Influence of Dietary Quercetin and Stocking Density on Stress Indicators and Meat Quality of Broiler Chickens in Tropical Production Systems

Influence of Dietary Quercetin and Stocking Density on Stress Indicators and Meat Quality of Broiler Chickens in Tropical Production Systems | 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 Research Article Influence of Dietary Quercetin and Stocking Density on Stress Indicators and Meat Quality of Broiler Chickens in Tropical Production Systems Adelaja Ariyo Abimbola, Victor Olusegun Sinkalu, Mohammed Musa Suleiman, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8295668/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 This study investigated influence of dietary quercetin and stocking density on stress indicators and meat quality of broiler chickens in tropical production systems. Broilers were allotted to four treatments combining two stocking densities (12 and 18 birds/m²) with or without oral quercetin administration (50 mg/kg). Stocking density had a marked effect on stress indicators, with birds kept at the lower density showing reduced erythrocyte fragility compared with those reared at higher density. Meat pH declined more rapidly in the high-density, non-supplemented group, suggesting early post-mortem quality deterioration, while quercetin and lower stocking density helped maintain more desirable pH trends during storage. Although quercetin did not improve erythrocyte stability at low stocking density, supplementation moderated meat quality losses in broilers previously exposed to high-density stress. These findings indicate that managing stocking density, alongside targeted antioxidant supplementation, may contribute to improved welfare and extended shelf-life of broiler meat under tropical production conditions. quercetin pH erythrocyte osmotic fragility stress shelf-life stress INTRODUCTION Stocking density remains one of the most debated management factors influencing broiler welfare and performance (Kim et al. 2024 ). While some studies report minimal or inconsistent effects on physiological stress and welfare indicators (Dawkins et al. 2004 ; Dozier et al. 2005 ; Shana et al. 2016 ; Fernanda et al. 2020), others describe measurable negative impacts when birds are kept at higher densities (Nordquist et al. 2020). Stocking density is typically defined either as the number of birds per square metre or as total live weight per unit area (Sanchez-Casanova et al. 2020), and deviations above or below recommended ranges can influence behavioural expression, physiological stability, and overall productivity. Concerns about stocking density are particularly relevant in tropical climates, where high ambient temperatures and extended heat periods intensify production challenges (Lopez-Lopez et al. 2021). In the Northern Guinea Savannah zone, for instance, the hot–dry season is characterized by extreme thermal conditions that amplify stress in poultry flocks (Sinkalu et al. 2015 ; Madkour et al. 2022 ). To minimize heat-related losses, farmers in these regions often adopt lower stocking densities than those commonly practiced in temperate production systems (Rambau et al. 2016 ; Sánchez-Casanova et al. 2021 ). Identifying density thresholds that maintain bird welfare without reducing the farmer’s economic return therefore remains a critical area of research (Skrbic et al. 2009 ; Ligaraba et al. 2016 ; Monica et al. 2019 ; Geng et al. 2020 ; Bergeron et al., 2020 ). Natural antioxidants, especially plant-derived flavonoids, have attracted interest as potential agents for mitigating stress and enhancing poultry performance under challenging environmental conditions (El-Ghareeb et al. 2023 ). Quercetin—found in foods such as onions, apples, tomatoes, garlic, tea, and citrus fruits (Srivastava et al. 2016 )—is recognized for its strong free-radical scavenging properties (Pietta 2000 ; Watjen et al. 2005; Leopoldini et al. 2006 ; Aghababaei and Hadidi, 2023 ). Previous studies have reported that quercetin can improve physiological resilience, production efficiency, and meat quality in broilers (Liu et al. 2014 ; Fayyaz et al. 2016 ; Saeed et al. 2017 ; Simitzis et al. 2018 ; Ballard and Marostica 2019 ). However, information is limited regarding its effects on specific indicators of physiological stress, such as erythrocyte osmotic fragility, and on post-mortem meat quality traits in birds reared at different stocking densities. In view of the need for practical strategies that support broiler welfare and carcass quality under tropical production conditions, this study examined the effects of oral quercetin supplementation (50 mg/kg) on erythrocyte osmotic fragility and meat pH in broiler chickens raised at two stocking densities. MATERIALS AND METHODS Experimental Site The experimental site was a poultry pen in Ahmadu Bello University, Zaria (11 o 10 / N, 07 o 38 / E), located in the Northern Guinea Savannah zone of Nigeria. Experimental Birds and Management A total of 60 one-day-old, Ross 308 unsexed broiler chickens, purchased from a reliable commercial hatchery at Mayo Belwa (9° 22 / 6 // N and 12° 42 / 0 // E), Adamawa State, served as subjects. The broiler chickens were housed in open-sided floor pens. The wall was 1.0 m from the floor, with 1.0 m high wire-mesh, extended from the peak of the wall to the roof. Wood shaving was used as bedding material. The broiler chickens were separated into respective compartments, made of 1 x 1 x 1 m wire-mesh with uniform feeder and drinker space within each compartment. Water and feed were provided ad libitum to the broiler chickens throughout the experimental period. On arrival, the broiler chickens were brooded under 24-h lighting for 6 days. Thereafter, they were exposed to 14-h lighting daily till 42 days of age. They were fed starter diet from day 1 to 21 of age, and finisher diet from 22–42 days of age. Experimental Design The experiment was carried out on the basis of a completely randomized design and strict biosecurity measures were observed. A total of 60 one-day-old unsexed broiler chicks were used for the experiment. At 14-day old, they were divided into separate pens, comprising four density groups: Group I, 18 birds/m2 without quercetin treatment; Group II, 18 birds/m2 with quercetin treatment; Group III, 12 birds/m2 without quercetin treatment and Group IV, 12 birds/m2 with quercetin treatment. Quercetin (50 mg/kg) was administered daily per os . All groups were given access to feed and water ad libitum and brooded under continuous lighting until the chicks were 6 days old. Thereafter, all the groups were subjected to 14:00 hours of light and 10:00 hours dark cycle. Due to logistical constraints, each treatment was represented by a single pen, and therefore pen-level values were considered the experimental unit for statistical interpretation. The limited replication reflects the preliminary nature of the study. Determination of Erythrocyte Osmotic Fragility The erythrocyte osmotic fragility (EOF) was determined following the procedure described by Olayemi and Oyewale ( 2002 ) on days 28, 35 and 42. Briefly, five broiler chickens were selected from each group and 2.0 mL of blood was aspirated from wing vein of each bird into potassium ethylenediaminetetraacetate (K2EDTA) sample bottles to determine the EOF. Bleeding procedure was limited to one minute or less to minimize the influence of handling stress. Exactly 0.02 mL of blood was added to tubes, containing increasing concentrations (0.1, 0.3, 0.5, 0.7 and 0.9%) of phosphate-buffered sodium chloride (NaCl) solution at pH 7.4. The tubes were gently mixed and incubated at room temperature (24.0 o C) for 30 minutes. The content of each tube was then centrifuged at 3000 g for 10 minutes and