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Full Research Article

Evaluating Oxidative Stress, Nitric Oxide Production and Mitochondrial Activity Trend in Broiler Blood Cells under Progressive Cold Stress

S
Siriluck Juntautsa1,2
K
Kiattisak Pimpjong1
W
Worapol Aengwanich1,3
N
Nitima Tatiya-aphiradee4
W
Watcharapon Promsut1,*
1Faculty of Veterinary Sciences, Mahasarakham University, Maha Sarakham, 44000, Thailand.
2Bioveterinary Research Unit, Mahasarakham University, Maha Sarakham, 44000, Thailand.
3Stress and Oxidative Stress in Animal Research Unit, Mahasarakham University, Maha Sarakham, 44000, Thailand.
4Program in Veterinary Technology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani, 41000, Thailand.

ABSTRACT

Background: Broiler production is crucial for global food security as a high-quality protein source. However, climate change causes extreme temperature fluctuations, including severe cold stress. Sudden ambient temperature drops impair broiler growth and health. This study investigates how continuously decreasing ambient temperature affects oxidative stress and mitochondrial activity in broiler blood cells.

Methods: Blood cells pooled from two broiler chickens were diluted and subjected to a gradual temperature reduction from 42oC to 0oC, with 3oC decrements at each step. Mitochondrial activity, malondialdehyde (MDA), total antioxidant capacity (TAC), hydrogen peroxide (H2O2), reduced glutathione (GSH), catalase activity and nitric oxide (NO) levels were subsequently measured.

Result: Mitochondrial activity and NO levels showed significant quadratic trends (P<0.05), increasing at moderate cold before declining at lower temperatures. From 42oC to 21oC, mitochondrial activity rose significantly at 39-33oC and 21oC (P<0.05), while MDA levels increased at 39-27oC and 21oC (P<0.05). TAC declined significantly from 42oC to colder points (P<0.05). Between 18oC and 0oC, mitochondrial activity and NO fluctuated, suggesting an adaptive response. Conversely, H2O2, GSH and catalase levels remained stable across all temperatures (P>0.05). These findings highlight a complex cellular response to cold stress, emphasizing the need for proper cold stress management in broiler production.

INTRODUCTION

Broiler production is a key contributor to global food supply. In 2022, global output reached 102.9 million tons and is expected to rise to 152.8 million tons by 2031 (Oke et al., 2024). Broilers provide high-quality protein at low cost due to rapid growth and superior feed efficiency, supporting rising demand, especially in developing regions (Nguyen et al., 2025). However, climate change, notably abrupt cold exposure, poses major challenges. Cold stress impairs growth, weakens immunity and elevates mortality in broilers (Aarif et al., 2014; Liu et al., 2022).
       
Cold stress induces oxidative stress, a condition in which the generation of reactive oxygen species (ROS) exceeds the antioxidant defense capacity (Afzal et al., 2023). ROS can damage cellular proteins, lipids and DNA, posing a threat to cell viability (Jomova et al., 2023). Understanding the oxidative stress response is therefore critical for mitigating cold-induced damage in broilers (Abo-Al-Ela et al., 2021; Sahib et al., 2024). Several biomarkers are used to assess oxidative stress: malondialdehyde (MDA), which indicates lipid peroxidation and cellular damage (Cordiano et al., 2023; Srinontong et al., 2024); total antioxidant capacity (TAC), which reflects the overall antioxidant status (Surai et al., 2019); hydrogen peroxide (H2O2) accumulation, which signals redox imbalance (Ponnampalam et al., 2022); reduced glutathione (GSH), which maintains redox homeostasis (Liu et al., 2021 and catalase, which detoxifies H2O2 (Baker et al., 2023). Nitric oxide (NO) regulates intracellular signaling and responds to oxidative cues (Xu et al., 2022), while mitochondria, as the central site of cellular energy metabolism, modulate ROS generation under stress conditions (Casanova et al., 2023).
       
Previous studies reported that dietary supplementation with glutamine, L-carnitine and betaine enhances TAC and GSH levels while reducing MDA concentration (Liu et al., 2021). In addition, Baker et al. (2023) emphasized that catalase and GSH are major antioxidants depleted during cold exposure. Alterations in NO during stress have been linked to cellular injury (Semenikhina et al., 2022) and Casanova et al. (2023) highlighted mitochondria as a key modulator of oxidative responses.
       
Although the effects of cold on oxidative stress, NO variation and mitochondrial activity are recognized, data on dynamic shifts in oxidative markers, NO and mitochondrial function under exposure to 0oC remain limited. Clarifying broiler cellular adaptation to extreme cold is essential. Therefore, the objective of this study was to investigate the responses of MDA, TAC, H2O2, GSH, catalase, NO and mitochondrial activity in broiler blood cells as ambient temperature declined from 42oC to 0oC. This study will reveal the impact of cold stress on broilers by assessing cellular responses and adaptations, offering insights to mitigate potential damage caused by climate change, particularly under extreme cold conditions.

MATERIALS AND METHODS

This experiment was conducted in 2025 and was approved by the Institutional Animal Experimentation Ethics Committee, Mahasarakham University (Approval No. IACUC-MSU-009-055/2025).
 
Animals
 
Ten 28-day-old broilers were obtained from a commercial farm in Maha Sarakham Province and housed at the Faculty of Veterinary Sciences, Mahasarakham University. After a 7-day acclimation under a 16L:8D photoperiod, birds were provided ad libitum access to a standard grower diet and clean water. No additional vaccines were given beyond the routine schedule. All broilers remained clinically healthy, showing no signs of disease or abnormality. At 35 days of age, birds were considered physiologically stable for experimentation.
 
Experimental design
 
This study comprised two parts. The first examined changes in key biochemical markers in broiler blood cells as ambient temperature gradually declined from 42oC (normal broiler body temperature) to 0oC. The markers analyzed included mitochondrial activity, H2O2, MDA, TAC, NO, GSH and catalase activity. The second part investigated how lower temperatures affected the same markers and was divided into two sub-studies: sub-study 2.1 compared 42oC with 39-21oC and sub-study 2.2 compared 42oC with 18-0oC.
 
Experimental procedure
 
The sample size was based on the method of Ilyas et al. (2017). Blood was collected from two broilers (randomly selected from ten broiler chickens; 2 mL each) in heparinized tubes. For washing, blood was mixed with phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 2,500 rpm (769 × g) for 5 minutes. The supernatant was discarded and the process repeated twice. The washed blood was then diluted 1:200 (v/v) with PBS and 10 mL of the diluted sample was aliquoted into test tubes. Tubes were arranged by temperature from 42oC to 0oC, decreasing 3oC per step, with four tubes per temperature. All tubes were placed in a temperature-controlled water bath monitored by a digital thermometer. Prior to cooling, blood cells were held at 42oC for 30 minutes. Then, the temperature was reduced stepwise to 0oC. At each step, cells were held for 20 minutes before collecting four tubes (replicates) for testing.
 
