d32","order_by":1,"name":"Dongying Bai","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dongying","middleName":"","lastName":"Bai","suffix":""},{"id":477088647,"identity":"d013da1a-673c-476c-bddc-50ef97550116","order_by":2,"name":"Caifang Guo","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Caifang","middleName":"","lastName":"Guo","suffix":""},{"id":477088648,"identity":"587d487b-8a3f-4ebf-9877-e408b7a49ecb","order_by":3,"name":"Penghui Ma","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Penghui","middleName":"","lastName":"Ma","suffix":""},{"id":477088649,"identity":"fa87a0f8-e533-4492-bd7b-5dab06147984","order_by":4,"name":"Ziwei Wang","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Wang","suffix":""},{"id":477088650,"identity":"46c28b90-3a62-4a7a-a034-eb3c446ec9a2","order_by":5,"name":"Xiaodie Zhao","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaodie","middleName":"","lastName":"Zhao","suffix":""},{"id":477088651,"identity":"dd2549dd-ce33-4a49-b7b2-16d6bf43d706","order_by":6,"name":"Wenrui Zhen","email":"","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenrui","middleName":"","lastName":"Zhen","suffix":""},{"id":477088652,"identity":"f9bdb2e9-c20e-4b39-9680-87ce9fa3f847","order_by":7,"name":"Xiqiang Ma","email":"","orcid":"","institution":"Longmen 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University","correspondingAuthor":false,"prefix":"","firstName":"Bingkun","middleName":"","lastName":"Zhang","suffix":""},{"id":477088656,"identity":"5bcc4d8b-137b-48b2-ab4b-feb0e844c24e","order_by":11,"name":"Yanbo Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYLACxgYGBgMwq+IAVIiNWC0HzoC0MJOi5WAbEVp0288Yfi7cYcNgzn7G+PPHeXfkDc6fP8DwoewwA//sBqxazM7kGEvPPJPGYNmTYyZxcNszww03khkYZ5w7zCBx5wB2LQdyDKR52w4zGBzIMWM4uO0w44YbzAzMYBGJBOxazr8x/g1WAGR8ODjnsP2G84cZmP/i03Ijxwxiy40cA4mDDYcTNxxIZmBmxKvlWZk1L9AvBkCGxJljh5Nn3kg2ONhzLp1H4gYuhyVvvs0LDDEDIONDRc1h277zBx8++FFmLcc/A7sWBgYOcIzUNyCLHQBiHhzqgYD9AW65UTAKRsEoGAUgAAD7/Wm0OCemBQAAAABJRU5ErkJggg==","orcid":"","institution":"Henan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yanbo","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2025-06-10 12:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6862999/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6862999/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85575606,"identity":"7a5d5649-50c0-47d3-9679-84056e7425f1","added_by":"auto","created_at":"2025-06-27 18:30:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106369,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations in breast muscle development and meat quality under HD and CGA supplementation. (A) Breast muscle weight. (B) Breast muscle yield (% of body weight) . (C) Serum CORT concentration. (D–E) Lightness (\u003cem\u003eL*\u003c/em\u003e) of breast muscle at 45 min and 24 h; (F–G) Redness (\u003cem\u003ea*\u003c/em\u003e) of breast muscle at 45 min and 24 h; (H–I) Yellowness (b*) of breast muscle at 45 min and 24 h; (J–K) pH values at 45 min and 24 h postmortem; (L–M) Drip loss at 24 h and 48 h; (N) Cooking loss; (O) Peak shear force. \u003cem\u003eL\u003c/em\u003e* ¼ lightness; \u003cem\u003ea\u003c/em\u003e* ¼ redness/greenness; \u003cem\u003eb\u003c/em\u003e* ¼ yellowness/blueness. ND = normal density; HD = high density; HD + 0.05 CGA= high density + 0.05% CGA; HD + 0.1 CGA= high density + 0.1% CGA; HD + 0.15 CGA= high density + 0.15% CGA. Results were expressed as mean ± SD (for breast muscle weight, breast muscle percentage, peak shear force, cooking loss, drip loss, pH value, \u003cem\u003eL\u003c/em\u003e*, a*, b* value, the \u003cem\u003en\u003c/em\u003e = 6). \u003csup\u003ea-b\u003c/sup\u003eDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/aea941a9f11f50ab905119a9.jpg"},{"id":85575604,"identity":"8b48373e-715e-42bc-886e-c4f304f50080","added_by":"auto","created_at":"2025-06-27 18:30:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123359,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CGA supplementation on antioxidant capacity in serum and breast muscle of broilers under HD. (A) MDA concentration in serum. (B–D) Activities of GSH-Px, T-SOD, and T-AOC in serum;. (E) MDA concentration in breast muscle. (F–H) GSH-Px, T-SOD, and T-AOC activities in breast muscle. ND = normal density; HD = high density; HD + 0.05 CGA = high density + 0.05 % CGA; HD + 0.1 CGA = high density + 0.1 % CGA; HD + 0.15 CGA = high density + 0.15 % CGA. Results were expressed as mean ± SD (for MDA concentration, GSH-Px activity, T-SOD activity, T-AOC activity, CORT concentration, ATP concentration, ROS concentrationthe, the n = 6). a-bDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/c70922b5b76e3b5214509d8b.jpg"},{"id":85575605,"identity":"8aa4c157-fa9e-49c6-aa63-4bae3c0015a3","added_by":"auto","created_at":"2025-06-27 18:30:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101220,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial homeostasis of breast muscle. (A) ATP concentration in breast muscle. (B) ROS concentration in breast muscle. (C) Related mRNA expression of mitochondrial homeostasis biomarkers in breast muscle. (D) Related protein expression of mitochondrial homeostasis biomarkers in breast muscle. ND = normal density; HD = high density; HD + 0.05 CGA= high density + 0.05% CGA; HD + 0.1 CGA= high density + 0.1% CGA; HD + 0.15 CGA= high density + 0.15% CGA. Results were expressed as mean ± SD (for HSP 60, HSP 70, CLPP, CLPX, the n = 6). a-bDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/6164b678f30c58f83e028edb.jpg"},{"id":85576101,"identity":"53e3b819-3d77-404f-9fee-b47cabb17a3e","added_by":"auto","created_at":"2025-06-27 18:46:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144294,"visible":true,"origin":"","legend":"\u003cp\u003eGlycolytic activity, mitochondrial metabolism, and muscle fiber type markers in breast muscle under HD and CGA supplementation. (A) mRNA and protein expression levels of slow-twitch (Slow-MyHC) and fast-twitch (Fast-MyHC) myosin heavy chain isoforms. (B–E) Concentrations of glycogen, glucose, lactic acid, and pyruvic acid in breast muscle tissue. (F) LDH enzymatic activity in breast muscle. (G) Relative mRNA expression of glycolysis-associated markers, including HK1, PFKM, PKM, PDK4, and LDHA. ND = normal density; HD = high density; HD + 0.05 CGA= high density + 0.05% CGA; HD + 0.1 CGA= high density + 0.1% CGA; HD + 0.15 CGA= high density + 0.15% CGA. Results were expressed as mean ± SD (for glycogen concentration, glucose concentration, lactic acid concentration, pyruvic acid concentration, Acetyl-CoA concentration, mitochondrial CA concentration, oxaloacetic acid concentration, activity of LDH, activity of CS, activity of ICD, activity of SDH, activity of MDH, the n = 6). a-bDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/dee961886f6c7642057af3e6.jpg"},{"id":85576103,"identity":"3b1577d4-95bb-4fe9-8543-adc71ff4f973","added_by":"auto","created_at":"2025-06-27 18:46:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114799,"visible":true,"origin":"","legend":"\u003cp\u003eTCA cycle activity and related metabolite levels in breast muscle under HD and CGA supplementation. (A) Overview of metabolite concentrations and enzymatic activity changes related to the TCA cycle. (B–D) Levels of acetyl-CoA, mitochondrial CA, and oxaloacetic acid in breast muscle;\u003c/p\u003e\n\u003cp\u003e(E–H) Enzyme activities of CS, ICD, SDH, and MDH in breast muscle tissue. ND = normal density; HD = high density; HD + 0.05 CGA= high density + 0.05% CGA; HD + 0.1 CGA= high density + 0.1% CGA; HD + 0.15 CGA= high density + 0.15% CGA. Results were expressed as mean ± SD (for glycogen concentration, glucose concentration, lactic acid concentration, pyruvic acid concentration, Acetyl-CoA concentration, mitochondrial CA concentration, oxaloacetic acid concentration, activity of LDH, activity of CS, activity of ICD, activity of SDH, activity of MDH, the n = 6). a-bDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/29bd4837ea3f183d250fd1c2.jpg"},{"id":85575611,"identity":"b65f2f39-25e0-4c9d-9bc7-017688857f0f","added_by":"auto","created_at":"2025-06-27 18:30:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156799,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of correlation analysis among key physiological and biochemical parameters. Asterisks (*) indicate statistically significant correlations (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Red shading represents positive correlations, while blue shading denotes negative correlations between variables.