the supernatant was decanted. The haemoglobin content of the supernatant was determined spectrophotometrically at a wavelength of 540 nm. Distilled water served as blank. The percentage haemolysis in each concentration of NaCl was evaluated taking the tube with maximum haemolysis (0%) as 100%. Determination of pH of Breast Meat Breast muscle ( M. pectoralis major and M. pectoralis minor ) was used to determine meat quality characteristics (Sekeroglu et al. 2011 ). Briefly, initial pH of breast meat was determined on day 1 and refrigerated at 4.0 o C. The meat was allowed to cool for 20 min prior to further determination of pH on days 2, 3 and day 4 post-slaughter; using a digital JENWAY 3505 pH metre equipped with a penetrating electrode. Data Analyses Data obtained were expressed as mean ± standard error of the mean (± SEM) and analysed using repeated-measures one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Pearson’s correlation analysis was used to evaluate the significance of the difference between the group means using statistical package for social sciences software (Version 20.0; New York, USA). Values of P < 0.05 were considered significant (Snedector and Cochran 1994 ). RESULTS Overall (Table 1 ), the erythrocyte osmotic fragility (EOF) was consistently and significantly (P < 0.05) lower in the untreated LSD group of 12 birds/m2. However, the erythrocytes were more fragile in the quercetin -treated LSD and both quercetin -treated and untreated HSD groups across all concentrations. The EOF value was lowest in the untreated LSD of 12 birds/m2 but the value of EOF was high in the quercetin -treated LSD group. Similarly, Irrespective of treatment, EOF was also higher in the HSD groups. The pH increased as the duration of meat storage increased. The earliest significant increase in pH was observed in the untreated HSD group (Table 2 ). Table 1 Between group variation in erythrocyte osmotic fragility in quercetin-treated broiler chickens reared at different stocking densities Density Groups NaCl Concentration (g/L) 0.5 0.3 0.1 18 Birds/m2 (n = 5) 7.34 ± 4.12 b 64.66 ± 3.91 b 89.35 ± 2.17 b 18 Birds/m2 + Quecertin (n = 5) 9.57 ± 4.53 b 67.28 ± 3.78 b 90.97 ± 1.82 b 12 Birds/m2 (n = 5) 0.24 ± 0.16 a 62.21 ± 4.22 a 85.5 ± 3.56 a 12 Birds/m2 + Quercetin (n = 5) 10.29 ± 6.09 b 67.41 ± 3.30 b 88.93 ± 3.47 b Means within columns with different superscript letters vary significantly (P < 0.05) Table 2 Effect of quercetin on pH of meat of 42 day-old-broiler chickens reared at different stocking densities Density Groups Day Day 1 Day 2 Day 3 Day 4 18 Birds/ m2 (n = 5) 5.47 ± 0.06 a 5.64 ± 0.10 a 5.96 ± 0.08 2,b 6.02 ± 0.04 1,b 18 Birds/m2 + Quercetin (n = 5) 5.51 ± 0.05 a 5.57 ± 0.05 a 5.73 ± 0.09 1,a 5.98 ± 0.03 1,b 12 Birds/ m2 (n = 5) 5.49 ± 0.10 a 5.61 ± 0.05 a 5.68 ± 0.07 1,a 85.5 ± 3.56 a 12 Birds/m2 + Quercetin (n = 5) 5.47 ± 0.08 a 5.54 ± 0.06 a 5.71 ± 0.09 1,a 6.09 ± 0.04 1,b Superscripts with different numbers vary significantly (P < 0.05) within columns. Superscripts with different letters vary significantly (p < 0.05) within rows. DISCUSSION This aligns with the observations of Watjen et al. (2005), who reported that the effects of flavonoids are strongly influenced by tissue type, the presence or absence of stressors, and dose level. The erythrocyte osmotic fragility (EOF) patterns observed in this study indicate that the quercetin dose administered may not have been sufficient to offset the physiological strain associated with high stocking density (HSD). Although quercetin is widely recognized for its antioxidant properties, it has not undergone the pharmacological or toxicological standardization seen with approved therapeutic agents. Consequently, it is commonly used as a dietary supplement (Devi et al., 2024 ). Evidence from human studies similarly demonstrates that quercetin can increase erythrocyte fragility under specific conditions (Yousif and Shtaywy 1998 ), possibly through its interactions with membrane proteins and lipids (Pawlikowska-Pawlega et al. 2003). Surprisingly, Bilto et al. ( 2012 ) showed quercetin can protect erythrocytes against increased osmotic fragility induced by hydrogen peroxide. However, Donaldson and Erlwanger ( 2019 ) found that quercetin did not have any significant effect on osmotic fragility in rat models. The present findings reinforce the likelihood of a dose-dependent response in broilers, with the biological impact of quercetin varying according to the degree of environmental stress and stocking density. These results underscore the need for detailed dose-response and risk-assessment studies, particularly in tropical regions where birds frequently experience heightened environmental pressures. Establishing an effective and reliable quercetin dose may remain a challenge because environmental stressors differ substantially across production zones. Nevertheless, such work is essential for developing practical antioxidant supplementation strategies capable of enhancing bird welfare and resilience under commercial tropical conditions. Changes in meat pH during chilled storage further illustrate the influence of stocking density and quercetin supplementation on carcass quality. Although all groups showed a steady pH increase during storage, values remained below the Food and Agriculture Organization (FAO) limit of 6.2, indicating that the rise reflected normal storage progression rather than pre-slaughter disease or excessive stress. Elevated pH values surpassing this threshold are typically associated with stressed or injured animals and with microbial-driven early spoilage (Fanatico et al. 2007 ; Wapi et al. 2013 ). The untreated HSD group exhibited a notable pH increase by day three (5.96 ± 0.08), suggesting that high stocking density accelerated post-mortem biochemical changes. In contrast, quercetin successfully delayed this shift in birds kept under HSD, maintaining lower pH values for a longer duration and thereby prolonging meat quality. This observation agrees with the findings of Goliomytis et al. ( 2014 ), who reported that quercetin reduces lipid peroxidation and extends meat shelf-life. Similar finding was also reported by Deng et al. ( 2024 ). Overall, these results indicate that quercetin may be valuable for limiting oxidative deterioration in broilers exposed to stressful stocking conditions. The broader biological relevance of these findings is supported by Boots et al. ( 2008 ), who described quercetin’s capacity to modulate oxidative damage and influence pathways involved in tissue integrity and cellular protection. These mechanisms suggest that quercetin could play a role in slowing tissue degradation and mitigating factors that predispose carcasses to quality loss. In production settings, such protective effects may help reduce spoilage-related defects and potentially lower the risk of meat-borne illnesses associated with oxidative deterioration. Taken together, the results of this study highlight the need for further investigation into the in-vivo protective roles of quercetin, particularly with respect to identifying optimal dosing strategies for birds raised under different environmental and management conditions. As stocking density continues to be a major determinant of stress in tropical poultry production (Gul et al. 2024), integrating an effective antioxidant strategy may provide a valuable tool for maintaining meat quality, supporting bird welfare, and improving production outcomes. Conclusion This study demonstrates that quercetin influences erythrocyte membrane stability and post-mortem meat quality in broiler chickens, with effects that differ according to stocking density. Under low stocking density, quercetin was associated with increased erythrocyte osmotic