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Full Research Article

Mitigating Effect of Phytogenic Plant Extracts on Heat Induced Stress in Broilers

H
Hritika Tushar Merchant1
https://orcid.org/0009-0007-3566-5933
P
Pratik Hivraj Barsinge1
https://orcid.org/0009-0008-4007-2961
B
Bhagyalaxmi Chinnavadu Junnuru1
https://orcid.org/0009-0005-9955-9748
A
Antara Anup Guha1
https://orcid.org/0009-0000-0717-5995
A
Amit Chandrakant Kini1,*
https://orcid.org/0000-0001-5152-8073
1Department of Research and Development, Nutranovel Additives Pvt. Ltd. Bhiwandi-421 302, Maharashtra, India.

ABSTRACT

Background: This study was aimed to evaluate the effectiveness of a phytogenic blend composed of extracts from Mangifera indica, Citrullus lanatus, Cymbopogon citratus, Allium cepa, Rosmarinus officinalis and Allium sativum, against induced heat stress in commercial broilers. These were studied by examining their effects on growth performance, haematological parameters and stress biomarkers. The biomarkers used were HSP70 and cortisol (COR) in feathers and blood serum.

Methods: A total of 1760 one day-old Cobb 430Y broiler chicks were randomly allocated into four treatment groups, consisting of 440 birds in each group. A phytogenic plant extract, referred as PPE, was used in this study. The experimental groups were as follows: Normal Control (NC, 36.05±0.32oC at 2 PM, no PPE), NC+PPE (36.05±0.32oC at 2 PM, with PPE), Heat Stress (HS, 42.20±1oC at 2 PM, no PPE), HS+PPE (42.20±1oC at 2 PM, with PPE). Heat stress was induced using portable electric heater for the HS groups. After a 14-day acclimatization period, PPE was administered through drinking water. Feed was withdrawn from 12:00 hrs. and 16:00 hrs, while water was provided ad libitum for all the birds.

Result: The NC+PPE and HS+PPE groups showed significantly higher (P<0.05) body weight and body weight gain compared to their respective control groups. The cumulative weekly mortality percentage was lowest in the NC+PPE group (1.36%, 6 birds). Feather HSP70 levels on day 41 were lowest (P<0.05) in the HS+PPE group. No significant differences were observed in the haematological parameters between the treatment groups. The NC+PPE and HS+PPE both groups performed comparatively better than other treatment groups. Both the groups had a comparatively better reduction of stress biomarkers, viz., feather HSP70, as compared to the respective control groups.

INTRODUCTION

India’s poultry industry is among the fastest-growing agricultural sectors, expanding at an annual rate of approximately 8% and currently valued at around USD 7.5 billion (Kumar et al., 2024). Global population is projected to reach 9.7 billion by 2050, driving a 60% increase in food demand and a 70% rise in animal-based product consumption (Ritchie et al., 2019). As a result, global meat production is expected to reach 347 million tonnes by 2030, with poultry accounting for approximately 52% (OECD-FAO Agricultural Outlook 2021-2030, 2021). Despite this growth, heat stress remains a major constraint, causing losses of up to USD 2.36 billion annually in the U.S. poultry sector (Abbas et al., 2025). Heat stress disrupts physiological balance by overwhelming thermoregulation, leading to oxidative damage and reduced performance (Greene et al., 2019). Broiler chickens are especially prone to heat stress due to dense feathers and absence of sweat glands, limiting heat loss (Song and King, 2015).
       
Heat shock proteins (HSPs) function as molecular chaperones that play a crucial role in preserving cellular homeostasis by inhibiting apoptosis and enhancing cell survival mechanisms (Madkour et al., 2022). Under stressful conditions, HSPs are induced to synthesize specific proteins that maintain cell viability and stabilize the internal environment, with HSP70 playing a central role in thermotolerance and serving as a key biomarker for heat stress in animals (Song and King, 2015). Feather-expressed HSP70 serves as a reliable, non-invasive biomarker for assessing thermal stress in broilers (Greene et al., 2019).
       
Phytogenic compounds from various plant parts act as bioactive agents that promote health, food safety and shelf-life. Mangifera indica provides mangiferin, a potent antioxidant (Okwu and Ezenagu, 2008), while Citrullus lanatus (watermelon) is rich in antioxidants like beta-carotene, vitamins C and E, arginine and citrulline, which help protect cells, DNA and tissues from oxidative damage (Collins et al., 2007). Lemongrass (Cymbopogon citratus) contains region-specific phytochemicals, including antimicrobial myrcene and flavonoids like luteolin, isoorientin 2'-O-rhamnoside, quercetin, kaempferol and apigenin (Shah et al., 2011). Onion (Allium cepa) naturally contains allicin and sulfur compounds such as S-methylcysteine sulfoxide and S-allylcysteine sulfoxide (Kothari et al., 2019).
       