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/db97f3e67f580d6b9dfd645e.jpg"},{"id":87446383,"identity":"7e82df65-7e9e-4221-bd70-7e98d371b468","added_by":"auto","created_at":"2025-07-24 00:31:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1967714,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/fa070bbe-aff4-4d7f-a031-31a7cd8fa6f1.pdf"},{"id":85575811,"identity":"350e6ccf-9362-4f9a-9620-2b8c309f2447","added_by":"auto","created_at":"2025-06-27 18:38:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":30127,"visible":true,"origin":"","legend":"","description":"","filename":"supplementdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6862999/v1/0ad20781b905739606378f73.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chlorogenic acid mitigates high stocking density-induced breast muscle quality decline in broilers via modulating mitochondrial redox homeostasis and glycolytic metabolism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global broiler industry has experienced a consistent growth trajectory over an extended period. In 2024, the global production volume reached an impressive 104.13\u0026nbsp;million tons (Livestock and Poultry, 2024). However, the escalating costs of feed and energy present significant challenges to the industry's continued profitability. To improve economic returns and production performance, breeders often adopt intensive rearing techniques, such as increasing stocking density to maximize yield (Pesti and Choct, 2023). As a critical parameter in poultry rearing, stocking density is usually represented by the live weight per unit area (kg/m\u0026sup2;) or the number of birds (broilers/m\u0026sup2;) (Dawkins et al., 2004; El Sabry MI et al., 2022). Based on modern feeding guidelines, a broiler density of 39 kg/m\u003csup\u003e2\u003c/sup\u003e or 16 broilers/m\u003csup\u003e2\u003c/sup\u003e is appropriate, taking into account growth performance, animal welfare, as well as other factors (Cengiz et al., 2015; Son et al., 2022). Nevertheless, to maximize profits, high stocking density (HD) is often chosen. Although HD can lower production costs and enhance profitability, it can also disrupt the physiology, metabolism, and behavior of broiler animals, leading to oxidative stress reactions (Jones et al., 2005; Nasr et al., 2021; Evans et al., 2023). Moreover, as we indicated in our previous study, HD stress-induced oxidative damage can disturb gut microbiota balance (Liu et al., 2023), exacerbate intestinal damage (Li et al., 2023; Zhang et al., 2024), particularly in the ileum and jejunum, and alter the levels of immunobiochemicals (Campbell et al., 2023) in serum and hypothalamic physiological metabolism. Despite the well-documented link between HD stress and oxidative damage, the molecular mechanisms underlying the connection between mitochondrial dysfunction and meat quality deterioration, especially through the dysregulation of glycolytic and TCA cycle pathways, remain poorly understood. Thus, effective interventions targeting mitochondrial homeostasis under HD conditions are urgently needed.\u003c/p\u003e \u003cp\u003eIn the poultry industry, muscle growth, development and meat quality are key components that are intimately related to the health and productivity of broilers (Mir et al., 2017). Effective husbandry and nutritional strategies can promote muscle development in broiler chickens, thereby optimizing meat quality and enhancing both animal health and meat production (Choi et al., 2023). Nevertheless, numerous studies have shown that elevated ambient temperatures, such as those causing heat stress, can decrease broiler growth performance along with meat quality (Liu et al., 2022; Liu et al., 2023). As mentioned earlier, heat stress also induces oxidative stress, which is mainly attributed to mitochondrial stress. Therefore, it is reasonable to suggest that oxidative stress due to heat stress may be the underlying cause of mitochondrial dysfunction or mammary muscle damage. ROS generation rises as a result of mitochondrial malfunction. This process not only results in low-quality meat but also reduces the pH and antioxidant capacity of broiler meat (Gray et al., 1996; Akbarian et al., 2016; Jing et al., 2024a). Excessive ROS generation and insufficient antioxidant capacity has been identified as a principal cause of oxidative stress (Ahmad et al., 2012; Lu et al., 2017). ROS in excess can alter protein structure, leading to protein polymerization or cross-linking, which can affect major meat qualities for instance juiciness and tenderness (Lu et al., 2017; Jing et al., 2024a). Additionally, further evidence indicates that oxidative stress induces mitochondrial dysfunction, which in turn causes mitochondrial damage. This disorder affects the broiler liver's ability to aerobically metabolize pyruvate due to damage to the mitochondrial tricarboxylic acid (\u003cb\u003eTCA\u003c/b\u003e) cycle (Jing et al., 2023). Therefore, alleviating mitochondrial stress may effectively improve redox homeostasis, promote pyruvate aerobic metabolism, and preserve meat quality of broilers under HD stress.\u003c/p\u003e \u003cp\u003eAntibiotic substitutes, including probiotics, antibacterial peptides, and traditional Chinese herbal formulations, have been utilized to promote broiler growth and enhance antioxidant capacity under HD conditions (Jiang and Xiong, 2016; Yue et al., 2024). As a key bioactive polyphenol in herbal extracts, CGA is generated from caffeic and quinic acids derived from green coffee bean extract by esterification (Liang and Kitts, 2015; Tajik et al., 2017). Various studies have confirmed the remarkable pharmacological properties and potent antioxidant capacity of CGA (Naveed et al., 2018; Lu et al., 2020; Xie et al., 2023). According to animal studies, CGA exerts multiple pharmacological effects, such as improving gut microbiota, enhancing immunity, and promoting antioxidant (Liang and Kitts, 2015), lipid metabolism-regulating activities (Huang et al., 2015), antiviral (Gamaleldin Elsadig Karar et al., 2016), anti-inflammatory (Wang et al., 2020) and antibacterial (Zhang et al., 2020). Therefore, CGA has become a prospective feed additive that enhances the quality of meat products and strengthens animal health. In particular, CGA-enriched diet facilitates muscle production in porcine longissimus dorsi muscle and makes its muscle fibers more susceptible to oxidation, thereby enhancing meat quality (Wang et al., 2021). Additionally, CGA may be beneficial in reducing the severity of colitis and controlling inflammation by inhibiting pro-inflammatory and apoptotic pathways, particularly those mediated by mitochondrial mechanisms (Hu et al., 2022; Qu et al., 2024). When coping with HD stress, the use of CGA may be a feasible therapeutic strategy to enhance meat quality and mitigate the damage from oxidative stress and mitochondrial dysfunction. To this end, a HD stress broiler model was established to assess the efficacy of CGA in improving breast muscle quality, mitigating mitochondrial stress while enhancing glycolytic activity, and identifying the optimal CGA concentration for preventing mitochondrial damage caused by HD.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal ethics statement\u003c/h2\u003e \u003cp\u003eAll of the experimental operations and animal handling followed the Guidelines for Experimental Animals issued by the Ministry of Science and Technology, China (Beijing), and authorized by the Experimental Animal Care and Utilization Committee of Henan University of Science and Technology (AW20602202-1-2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals, diets and experimental design\u003c/h2\u003e \u003cp\u003eIn total, 510 male Arbor Acres broiler chicks, each weighing around 42\u0026thinsp;\u0026plusmn;\u0026thinsp;3 g at one day of age were supplied by a commercial hatchery (Henan Quanda Poultry Breeding Co., Ltd., Hebi, China). All trials were implemented at the Animal Research Center of Henan University of Science and Technology. On day seven, broilers were divided into five treatment groups at random after being stratified by body weight: (1) ND (14 birds/m\u0026sup2;, basal diet), (2) HD (22 birds/m\u0026sup2;, basal diet), (3) HD\u0026thinsp;+\u0026thinsp;0.5 g/kg CGA, (4) HD\u0026thinsp;+\u0026thinsp;1.0 g/kg CGA, and (5) HD\u0026thinsp;+\u0026thinsp;1.5 g/kg CGA. Each group included 10 replicates with 7 (ND) or 11 (HD) birds in each replicate. The standard basal diet for the ND was based on the NRC (NRC, 1994), as listed in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. 0.0, 0.5, 1.0 and 1.5 g/kg CGA (98% purity, obtained from Changsha Staerb Natural Ingredients Ltd (Changsha, China) was added to HD group the basal diet and directly to the basic diet for 42 days of pretreatment.