fragility, suggesting a direct interaction with cell membranes. In contrast, under high stocking density, quercetin supplementation contributed to a slower rate of lipid peroxidation during storage, thereby enhancing meat quality. These results indicate that the physiological effects of quercetin are shaped by the birds’ rearing environment and stress load. Further research is needed to clarify dose–response relationships across a wider range of stocking densities and under both stressful and low-stress conditions. Defining these parameters will be crucial for determining the practical application of quercetin as a nutritional strategy aimed at supporting welfare and improving carcass quality in broiler production systems in tropical climates. Statements and Declaration Funding The authors declare that no funds, grants or other support were received during preparation of this manuscript. Competing interest The authors declare that they have no conflict of interest. Author Contributions All authors contributed to the study concept and design. Material preparation, data collection and analysis were performed by Adelaja Ariyo Abimbola. First draft was written by Adelaja Ariyo Abimbola. All authors commented on previous versions of the manuscript. All authors read and approved final manuscript. Data Availability The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethical Approval All animal procedures were approved by the Ethical Committee on Animal Use and Care of the Ahmadu Bello University, Zaria, Nigeria (P13/VTPP/8008). The authors confirm that the experiment was conducted in accordance with the principles of the Declaration of Helsinki. ACKNOWLEDGEMENTS The authors’ sincere appreciations go to the staff and technical personnel of the Departments of Veterinary Physiology, Veterinary Pharmacology and toxicology and Veterinary Pathology, Ahmadu Bello University, Zaria, Nigeria. Also, appreciations go to Prof. J.J. Omage of the Department of Animal Science for his professional input and generous provision of farm space for this research. References Aghababaei F, Hadidi M (2023) Recent Advances in Potential Health Benefits of Quercetin. Pharmaceuticals, 16, 1020. https://doi.org/10.3390/ph16071020 Ballard CR, Marostica MR (2019) Health Benefits of Flavonoids. Bioactive Compounds 185 – 201. https://doi.org/10.1016/B978-0-12-814774-0.00010-4 Bergeron S, Pouliot E, Doyon M (2020). Commercial Poultry Production Stocking Density Influence on Bird Health and Performance Indicators. Animals 10. https://doi:10.3390/ani10081253 Bilto YY, Suboh S, Aburjai T, Abdalla SS (2012) Structure-activity relationships regarding the antioxidant effects of the flavonoids on human erythrocytes. Natural Science4: 740-747. Boots AW, Haenen RM, Bast A (2008) Health effects of quercetin: From antioxidant to neutraceutical. Eur J Pharmacol 585 : 325-337 Dawkins MS, Donelly S, Jones TA, (2004) Chicken welfare is influenced more by housing conditions than by stocking density. Nature 427 : 342–344 Deng C, Zou H, Wu Y, Lou A, Liu Y, Luo J, Quan W, Shen Q (2024) Dietary supplementation with quercetin: an ideal approach for improving meat quality and oxidative stability of broiler chickens. Poultry Science, 103. Devi, V., Deswal, G., Dass, R., Chopra, B., Kriplani, P., Grewal, A. S., Guarve, K., & Dhingra, A. K. (2024). Therapeutic Potential and Clinical Effectiveness of Quercetin: A Dietary Supplement. Recent advances in food, nutrition & agriculture, 15: 13–32. https://doi.org/10.2174/012772574X269376231107095831 Donaldson J, Erlwanger KH (2019) Quercetin does not alter erythrocyte osmotic fragility in female, sprague dawley rats fed a high-fructose, high-cholesterol diet. J. Biol. Sci. 19: 347-353. Dozier WA, Thaxton III JP, Branton SL, Morgan GW, Miles DM, Roush WB, Lott BD, Vizzier-Thaxton Y (2005) Stocking density effects on growth performance and processing yields of heavy broilers. Poult. Sci. J. 84:1332–1338 El-Ghareeb WR, Kishawy ATY, Anter RG, Aboelabbas GA, Abdelaziz WS, Alhawas B, Meligy AMA, Abdel-Raheem SM, Ismail HI (2023). Novel Antioxidant Insights of Myricetin on the Performance of Broiler Chickens and Alleviating Experimental Infection with Eimeria spp.: Crosstalk between Oxidative Stress and Inflammation. Antioxidants 12 1026 https://doi.org/10.3390/ antiox12051026 Fanatico AC, Pillai PB, Emmert JL, Owens CM (2007) Meat quality of slow- and fast-growing chicken genotypes fed low-nutrient or standard diets and raised indoors or with outdoor access. Poult. Sci. J. 86:2245-2255 Fayyaz S, Saddique S, Khalid H, Naureen S, Salman M, Salman M, Islam M, Zahid F, Naheed S, Khan MT, Amjad S, Qamar S, Ahmad S (2016) Is quercetin accumulating in eggs using flavonoids-enriched poultry feed? J Anim Plant Sci 26:1479 – 1485 Geng AL, Liu HG, Zhang Y, Zhang J, Wang HH, Chu Q, Yan ZX (2020) Effects of indoor stocking density on performance, egg quality, and welfare status of a native chicken during 22 to 38 weeks. Poult. Sci. J. 99:163 – 171 Goliomytis, M, Tsoureki D, Simitzis PE, Charismiadou MA, Hager-Theodorides AL, Deligeorgis SG (2014) The effects of quercetin dietary supplementation on broiler growth performance, meat quality, and oxidative stability. Poult. Sci. J. 93:1957–1962 Gül ET, Yıldız A, Olgun O (2022) The importance of nutrition in alleviating high stocking density stress in poultry - A Review. Annals of Animal Science 22 : 855 – 863.· Kim C.H, Kim KH, Chun J, Lim S, Jeon JH (2024) Animal welfare indicators and stress response of broiler chickens raised at low and high stocking density. Journal of Animal Science and Technology, https://doi.org/10.5187/jast.2024.e64 Leopoldini M, Russo N, Chiodo S, Toscano M (2006) Iron chelation by the powerful antioxidant flavonoid quercetin . J Agr Food Chem 54:6343–6351 Ligaraba TJ, Benyi K, Baloyi JJ. (2016) Effects of genotype and stocking density on broiler performance under three feeding regimes. Trop Anim Health Prod 46:1227 -1234 https://Doi:1007/s11250-016- 1060-z Liu HN, Liu Y, Hu LL, Suo YL, Zhang L, Jin F, Feng XA, Teng N, Li Y (2014) Effects of dietary supplementation of quercetin on performance, egg quality, cecal microflora populations, and antioxidant status in laying hens. Poult. Sci. J. 93:347–353 López-López P, Sarmiento-Franco LA, Santos-Ricalde RH (2021) Effect of stocking density on performance, infection by Eimeria spp., intestinal lesions and foot pad injuries in broilers with outdoor access under tropical conditions. British Poultry Science63: 108 - 114. Madkour M, Salman FM, El-Wardany I, Abdel-Fattah SA, Alagawany M, Hashem NM, Abdelnour SA, El-Kholy MS, Dhama K (2022) Mitigating the detrimental effects of heat stress in poultry through thermal conditioning and nutritional manipulation. Journal of thermal biology 103 103169 . Monica HB, Fernanda MT, Ida JP, Anja BR (2019) Effects of environmental complexity on behaviour in fast-growing broiler chickens. Appl. Anim. Behav. Sci. 219. https://doi.org/10.1016/j.applanim.2019.104840 Nordquist RE, Staay FJ, Eerdenburg CM, Velkers FC, Fijn L, Arndt SS, (2017) Mutilating procedures, management practices, and housing conditions that may affect the welfare of farm animals: implications for welfare research. Animals 7:1–22 Olayemi FO, Oyewale JO (2002) Comparative assessment of erythrocyte osmotic fragility and of haematological and plasma biochemical values in the Nigerian white Fulani and N’dama breeds of cattle. Trop Anim Health Prod 34:181–187 Pawlikowska-Pawlęga B, Gruszecki WI, Misiak LE, Gawron A (2003) The study of the quercetin action on human erythrocyte membranes. Biochem Pharmacol. 