Determination of biochemical indicators
 
Total antioxidant capacity
 
TAC was assessed by the FRAP assay (Srinontong et al., 2023). The working solution combined 10 mL of 300 mmol sodium acetate buffer (pH 3.6), 1 mL of 10 mmol TPTZ and 1 mL of 20 mmol FeCl3·6H2O. Then, 20 µL of sample was mixed with 180 µL of the solution and incubated for 5 min at room temperature. Absorbance was read at 595 nm. Ferrous sulfate heptahydrate was used as standard.
 
Malondialdehyde
 
MDA was measured using the TBARS assay. A 0.1 mL sample was mixed with 0.45 mL of 0.09% NaCl, 0.2 mL of 0.67% TBA and 1 mL of 10% TCA in 0.6 M HCl. The mixture was heated at 100oC for 30 min, cooled, then 2 mL of deionized water was added, vortexed and centrifuged at 3,000 rpm (1,008 × g) for 10 min. Absorbance was read at 532 nm (Sürmen-Gür et al., 2003).
 
Nitric oxide
 
NO levels were determined using Griess reagent, composed of 1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 2.5% phosphoric acid. Equal volumes of reagent and supernatant were mixed and incubated for 15 minutes. Absorbance was read at 540 nm. Sodium nitrite served as the standard (Giustarini et al., 2008).
 
Hydrogen peroxide
 
H2O2 was measured per Orprayoon et al. (2020), with slight modifications. Sample was mixed with 2.25 mmol/L FeSO4, incubated 5 min, then 4 mmol/L norfloxacin was added and incubated 3 min. Absorbance was read at 440 nm. H2O2 (0-40 µmol/L) was used for the standard curve.
 
Mitochondrial activity
 
Mitochondrial activity in blood cells was measured using the MTT assay. MTT (5 mg/mL in acetone) was filtered. One milliliter of diluted blood was centrifuged, the supernatant removed and the pellet incubated with MTT at 41.5oC for 75 minutes. Dimethyl sulfoxide (150 µL) was added and absorbance read at 540 nm (Bahuguna et al., 2017).
 
Reduced glutathione and catalase
 
GSH and catalase levels were measured using colorimetric assay kits (Abbkine; KTB1600 for GSH, KTB1040 for catalase), following the manufacturer’s instructions. Absorbance was read using a microplate reader.
 
Statistical analysis
 
Normality was checked before the analysis using PROC GLM. The differences were tested for significance using Duncan’s multiple range test and results were considered significant if the P-value was less than 0.05 (SAS® Studio).

RESULTS AND DISCUSSION

Trends in the changes of biochemical parameters in broiler blood cells when ambient temperature is reduced from 42oC to 0oC
 
As shown in Table 1, mitochondrial activity and NO levels showed significant quadratic trends (P<0.05), indicating complex response patterns. Mitochondrial activity rose at 39-33oC, dropped at 30oC, increased again at 18-15oC, declined at 12-6oC, then sharply increased at 3-0oC (P<0.05). The rise at 39-33oC may help broiler blood cells sustain energy under mild cold stress (Gong et al., 2023). The drop at 30-27oC may reflect impaired electron transport and reduced energy, leading to ROS overproduction and oxidative stress (Casanova et al., 2023). Activity rose again as temperature neared 0oC, suggesting adaptive responses. NO levels fell at 39-36oC, rose at 33-21oC, dropped at 18oC and peaked at 6oC (P<0.05), consistent with its signaling role (Semenikhina et al., 2022). These changes may reflect impaired NO synthesis or oxidative stress response (Pappas et al., 2023). In contrast, H2O2, MDA, TAC, GSH and catalase levels showed no significant changes (P>0.05; Table 1). Overall, broiler blood cells retained function and showed dynamic biochemical shifts even at 0oC.

Table 1: Changing of mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells when the ambient temperature continuously decreased from 42oC until to 0oC (3oC each time).


 
The effects of decreasing environmental temperatures from 42oC to 21oC on biochemical parameters in broiler blood cells
       
Mitochondrial activity at 42oC was significantly lower than at 39-33oC and 21oC (P<0.05). Activity at 39-33oC was significantly higher than at 30oC and 27oC, which in turn were lower than at 21oC (P<0.05). No significant differences were found between 42oC and 30-24oC, or between 39-33oC and 21oC (P>0.05; Table 2). Elevated activity at 39-33oC suggests an adaptive response to moderate cold, aligning with reports of mitochondrial energy upregulation under mild stress (Park et al., 2021). The drop at 30-27oC may indicate overwhelmed adaptive capacity, reducing efficiency (Flensted-Jensen et al., 2024). This supports findings that severe cold impairs mitochondrial function via oxidative damage and electron transport disruption (Timkova et al., 2016). The rebound at 21oC may reflect a compensatory mechanism for energy restoration (Mohan et al., 2023).

Table 2: Effect of decreasing ambient temperature on mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells when the ambient temperature continuously decreased from 42oC until to 21oC (3oC each time).


       
MDA levels at 42oC were significantly lower than those at 39-27oC and 21oC (P<0.05). Likewise, levels at 24oC were significantly lower than at 39oC and 21oC (P<0.05; Table 2). However, no significant differences were found between 42oC and 24oC or among 39-27oC and 21oC (P>0.05). These findings support earlier studies reporting increased lipid damage and oxidative stress under cold conditions (Aksit et al., 2008). The elevated MDA at 39-27oC suggests rising oxidative stress as temperatures fall, likely due to increased ROS production beyond antioxidant control (Cordiano et al., 2023). Interestingly, MDA was lower at 24oC than at 39oC and 21oC, possibly reflecting an antioxidant response at moderate cold. However, this response seems limited at colder temperatures, as indicated by the higher MDA at 21oC.
       
TAC was significantly higher at 42oC than at 39-27oC and 21oC and higher at 24oC than at 21oC (P<0.05). No significant difference was found between 42oC and 24oC, or among 39-24oC (P>0.05). These findings suggest that TAC decreased as temperature dropped, likely due to antioxidant depletion from elevated oxidative stress (Liu et al., 2021). This aligns with previous reports showing oxidative stress lowers TAC by consuming antioxidants like GSH and catalase (Saracila et al., 2023).
       
NO levels at 39oC and 36oC were significantly lower than at 42oC and between 33oC and 21oC (P<0.05). The decrease at 39-36oC may reflect reduced production during mild cold stress. These findings contrast with Zhang et al. (2011), who observed NO elevation under cold stress. However, in this study, NO increased as the temperature dropped further, suggesting an adaptive response. The elevated NO at 42oC and 33-21oC supports its role in maintaining physiological stability under both normal and cold conditions.
       