\u003c/p\u003e \u003cp\u003ePrior to the study, all chicken houses and feeding equipment were thoroughly sanitized following standard biosecurity procedures. The broilers were reared in a ventilation-assisted facility using a three-tier stacked cage system with dimensions of 70 cm \u0026times; 70 cm \u0026times; 50 cm (L \u0026times; W \u0026times; H) per cage. During the entire duration of the experiment, they had free access to water and feed. The feeding program contains two stages: days 1 to 21 and days 22 to 42, with formulation details illustrated in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. For the first five days, the temperature was maintained at 32\u0026ndash;34\u0026deg;C, then dropped by 2\u0026deg;C per week before reaching 22\u0026ndash;24\u0026deg;C. The relative humidity was kept constant at 40\u0026ndash;60%. For the first three days, the lighting was constant. After that, the photoperiod was 23 hrs of light and 1 hr of darkness. All birds were provided with adequate nutritional support and routine immunizations, while housing hygiene was maintained through regular cleaning. Their health status and growth metrics were monitored carefully and documented in detail throughout the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Growth performance assessment\u003c/h2\u003e \u003cp\u003eOn days 21, 28, 35 and 42, feed intake and body weight of broilers were recorded after 8 hours of fasting during the experiment. Based on these measurements, key growth performance indicators, including ADG, ADFI, as well as the FCR were computed with the formulas :\u003c/p\u003e \u003cp\u003eADG\u0026thinsp;=\u0026thinsp;total body gain (g) / test days / number of broilers in each replicate\u003c/p\u003e \u003cp\u003eADFI\u0026thinsp;=\u0026thinsp;total feed intake (g) / test days / number of broilers in each replicate\u003c/p\u003e \u003cp\u003eFCR\u0026thinsp;=\u0026thinsp;ADFI / ADG\u003c/p\u003e \u003cp\u003eThroughout the experiment, the broiler's daily growth performance was monitored and recorded closely. In cases of mortality, relevant data including the time of death, leftover feed quantity, and the bird\u0026rsquo;s final body weight were systematically documented for subsequent analysis.. Any resulting deviations due to mortality were subsequently analyzed, and data from deceased animals were excluded during the final data processing to ensure accuracy and consistency of growth performance evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Muscle weight and sample collection\u003c/h2\u003e \u003cp\u003eSix broilers were chosen from each treatment group at the conclusion of the 42-day period, and they fasted overnight. All of the broilers in each group weighed almost the same on average. The broilers were weighed prior to slaughter, and the weight was recorded as the live weight. Following that, the complete pectoral muscle of each broiler was removed, weighed, and its weight was noted, which was calculated using the formula as below:\u003c/p\u003e \u003cp\u003ePectoral muscle percentage (%) = (breast muscle weight / live weight) \u0026times; 100\u003c/p\u003e \u003cp\u003eAdditionally, a small sample of right pectoral muscle tissue was gathered in a sterile test tube and kept immediately at -80\u0026deg;C for follow-up analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Meat quality of breast muscle\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Meat color and pH\u003c/h2\u003e \u003cp\u003eA portion of the left breast muscle was taken from each selected broiler in order to assess the meat color and pH. Measurements were conducted with a portable pH meter (pH-Star, Matth\u0026auml;us, P\u0026ouml;ttmes, Germany) and a Minolta Chromameter CR-300 (Minolta Camera, Osaka, Japan). Yellowness/blueness (\u003cem\u003eb*\u003c/em\u003e), redness/greenness (\u003cem\u003ea*\u003c/em\u003e), and lightness (\u003cem\u003eL*\u003c/em\u003e) were the parameters utilized to assess the flesh color. First, the samples of breast muscle were cut, left to rest for 45 minutes, and then three separate pH and color measurements were made. After that, the samples were maintained at 4\u0026deg;C in a Ziploc bags. The pH and color measurements were repeated after a 24-hour period. The average of three measurement points per sample was utilized in the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Cooking loss\u003c/h2\u003e \u003cp\u003eIn brief, from each chosen broiler, 30 g of left-side breast muscle tissue was removed and originally kept at 4\u0026deg;C. Samples were taken out and weighed to determine the initial mass (Wa) following a 24-hour period of refrigeration. For the purpose of thermal processing, 10 samples in all were individually sealed in Ziplock bags and submerged in a water bath set at 90\u0026deg;C. A kerosene thermometer was utilized to monitor the internal temperature of each sample and when the temperature reached 70\u0026deg;C, the samples were immediately removed and cooled naturally at RT. Filter paper was applied to properly remove surface moisture. After that, the post-cooking weight (Wb) was noted. The following formula was utilized to compute cooking loss (%):\u003c/p\u003e \u003cp\u003eCooking loss (%) = (Wa\u0026thinsp;\u0026minus;\u0026thinsp;Wb) / Wa \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Drip loss\u003c/h2\u003e \u003cp\u003eFrom the right pectoral muscle, a fresh thick piece (5 cm \u0026times; 5 cm \u0026times; 1 cm) was taken, weighed, and then designated as W1 according to the initial breast muscle weighing results. Ten of these slices were prepared, sealed in Ziplock bags one at a time, and kept at 4\u0026deg;C. Each sample was reweighed after being carefully blotted dry using filter paper to eliminate surface moisture after 24 and 48 hours. These subsequent weights were recorded as W2 and W3, respectively. The following formulas were applied to calculated drip loss:\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e24 - hour drip loss (percent) = (W1 − W2) / W1 × 100\u003c/h3\u003e\n\n\u003ch3\u003e48 - hour drip loss (percent) = (W1 − W3) / W1 × 100\u003c/h3\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5.4. Peak shear force\u003c/h2\u003e \u003cp\u003eFollowing the 24-hour measurements of pH and meat color, the peak shear force was measured using the remaining muscle samples. Each piece was sealed in a Ziplock bag and thermally processed in a water bath at 90\u0026deg;C. Once removed, the samples were let cool naturally to RT and then placed in a 4\u0026deg;C refrigerator from which the internal temperature reached 70\u0026deg;C. After 24 hours, 5 cores (with a diameter of 1.5 cm) were taken from each sample utilizing the same sampler. Ten of these cores underwent shear testing using a Texture Analyzer (XT2, Stable Micro Systems Ltd., Godalming, Surrey, UK). The findings of the five cores extracted from the same pectoral muscle were averaged to establish the peak shear force value for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Assessment of antioxidant capacity and enzymatic activity\u003c/h2\u003e \u003cp\u003eTBA test was exploited to determine the serum levels of MDA. WST-1 test and Colorimetric analysis were employed to assess the activity of T-SOD and serum GSH-PX, separately. The ABTS test was also applied to measure the serum's T-AOC. All tests were carried out with commercially available kits provided by the Nanjing Jiancheng Bioengineering Institute Co., Ltd. (Nanjing, China).\u003c/p\u003e \u003cp\u003eUsing certain test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the levels of lactic acid, glucose, glycogen, mitochondrial citric acid, pyruvate, ATP and MDA in breast muscle samples were measured. ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) were applied to measure the levels of oxaloacetate (\u003cb\u003eOA\u003c/b\u003e) and acetyl-CoA, along with the activity of the enzymes isocitrate dehydrogenase (\u003cb\u003eICD\u003c/b\u003e) and citroyl synthetase (\u003cb\u003eCS\u003c/b\u003e) in breast muscle samples. Via applying an ELISA kit (Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China), the ROS concentration in the identical samples was measured. Furthermore, through the appropriate test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the enzyme activity of MDH, SDH, LDH, T-SOD, T-AOC, and GSH-Px in breast muscle samples were assessed. With a commercial test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the protein content of the sample extract was identified. The instructions included with the kit were followed when performing sample pretreatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7. RT-PCR analysis of mRNA expression in breast muscle\u003c/h2\u003e \u003cp\u003eSamples of muscle tissue were thawed on ice as described previously (Zhang et al., 2024), and then ~\u0026thinsp;100 mg of tissue was transferred into 1.5 ml sterile test tubes. Extraction of total RNA was carried out from each sample through TRIzol reagent (Invitrogen Inc., Carlsbad, CA), and Nanodrop 2000 spectrophotometry (Thermo Scientific, Ottawa, Canada) was applied to evaluate the RNA purity. Only samples with an A260/A280 ratio\u0026thinsp;\u0026gt;\u0026thinsp;1.9 were considered suitable for downstream analysis. RNA concentrations were determined by absorbance, afterwards, the RNA samples were aliquoted and preserved at -80\u0026deg;C before use. A reverse transcription kit (Vazyme, Nanjing, China) was exploited to create cDNA from the isolated RNA. The primers (Supplementary \u003cb\u003eTable S2\u003c/b\u003e) were designed with Primer 3.0 and generated from Shanghai Shenggong Bioengineering Co., Ltd., and the housekeeping gene GAPDH served as an internal reference for normalization. On a CFX Connect Real-Time PCR system, qRT-PCR was implemented with the SYBR Green PCR kit (Vazyme Biotechnology Co., Ltd., Nanjing, China). Thermocycling conditions included initial denaturation at 95\u0026deg;C for 5 min, denaturation at 95\u0026deg;C for 15 s, and annealing/extension at 60\u0026deg;C for 30 s, for a total of 40 cycles. 