66:605–612 Pietta P (2000) Flavonoids as antioxidants: Reviews. J. Nat. Prod. 63:1035–1042 Rambau MD, Mudau ML, Makhanya SD, Benyi K. (2016) Effects of stocking density and feed withdrawal periods on the performance of broiler chickens in a semi-arid environment. Trop Anim Health Prod 48:1547–1554. Saeed M, Naveed M, Arain MA, Arif M, Abd El-Hack ME, Alagawany M, Siyal FA, Soomro RN, Sun C (2017) Quercetin : Nutritional and beneficial effects in poultry. World Poultry Sci J73:355–364 Sánchez-Casanova R, Sarmiento-Franco L, Phillips CJ, Zulkifli I (2020) Do free-range systems have potential to improve broiler welfare in the tropics? World Poultry Sci J https://doi.org/ 10.1080/00439339.2020.1707389 Sánchez-Casanova RE, Sarmiento-Franco LA, Phillips CJ (2021) The effects of outdoor access and stocking density on the performance of broilers reared under tropical conditions. British Poultry Science, 62, 632 - 637. Sekeroglu A, Sarica M, Gulay MS, Duman M (2011) Effect of stocking density on chick performance, internal organ weights and blood parameters in broilers. J Anim Vet Adv,10:246–250 Shana B, Helen L, Christine W, Katharina WA, Andreas B, Josef B, Michael HE, Elke R (2016) Field trial on animal-based measures for animal welfare in slow growing broilers reared under an alternative concept suitable for the German market. Berl Munch Tierarztl Wochenschr 126:149– 155. Simitzis P, Spanou D, Glastra N, Goliomytis M (2018) Impact of dietary quercetin on laying hen 308 performance, egg quality and yolk oxidative stability. Anim Feed Sci Tech 239:27–32 Sinkalu VO, Ayo JO, Abimbola AA, Ibrahim JE (2015) Effects of melatonin on cloacal temperature and erythrocyte osmotic fragility in layer hens during the hot-dry season. J Appl Anim Res 43:52–60 Skrbic Z, Pavlovski Z, Lukic M (2009) Stocking density–factor of production performance, quality and 312 broiler welfare. Biotechnol. Anim. Husb . 25:359–372 Snedector GW, Cochran WG (1994) Statistical Methods. Oxford and IBH Publishing, Calcutta, pp 509 Srivastava S, Somasagara RR, Hedge M, Nishana M, Tadi SK, srivastava M, Choudhary B, Raghavan SC. (2016) Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis. Scientific Reports pp 1 – 13 https://DOI: 10.1038/srep24049 Wapi C, Nkukwana TT, Hoffman LC, Dzama K, Pieterse E, Mabusela T, Muchenje V (2013) Physico-chemical shelf life indicators of meat from broilers given Moringa oleifera leaf meal. South African Journal of Animal Science, 43:543 – 547 Wätjen W, Michels G, Steffan B, Niering P, Chovolou Y, Kampkötter A, Tran-Thi Q, Proksch P, Kahl R (2005) Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J. Nutr. 135:525–531 Yousif B, Shtaywy AS (1998) Effects of selected flavonoids on deformability, osmotic fragility and aggregation of human erythrocytes. Clinical Haemorheology and Microcirculation 18:165–173 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8295668","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":568358046,"identity":"43ac1b73-aa07-474d-b59a-ccfa7e9655ee","order_by":0,"name":"Adelaja Ariyo Abimbola","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBACNgbGBhCdwMbAw8DwgMGGgUGCgBZ+FC0JDGmEtUg2QOgEBoiWw4S1GNw+3Pzh4w67PD6xswcfJNScT+yf3XzwAUONTTROLecS2yRnnkkuZpPOSzZIOHY7ccadY8kGDMfSchtwaTnD2MbM28ac2CadYyaRwHY7seEGkMHYcBinFvszjM2fedvqoVr+nUucT0gL0JYGad62wxAtiW0HEjcQoQXol7bjEL8k9iUbb7yRBvIUPr+wP/7wsa06T3527sEHH77Zyc67kQxk1Njg1IIBHMEqE4hVDgL2pCgeBaNgFIyCkQEA7w9f7CGZ9UYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5896-326X","institution":"Ahmadu Bello University Faculty of Veterinary Medicine","correspondingAuthor":true,"prefix":"","firstName":"Adelaja","middleName":"Ariyo","lastName":"Abimbola","suffix":""},{"id":568358047,"identity":"1917f205-d9e1-4a1c-bd03-4266f5a5ac5a","order_by":1,"name":"Victor Olusegun Sinkalu","email":"","orcid":"","institution":"Ahmadu Bello University Faculty of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"Olusegun","lastName":"Sinkalu","suffix":""},{"id":568358048,"identity":"940b35f2-3f66-42e5-81ea-e36ef773b396","order_by":2,"name":"Mohammed Musa Suleiman","email":"","orcid":"","institution":"Ahmadu Bello University Faculty of Physical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"Musa","lastName":"Suleiman","suffix":""},{"id":568358049,"identity":"1733b290-4dfa-48bd-aca8-fc27cc676b75","order_by":3,"name":"Joseph Olusegun Ayo","email":"","orcid":"","institution":"Ahmadu Bello University Faculty of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"Olusegun","lastName":"Ayo","suffix":""}],"badges":[],"createdAt":"2025-12-06 15:37:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8295668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8295668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99790775,"identity":"453426a0-7fa1-4ddb-afdd-9571ae6589bd","added_by":"auto","created_at":"2026-01-08 12:58:41","extension":"xml","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9125,"visible":true,"origin":"","legend":"","description":"","filename":"tropTROPD2502385.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/358fdc4242f6880e8cc98afa.xml"},{"id":99497789,"identity":"9772b11b-8635-46e6-a2b1-ed652f3ababb","added_by":"auto","created_at":"2026-01-05 06:58:35","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":944,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD250238553215.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/a3db0cbb7344b95510b3a4bd.xml"},{"id":99497792,"identity":"f17d4037-c41c-4fbb-91a3-f6c6ed1544ef","added_by":"auto","created_at":"2026-01-05 06:58:35","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":949,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD2502385Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/7548327795d5532531765f35.xml"},{"id":99497793,"identity":"d515a617-70af-44ff-a2ce-684015e416cf","added_by":"auto","created_at":"2026-01-05 06:58:35","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87951,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD25023850enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/ca7e97efc9a4f4e377c84943.xml"},{"id":99497791,"identity":"7efbca3b-2dab-439a-908b-dff31059426c","added_by":"auto","created_at":"2026-01-05 06:58:35","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86657,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD25023850structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/fc730c53cef91db52d49ee5f.xml"},{"id":99497794,"identity":"14c41af3-87b8-414b-8cc3-e184a169b37e","added_by":"auto","created_at":"2026-01-05 06:58:35","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92580,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/946779c61cf3bfc3a61896a5.html"},{"id":100368213,"identity":"7c6232d2-d66c-4e77-afec-d30e183cfb0b","added_by":"auto","created_at":"2026-01-16 07:57:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":504840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8295668/v1/b0f78733-20a5-4126-8af3-d73a94a034b6.pdf"}],"financialInterests":"","formattedTitle":"Influence of Dietary Quercetin and Stocking Density on Stress Indicators and Meat Quality of Broiler Chickens in Tropical Production Systems","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eStocking density remains one of the most debated management factors influencing broiler welfare and performance (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While some studies report minimal or inconsistent effects on physiological stress and welfare indicators (Dawkins et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dozier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Shana et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fernanda et al. 2020), others describe measurable negative impacts when birds are kept at higher densities (Nordquist et al. 2020). Stocking density is typically defined either as the number of birds per square metre or as total live weight per unit area (Sanchez-Casanova et al. 2020), and deviations above or below recommended ranges can influence behavioural expression, physiological stability, and overall productivity.