However, H2O2, GSH and catalase activity remained stable between 42oC and 21oC (P>0.05; Table 2). This suggests that GSH and catalase defense may sufficiently control H2O2 during moderate cold stress (Ponnampalam et al., 2022). The consistent levels of these markers indicate that broiler blood cells can maintain antioxidant capacity, helping to limit cold-induced damage.
 
Comparative analysis of biochemical parameter changes in broiler blood cells at 42oC and reduced temperatures from 18oC to 0oC
 
Mitochondrial activity at 42oC was significantly lower than at 18-15oC and 3-0oC (P<0.05), but not different from 12-6oC (P>0.05). Activity at 18-15oC and 3-0oC also did not differ (P>0.05). The pattern differed from the steady decline between 42-21oC and may reflect reduced mitochondrial respiration under cold. The increased activity at 18-15oC and 3-0oC may indicate a compensatory response to generate energy during cold stress (Casanova et al., 2023), though prolonged activation may impair mitochondria via excess ROS (Lennicke and Cochemé, 2021).
       
MDA levels at 42oC were significantly lower than those at 18-0oC (P<0.05), indicating greater oxidative damage under cold. The rise in MDA reflects excess ROS production during cold stress, causing lipid peroxidation as antioxidant reserves decline (Wei et al., 2024).
       
NO levels at 6-3oC were significantly higher than at 18-12oC (P<0.05), while levels at 15oC were significantly lower than at 42oC and 9-0oC (P<0.05). No differences were found between 42oC and 12-0oC or between 18oC and 12oC (P>0.05). The drop at 18-15oC may reflect protection against NO-induced oxidative damage (Pappas et al., 2023). The lower NO at 15oC versus 42oC and 9-0oC suggests temperature-dependent synthesis. In contrast to Su et al. (2020), who reported increased NO under cold, our results reveal a cyclic fluctuation, suggesting adaptive responses to falling temperatures.
       
Catalase activity at 42oC was significantly higher than at 18-15oC and 9-0oC (P<0.05) and activity at 12oC was higher than at 3-0oC (P<0.05). No differences were found between 42oC and 12oC or between 18-15oC and 9-0oC (P>0.05). The high activity at 42oC highlights catalase’s role in H2O2 detoxification (Baker et al., 2023), while reduced activity at lower temperatures may reflect weakened antioxidant defense. The peak at 12oC, followed by decline at 3-0oC, may indicate a short-lived adaptive response before enzyme inactivation or antioxidant depletion.
               
In contrast, the levels of H2O2, TAC and GSH remained constant from 18-0oC (Table 3), indicating that the GSH antioxidant system and TAC can counteract cold stress and reduce the production of H2O2.

Table 3: The effects of environmental temperatures of 42oC and a decrease in ambient temperature from 18oC to 0oC (3oC reduction each time) on the mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells.

CONCLUSION

This study examined how broiler blood cells respond to cold stress by analyzing key biochemical markers as ambient temperatures dropped from 42oC to 0oC. Cold exposure caused notable changes in mitochondrial activity, MDA, NO, H2O2, TAC, GSH and catalase activity. Quadratic trend analysis revealed complex cellular responses. Mitochondrial activity and NO increased under moderate cold but declined in severe cold, reflecting adaptive mechanisms. In contrast, H2O2, GSH and catalase levels stayed stable, indicating strong antioxidant defense. These results enhance our understanding of cold stress effects and support strategies to reduce its impact in poultry.

ACKNOWLEDGEMENT

This research project was financially supported by Mahasarakham University. 

Disclaimers
 
The views and conclusions expressed in this article are solely those of the author and do not necessarily represent the views of their affiliated institutions. The author is responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

REFERENCES


  1. Aarif, O., Shergojry, S.A., Dar, S.A., Khan, N., Mir, N.A. and Sheikh, A.A. (2014). Impact of cold stress on blood biochemical and immune status in male and female Vanaraja chickens. Indian Journal of Animal Research. 48(2): 139-142. doi: 10.5958/j.0976-0555.48.2.030.

  2. Abo-Al-Ela, H.G., El-Kassas, S., El-Naggar, K., Abdo, S.E., Jahejo, A.R. and Al Wakeel, R.A. (2021).  Stress and immunity in poultry: Light management and nanotechnology as effective immune enhancers to fight stress. Cell Stress and Chaperones. 26(3): 457-472. 

  3. Afzal, S., Abdul Manap, A. S., Attiq, A., Albokhadaim, I., Kandeel, M. and Alhojaily, S.M. (2023). From imbalance to impairment: The central role of reactive oxygen species in oxidative stress-induced disorders and therapeutic exploration. Frontiers in Pharmacology. 14: 1269581.

  4. Aksit, M., Altan, O., Buyukozturk, A., Balkaya, M. and Ozdemir, D.E.M.İ.R. (2008). Effects of cold temperature and vitamin E supplementation on oxidative stress, Troponin-T level and other ascites related traits in broilers. Archiv für Geflügelkunde/European Poultry Science. 72(5): 221.

  5. Bahuguna, A., Khan, I., Bajpai, V.K. and Kang, S.C. (2017). MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh Journal of Pharmacology. 12(2): 115-118.

  6. Baker, A., Lin, C. C., Lett, C., Karpinska, B., Wright, M.H. and Foyer, C.H. (2023). Catalase: A critical node in the regulation of cell fate. Free Radical Biology and Medicine. 199: 56-66.

  7. Casanova, A., Wevers, A., Navarro-Ledesma, S. and Pruimboom, L. (2023). Mitochondria: It is all about energy. Frontiers in physiology. 14: 1114231.

  8. Cordiano, R., Di Gioacchino, M., Mangifesta, R., Panzera, C., Gangemi, S. and Minciullo, P.L. (2023). Malondialdehyde as a potential oxidative stress marker for allergy-oriented diseases: An update. Molecules. 28(16): 5979.

  9. Flensted-Jensen, M., Kleis-Olsen, A.S., Hassø, R.K., Lindtofte, S., Corral Pérez, J., Ortega-Gómez, S. and Larsen, S. (2024). Combined changes in temperature and pH mimicking exercise result in decreased efficiency in muscle mitochondria. Journal of Applied Physiology. 136(1): 79-88.

  10. Giustarini, D., Rossi, R., Milzani, A. and Dalle Donne, I. (2008). Nitrite and nitrate measurement by Griess reagent in human plasma: Evaluation of interferences and standardization. Methods in enzymology. 440: 361-380.

  11. Gong, R., Xing, L., Yin, J., Ding, Y., Liu, X., Bao, J. and Li, J. (2023). Appropriate cold stimulation changes energy distribution to improve stress resistance in broilers. Journal of Animal Science. 101: skad185.

