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

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 0^oC 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 42^oC to 0^oC. 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.

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 42^oC (normal broiler body temperature) to 0^oC. 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 42^oC with 39-21^oC and sub-study 2.2 compared 42^oC with 18-0^oC.
 
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 42^oC to 0^oC, decreasing 3^oC 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 42^oC for 30 minutes. Then, the temperature was reduced stepwise to 0^oC. 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 100^oC 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 41.5^oC 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).

Trends in the changes of biochemical parameters in broiler blood cells when ambient temperature is reduced from 42^oC to 0^oC
 
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 39-33^oC, dropped at 30^oC, increased again at 18-15^oC, declined at 12-6^oC, then sharply increased at 3-0^oC (P<0.05). The rise at 39-33^oC may help broiler blood cells sustain energy under mild cold stress (Gong et al., 2023). The drop at 30-27^oC 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 0^oC, suggesting adaptive responses. NO levels fell at 39-36^oC, rose at 33-21^oC, dropped at 18^oC and peaked at 6^oC (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 0^oC.


 
The effects of decreasing environmental temperatures from 42^oC to 21^oC on biochemical parameters in broiler blood cells
       
Mitochondrial activity at 42^oC was significantly lower than at 39-33^oC and 21^oC (P<0.05). Activity at 39-33^oC was significantly higher than at 30^oC and 27^oC, which in turn were lower than at 21^oC (P<0.05). No significant differences were found between 42^oC and 30-24^oC, or between 39-33^oC and 21^oC (P>0.05; Table 2). Elevated activity at 39-33^oC suggests an adaptive response to moderate cold, aligning with reports of mitochondrial energy upregulation under mild stress (Park et al., 2021). The drop at 30-27^oC 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 21^oC may reflect a compensatory mechanism for energy restoration (Mohan et al., 2023).


       
MDA levels at 42^oC were significantly lower than those at 39-27^oC and 21^oC (P<0.05). Likewise, levels at 24^oC were significantly lower than at 39^oC and 21^oC (P<0.05; Table 2). However, no significant differences were found between 42^oC and 24^oC or among 39-27^oC and 21^oC (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 39-27^oC 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 24^oC than at 39^oC and 21^oC, possibly reflecting an antioxidant response at moderate cold. However, this response seems limited at colder temperatures, as indicated by the higher MDA at 21^oC.

       
TAC was significantly higher at 42^oC than at 39-27^oC and 21^oC and higher at 24^oC than at 21^oC (P<0.05). No significant difference was found between 42^oC and 24^oC, or among 39-24^oC (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 39^oC and 36^oC were significantly lower than at 42^oC and between 33^oC and 21^oC (P<0.05). The decrease at 39-36^oC 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 42^oC and 33-21^oC supports its role in maintaining physiological stability under both normal and cold conditions.
       
However, H₂O₂, GSH and catalase activity remained stable between 42^oC and 21^oC (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 42^oC and reduced temperatures from 18^oC to 0oC
 
Mitochondrial activity at 42^oC was significantly lower than at 18-15^oC and 3-0^oC (P<0.05), but not different from 12-6^oC (P>0.05). Activity at 18-15^oC and 3-0^oC also did not differ (P>0.05). The pattern differed from the steady decline between 42-21^oC and may reflect reduced mitochondrial respiration under cold. The increased activity at 18-15^oC and 3-0^oC 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 42^oC were significantly lower than those at 18-0^oC (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 6-3^oC were significantly higher than at 18-12^oC (P<0.05), while levels at 15^oC were significantly lower than at 42^oC and 9-0^oC (P<0.05). No differences were found between 42^oC and 12-0^oC or between 18^oC and 12^oC (P>0.05). The drop at 18-15^oC may reflect protection against NO-induced oxidative damage (Pappas et al., 2023). The lower NO at 15^oC versus 42^oC and 9-0^oC 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 42^oC was significantly higher than at 18-15^oC and 9-0^oC (P<0.05) and activity at 12^oC was higher than at 3-0^oC (P<0.05). No differences were found between 42^oC and 12^oC or between 18-15^oC and 9-0^oC (P>0.05). The high activity at 42^oC 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 12^oC, followed by decline at 3-0^oC, 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 18-0^oC (Table 3), indicating that the GSH antioxidant system and TAC can counteract cold stress and reduce the production of H₂O₂.

This study examined how broiler blood cells respond to cold stress by analyzing key biochemical markers as ambient temperatures dropped from 42^oC to 0^oC. 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.

This research project was financially supported by Mahasarakham University. 

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