\u003c/p\u003e \u003cp\u003eConcerns about stocking density are particularly relevant in tropical climates, where high ambient temperatures and extended heat periods intensify production challenges (Lopez-Lopez et al. 2021). In the Northern Guinea Savannah zone, for instance, the hot\u0026ndash;dry season is characterized by extreme thermal conditions that amplify stress in poultry flocks (Sinkalu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Madkour et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To minimize heat-related losses, farmers in these regions often adopt lower stocking densities than those commonly practiced in temperate production systems (Rambau et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S\u0026aacute;nchez-Casanova et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Identifying density thresholds that maintain bird welfare without reducing the farmer\u0026rsquo;s economic return therefore remains a critical area of research (Skrbic et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ligaraba et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Monica et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Geng et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bergeron et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNatural antioxidants, especially plant-derived flavonoids, have attracted interest as potential agents for mitigating stress and enhancing poultry performance under challenging environmental conditions (El-Ghareeb et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Quercetin\u0026mdash;found in foods such as onions, apples, tomatoes, garlic, tea, and citrus fruits (Srivastava et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u0026mdash;is recognized for its strong free-radical scavenging properties (Pietta \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Watjen et al. 2005; Leopoldini et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Aghababaei and Hadidi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies have reported that quercetin can improve physiological resilience, production efficiency, and meat quality in broilers (Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fayyaz et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Saeed et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Simitzis et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ballard and Marostica \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, information is limited regarding its effects on specific indicators of physiological stress, such as erythrocyte osmotic fragility, and on post-mortem meat quality traits in birds reared at different stocking densities.\u003c/p\u003e \u003cp\u003eIn view of the need for practical strategies that support broiler welfare and carcass quality under tropical production conditions, this study examined the effects of oral quercetin supplementation (50 mg/kg) on erythrocyte osmotic fragility and meat pH in broiler chickens raised at two stocking densities.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental Site\u003c/h2\u003e\n \u003cp\u003eThe experimental site was a poultry pen in Ahmadu Bello University, Zaria (11\u003csup\u003eo\u003c/sup\u003e 10\u003csup\u003e/\u003c/sup\u003e N, 07\u003csup\u003eo\u003c/sup\u003e 38\u003csup\u003e/\u003c/sup\u003e E), located in the Northern Guinea Savannah zone of Nigeria.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental Birds and Management\u003c/h3\u003e\n\u003cp\u003eA total of 60 one-day-old, Ross 308 unsexed broiler chickens, purchased from a reliable commercial hatchery at Mayo Belwa (9\u0026deg; 22\u003csup\u003e/\u003c/sup\u003e 6\u003csup\u003e//\u003c/sup\u003e N and 12\u0026deg; 42\u003csup\u003e/\u003c/sup\u003e 0\u003csup\u003e//\u003c/sup\u003e E), Adamawa State, served as subjects. The broiler chickens were housed in open-sided floor pens. The wall was 1.0 m from the floor, with 1.0 m high wire-mesh, extended from the peak of the wall to the roof. Wood shaving was used as bedding material. The broiler chickens were separated into respective compartments, made of 1 x 1 x 1 m wire-mesh with uniform feeder and drinker space within each compartment. Water and feed were provided \u003cem\u003ead libitum\u003c/em\u003e to the broiler chickens throughout the experimental period. On arrival, the broiler chickens were brooded under 24-h lighting for 6 days. Thereafter, they were exposed to 14-h lighting daily till 42 days of age. They were fed starter diet from day 1 to 21 of age, and finisher diet from 22\u0026ndash;42 days of age.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eThe experiment was carried out on the basis of a completely randomized design and strict biosecurity measures were observed. A total of 60 one-day-old unsexed broiler chicks were used for the experiment. At 14-day old, they were divided into separate pens, comprising four density groups: Group I, 18 birds/m2 without \u003cem\u003equercetin\u003c/em\u003e treatment; Group II, 18 birds/m2 with \u003cem\u003equercetin\u003c/em\u003e treatment; Group III, 12 birds/m2 without \u003cem\u003equercetin\u003c/em\u003e treatment and Group IV, 12 birds/m2 with \u003cem\u003equercetin\u003c/em\u003e treatment. \u003cem\u003eQuercetin\u003c/em\u003e (50 mg/kg) was administered daily \u003cem\u003eper os\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAll groups were given access to feed and water \u003cem\u003ead libitum\u003c/em\u003e and brooded under continuous lighting until the chicks were 6 days old. Thereafter, all the groups were subjected to 14:00 hours of light and 10:00 hours dark cycle.\u003c/p\u003e\n\u003cp\u003eDue to logistical constraints, each treatment was represented by a single pen, and therefore pen-level values were considered the experimental unit for statistical interpretation. The limited replication reflects the preliminary nature of the study.\u003c/p\u003e\n\u003ch3\u003eDetermination of Erythrocyte Osmotic Fragility\u003c/h3\u003e\n\u003cp\u003eThe erythrocyte osmotic fragility (EOF) was determined following the procedure described by Olayemi and Oyewale (\u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e) on days 28, 35 and 42. Briefly, five broiler chickens were selected from each group and 2.0 mL of blood was aspirated from wing vein of each bird into potassium ethylenediaminetetraacetate (K2EDTA) sample bottles to determine the EOF. Bleeding procedure was limited to one minute or less to minimize the influence of handling stress. Exactly 0.02 mL of blood was added to tubes, containing increasing concentrations (0.1, 0.3, 0.5, 0.7 and 0.9%) of phosphate-buffered sodium chloride (NaCl) solution at pH 7.4. The tubes were gently mixed and incubated at room temperature (24.0 \u003csup\u003eo\u003c/sup\u003eC) for 30 minutes. The content of each tube was then centrifuged at 3000 \u003cem\u003eg\u003c/em\u003e for 10 minutes and the supernatant was decanted. The haemoglobin content of the supernatant was determined spectrophotometrically at a wavelength of 540 nm. Distilled water served as blank. The percentage haemolysis in each concentration of NaCl was evaluated taking the tube with maximum haemolysis (0%) as 100%.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003ch3\u003eDetermination of pH of Breast Meat\u003c/h3\u003e\n\u003cp\u003eBreast muscle (\u003cem\u003eM. pectoralis major\u003c/em\u003e and \u003cem\u003eM. pectoralis minor\u003c/em\u003e) was used to determine meat quality characteristics (Sekeroglu et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Briefly, initial pH of breast meat was determined on day 1 and refrigerated at 4.0 \u003csup\u003eo\u003c/sup\u003eC. The meat was allowed to cool for 20 min prior to further determination of pH on days 2, 3 and day 4 post-slaughter; using a digital JENWAY 3505 pH metre equipped with a penetrating electrode.