  12. Ilyas, M., Adzim, M., Simbak, N. and Atif, A. (2017). Sample size calculation for animal studies using degree of freedom (E); an easy and statistically defined approach for metabolomics and genetic research. ILAR J. 43: 207-213.

  13. Jomova, K., Raptova, R., Alomar, S. Y., Alwasel, S. H., Nepovimova, E., Kuca, K. and Valko, M. (2023). Reactive oxygen species, toxicity, oxidative stress and antioxidants: Chronic diseases and aging. Archives of toxicology. 97(10): 2499-2574.

  14. Lennicke, C. and Cochemé, H.M. (2021). Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Molecular cell. 81(18): 3691-3707.

  15. Liu, X., Li, S., Zhao, N., Xing, L., Gong, R., Li, T.  and Bao, J. (2022). Effects of acute cold stress after intermittent cold stimulation on immune-related molecules, intestinal barrier genes and heat shock proteins in broiler ileum. Animals. 12(23): 3260. 

  16. Liu, Y., Yang, Y., Yao, R., Hu, Y., Liu, P., Lian, S. and Li, S. (2021). Dietary supplementary glutamine and L-carnitine enhanced the anti-cold stress of Arbor Acres broilers. Archives Animal Breeding. 64(1): 231-243.

  17. Mohan, M.S., Aswani, S.S., Aparna, N.S., Boban, P.T., Sudhakaran, P.R. and Saja, K. (2023). Effect of acute cold exposure on cardiac mitochondrial function: role of sirtuins. Molecular and Cellular Biochemistry. 478(10): 2257-2270.

  18. Nguyen, H.D., Moss, A.F., Yan, F., Romero-Sanchez, H. and Dao, T.H. (2025). Effects of feeding methionine hydroxyl analogue chelated zinc, copper and manganese on growth performance, nutrient digestibility, mineral excretion and welfare conditions of broiler chickens: Part 2: Sustainability and welfare aspects. Animals. 15(3): 419. 

  19. Oke, O.E., Akosile, O.A., Uyanga, V.A., Oke, F.O., Oni, A.I., Tona, K. and Onagbesan, O.M. (2024). Climate change and broiler production. Veterinary Medicine and Science. 10(3): e1416. 

  20. Orprayoon, T., Ratanawimanwong, N. and Donpudsa, S. (2020). A simple method for hydrogen peroxide determination using Fe (II) with norfloxacin. In The Proceedings of National Conference of Rungsit University, Bangkok. pp: 137-147.

  21. Pappas, G., Wilkinson, M.L. and Gow, A.J. (2023). Nitric oxide regulation of cellular metabolism: Adaptive tuning of cellular energy. Nitric Oxide. 131: 8-17.

  22. Park, J.H., Lo, E.H. and Hayakawa, K. (2021). Endoplasmic reticulum interaction supports energy production and redox homeostasis in mitochondria released from astrocytes. Translational stroke research. 12(6): 1045-1054.

  23. Ponnampalam, E.N., Kiani, A., Santhiravel, S., Holman, B.W., Lauridsen, C. and Dunshea, F.R. (2022). The importance of dietary antioxidants on oxidative stress, meat and milk production and their preservative aspects in farm animals: Antioxidant action, animal health and product quality-Invited review. Animals. 12(23): 3279.

  24. Sahib, Q.S., Aafaq, I., Ahmed, H.A., Sheikh, G.G. and Ganai, I.A. (2024). Mitigating cold stress in livestock by nutritional interventions: A comprehensive review. Indian Journal of Animal Research. 58(3): 353-363. doi: 10.18805/ IJAR.B-5298.

  25. Saracila, M., Panaite, T.D., Predescu, N.C., Untea, A.E. and Vlaicu, P.A. (2023). Effect of dietary salicin standardized extract from salix alba bark on oxidative stress biomarkers and intestinal microflora of broiler chickens exposed to heat stress. Agriculture. 13(3): 698.

  26. Semenikhina, M., Stefanenko, M., Spires, D.R., Ilatovskaya, D.V.  and Palygin, O. (2022). Nitric-oxide-mediated signaling in podocyte pathophysiology. Biomolecules. 12(6): 745.

  27. Srinontong, P., Wandee, J. and Aengwanich, W. (2023). The effect of gallic acid on malondialdehyde, hydrogen peroxide and nitric oxide that influence viability of broiler blood cells at high ambient temperatures. British Poultry Science. 64(4): 512-517.

  28. Srinontong, P., Wandee, J. and Aengwanich, W. (2024). The effect of vitamin C on oxidative stress, nitric oxide formation and viability of broiler finisher blood cells subjected to high ambient temperature. Indian Journal of Animal Research. 58(7): 1117-1121. doi: 10.18805/IJAR.BF-1576. 

  29. Su, Y., Li, S., Xin, H., Li, J., Li, X., Zhang, R. and Bao, J. (2020). Proper cold stimulation starting at an earlier age can enhance immunity and improve adaptability to cold stress in broilers. Poultry science. 99(1): 129-141.

  30. Surai, P.F., Kochish, I.I., Fisinin, V.I. and Kidd, M.T. (2019). Antioxidant defence systems and oxidative stress in poultry biology: An update. Antioxidants. 8(7): 235.

  31. Sürmen-Gür, E., Erdinc, A., Serdar, Z. and Gür, H. (2003). Influence of acute exercise on oxidative stress in chronic smokers. Journal of Sports Science and Medicine. 2(3): 98-105. 

  32. Timkova, V., Tatarkova, Z., Lehotsky, J., Racay, P., Dobrota, D.  and Kaplan, P. (2016). Effects of mild hyperhomocysteinemia on electron transport chain complexes, oxidative stress and protein expression in rat cardiac mitochondria. Molecular and Cellular Biochemistry. 411: 261-270.

  33. Wei, H., Li, H., Miao, D., Wang, H., Liu, Y., Xing, L. and Li, J. (2024). Dietary resveratrol supplementation alleviates cold exposure-induced pyroptosis and inflammation in broiler heart by modulating oxidative stress and endoplasmic reticulum stress. Poultry Science. 103(11): 104203.

  34. Xu, H.L., Li, H., Bao, R.K., Tang, Y.X., Elsherbeni, A.I.A., Gharib, H.B.A. and Li, J.L. (2022). Transport stress induced cardiac NO-NOS disorder is mitigated by activating Nrf2/ HO-1/NQO1 antioxidant defense response in newly hatched chicks. Frontiers in Veterinary Science. 9: 938826.