Rosemary (Rosmarinus officinalis) contains bioactives like camphor, ursolic acid, carnosol, carnosic acid and caffeine (Ulbricht et al., 2010). Garlic (Allium sativum) is rich in organosulfur compounds such as allicin and diallylsulfide, contributing to anti-inflammatory and immune-enhancing effects (Jang et al., 2018). However, evidence on the role of phytogenic in reducing heat stress in broilers remains limited (Chiwaya et al., 2025). Therefore, our current objective is to investigate the potential of phytogenic plant extracts (PPE), comprising blends of Mangifera indica, Citrullus lanatus, Cymbopogon citratus, Allium cepa, Rosmarinus officinalis and Allium sativum, in alleviating heat stress in broilers and to evaluate their synergistic effects.

MATERIALS AND METHODS

The research trial was conducted following ethical guidelines at Dr. B.V. Rao Institute of Poultry Management and Technology (IPMT), Pune, using 1,760 one-day-old Cobb 430Y broiler chicks (mixed sex) procured from Venkateshwara Hatcheries, Pune. The 42-day trial was conducted from 17 January to 27 February 2025. It involved four groups: NC (normal temperature, no PPE), NC+PPE (normal temperature with PPE), HS (heat stress at 42oC, no PPE) and HS+PPE (heat stress with PPE). Each group consisting of 440 birds, subdivided into four replicates of 110 birds each. Groups HS and HS+PPE were housed in shed C1, while NC and NC+PPE were in shed C2.
       
An acclimatization period (day 0-14) without heat stress or test treatments was provided. Venky’s Aminovit Gold was supplemented via drinking water for the first five days. Heat stress was induced from day 15 to 42 using a portable electric heater from 12:00 to 16:00 hrs daily. Recorded average temperatures in the HS shed were 26.71±0.50 oC at 08:00, 42.20±0.10 oC at 14:00 and 34.31 ±0.39 oC at 17:00.
       
Vaccination was performed as follows: Newcastle disease (Super LaSota™) intranasally on day 7, booster orally on day 21; IBD vaccine on day 14 and day 28 (intermediate strain). Birds were fed in-house formulated mash diets: pre-starter (weeks 1-2), starter (weeks 3-4) and finisher (weeks 5-6) with ad libitum water. Proximate analysis was performed by Envirocare Labs Pvt. Ltd., Thane, as presented in Table 1. Feed was restricted daily from 12:00 to 16:00 hrs. Growth performance (body weight, weight gain, feed intake and FCR) was recorded weekly from 20 randomly selected birds per replicate.

Table 1: Nutrient composition of pre-starter, starter and finisher broiler diets.


       
The heat stress-relieving agent (PPE) was a blend of plant extracts: Rosmarinus officinalis, Cymbopogon citratus, Mangifera indica, Citrullus lanatus, Allium sativum and Allium cepa, administered via drinking water at 0.25  mL/L from day 15 to 42 for 12 hours daily (6 AM-6 PM).
 
Sample collection and analysis
 
On day 13 and 41, blood and feather samples were collected for cortisol and HSP70 estimation. Feather samples were collected twice daily morning (9:30-10:30 AM) and afternoon (2:30 PM), while blood was sampled only in the morning. Eight birds per replicate were marked; serum was collected from three birds/replicate without EDTA. Feather samples from all eight birds were pooled. Morning sampling: HS and HS+PPE (9:30-10:30 AM), then NC and NC+PPE (10:45–11:45 AM). On day 13, feather collection was done outside the shed; on day 41, inside. Sampling followed shed order: C1 then C2. Cortisol and HSP70 were quantified using chicken-specific sandwich ELISA kits (Cortisol: Cat. No. EA0017Ch; HSP70: Cat. No. E0326Ch; BT LAB, China). Tests were done according to the manufacturer’s protocol. For haematological analysis (day 41), 2 mL blood was collected from three birds per replicate (n=48) into 4% EDTA vacutainers. Hematological parameters were assessed using an automated veterinary hematology analyser (MicroCC Vet, High Technology Inc., USA). Differential leukocyte counts were performed on Field stain. Field Stains A (Molychem 31680) and B (Molychem 31720) processed blood smears.
 