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eData Analyses\u003c/h2\u003e\n \u003cp\u003eData obtained were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (\u0026plusmn;\u0026thinsp;SEM) and analysed using repeated-measures one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s \u003cem\u003epost-hoc\u003c/em\u003e test. Pearson\u0026rsquo;s correlation analysis was used to evaluate the significance of the difference between the group means using statistical package for social sciences software (Version 20.0; New York, USA). Values of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant (Snedector and Cochran \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOverall (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the erythrocyte osmotic fragility (EOF) was consistently and significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower in the untreated LSD group of 12 birds/m2. However, the erythrocytes were more fragile in the \u003cem\u003equercetin\u003c/em\u003e-treated LSD and both \u003cem\u003equercetin\u003c/em\u003e-treated and untreated HSD groups across all concentrations. The EOF value was lowest in the untreated LSD of 12 birds/m2 but the value of EOF was high in the \u003cem\u003equercetin\u003c/em\u003e-treated LSD group. Similarly, Irrespective of treatment, EOF was also higher in the HSD groups. The pH increased as the duration of meat storage increased. The earliest significant increase in pH was observed in the untreated HSD group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBetween group variation in erythrocyte osmotic fragility in quercetin-treated broiler chickens reared at different stocking densities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDensity Groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNaCl Concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 Birds/m2 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.34\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 Birds/m2\u0026thinsp;+\u0026thinsp;Quecertin (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 Birds/m2 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 Birds/m2\u0026thinsp;+\u0026thinsp;Quercetin (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eMeans within columns with different superscript letters vary significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of quercetin on pH of meat of 42 day-old-broiler chickens reared at different stocking densities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDensity Groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 Birds/ m2 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003e2,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e1,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 Birds/m2\u0026thinsp;+\u0026thinsp;Quercetin (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e1,a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003e1,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 Birds/ m2 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003e1,a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 Birds/m2\u0026thinsp;+\u0026thinsp;Quercetin (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e1,a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e1,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eSuperscripts with different numbers vary significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) within columns. Superscripts with different letters vary significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) within rows.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis aligns with the observations of Watjen et al. (2005), who reported that the effects of flavonoids are strongly influenced by tissue type, the presence or absence of stressors, and dose level. The erythrocyte osmotic fragility (EOF) patterns observed in this study indicate that the quercetin dose administered may not have been sufficient to offset the physiological strain associated with high stocking density (HSD). Although quercetin is widely recognized for its antioxidant properties, it has not undergone the pharmacological or toxicological standardization seen with approved therapeutic agents. Consequently, it is commonly used as a dietary supplement (Devi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEvidence from human studies similarly demonstrates that quercetin can increase erythrocyte fragility under specific conditions (Yousif and Shtaywy \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), possibly through its interactions with membrane proteins and lipids (Pawlikowska-Pawlega et al. 2003). Surprisingly, Bilto et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) showed quercetin can protect erythrocytes against increased osmotic fragility induced by hydrogen peroxide. However, Donaldson and Erlwanger (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that quercetin did not have any significant effect on osmotic fragility in rat models.\u003c/p\u003e \u003cp\u003eThe present findings reinforce the likelihood of a dose-dependent response in broilers, with the biological impact of quercetin varying according to the degree of environmental stress and stocking density. These results underscore the need for detailed dose-response and risk-assessment studies, particularly in tropical regions where birds frequently experience heightened environmental pressures. Establishing an effective and reliable quercetin dose may remain a challenge because environmental stressors differ substantially across production zones. Nevertheless, such work is essential for developing practical antioxidant supplementation strategies capable of enhancing bird welfare and resilience under commercial tropical conditions.\u003c/p\u003e \u003cp\u003eChanges in meat pH during chilled storage further illustrate the influence of stocking density and quercetin supplementation on carcass quality. Although all groups showed a steady pH increase during storage, values remained below the Food and Agriculture Organization (FAO) limit of 6.2, indicating that the rise reflected normal storage progression rather than pre-slaughter disease or excessive stress. Elevated pH values surpassing this threshold are typically associated with stressed or injured animals and with microbial-driven early spoilage (Fanatico et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wapi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe untreated HSD group exhibited a notable pH increase by day three (5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08), suggesting that high stocking density accelerated post-mortem biochemical changes. In contrast, quercetin successfully delayed this shift in birds kept under HSD, maintaining lower pH values for a longer duration and thereby prolonging meat quality. This observation agrees with the findings of Goliomytis et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), who reported that quercetin reduces lipid peroxidation and extends meat shelf-life. Similar finding was also reported by Deng et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Overall, these results indicate that quercetin may be valuable for limiting oxidative deterioration in broilers exposed to stressful stocking conditions.