  35. Zhang, Z.W., Lv, Z.H., Li, J.L., Li, S., Xu, S.W. and Wang, X.L. (2011). Effects of cold stress on nitric oxide in duodenum of chicks. Poultry Science. 90(7): 1555-1561.
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    Full Research Article

    Evaluating Oxidative Stress, Nitric Oxide Production and Mitochondrial Activity Trend in Broiler Blood Cells under Progressive Cold Stress

    S
    Siriluck Juntautsa1,2
    K
    Kiattisak Pimpjong1
    W
    Worapol Aengwanich1,3
    N
    Nitima Tatiya-aphiradee4
    W
    Watcharapon Promsut1,*
    1Faculty of Veterinary Sciences, Mahasarakham University, Maha Sarakham, 44000, Thailand.
    2Bioveterinary Research Unit, Mahasarakham University, Maha Sarakham, 44000, Thailand.
    3Stress and Oxidative Stress in Animal Research Unit, Mahasarakham University, Maha Sarakham, 44000, Thailand.
    4Program in Veterinary Technology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani, 41000, Thailand.

    ABSTRACT

    Background: Broiler production is crucial for global food security as a high-quality protein source. However, climate change causes extreme temperature fluctuations, including severe cold stress. Sudden ambient temperature drops impair broiler growth and health. This study investigates how continuously decreasing ambient temperature affects oxidative stress and mitochondrial activity in broiler blood cells.

    Methods: Blood cells pooled from two broiler chickens were diluted and subjected to a gradual temperature reduction from 42oC to 0oC, with 3oC decrements at each step. Mitochondrial activity, malondialdehyde (MDA), total antioxidant capacity (TAC), hydrogen peroxide (H2O2), reduced glutathione (GSH), catalase activity and nitric oxide (NO) levels were subsequently measured.

    Result: Mitochondrial activity and NO levels showed significant quadratic trends (P<0.05), increasing at moderate cold before declining at lower temperatures. From 42oC to 21oC, mitochondrial activity rose significantly at 39-33oC and 21oC (P<0.05), while MDA levels increased at 39-27oC and 21oC (P<0.05). TAC declined significantly from 42oC to colder points (P<0.05). Between 18oC and 0oC, mitochondrial activity and NO fluctuated, suggesting an adaptive response. Conversely, H2O2, GSH and catalase levels remained stable across all temperatures (P>0.05). These findings highlight a complex cellular response to cold stress, emphasizing the need for proper cold stress management in broiler production.

    INTRODUCTION

    Broiler production is a key contributor to global food supply. In 2022, global output reached 102.9 million tons and is expected to rise to 152.8 million tons by 2031 (Oke et al., 2024). Broilers provide high-quality protein at low cost due to rapid growth and superior feed efficiency, supporting rising demand, especially in developing regions (Nguyen et al., 2025). However, climate change, notably abrupt cold exposure, poses major challenges. Cold stress impairs growth, weakens immunity and elevates mortality in broilers (Aarif et al., 2014; Liu et al., 2022).
           
    Cold stress induces oxidative stress, a condition in which the generation of reactive oxygen species (ROS) exceeds the antioxidant defense capacity (Afzal et al., 2023). ROS can damage cellular proteins, lipids and DNA, posing a threat to cell viability (Jomova et al., 2023). Understanding the oxidative stress response is therefore critical for mitigating cold-induced damage in broilers (Abo-Al-Ela et al., 2021; Sahib et al., 2024). Several biomarkers are used to assess oxidative stress: malondialdehyde (MDA), which indicates lipid peroxidation and cellular damage (Cordiano et al., 2023; Srinontong et al., 2024); total antioxidant capacity (TAC), which reflects the overall antioxidant status (Surai et al., 2019); hydrogen peroxide (H2O2) accumulation, which signals redox imbalance (Ponnampalam et al., 2022); reduced glutathione (GSH), which maintains redox homeostasis (Liu et al., 2021 and catalase, which detoxifies H2O2 (Baker et al., 2023). Nitric oxide (NO) regulates intracellular signaling and responds to oxidative cues (Xu et al., 2022), while mitochondria, as the central site of cellular energy metabolism, modulate ROS generation under stress conditions (Casanova et al., 2023).
           
    Previous studies reported that dietary supplementation with glutamine, L-carnitine and betaine enhances TAC and GSH levels while reducing MDA concentration (Liu et al., 2021). In addition, Baker et al. (2023) emphasized that catalase and GSH are major antioxidants depleted during cold exposure. Alterations in NO during stress have been linked to cellular injury (Semenikhina et al., 2022) and Casanova et al. (2023) highlighted mitochondria as a key modulator of oxidative responses.
           
    Although the effects of cold on oxidative stress, NO variation and mitochondrial activity are recognized, data on dynamic shifts in oxidative markers, NO and mitochondrial function under exposure to 0oC remain limited. Clarifying broiler cellular adaptation to extreme cold is essential. Therefore, the objective of this study was to investigate the responses of MDA, TAC, H2O2, GSH, catalase, NO and mitochondrial activity in broiler blood cells as ambient temperature declined from 42oC to 0oC. This study will reveal the impact of cold stress on broilers by assessing cellular responses and adaptations, offering insights to mitigate potential damage caused by climate change, particularly under extreme cold conditions.

    MATERIALS AND METHODS

    This experiment was conducted in 2025 and was approved by the Institutional Animal Experimentation Ethics Committee, Mahasarakham University (Approval No. IACUC-MSU-009-055/2025).
     
    Animals
     
    Ten 28-day-old broilers were obtained from a commercial farm in Maha Sarakham Province and housed at the Faculty of Veterinary Sciences, Mahasarakham University. After a 7-day acclimation under a 16L:8D photoperiod, birds were provided ad libitum access to a standard grower diet and clean water. No additional vaccines were given beyond the routine schedule. All broilers remained clinically healthy, showing no signs of disease or abnormality. At 35 days of age, birds were considered physiologically stable for experimentation.
     
    Experimental design
     
    This study comprised two parts. The first examined changes in key biochemical markers in broiler blood cells as ambient temperature gradually declined from 42oC (normal broiler body temperature) to 0oC. The markers analyzed included mitochondrial activity, H2O2, MDA, TAC, NO, GSH and catalase activity. The second part investigated how lower temperatures affected the same markers and was divided into two sub-studies: sub-study 2.1 compared 42oC with 39-21oC and sub-study 2.2 compared 42oC with 18-0oC.
     
    Experimental procedure
     
    The sample size was based on the method of Ilyas et al. (2017). Blood was collected from two broilers (randomly selected from ten broiler chickens; 2 mL each) in heparinized tubes. For washing, blood was mixed with phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 2,500 rpm (769 × g) for 5 minutes. The supernatant was discarded and the process repeated twice. The washed blood was then diluted 1:200 (v/v) with PBS and 10 mL of the diluted sample was aliquoted into test tubes. Tubes were arranged by temperature from 42oC to 0oC, decreasing 3oC per step, with four tubes per temperature. All tubes were placed in a temperature-controlled water bath monitored by a digital thermometer. Prior to cooling, blood cells were held at 42oC for 30 minutes. Then, the temperature was reduced stepwise to 0oC. At each step, cells were held for 20 minutes before collecting four tubes (replicates) for testing.
     