Statistical analysis
 
One-way ANOVA (Quirk, 2012) was used to analyze treatment effects followed by Tukey’s multiple comparison test (Abdi and Williams, 2010). All analyses were performed using GraphPad Prism (Version 10.3.0). 

RESULTS AND DISCUSSION

Growth performance parameters and mortality
 
The results of various growth performance parameters are presented in the Table 2,3,4,5 and 6, respectively. The average live body weight (g) and cumulative body weight gain (g) were significantly (p<0.05) higher in the NC+PPE and HS+PPE groups supplemented with PPE, compared to their respective control groups, NC and HS, which did not receive PPE. Throughout the research trial, the NC+PPE group exhibited a significantly (p<0.05) higher LBW and BWG compared to the other three groups. No significant (p>0.05) difference was observed in the 5th and 6th weeks between the NC and HS+PPE groups, indicating that both groups was relatively performed similarly in terms of LBW and BWG. At the end of the experimental period, all four groups showed no significant (p>0.05) difference in average cumulative feed consumption (g). After one week of PPE administration, in the 3rd week, both the NC+PPE and HS+PPE groups showed a significantly (p<0.05) better average cumulative feed conversion ratio compared to their respective control groups. In the 4th and 5th weeks, the HS+ PPE group continued to show a better FCR compared to the HS group. In the 6th week the average cumulative mortality was lowest in normal control group (Table 4) supplemented with PPE and second lowest seen in the heat stress group supplemented with PPE.

Table 2: Average weekly live body weight (g) of experimental birds from different groups.



Table 3: Average cumulative weekly live body weight gain (g) of experimental birds from different groups.



Table 4: Average cumulative weekly feed consumption (g/bird) of experimental birds from different groups.



Table 5: Average cumulative weekly feed conversion ratio (FCR) of experimental birds from different groups.



Table 6: Average cumulative weekly mortality (%) of birds from different experimental groups.


       
Lemongrass essential oil may improve broiler growth under heat stress through its antioxidant, digestive and antimicrobial properties (Alagawany et al., 2021). Thymol enhances trypsin and pancreatic enzyme activity (Lee et al., 2003). Supplementation with garlic, lemon and their combination (200 mg/kg) significantly increased body weight gain, improved FCR and mortality, without affecting feed intake (Elbaz et al., 2022). Similarly, rosemary extract at 50 mg/kg improved ABW and BWG without affecting feed intake, likely due to carnosic acid, a potent antioxidant derived from ferruginol, scavenging ROS and reducing lipid peroxidation. Conversely, 100 mg/kg under heat stress significantly improved FCR, while 50 mg/kg showed no such effect (Madkour et al., 2024). Similarly, the supplementation of a mixture of 1.5 g/kg lemongrass leaf and wormwood powders significantly improved live weight gain in heat-stressed birds compared to the control group (Al-Gburi, 2025). In contrast, Rokade et al. (2017) reported improved FCR (p<0.001) in broilers under hot summer conditions with mannan oligosaccharide (3% and 5%) supplementation. The mitigation of heat-induced reductions in broiler performance with PPE supplementation may be due to lemongrass essential oils’ antioxidant, digestive enzyme-stimulating and antimicrobial effects (Alagawany et al., 2021). The results are consistent with our findings, supplementation with 40% watermelon juice (WJ) resulted in 0% mortality (100% survival rate) compared to the control group (Jimoh et al., 2018). Similarly, provision of Amla juice, Aloe vera gel and rough lemon juice (2% each) through drinking water reduced mortality in broilers during summer (Gowri et al., 2022).
 
Hematological parameters
 
PPE significantly (p<0.05) increased red blood cell counts under heat stress and improved (P<0.05) the heterophil-to-lymphocyte ratio (H:L) in the NC+PPE group compared to the NC group. While all other hematological parameters were showed no significant (p>0.05) difference (Table 7). The higher H:L ratio in the NC group may be due to differences in blood collection timing, as noted in the materials and methods. Similarly, Rabadan (2022) reported no significant (p>0.05) changes in total leukocyte counts, hemoglobin, packed cell volume, or mean corpuscular volume in broilers given various concentrations of lemongrass hydrosol extract. Likewise, El-Sahn et al., (2024) observed that 200-300 mL/L lemongrass extract significantly (P<0.05) improved RBC count. On contrary, they also reported significantly (P<0.05) improved haemoglobin, lymphocytes, PCV, MCV and MCH in broilers.