\u003c/p\u003e \u003cp\u003eThe broader biological relevance of these findings is supported by Boots et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), who described quercetin\u0026rsquo;s capacity to modulate oxidative damage and influence pathways involved in tissue integrity and cellular protection. These mechanisms suggest that quercetin could play a role in slowing tissue degradation and mitigating factors that predispose carcasses to quality loss. In production settings, such protective effects may help reduce spoilage-related defects and potentially lower the risk of meat-borne illnesses associated with oxidative deterioration.\u003c/p\u003e \u003cp\u003eTaken together, the results of this study highlight the need for further investigation into the in-vivo protective roles of quercetin, particularly with respect to identifying optimal dosing strategies for birds raised under different environmental and management conditions. As stocking density continues to be a major determinant of stress in tropical poultry production (Gul et al. 2024), integrating an effective antioxidant strategy may provide a valuable tool for maintaining meat quality, supporting bird welfare, and improving production outcomes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that quercetin influences erythrocyte membrane stability and post-mortem meat quality in broiler chickens, with effects that differ according to stocking density. Under low stocking density, quercetin was associated with increased erythrocyte osmotic fragility, suggesting a direct interaction with cell membranes. In contrast, under high stocking density, quercetin supplementation contributed to a slower rate of lipid peroxidation during storage, thereby enhancing meat quality.\u003c/p\u003e \u003cp\u003eThese results indicate that the physiological effects of quercetin are shaped by the birds\u0026rsquo; rearing environment and stress load. Further research is needed to clarify dose\u0026ndash;response relationships across a wider range of stocking densities and under both stressful and low-stress conditions. Defining these parameters will be crucial for determining the practical application of quercetin as a nutritional strategy aimed at supporting welfare and improving carcass quality in broiler production systems in tropical climates.\u003c/p\u003e"},{"header":"Statements and Declaration ","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants or other support were received during preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study concept and design. Material preparation, data collection and analysis were performed by Adelaja Ariyo Abimbola. First draft was written by Adelaja Ariyo Abimbola. All authors commented on previous versions of the manuscript. All authors read and approved final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Ethical Committee on Animal Use and Care of the Ahmadu Bello University, Zaria, Nigeria (P13/VTPP/8008). The authors confirm that the experiment was conducted in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors’ sincere appreciations go to the staff and technical personnel of the Departments of Veterinary Physiology, Veterinary Pharmacology and toxicology and Veterinary Pathology, Ahmadu Bello University, Zaria, Nigeria. Also, appreciations go to Prof. J.J. Omage of the Department of Animal Science for his professional input and generous provision of farm space for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAghababaei F, Hadidi M (2023) Recent Advances in Potential Health Benefits of Quercetin. Pharmaceuticals, 16, 1020. https://doi.org/10.3390/ph16071020\u003c/li\u003e\n\u003cli\u003eBallard CR, Marostica MR (2019) Health Benefits of Flavonoids. Bioactive Compounds 185 \u0026ndash; 201. https://doi.org/10.1016/B978-0-12-814774-0.00010-4\u003c/li\u003e\n\u003cli\u003eBergeron S, Pouliot E, Doyon M (2020). Commercial Poultry Production Stocking Density Influence on Bird Health and Performance Indicators. Animals 10. https://doi:10.3390/ani10081253 \u003c/li\u003e\n\u003cli\u003eBilto YY, Suboh S, Aburjai T, Abdalla SS (2012) Structure-activity relationships regarding the antioxidant effects of the flavonoids on human erythrocytes. Natural Science4: 740-747.\u003c/li\u003e\n\u003cli\u003eBoots AW, Haenen RM, Bast A (2008) Health effects of quercetin: From antioxidant to neutraceutical. Eur J Pharmacol 585 : 325-337\u003c/li\u003e\n\u003cli\u003eDawkins MS, Donelly S, Jones TA, (2004) Chicken welfare is influenced more by housing conditions than by stocking density. Nature 427 : 342\u0026ndash;344\u003c/li\u003e\n\u003cli\u003eDeng C, Zou H, Wu Y, Lou A, Liu Y, Luo J, Quan W, Shen Q (2024) Dietary supplementation with quercetin: an ideal approach for improving meat quality and oxidative stability of broiler chickens. Poultry Science, 103.\u003c/li\u003e\n\u003cli\u003eDevi, V., Deswal, G., Dass, R., Chopra, B., Kriplani, P., Grewal, A. S., Guarve, K., \u0026amp; Dhingra, A. K. (2024). Therapeutic Potential and Clinical Effectiveness of Quercetin: A Dietary Supplement. Recent advances in food, nutrition \u0026amp; agriculture, 15: 13\u0026ndash;32. https://doi.org/10.2174/012772574X269376231107095831\u003c/li\u003e\n\u003cli\u003eDonaldson J, Erlwanger KH (2019) Quercetin does not alter erythrocyte osmotic fragility in female, sprague dawley rats fed a high-fructose, high-cholesterol diet. J. Biol. Sci. 19: 347-353.\u003c/li\u003e\n\u003cli\u003eDozier WA, Thaxton III JP, Branton SL, Morgan GW, Miles DM, Roush WB, Lott BD, Vizzier-Thaxton Y (2005) Stocking density effects on growth performance and processing yields of heavy broilers. Poult. Sci. J. 84:1332\u0026ndash;1338\u003c/li\u003e\n\u003cli\u003eEl-Ghareeb WR, Kishawy ATY, Anter RG, Aboelabbas GA, Abdelaziz WS, Alhawas B, Meligy AMA, Abdel-Raheem SM, Ismail HI (2023). Novel Antioxidant Insights of Myricetin on the Performance of Broiler Chickens and Alleviating Experimental Infection with Eimeria spp.: Crosstalk between Oxidative Stress and Inflammation. Antioxidants 12 1026 https://doi.org/10.3390/ antiox12051026\u003c/li\u003e\n\u003cli\u003eFanatico AC, Pillai PB, Emmert JL, Owens CM (2007) Meat quality of slow- and fast-growing chicken genotypes fed low-nutrient or standard diets and raised indoors or with outdoor access. Poult. Sci. J. 86:2245-2255 \u003c/li\u003e\n\u003cli\u003eFayyaz S, Saddique S, Khalid H, Naureen S, Salman M, Salman M, Islam M, Zahid F, Naheed S, Khan MT, Amjad S, Qamar S, Ahmad S (2016) Is \u003cem\u003equercetin \u003c/em\u003eaccumulating in eggs using flavonoids-enriched poultry feed? J Anim Plant Sci 26:1479 \u0026ndash; 1485\u003c/li\u003e\n\u003cli\u003eGeng AL, Liu HG, Zhang Y, Zhang J, Wang HH, Chu Q, Yan ZX (2020) Effects of indoor stocking density on performance, egg quality, and welfare status of a native chicken during 22 to 38 weeks. Poult. Sci. J. 99:163 \u0026ndash; 171\u003c/li\u003e\n\u003cli\u003eGoliomytis, M, Tsoureki D, Simitzis PE, Charismiadou MA, Hager-Theodorides AL, Deligeorgis SG (2014) The effects of \u003cem\u003equercetin \u003c/em\u003edietary supplementation on broiler growth performance, meat quality, and oxidative stability. Poult. Sci. J. 93:1957\u0026ndash;1962\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;l ET, Yıldız A, Olgun O (2022) The importance of nutrition in alleviating high stocking density stress in poultry - A Review. Annals of Animal Science 22 : 855 \u0026ndash; 863.