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was utilized to determine the levels of relative gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blot analysis of protein abundance in breast muscle\u003c/h2\u003e \u003cp\u003eWestern blotting was implemented based on the procedures described by Liu et al. (2022). In brief, a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), manufactured by Beo Tianmei in Shanghai, China, was added to pre-chilled RIPA lysis buffer buffer prior to homogenizing the breast muscle samples. After centrifuging the resultant homogenates for 30 minutes at 4\u0026deg;C at 13,000 \u0026times; g, the supernatants were gathered. A BCA assay kit (catalog no. A045-3 from Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was exploited to measure the total content of protein in each sample after the supernatant was gathered, and the results were then standardized to 6 \u0026micro;g/\u0026micro;L. 400 \u0026micro;L of each protein sample was then combined with 100 \u0026micro;L of 5 \u0026times; SDS-PAGE sample loading buffer (catalog no. P0015L from Beyotime, Shanghai, China), and the mixture was denaturated in a water bath at 95\u0026deg;C for 10 minutes. Proteins were resolved on SDS-PAGE gels with concentrations ranging from 8\u0026ndash;12%, and then electrotransferred onto PVDF membranes (Bio-Rad, CA, USA). For lowering non-specific binding, membranes were blocked for 120 min utilizing 5% non-fat dry milk. This was followed by three TBST buffer washes. The membranes were incubated with primary antibody after blocking (Supplementary \u003cb\u003eTable S3\u003c/b\u003e). for a whole night at 4\u0026deg;C. HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG, 1:5000 dilution; Proteintech Group, IL, USA) were then added. The bands were visualized via an enhanced chemiluminescence system (Bio-Rad, CA, USA), and the quantification was done through the Image Lab software system (Bio-Rad, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eEach cage was regarded as an experimental unit in order to assess growth performance. All other parameters were taken as experimental units of a single bird in each replicate. One-way ANOVA with SPSS 20.0 (SPSS Inc., Chicago, Illinois) was employed for the statistical analysis. With Tukey's multiple range test, significant differences across groups were determined; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant. GraphPad Prism Version 9 (GraphPad Software Inc., San Diego, CA) was employed for data visualization and result graphing.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Growth performance of broilers\u003c/h2\u003e \u003cp\u003eThe results demonstrate how high-density (\u003cb\u003eHD\u003c/b\u003e) stress and chlorogenic acid (\u003cb\u003eCGA\u003c/b\u003e) dietary conditions influenced production performance throughout the trial, as depicted in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. No significant difference in growth metrics, including FCR, ADG, and ADFI, were observed between the HD and normal-density (\u003cb\u003eND\u003c/b\u003e) groups or among the three CGA concentration levels during the initial feeding phase (\u003cb\u003edays 1\u0026ndash;21\u003c/b\u003e). However, exposure to HD circumstances from day 22 to day 42 remarkably affected the development of broiler chicks versus the ND group. In the HD group, broiler body weight dropped by 12% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and broiler FCR increased while ADG and ADFI decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) under HD. Supplementation with dietary CGA, especially at concentrations of 0.1% and 0.15%, partially mitigated the adverse effects of HD, leading to a rise (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in final weight of body and improvements (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in ADFI and ADG for broilers. Only a few broilers died in the ND group, while the mortality rate in the HD group was 2.56%. The addition of CGA remarkably raised the broiler mortality rate under HD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Breast muscle integrity and meat quality alterations\u003c/h2\u003e \u003cp\u003eUnder HD, broilers reared revealed a substantial (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in the weight of the breast muscles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) together with breast muscle index (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) in contrast to the ND group. In contrast to ND group, the serum CORT concentration in HD group was considerably higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating a physiological stress response. Additionally, HD conditions adversely affected the meat quality of breast muscle, resulting in a considerable decline (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the \u003cem\u003eL*\u003c/em\u003e values at both 45 minutes and 24 hours, as well as a rise (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the \u003cem\u003eb*\u003c/em\u003e values at the same time points. Conversely, the \u003cem\u003ea*\u003c/em\u003e value at 45 minutes was remarkably decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-I). Furthermore, at 24 and 48 hours, under HD, drip loss markedly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as did cooking loss together with peak shear (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Yet, the pH showed a substantial drop after 24 hours (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-O). Incorporation of CGA to the diet led to improved broiler meat quality under HD, resulting in a reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in 24 h \u003cem\u003eL*\u003c/em\u003e, both 45 min and 24 h \u003cem\u003eb*\u003c/em\u003e as well as the peak shear force, cooking and drip losses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antioxidant status of serum and breast muscle\u003c/h2\u003e \u003cp\u003eBroilers exposed to HD displayed a considerably lowered (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) amount of MDA in their serum than the ND group, similar differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06) were noted in the tissue of breast muscle (\u003cb\u003eFig.\u0026nbsp;2A and E\u003c/b\u003e). Additionally, the antioxidant status of breast muscle meat was adversely affected by HD, resulting in a substantial decrease (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the blood levels of T-AOC and GSH-Px (\u003cb\u003eFig.\u0026nbsp;2B and D\u003c/b\u003e). At the same time, the amount of T-SOD (\u003cb\u003eFig.\u0026nbsp;2G\u003c/b\u003e) in the breast muscle declined (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When in contrast to the ND group, the ROS content was considerably raised (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the level of ATP in the pectoral muscles was lowered (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in HD group (\u003cb\u003eFig.\u0026nbsp;2J and K\u003c/b\u003e). As demonstrated by lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) levels of MDA and higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) serum levels of T-AOC and GSH-Px, together with higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) levels of ATP and T-SOD in breast muscle, dietary with CGA enhanced the antioxidant status of broilers under HD (\u003cb\u003eFig.\u0026nbsp;2A, B, D, G, J, and K\u003c/b\u003e). It is worth noting that a 0.1% concentration of CGA boosted (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ATP and T-SOD levels in breast muscle and considerably raised (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) blood levels of T-AOC and GSH-Px (\u003cb\u003eFig.