    Determination of biochemical indicators
     
    Total antioxidant capacity
     
    TAC was assessed by the FRAP assay (Srinontong et al., 2023). The working solution combined 10 mL of 300 mmol sodium acetate buffer (pH 3.6), 1 mL of 10 mmol TPTZ and 1 mL of 20 mmol FeCl3·6H2O. Then, 20 µL of sample was mixed with 180 µL of the solution and incubated for 5 min at room temperature. Absorbance was read at 595 nm. Ferrous sulfate heptahydrate was used as standard.
     
    Malondialdehyde
     
    MDA was measured using the TBARS assay. A 0.1 mL sample was mixed with 0.45 mL of 0.09% NaCl, 0.2 mL of 0.67% TBA and 1 mL of 10% TCA in 0.6 M HCl. The mixture was heated at 100oC for 30 min, cooled, then 2 mL of deionized water was added, vortexed and centrifuged at 3,000 rpm (1,008 × g) for 10 min. Absorbance was read at 532 nm (Sürmen-Gür et al., 2003).
     
    Nitric oxide
     
    NO levels were determined using Griess reagent, composed of 1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 2.5% phosphoric acid. Equal volumes of reagent and supernatant were mixed and incubated for 15 minutes. Absorbance was read at 540 nm. Sodium nitrite served as the standard (Giustarini et al., 2008).
     
    Hydrogen peroxide
     
    H2O2 was measured per Orprayoon et al. (2020), with slight modifications. Sample was mixed with 2.25 mmol/L FeSO4, incubated 5 min, then 4 mmol/L norfloxacin was added and incubated 3 min. Absorbance was read at 440 nm. H2O2 (0-40 µmol/L) was used for the standard curve.
     
    Mitochondrial activity
     
    Mitochondrial activity in blood cells was measured using the MTT assay. MTT (5 mg/mL in acetone) was filtered. One milliliter of diluted blood was centrifuged, the supernatant removed and the pellet incubated with MTT at 41.5oC for 75 minutes. Dimethyl sulfoxide (150 µL) was added and absorbance read at 540 nm (Bahuguna et al., 2017).
     
    Reduced glutathione and catalase
     
    GSH and catalase levels were measured using colorimetric assay kits (Abbkine; KTB1600 for GSH, KTB1040 for catalase), following the manufacturer’s instructions. Absorbance was read using a microplate reader.
     
    Statistical analysis
     
    Normality was checked before the analysis using PROC GLM. The differences were tested for significance using Duncan’s multiple range test and results were considered significant if the P-value was less than 0.05 (SAS® Studio).

    RESULTS AND DISCUSSION

    Trends in the changes of biochemical parameters in broiler blood cells when ambient temperature is reduced from 42oC to 0oC
     
    As shown in Table 1, mitochondrial activity and NO levels showed significant quadratic trends (P<0.05), indicating complex response patterns. Mitochondrial activity rose at 39-33oC, dropped at 30oC, increased again at 18-15oC, declined at 12-6oC, then sharply increased at 3-0oC (P<0.05). The rise at 39-33oC may help broiler blood cells sustain energy under mild cold stress (Gong et al., 2023). The drop at 30-27oC may reflect impaired electron transport and reduced energy, leading to ROS overproduction and oxidative stress (Casanova et al., 2023). Activity rose again as temperature neared 0oC, suggesting adaptive responses. NO levels fell at 39-36oC, rose at 33-21oC, dropped at 18oC and peaked at 6oC (P<0.05), consistent with its signaling role (Semenikhina et al., 2022). These changes may reflect impaired NO synthesis or oxidative stress response (Pappas et al., 2023). In contrast, H2O2, MDA, TAC, GSH and catalase levels showed no significant changes (P>0.05; Table 1). Overall, broiler blood cells retained function and showed dynamic biochemical shifts even at 0oC.

    Table 1: Changing of mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells when the ambient temperature continuously decreased from 42oC until to 0oC (3oC each time).


     
    The effects of decreasing environmental temperatures from 42oC to 21oC on biochemical parameters in broiler blood cells
           
    Mitochondrial activity at 42oC was significantly lower than at 39-33oC and 21oC (P<0.05). Activity at 39-33oC was significantly higher than at 30oC and 27oC, which in turn were lower than at 21oC (P<0.05). No significant differences were found between 42oC and 30-24oC, or between 39-33oC and 21oC (P>0.05; Table 2). Elevated activity at 39-33oC suggests an adaptive response to moderate cold, aligning with reports of mitochondrial energy upregulation under mild stress (Park et al., 2021). The drop at 30-27oC may indicate overwhelmed adaptive capacity, reducing efficiency (Flensted-Jensen et al., 2024). This supports findings that severe cold impairs mitochondrial function via oxidative damage and electron transport disruption (Timkova et al., 2016). The rebound at 21oC may reflect a compensatory mechanism for energy restoration (Mohan et al., 2023).

    Table 2: Effect of decreasing ambient temperature on mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells when the ambient temperature continuously decreased from 42oC until to 21oC (3oC each time).


           
    MDA levels at 42oC were significantly lower than those at 39-27oC and 21oC (P<0.05). Likewise, levels at 24oC were significantly lower than at 39oC and 21oC (P<0.05; Table 2). However, no significant differences were found between 42oC and 24oC or among 39-27oC and 21oC (P>0.05). These findings support earlier studies reporting increased lipid damage and oxidative stress under cold conditions (Aksit et al., 2008). The elevated MDA at 39-27oC suggests rising oxidative stress as temperatures fall, likely due to increased ROS production beyond antioxidant control (Cordiano et al., 2023). Interestingly, MDA was lower at 24oC than at 39oC and 21oC, possibly reflecting an antioxidant response at moderate cold. However, this response seems limited at colder temperatures, as indicated by the higher MDA at 21oC.
           
    TAC was significantly higher at 42oC than at 39-27oC and 21oC and higher at 24oC than at 21oC (P<0.05). No significant difference was found between 42oC and 24oC, or among 39-24oC (P>0.05). These findings suggest that TAC decreased as temperature dropped, likely due to antioxidant depletion from elevated oxidative stress (Liu et al., 2021). This aligns with previous reports showing oxidative stress lowers TAC by consuming antioxidants like GSH and catalase (Saracila et al., 2023).
           
    NO levels at 39oC and 36oC were significantly lower than at 42oC and between 33oC and 21oC (P<0.05). The decrease at 39-36oC may reflect reduced production during mild cold stress. These findings contrast with Zhang et al. (2011), who observed NO elevation under cold stress. However, in this study, NO increased as the temperature dropped further, suggesting an adaptive response. The elevated NO at 42oC and 33-21oC supports its role in maintaining physiological stability under both normal and cold conditions.
           