\u0026middot;\u003c/li\u003e\n\u003cli\u003eKim C.H, Kim KH, Chun J, Lim S, Jeon JH (2024) Animal welfare indicators and stress response of broiler chickens raised at low and high stocking density. Journal of Animal Science and Technology, https://doi.org/10.5187/jast.2024.e64\u003c/li\u003e\n\u003cli\u003eLeopoldini M, Russo N, Chiodo S, Toscano M (2006) Iron chelation by the powerful antioxidant flavonoid\u003cem\u003e quercetin\u003c/em\u003e. J Agr Food Chem 54:6343\u0026ndash;6351 \u003c/li\u003e\n\u003cli\u003eLigaraba TJ, Benyi K, Baloyi JJ. (2016) Effects of genotype and stocking density on broiler performance under three feeding regimes. Trop Anim Health Prod 46:1227 -1234 https://Doi:1007/s11250-016- 1060-z\u003c/li\u003e\n\u003cli\u003eLiu HN, Liu Y, Hu LL, Suo YL, Zhang L, Jin F, Feng XA, Teng N, Li Y (2014) Effects of dietary supplementation of \u003cem\u003equercetin \u003c/em\u003eon performance, egg quality, cecal microflora populations, and antioxidant status in laying hens. Poult. Sci. J. 93:347\u0026ndash;353\u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-L\u0026oacute;pez P, Sarmiento-Franco LA, Santos-Ricalde RH (2021) Effect of stocking density on performance, infection by Eimeria spp., intestinal lesions and foot pad injuries in broilers with outdoor access under tropical conditions. British Poultry Science63: 108 - 114.\u003c/li\u003e\n\u003cli\u003eMadkour M, Salman FM, El-Wardany I, Abdel-Fattah SA, Alagawany M, Hashem NM, Abdelnour SA, El-Kholy MS, Dhama K (2022) Mitigating the detrimental effects of heat stress in poultry through thermal conditioning and nutritional manipulation. Journal of thermal biology 103 103169 .\u003c/li\u003e\n\u003cli\u003eMonica HB, Fernanda MT, Ida JP, Anja BR (2019) Effects of environmental complexity on behaviour in fast-growing broiler chickens. Appl. Anim. Behav. Sci. 219. https://doi.org/10.1016/j.applanim.2019.104840\u003c/li\u003e\n\u003cli\u003eNordquist RE, Staay FJ, Eerdenburg CM, Velkers FC, Fijn L, Arndt SS, (2017) Mutilating procedures, management practices, and housing conditions that may affect the welfare of farm animals: implications for welfare research. Animals 7:1\u0026ndash;22\u003c/li\u003e\n\u003cli\u003eOlayemi FO, Oyewale JO (2002) Comparative assessment of erythrocyte osmotic fragility and of haematological and plasma biochemical values in the Nigerian white Fulani and N\u0026rsquo;dama breeds of cattle. Trop Anim Health Prod 34:181\u0026ndash;187\u003c/li\u003e\n\u003cli\u003ePawlikowska-Pawlęga B, Gruszecki WI, Misiak LE, Gawron A (2003) The study of the \u003cem\u003equercetin \u003c/em\u003eaction on human erythrocyte membranes. Biochem Pharmacol. 66:605\u0026ndash;612\u003c/li\u003e\n\u003cli\u003ePietta P (2000) Flavonoids as antioxidants: Reviews. J. Nat. Prod. 63:1035\u0026ndash;1042\u003c/li\u003e\n\u003cli\u003eRambau MD, Mudau ML, Makhanya SD, Benyi K. (2016) Effects of stocking density and feed withdrawal periods on the performance of broiler chickens in a semi-arid environment. Trop Anim Health Prod 48:1547\u0026ndash;1554.\u003c/li\u003e\n\u003cli\u003eSaeed M, Naveed M, Arain MA, Arif M, Abd El-Hack ME, Alagawany M, Siyal FA, Soomro RN, Sun C (2017) \u003cem\u003eQuercetin\u003c/em\u003e: Nutritional and beneficial effects in poultry. World Poultry Sci J73:355\u0026ndash;364\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Casanova R, Sarmiento-Franco L, Phillips CJ, Zulkifli I (2020) Do free-range systems have potential to improve broiler welfare in the tropics? World Poultry Sci J https://doi.org/ 10.1080/00439339.2020.1707389\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Casanova RE, Sarmiento-Franco LA, Phillips CJ (2021) The effects of outdoor access and stocking density on the performance of broilers reared under tropical conditions. British Poultry Science, 62, 632 - 637.\u003c/li\u003e\n\u003cli\u003eSekeroglu A, Sarica M, Gulay MS, Duman M (2011) Effect of stocking density on chick performance, internal organ weights and blood parameters in broilers. J Anim Vet Adv,10:246\u0026ndash;250\u003c/li\u003e\n\u003cli\u003eShana B, Helen L, Christine W, Katharina WA, Andreas B, Josef B, Michael HE, Elke R (2016) Field trial on animal-based measures for animal welfare in slow growing broilers reared under an alternative concept suitable for the German market. Berl Munch Tierarztl Wochenschr 126:149\u0026ndash; 155.\u003c/li\u003e\n\u003cli\u003eSimitzis P, Spanou D, Glastra N, Goliomytis M (2018) Impact of dietary \u003cem\u003equercetin \u003c/em\u003eon laying hen 308 performance, egg quality and yolk oxidative stability. Anim Feed Sci Tech 239:27\u0026ndash;32\u003c/li\u003e\n\u003cli\u003eSinkalu VO, Ayo JO, Abimbola AA, Ibrahim JE (2015) Effects of melatonin on cloacal temperature and erythrocyte osmotic fragility in layer hens during the hot-dry season. J Appl Anim Res 43:52\u0026ndash;60 \u003c/li\u003e\n\u003cli\u003eSkrbic Z, Pavlovski Z, Lukic M (2009) Stocking density\u0026ndash;factor of production performance, quality and 312 broiler welfare. Biotechnol. Anim. Husb\u003cem\u003e. \u003c/em\u003e25:359\u0026ndash;372\u003c/li\u003e\n\u003cli\u003eSnedector GW, Cochran WG (1994) Statistical Methods. Oxford and IBH Publishing, Calcutta, pp 509\u003c/li\u003e\n\u003cli\u003eSrivastava S, Somasagara RR, Hedge M, Nishana M, Tadi SK, srivastava M, Choudhary B, Raghavan SC. (2016) Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis. Scientific Reports pp 1 \u0026ndash; 13 https://DOI: 10.1038/srep24049\u003c/li\u003e\n\u003cli\u003eWapi C, Nkukwana TT, Hoffman LC, Dzama K, Pieterse E, Mabusela T, Muchenje V (2013) Physico-chemical shelf life indicators of meat from broilers given \u003cem\u003eMoringa oleifera \u003c/em\u003eleaf meal. South African Journal of Animal Science, 43:543 \u0026ndash; 547\u003c/li\u003e\n\u003cli\u003eWätjen W, Michels G, Steffan B, Niering P, Chovolou Y, Kampkötter A, Tran-Thi Q, Proksch P, Kahl R (2005) Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J. Nutr. 135:525\u0026ndash;531\u003c/li\u003e\n\u003cli\u003eYousif B, Shtaywy AS (1998) Effects of selected flavonoids on deformability, osmotic fragility and aggregation of human erythrocytes. \u003cem\u003eClinical Haemorheology and Microcirculation \u003c/em\u003e18:165\u0026ndash;173\u003c/li\u003e\n\u003c/ol\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":"quercetin, pH, erythrocyte osmotic fragility, stress, shelf-life, stress","lastPublishedDoi":"10.21203/rs.3.rs-8295668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8295668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated influence of dietary quercetin and stocking density on stress indicators and meat quality of broiler chickens in tropical production systems. Broilers were allotted to four treatments combining two stocking densities (12 and 18 birds/m\u0026sup2;) with or without oral quercetin administration (50 mg/kg). Stocking density had a marked effect on stress indicators, with birds kept at the lower density showing reduced erythrocyte fragility compared with those reared at higher density. Meat pH declined more rapidly in the high-density, non-supplemented group, suggesting early post-mortem quality deterioration, while quercetin and lower stocking density helped maintain more desirable pH trends during storage. Although quercetin did not improve erythrocyte stability at low stocking density, supplementation moderated meat quality losses in broilers previously exposed to high-density stress. These findings indicate that managing stocking density, alongside targeted antioxidant supplementation, may contribute to improved welfare and extended shelf-life of broiler meat under tropical production conditions.\u003c/p\u003e","manuscriptTitle":"Influence of Dietary Quercetin and Stocking Density on Stress Indicators and Meat Quality of Broiler Chickens in Tropical Production Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 06:58:30","doi":"10.21203/rs.3.rs-8295668/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":"a075cdd8-c1df-492a-86aa-edabb3003b21","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T15:32:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 06:58:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8295668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8295668","identity":"rs-8295668","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}