\u0026nbsp;2B, D, G, and J\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Mitochondria homoeostasis in breast muscle\u003c/h2\u003e \u003cp\u003eTo assess mitochondrial stress induced by HD and the potential protective effects of varying doses of CGA, we analyzed several mitochondrial function indicators in broiler breast muscle. The findings suggested that, under HD, the levels of ATP were markedly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the that of ROS were considerably higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Further analysis showed that HD resulted in a marked upregulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the mRNA expression of heat shock proteins HSP60 and HSP70, as well as mitochondrial quality control markers CLPP and CLPX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The expression of CLPX and CLPP at the protein level was also notably raised in the HD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting activation of mitochondrial stress response pathways. Dietary CGA supplementation effectively ameliorated mitochondrial dysfunction, as evidenced by increased ATP concentrations and reduced ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), highlighting its protective role under HD stress conditions. Furthermore, different concentrations of CGA have demonstrated a reduction or a tendency to decline the mRNA expression of HSP60, HSP70, CLPP, and CLPX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The findings also suggested that CGA supplementation decreased the CLPX and CLPP protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In comparison to the other two concentrations of CGA, broilers administered 0.1% CGA displayed the lowest levels of CLPP in the breast muscle, as displayed by both protein and mRNA expression levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Myofiber types and glycolysis status in breast muscle\u003c/h2\u003e \u003cp\u003eGlycolytic activity and muscle fiber types in broiler breast muscle were assessed. The HD group considerably increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the protein expression of Fast-MyHC in breast muscle while dramatically reducing (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) that of Slow-MyHC versus the ND group(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Regarding glycolysis activity, broilers under HD conditions revealed a remarkable rise (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in breast muscle concentrations of lactic acid, glucose, and glycogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). In addition, the enzymatic activity of lactate dehydrogenase (\u003cb\u003eLDH\u003c/b\u003e) was evidently raised (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the HD group versus ND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The findings also suggested that the LDH activity was obviously higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in HD broilers versus ND broilers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Furthermore, Supplementation with CGA caused a marked decline (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in LDH activity, glucose, and glycogen, alongside a marked rise (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the content of pyruvic acid. Follow-up analysis exhibited that HD considerably elevated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the glycolysis-associated enzyme mRNA expression, encompassing PKM, PFKM, HK1, LDHA and PDK4, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Additionally, dietary CGA supplementation potentially decreases the expression of biomarkers related to glycolysis, including PFKM, PGK, PKM, PDK4, and LDHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In contrast, birds receiving CGA-supplemented diets demonstrated a downregulation of several glycolytic markers, notably PFKM, PGK, PKM, PDK4, and LDHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Additionally, in contrast, the mTOR pathway components such as S6K1, 4EBP1, and mTOR were not appreciably altered in the ND group in terms of their expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Notably, CGA supplementation, particularly at a 0.1% inclusion rate, was found to enhance the transcription of mTOR and S6K1, indicating potential involvement in protein synthesis regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Mitochondria tricarboxylic acid cycle in breast muscle\u003c/h2\u003e \u003cp\u003ePyruvate, a byproduct of glycolysis, is used by muscle tissue primarily through the TCA metabolic pathway. In the present investigation, HD substantially decreased the acetyl-CoA concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by 12.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e) along with the levels of mitochondrial CA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and oxaloacetic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by 17.4% and 35.7%, separately (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C, \u003cb\u003eand D\u003c/b\u003e). Follow-up analysis suggested that CS, ICD, SDH, and MDH enzyme activities were decreased by 19.4%, 47.2%, 39.6%, and 13.9%, separately, in the breast muscle of broilers exposed to HD conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, E\u0026ndash;H). The dietary supplementation of CGA exhibited mitigating effects, resulting in a rise in the acetyl-CoA concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and a trend towards a raised oxaloacetic acid concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, it strengthened MDH, SDH and ICD activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG \u003cb\u003eand H\u003c/b\u003e). In the meantime, when compared to the three concentrations of CGA, broilers that received 0.1% CGA exhibited higher Acetyl-CoA activity in the breast muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additionally, compared to the 0.05% and 0.15% concentrations, supplementation with 0.1% CGA showed a tendency to increase the MDH and SDH concentrations in HD-stressed broiler breast muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG \u003cb\u003eand H\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Correlation analysis\u003c/h2\u003e \u003cp\u003eFor the exploration of the intrinsic mechanism of the CGA's protective effect on the meat quality under HD, we correlated the parameters of meat quality with a range of physiological indices, including antioxidant status, glycometabolic activity, and mitochondrial function. This analysis aimed to identify key factors associated with variations in meat quality in broilers stressed by HD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Regarding the meat color value of breast muscle, glycogen levels displayed a negative correlation with the 24-hour \u003cem\u003eL*\u003c/em\u003e value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the glucose and GSH-Px levels were positively correlated with the 24-hour \u003cem\u003ea*\u003c/em\u003e value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After 24 hours, drip loss in breast muscle had a negative relationship with muscle ROS and lactate levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Likewise, muscle ATP and T-SOD levels showed a negative association with 48 hr values of drip loss (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Peak shear in breast muscle has a positive association with the levels of glucose in breast muscle (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, muscle ROS and GSH-Px levels positively correlated with glucose and lactic acid, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe created a broiler model that was exposed to HD stress and examined the possible protective function of CGA in promoting growth and preserving meat quality. Furthermore, we examined potential differences in the effectiveness of CGA from various sources with varying concentrations. During the initial 21 days of HD exposure, no significant differences in production performance were observed. However, by day 42, broilers subjected to HD conditions exhibited a decrease of 22.0% in BW, 8.2% in ADFI, and 15.5% in ADG, in contrast to the ND group. This discovery is in line with previous investigations (Liu et al., 2023; Li et al., 2023; Zhang et al., 2024), which indicate that the onset of density stress is associated with a phase of accelerated growth. The current study demonstrates that CGA supplementation enhances production performance, as evidenced by increased BW, ADFI, and ADG while reducing mortality rates. Although CGA supplementation at 0.1% and 0.15% did not completely restore performance or quality indicators to levels of the ND group, these concentrations still exerted a significant mitigating effect on the physiological stress induced by HD.