    However, H2O2, GSH and catalase activity remained stable between 42oC and 21oC (P>0.05; Table 2). This suggests that GSH and catalase defense may sufficiently control H2O2 during moderate cold stress (Ponnampalam et al., 2022). The consistent levels of these markers indicate that broiler blood cells can maintain antioxidant capacity, helping to limit cold-induced damage.
     
    Comparative analysis of biochemical parameter changes in broiler blood cells at 42oC and reduced temperatures from 18oC to 0oC
     
    Mitochondrial activity at 42oC was significantly lower than at 18-15oC and 3-0oC (P<0.05), but not different from 12-6oC (P>0.05). Activity at 18-15oC and 3-0oC also did not differ (P>0.05). The pattern differed from the steady decline between 42-21oC and may reflect reduced mitochondrial respiration under cold. The increased activity at 18-15oC and 3-0oC may indicate a compensatory response to generate energy during cold stress (Casanova et al., 2023), though prolonged activation may impair mitochondria via excess ROS (Lennicke and Cochemé, 2021).
           
    MDA levels at 42oC were significantly lower than those at 18-0oC (P<0.05), indicating greater oxidative damage under cold. The rise in MDA reflects excess ROS production during cold stress, causing lipid peroxidation as antioxidant reserves decline (Wei et al., 2024).
           
    NO levels at 6-3oC were significantly higher than at 18-12oC (P<0.05), while levels at 15oC were significantly lower than at 42oC and 9-0oC (P<0.05). No differences were found between 42oC and 12-0oC or between 18oC and 12oC (P>0.05). The drop at 18-15oC may reflect protection against NO-induced oxidative damage (Pappas et al., 2023). The lower NO at 15oC versus 42oC and 9-0oC suggests temperature-dependent synthesis. In contrast to Su et al. (2020), who reported increased NO under cold, our results reveal a cyclic fluctuation, suggesting adaptive responses to falling temperatures.
           
    Catalase activity at 42oC was significantly higher than at 18-15oC and 9-0oC (P<0.05) and activity at 12oC was higher than at 3-0oC (P<0.05). No differences were found between 42oC and 12oC or between 18-15oC and 9-0oC (P>0.05). The high activity at 42oC highlights catalase’s role in H2O2 detoxification (Baker et al., 2023), while reduced activity at lower temperatures may reflect weakened antioxidant defense. The peak at 12oC, followed by decline at 3-0oC, may indicate a short-lived adaptive response before enzyme inactivation or antioxidant depletion.
                   
    In contrast, the levels of H2O2, TAC and GSH remained constant from 18-0oC (Table 3), indicating that the GSH antioxidant system and TAC can counteract cold stress and reduce the production of H2O2.

    Table 3: The effects of environmental temperatures of 42oC and a decrease in ambient temperature from 18oC to 0oC (3oC reduction each time) on the mitochondrial activity, hydrogen peroxide, malondialdehyde, total antioxidant capacity, nitric oxide, reduced glutathione and catalase activity of broiler blood cells.

    CONCLUSION

    This study examined how broiler blood cells respond to cold stress by analyzing key biochemical markers as ambient temperatures dropped from 42oC to 0oC. Cold exposure caused notable changes in mitochondrial activity, MDA, NO, H2O2, TAC, GSH and catalase activity. Quadratic trend analysis revealed complex cellular responses. Mitochondrial activity and NO increased under moderate cold but declined in severe cold, reflecting adaptive mechanisms. In contrast, H2O2, GSH and catalase levels stayed stable, indicating strong antioxidant defense. These results enhance our understanding of cold stress effects and support strategies to reduce its impact in poultry.

    ACKNOWLEDGEMENT

    This research project was financially supported by Mahasarakham University. 

    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the author and do not necessarily represent the views of their affiliated institutions. The author is responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

    REFERENCES


    1. Aarif, O., Shergojry, S.A., Dar, S.A., Khan, N., Mir, N.A. and Sheikh, A.A. (2014). Impact of cold stress on blood biochemical and immune status in male and female Vanaraja chickens. Indian Journal of Animal Research. 48(2): 139-142. doi: 10.5958/j.0976-0555.48.2.030.

    2. Abo-Al-Ela, H.G., El-Kassas, S., El-Naggar, K., Abdo, S.E., Jahejo, A.R. and Al Wakeel, R.A. (2021).  Stress and immunity in poultry: Light management and nanotechnology as effective immune enhancers to fight stress. Cell Stress and Chaperones. 26(3): 457-472. 

    3. Afzal, S., Abdul Manap, A. S., Attiq, A., Albokhadaim, I., Kandeel, M. and Alhojaily, S.M. (2023). From imbalance to impairment: The central role of reactive oxygen species in oxidative stress-induced disorders and therapeutic exploration. Frontiers in Pharmacology. 14: 1269581.

    4. Aksit, M., Altan, O., Buyukozturk, A., Balkaya, M. and Ozdemir, D.E.M.İ.R. (2008). Effects of cold temperature and vitamin E supplementation on oxidative stress, Troponin-T level and other ascites related traits in broilers. Archiv für Geflügelkunde/European Poultry Science. 72(5): 221.

    5. Bahuguna, A., Khan, I., Bajpai, V.K. and Kang, S.C. (2017). MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh Journal of Pharmacology. 12(2): 115-118.

    6. Baker, A., Lin, C. C., Lett, C., Karpinska, B., Wright, M.H. and Foyer, C.H. (2023). Catalase: A critical node in the regulation of cell fate. Free Radical Biology and Medicine. 199: 56-66.

    7. Casanova, A., Wevers, A., Navarro-Ledesma, S. and Pruimboom, L. (2023). Mitochondria: It is all about energy. Frontiers in physiology. 14: 1114231.

    8. Cordiano, R., Di Gioacchino, M., Mangifesta, R., Panzera, C., Gangemi, S. and Minciullo, P.L. (2023). Malondialdehyde as a potential oxidative stress marker for allergy-oriented diseases: An update. Molecules. 28(16): 5979.

    9. Flensted-Jensen, M., Kleis-Olsen, A.S., Hassø, R.K., Lindtofte, S., Corral Pérez, J., Ortega-Gómez, S. and Larsen, S. (2024). Combined changes in temperature and pH mimicking exercise result in decreased efficiency in muscle mitochondria. Journal of Applied Physiology. 136(1): 79-88.

    10. Giustarini, D., Rossi, R., Milzani, A. and Dalle Donne, I. (2008). Nitrite and nitrate measurement by Griess reagent in human plasma: Evaluation of interferences and standardization. Methods in enzymology. 440: 361-380.

    11. Gong, R., Xing, L., Yin, J., Ding, Y., Liu, X., Bao, J. and Li, J. (2023). Appropriate cold stimulation changes energy distribution to improve stress resistance in broilers. Journal of Animal Science. 101: skad185.

    12. Ilyas, M., Adzim, M., Simbak, N. and Atif, A. (2017). Sample size calculation for animal studies using degree of freedom (E); an easy and statistically defined approach for metabolomics and genetic research. ILAR J. 43: 207-213.

    13. Jomova, K., Raptova, R., Alomar, S. Y., Alwasel, S. H., Nepovimova, E., Kuca, K. and Valko, M. (2023). Reactive oxygen species, toxicity, oxidative stress and antioxidants: Chronic diseases and aging. Archives of toxicology. 97(10): 2499-2574.

    14. Lennicke, C. and Cochemé, H.M. (2021). Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Molecular cell. 81(18): 3691-3707.

    15. Liu, X., Li, S., Zhao, N., Xing, L., Gong, R., Li, T.  and Bao, J. (2022). Effects of acute cold stress after intermittent cold stimulation on immune-related molecules, intestinal barrier genes and heat shock proteins in broiler ileum. Animals. 12(23): 3260. 

    16. Liu, Y., Yang, Y., Yao, R., Hu, Y., Liu, P., Lian, S. and Li, S. (2021). Dietary supplementary glutamine and L-carnitine enhanced the anti-cold stress of Arbor Acres broilers. Archives Animal Breeding. 64(1): 231-243.

    17. Mohan, M.S., Aswani, S.S., Aparna, N.S., Boban, P.T., Sudhakaran, P.R. and Saja, K. (2023). Effect of acute cold exposure on cardiac mitochondrial function: role of sirtuins. Molecular and Cellular Biochemistry. 478(10): 2257-2270.

    18. Nguyen, H.D., Moss, A.F., Yan, F., Romero-Sanchez, H. and Dao, T.H. (2025). Effects of feeding methionine hydroxyl analogue chelated zinc, copper and manganese on growth performance, nutrient digestibility, mineral excretion and welfare conditions of broiler chickens: Part 2: Sustainability and welfare aspects. Animals. 15(3): 419. 

    19. Oke, O.E., Akosile, O.A., Uyanga, V.A., Oke, F.O., Oni, A.I., Tona, K. and Onagbesan, O.M. (2024). Climate change and broiler production. Veterinary Medicine and Science. 10(3): e1416. 

    20. Orprayoon, T., Ratanawimanwong, N. and Donpudsa, S. (2020). A simple method for hydrogen peroxide determination using Fe (II) with norfloxacin. In The Proceedings of National Conference of Rungsit University, Bangkok. pp: 137-147.

    21. Pappas, G., Wilkinson, M.L. and Gow, A.J. (2023). Nitric oxide regulation of cellular metabolism: Adaptive tuning of cellular energy. Nitric Oxide. 131: 8-17.

    22. Park, J.H., Lo, E.H. and Hayakawa, K. (2021). Endoplasmic reticulum interaction supports energy production and redox homeostasis in mitochondria released from astrocytes. Translational stroke research. 12(6): 1045-1054.

    23. Ponnampalam, E.N., Kiani, A., Santhiravel, S., Holman, B.W., Lauridsen, C. and Dunshea, F.R. (2022). The importance of dietary antioxidants on oxidative stress, meat and milk production and their preservative aspects in farm animals: Antioxidant action, animal health and product quality-Invited review. Animals. 12(23): 3279.

    24. Sahib, Q.S., Aafaq, I., Ahmed, H.A., Sheikh, G.G. and Ganai, I.A. (2024). Mitigating cold stress in livestock by nutritional interventions: A comprehensive review. Indian Journal of Animal Research. 58(3): 353-363. doi: 10.18805/ IJAR.B-5298.

    25. Saracila, M., Panaite, T.D., Predescu, N.C., Untea, A.E. and Vlaicu, P.A. (2023). Effect of dietary salicin standardized extract from salix alba bark on oxidative stress biomarkers and intestinal microflora of broiler chickens exposed to heat stress. Agriculture. 13(3): 698.

    26. Semenikhina, M., Stefanenko, M., Spires, D.R., Ilatovskaya, D.V.  and Palygin, O. (2022). Nitric-oxide-mediated signaling in podocyte pathophysiology. Biomolecules. 12(6): 745.

    27. Srinontong, P., Wandee, J. and Aengwanich, W. (2023). The effect of gallic acid on malondialdehyde, hydrogen peroxide and nitric oxide that influence viability of broiler blood cells at high ambient temperatures. British Poultry Science. 64(4): 512-517.

    28. Srinontong, P., Wandee, J. and Aengwanich, W. (2024). The effect of vitamin C on oxidative stress, nitric oxide formation and viability of broiler finisher blood cells subjected to high ambient temperature. Indian Journal of Animal Research. 58(7): 1117-1121. doi: 10.18805/IJAR.BF-1576. 

    29. Su, Y., Li, S., Xin, H., Li, J., Li, X., Zhang, R. and Bao, J. (2020). Proper cold stimulation starting at an earlier age can enhance immunity and improve adaptability to cold stress in broilers. Poultry science. 99(1): 129-141.

    30. Surai, P.F., Kochish, I.I., Fisinin, V.I. and Kidd, M.T. (2019). Antioxidant defence systems and oxidative stress in poultry biology: An update. Antioxidants. 8(7): 235.

    31. Sürmen-Gür, E., Erdinc, A., Serdar, Z. and Gür, H. (2003). Influence of acute exercise on oxidative stress in chronic smokers. Journal of Sports Science and Medicine. 2(3): 98-105. 

    32. Timkova, V., Tatarkova, Z., Lehotsky, J., Racay, P., Dobrota, D.  and Kaplan, P. (2016). Effects of mild hyperhomocysteinemia on electron transport chain complexes, oxidative stress and protein expression in rat cardiac mitochondria. Molecular and Cellular Biochemistry. 411: 261-270.

    33. Wei, H., Li, H., Miao, D., Wang, H., Liu, Y., Xing, L. and Li, J. (2024). Dietary resveratrol supplementation alleviates cold exposure-induced pyroptosis and inflammation in broiler heart by modulating oxidative stress and endoplasmic reticulum stress. Poultry Science. 103(11): 104203.

    34. Xu, H.L., Li, H., Bao, R.K., Tang, Y.X., Elsherbeni, A.I.A., Gharib, H.B.A. and Li, J.L. (2022). Transport stress induced cardiac NO-NOS disorder is mitigated by activating Nrf2/ HO-1/NQO1 antioxidant defense response in newly hatched chicks. Frontiers in Veterinary Science. 9: 938826.

    35. Zhang, Z.W., Lv, Z.H., Li, J.L., Li, S., Xu, S.W. and Wang, X.L. (2011). Effects of cold stress on nitric oxide in duodenum of chicks. Poultry Science. 90(7): 1555-1561.
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