\u003c/p\u003e \u003cp\u003eBreast muscle is the primary edible portion and a source of high-quality protein in broilers. This research observed that HD can decrease the proportion of pectoral muscles in broilers. In agreement with the previous findings, restricted growth in broilers after exposure to oxidative stress, including heat stress, is generally linked to a reduction in the percentage of breast muscle (Liu et al., 2023; Jing et al., 2024). The findings display that the progressive onset of HD may have triggered oxidative stress responses in broiler chickens, eventually causing a loss of breast muscle mass. Additionally, the findings present that HD-stress considerably elevated the CORT index. The CORT is regarded as a cellular stress sensor and is commonly utilized to evaluate damage induced by oxidative stress (Lee et al., 2018; Ma et al., 2020). This suggests that in broiler chickens, HD prevents the growth of skeletal muscle and damages the breast muscle. CGA has been proved to have strong antioxidant performance (Chen et al., 2023). When oxidative stress is present, dietary containing CGA can support the growth and development of skeletal muscle (Zhao et al., 2019). In this work, CGA reduced the concentration of CORT in serum and numerically increased the breast muscle mass in broilers under HD conditions. These findings indicate that CGA may mitigate oxidative stress, thereby safeguarding the quality and quantity of breast muscle against damage induced by HD.\u003c/p\u003e \u003cp\u003eHD stress negatively influences broiler meat quality, mainly by lowering the pH and color of the meat, while elevating peak shear force together with drip loss (Choi et al., 2023; Yue et al., 2024). In this research, we examined the influence of HD on meat quality in broilers, as well as the potential protective effects of CGA. As a result, HD elevated the drip loss, \u003cem\u003eb\u003c/em\u003e* value, peak shear force and cooking loss of breast muscle while decreasing the pH and both the \u003cem\u003ea*\u003c/em\u003e and \u003cem\u003eL*\u003c/em\u003e values. The color of meat is a critical quality characteristic because consumers associate its appearance with freshness, which influences purchasing choices (Yue et al., 2024). The \u003cem\u003eL*\u003c/em\u003e value indicates paleness, with higher values representing lighter meat (Tong et al., 2015). Our results align with research reported that HD stress leads to paler meat, which is linked to decreased meat quality and lower muscle pH (Pekel et al., 2020; Weng et al., 2022). The percentages of drip and cooking loss in this work is in line with the results of Wu et al. (2020), who reported elevated water loss from thigh and breast muscles of Arbor Acres broilers raised under HD. Furthermore, their findings indicate that in line with the current investigation, final muscle pH dropped under HD circumstances as drip and cooking losses increased. This may be explained by the substantial negative association between pH and drip and cooking loss, which is due to the accelerated deposition of lactate, leading to a subsequent increase in meat toughness (Huff-Lonergan and Lonergan, 2005; Li et al., 2019). The experimentally obtained data, which are logically consistent, further indicate that HD stress significantly compromises chicken breast muscle quality. According to this investigation, adding CGA to the breast meat of broilers under HD stress decreased both cooking and drip losses. This implies that CGA may be useful in improving meat's ability to retain water. The water-holding capacity of broiler breast meat has also been displayed to be improved by dietary supplementation with E. ulmoides extract, which contains 8% CGA, according to several studies (Jiang and Xiong, 2016; Zhao et al., 2019). A number of variables, encompassing final pH and oxidation of lipids and proteins, affect water-holding capacity of meat (Zhao et al., 2019).\u003c/p\u003e \u003cp\u003ePrevious studies, including our own, have shown that stress related to HD induces oxidative stress, which can disrupt mitochondrial homeostasis (Lu et al., 2017; Zhang et al., 2024). Typical features of this stress are elevated ROS levels and diminished antioxidant capacity (Lu et al., 2017; Jing et al., 2023). The reduced antioxidant capacity in muscle tissue is frequently associated with a decline in meat quality (Jing et al., 2024a). The efficient functioning of the TCA cycle relies on the preservation of mitochondrial homeostasis, which, in turn, affects the antioxidant capacity (Mart\u0026iacute;nez-Reyes and Chandel, 2020). In this study, it was discovered that HD lowers the antioxidant capacity in blood and breast muscle and disturbs mitochondrial homeostasis. As a result, ROS and MDA levels elevated, T-SOD and GSH-Px activity decreased, the concentration of ATP dropped, and HSP60, HSP70, CLPP and CLPX gene and protein expression raised. Through mitochondrial oxidative phosphorylation, ATP and ROS are produced. However, mitochondrial dysfunction results in an elevated production of ROS and a concomitant decrease in ATP synthesis (Murphy, 2013). Hsp70 is essential for proteostasis and alleviates damage from various stressors, including oxidative stress (Zhang et al., 2022). HSP60 and CLPP are key mitochondrial chaperones and proteases, involved in maintaining mitochondrial homeostasis and serving as biomarkers of mitochondrial stress (Venkatesh, S., \u0026amp; Suzuki, 2017; Luo et al., 2021). CLPX is known to interact with the mitochondrial complex II subunit, SDHB. Mitochondrial dysfunction is commonly associated with raised CLPX, CLPP, and HSP60 levels. This research revealed that HD enhanced the abundance of these proteins in breast muscle, suggesting that HD induces mitochondrial dysfunction. Dietary supplementation with CGA restored ROS levels, decreased the HSP70, HSP60, CLPX and CLPP expression, and elevated the levels of ATP, while also elevating T-SOD levels in breast muscle. These findings indicate that CGA enhances mitochondrial stability and boosts antioxidant capacity in broiler skeletal muscle under heat distress..\u003c/p\u003e \u003cp\u003eTo investigate the mechanisms by which CGA preserves broiler meat quality under HD conditon, we initially assessed muscle fiber type expression in the breast muscle. From a physiological metabolic standpoint, myofibers with higher fast myosin heavy chain (\u003cb\u003eMyHC\u003c/b\u003e) expression typically display a lower pH and increased drip loss, whereas those with elevated slow MyHC levels show reduced drip loss and a higher pH (Wang et al., 2021; Chauhan and England, 2018). This study revealed that in the breast muscle, HD boosted fast MyHC protein expression while reducing protein expression of slow MyHC. In broiler breast muscle under HD stress, dietary CGA supplementation maintained a larger proportion of fast MyHC fibers and declined expression of slow MyHC fibers by preventing muscle fiber type conversion. Research indicates that myofibers with higher concentrations of fast MyHC exhibit increased level of glycogen and lactic acid, which reduced the pH and weaken the water retention capacity of the meat (Lu et al., 2017; Huo et al., 2022). Research has discovered that HDL raises the concentration of lactate, glucose, and glycogen in the pectoral muscles while lowering the concentration of pyruvate. Subsequent analysis demonstrated that HD raised mRNA expression of glycolysis-associated enzymes together with LDHA, and strengthened the LDH enzymatic activity. PKM and HK1 are essential rate-limiting enzymes in the glycolytic pathway. Specifically, via catalyzing the glucose phosphorylation, HK1 initiates glucose metabolism, while PKM facilitates the final step, converting phosphoenolpyruvate into pyruvate (De Jesus et al., 2022; Horemans et al., 2022). Furthermore, via regulating the activity of the pyruvate dehydrogenase complex, PDK4 regulates the irreversible conversion of pyruvate to acetyl-CoA (Lu et al., 2017; Jing et al., 2024). According to research, pyruvate is rapidly converted to lactic acid in avian breast muscle by LDH (Wilson, Cahn, Kaplan, 1963). The raised PDK4 mRNA, increased LDH activity, and reduced mTOR expression suggest a diminished glucose entry into the tricarboxylic acid cycle, indicating a shift toward anaerobic glycolysis. These findings imply that HD adversely influences meat quality through the enhancement of glycolysis and modulation of muscle fiber type conversion. CGA supplementation lowered lactate and glycogen levels. Furthermore, it was linked to a rise in the expression of protein synthesis-associated genes (S6K1 and mTOR) and a reduction in the activity of glycolysis-associated enzymes (PDK4 and PFKM), which is in line with the report of Wang et al., (2021). Plant extracts rich in specific polyphenols (e.g., CGA) have been reported to have potential anabolic actions that may facilitate muscle production and produce anti-catabolic and protein-sparing effects on muscle tissue. The results indicate that CGA treatment significantly enhanced the breast muscle quality in broilers, although the effect of the CGA concentration gradient was not highly significant. In the expression of genes linked to protein synthesis (S6K1 and mTOR), the 0.01% CGA concentration was most effective, suggesting the need for further clarification of the concentration gradient.\u003c/p\u003e \u003cp\u003eAs previously mentioned, the efficiency of the mitochondrial TCA cycle relies on the maintenance of mitochondrial homeostasis, and pyruvate catabolism proceeds through two primary metabolic routes: anaerobic metabolism, leading to lactate production, or entry into the mitochondria for aerobic metabolism (Pithukpakorn, 2005; Che et al., 2024). Previous studies, primarily conducted using heat stress broiler models, have shown that HS causes mitochondrial stress, disrupts TCA cycle, and compromises metabolism of aerobic pyruvate (Jing et al., 2024). Furthermore, lactic acid concentration in breast muscle increased by 66.7%, indicating that HD enhances anaerobic pyruvate metabolism in the muscle. As a central pathway for aerobic pyruvate oxidation, the TCA cycle is dependent on enzymes for instance ICD, CS, MDH and SDH (Mart\u0026iacute;nez-Reyes and Chandel, 2020). In this study, HD markedly inhibited the ICD, CS, MDH and SDH activities, and lowered the concentrations of crucial metabolic intermediates like oxaloacetate and CA in the TCA cycle. Based on the previously described findings, HD inhibits the TCA cycle, which prevents pyruvate from being aerobically metabolized. This causes the breast muscle to produce more lactic acid and accumulate glycogen. The results of this experiment align with conclusions observed in heat stress studies, suggesting that there was less glucose entering the tricarboxylic acid cycle, cells became more dependent on anaerobic glycolysis (Lu et al., 2017; Jing et al., 2023). In comparison to the two other concentrations of CGA, supplementation with 0.1% CGA revealed the most protective effects on the mitochondrial TCA cycle in the breast muscle of broilers exposed to HD stress, as indicated by the increased activities of Acetyl-CoA, SDH, and MDH. Previous research on CGA has mainly concentrated on its role in enhancing oxidative stress resistance (Bao et al., 2018; Wang et al., 2021). This study proves for the first time that CGA augments TCA cycling in broiler muscle mitochondria, highlighting its targeted effect on mitochondrial function.\u003c/p\u003e \u003cp\u003eThis study focused on the correlation analysis, which uncovered complex relationships between meat quality parameters and biochemical factors in broilers under HD conditions. It also assays pectoral muscle meat color, shear and drip loss. The factors taken into account included antioxidant indicators (such as GSH-Px, T-SOD, ROS), glycometabolism-related substances (glycogen, glucose, lactic acid), and mitochondrial products (ATP). Correlations between these factors and meat quality parameters were tested at various time points (24 and 48 hours). These findings highlight the potential mechanisms by which CGA may exert protective effects. Higher glycogen levels are closely associated with darker-colored meat, as manifested by a lower \u003cem\u003eL*\u003c/em\u003e value at 24 hours post-mortem. This phenomenon can be primarily ascribed to the significant role of glycogen in post-mortem glycolysis (Petracci and Cavani, 2012). Glycogen-driven glycolysis exerts a profound influence on the decline in pH levels and the stability of myoglobin, which are crucial factors determining meat color (Yue et al., 2024). Additionally, elevated concentrations of the antioxidant enzyme GSH-Px and glucose are positively correlated with an enhancement in meat redness. GSH-Px, with its potent antioxidant capacity, likely mitigates oxidative damage to heme pigments, thereby effectively maintaining the natural red color of the meat (Yue et al., 2024). Meanwhile, glucose may serve to support the energy metabolism process, which in turn stabilizes the color-related chemical compounds within the meat.\u003c/p\u003e \u003cp\u003eIn contrast, negative correlations are observed between meat quality parameters and lactic acid as well as ROS (Schieber \u0026amp; Chandel, 2014). A greater lactic acid concentration can accelerate the pH drop, which improves the meat's ability to retain water through protein denaturation (Yue et al., 2024). Notably, a decrease in ROS levels is unexpectedly associated with higher drip loss. This finding implies that oxidative stress, despite its common perception of causing damage, may have an unpredictable impact on cell membrane integrity, potentially leading to increased fluid leakage from the muscle tissue. Negative correlations also exist between meat quality and ATP and T-SOD levels. Higher levels of ATP serve a key role in preserving the cellular energy state, which is vital for maintaining the cell membrane integrity (Jing et al., 2024a). The antioxidant activity of T-SOD effectively reduces oxidative damage to proteins, contributing to enhanced water retention over time. Moreover, higher glucose levels are correlated with increased meat toughness. This is likely due to the accelerated post-mortem glycolysis process triggered by high glucose. The rapid glycolysis leads to a sharp drop in pH, followed by protein denaturation, which ultimately impairs the tenderization process of the meat (Yue et al., 2024). Furthermore, a positive relationship is detected between GSH-Px and glucose levels. This positive correlation suggests that the GSH-Px antioxidant activity may have a regulatory effect on glucose metabolism. Similarly, ROS and lactic acid are positively correlated, indicating that oxidative stress can exacerbate the glycolytic flux. This exacerbation not only affects the pH balance within the muscle tissue but also intensifies the oxidative damage, further influencing the overall quality of the meat.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study developed a HD stress model for broilers and examined the effects of CGA supplementation. Results indicate that HD stress lowers the body weight of broilers, improves feed conversion, and negatively affects the mass of breast muscle, including reduced pH, higher drip and cooking losses, and altered color. However, CGA supplementation, especially at 0.1%, alleviates these negative effects by enhancing growth, increasing body weight, feed intake, and daily gain, while lowering mortality. In terms of meat quality, CGA reduces drip and cooking losses, improving water-holding capacity. Mechanistically, CGA alleviates mitochondrial stress, restores redox balance by reducing ROS and MDA, and boosts antioxidant enzyme activity and ATP levels. Additionally, CGA regulates glycolytic metabolism, preventing fiber-type conversion, lowering glycogen and lactic acid levels, and reducing glycolytic enzyme activity. It also enhances mitochondrial TCA cycle activity and metabolite concentrations. In conclusion, although further study is required to adjust its concentration and examine long-term impacts on meat quality and broiler health, CGA exhibits potential as a feed addition to enhance meat quality of broiler under HD stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe supporting information is available free of charge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eAnimal experiments were approved by the Animal Care and Use Committee of Henan University of Science and Technology (DWFL36891-2023) on October 1, 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eInvestigation, Y.Z. and D.B.; Methodology, Y.Z., D.B., C.G., P.M., Z.W. and X.Z.; Software, Y.Z. and D.B. and C.G.; Validation, X.X. and X.M.; Formal analysis, Y.Z., W.Z. and D.B.; Resources, Y.M., D.B., B.Z., X.X. and X.M.; Data curation, C.G., P.M., Z.W. and X.Z.; Writing the original draft preparation, Y.Z. and D.B.; Reviewing and editing, D.B., K.I., B.Z. and Y.M; Visualization, Y.Z.; Supervision, Y.Z., W.Z. and Y.M.; Project administration, Y.M.; Funding acquisition, Y.M. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources:\u0026nbsp;\u003c/strong\u003eThe research was supported in part by the National Key Research and Development Program of China (Grant Number 2022YFE0111100), the Key Research and Development Program of Henan Province (Grant Number 241111113800), the Program for International S\u0026amp;T Cooperation Projects of Henan (Grant Number 232102521012), the Key Scientific Research Foundation of the Higher Education Institutions of Henan Province (Grant Number 22A230001), the Frontier exploration Projects of Longmen Laboratory (Grant Number LMQYTSKT037)，and the Doctoral Research Grant of Henan University of Science and Technology (13480103).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe raw data supporting the conclusions of this article will be made available by the authors on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors extend their gratitude to the College of Animal Science and Technology, Henan University of Science and Technology for providing access to experimental facilities. Gratitude is also extended to the Animal Welfare and Health Breeding of Henan Province and the Expat Scientist Studio for Animal Stress and Health Breeding of Henan Province for their valuable academic guidance throughout this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad H, Tian J, Wang J, et al. Effects of dietary sodium selenite and selenium yeast on antioxidant enzyme activities and oxidative stability of chicken breast meat. J Agric Food Chem. 2012; 60(29): 7111\u0026ndash;7120. https://doi.org/10.1021/jf3017207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkbarian A, Michiels J, Degroote J, Majdeddin M, Golian A, De Smet S. 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Poult Sci. 2019; 98(7): 3040\u0026ndash;3049. https://doi.org/10.3382/ps/pez081.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"943\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eEffects of HD stress and CGA supplementation on broilers growth performance.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eItem\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHD +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHD +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHD +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSEM/ᵪ2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.05 CGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1 CGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.15 CGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1-21d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;BW, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.020\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.892\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;21 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e654.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e634.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e644.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e659.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e649.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.010\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.437\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;ADFI, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.121\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.331\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;ADG, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.235\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.773\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;FCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.51\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.014\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e22-42d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;BW, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;22 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e699.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e680.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e690.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e704.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e695.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.230\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.243\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;42 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2527.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2225.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2429.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2477.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2459.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.780\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;ADFI, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e142.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e130.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e133.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e138.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e135.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.822\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;ADG, g/bird\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e73.57\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.074\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;FCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.023\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMortality, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 1.323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.216\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 943px;\"\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eBW = body weight; ADFI = average daily feed intake; ADG = average daily gain; FCR = feed conversion rate. ND = normal stocking density fed basal diet group; HD = high stocking density fed basal diet group; HD + 0.05 CGA = high stocking density fed with 0.05% CGA; HD + 0.1 CGA = high stocking density fed with 0.1% CGA; HD + 0.15 CGA = high stocking density fed with 0.15% CGA. Results for BW, ADFI, ADG and FCR were expressed as mean with SEM (n = 10). \u003csup\u003ea-c\u003c/sup\u003eDifferent letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"broilers, CGA, high stocking density, meat quality, mitochondrial redox homeostasis, glycolytic metabolism","lastPublishedDoi":"10.21203/rs.3.rs-6862999/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6862999/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh stocking density (\u003cb\u003eHD\u003c/b\u003e) in intensive poultry farming induces oxidative stress and mitochondrial dysfunction, leading to compromising meat quality. This study explored the potential role of chlorogenic acid (CGA) as a protective dietary supplement for improving breast muscle quality under HD conditions. In total, 510 broilers were reared under either normal density (14 birds/m\u0026sup2;, ND) or HD (22 birds/m\u0026sup2;), with 0.0, 0.5, 1.0 or 1.5 g/kg CGA added to the HD group. After 42 days, exposure to HD led to a substantial 12% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in body weight, a significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) rise in feed conversion ratio (\u003cb\u003eFCR\u003c/b\u003e), and deterioration in meat quality as evidenced by a marked decrease in pH, a rise in cooking and dripping losses, and unfavorable alterations in meat color (\u003cem\u003eL*\u003c/em\u003e, \u003cem\u003ea*\u003c/em\u003e, \u003cem\u003eb*\u003c/em\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Supplementation with 0.1% CGA significantly alleviated HD-induced growth suppression and enhanced antioxidant defenses by reducing the levels of malondialdehyde (\u003cb\u003eMDA\u003c/b\u003e) and reactive oxygen species (\u003cb\u003eROS\u003c/b\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while boosting glutathione peroxidase (\u003cb\u003eGSH-Px\u003c/b\u003e) and superoxide dismutase (\u003cb\u003eSOD\u003c/b\u003e) activities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).. Mechanistically, CGA alleviated mitochondrial dysfunction by downregulating heat shock proteins (\u003cb\u003eHSP60\u003c/b\u003e, \u003cb\u003eHSP70\u003c/b\u003e) and mitochondrial proteases (\u003cb\u003eCLPP\u003c/b\u003e, \u003cb\u003eCLPX\u003c/b\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), restored tricarboxylic acid (\u003cb\u003eTCA\u003c/b\u003e) cycle activity (via increased \u003cb\u003eSDH\u003c/b\u003e and \u003cb\u003eMDH\u003c/b\u003e), and shifted glycolytic flux toward aerobic metabolism. These findings highlight CGA as a promising feed additive for improving meat quality under HD stress through mitochondrial protection and metabolic reprogramming.\u003c/p\u003e","manuscriptTitle":"Chlorogenic acid mitigates high stocking density-induced breast muscle quality decline in broilers via modulating mitochondrial redox homeostasis and glycolytic metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 18:29:58","doi":"10.21203/rs.3.rs-6862999/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b594d644-cd64-40b1-a78a-5c93684e9bb2","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50662011,"name":"Biological sciences/Biochemistry"},{"id":50662012,"name":"Scientific community and society/Agriculture"}],"tags":[],"updatedAt":"2025-07-24T00:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-27 18:29:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6862999","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6862999","identity":"rs-6862999","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}