Table 7: Average haematological parameters of birds from different experimental groups.


 
Stress biomarkers
 
HSP70 (ng/mL) and COR (ng/mL) levels in feathers and serum are presented in Table 8. No significant (P>0.05) difference was observed in blood serum HSP70 (ng/mL) and COR (ng/mL) levels in different experimental groups, on both 13th and 41st days. On day 41 (morning and afternoon), significantly lower (P<0.05) feather HSP70 levels (ng/mL) were observed in the NC+PPE and HS+ PPE groups compared to their respective controls. Additionally, the HS+PPE group recorded significantly (P<0.05) lower levels than the other three treatment groups.

Table 8: Average HSP70 and COR testing (ng/ml) of birds of various experimental groups.


 
Serum HSP70 and COR
 
Heat stress was applied from 12:00 PM to 4:00 PM, allowing birds overnight recovery, which may have helped maintain physiological stability and contributed to the non-significant differences in serum HSP70 and COR levels. Sarma et al., (2022) reported higher HSP70 in 13-day-old broilers compared to 41-day-olds, suggesting age-related regulation, while developed feathers in older birds may stabilize HSP70 during winter. Their study also reported no significant (p>0.05) difference in COR levels across agroclimatic zones in Assam during winter, corroborated with our findings. In contrast, Jimoh et al., (2024) observed a significant (p<0.05) reduction in HSP70 levels when garlic essential oil (30% v/v) was administered via drinking water for 49 days. In divergence, supplementation with antioxidants such as vitamin E and selenium significantly (p<0.001) lowered serum HSP70 levels (ng/mL) in broilers during heat stress (Bora et al., 2024).
 
Feather HSP70 and COR
 
In this study, ELISA was used to quantify HSP70 and cortisol (COR) levels (ng/mL). Heat stress increased HSP70 expression. On day 41, both HSP70 and COR levels were lower in the afternoon than in the morning. This may be due to sampling conditions as explained in materials and methods: morning samples were collected outdoors at ~37oC, while afternoon samples were taken indoors at ~42.5oC. Both temperatures exceeded the thermoneutral range. On the contrary, Greene et al., (2019) reported significantly (p<0.01) higher HSP70 levels, both in mRNA expression and protein content, in growing feathers collected in the afternoon compared to the morning from the same birds. Feather HSP70 expression reflects systemic and cellular stress, serving as a reliable, non-invasive biomarker for longitudinal stress monitoring. Sampling is quick, low-stress and suitable for repeated measures (Greene et al., 2019).

CONCLUSION

Based on the findings of the present study, it is concluded that inclusion of phytogenic plant extracts under both normal and heat-stressed conditions improved growth performance and reduced mortality in broilers. The NC+PPE and HS+ PPE groups showed greater reductions in stress biomarkers, such as feather HSP70, compared to their respective control groups. PPE supplementation shows potential in mitigating the adverse effects of heat stress in current fast growing commercial broilers raised under high-temperature conditions.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

REFERENCES


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    Full Research Article

    Mitigating Effect of Phytogenic Plant Extracts on Heat Induced Stress in Broilers

    H
    Hritika Tushar Merchant1
    https://orcid.org/0009-0007-3566-5933
    P
    Pratik Hivraj Barsinge1
    https://orcid.org/0009-0008-4007-2961
    B
    Bhagyalaxmi Chinnavadu Junnuru1
    https://orcid.org/0009-0005-9955-9748
    A
    Antara Anup Guha1
    https://orcid.org/0009-0000-0717-5995
    A
    Amit Chandrakant Kini1,*
    https://orcid.org/0000-0001-5152-8073
    1Department of Research and Development, Nutranovel Additives Pvt. Ltd. Bhiwandi-421 302, Maharashtra, India.

    ABSTRACT

    Background: This study was aimed to evaluate the effectiveness of a phytogenic blend composed of extracts from Mangifera indica, Citrullus lanatus, Cymbopogon citratus, Allium cepa, Rosmarinus officinalis and Allium sativum, against induced heat stress in commercial broilers. These were studied by examining their effects on growth performance, haematological parameters and stress biomarkers. The biomarkers used were HSP70 and cortisol (COR) in feathers and blood serum.

    Methods: A total of 1760 one day-old Cobb 430Y broiler chicks were randomly allocated into four treatment groups, consisting of 440 birds in each group. A phytogenic plant extract, referred as PPE, was used in this study. The experimental groups were as follows: Normal Control (NC, 36.05±0.32oC at 2 PM, no PPE), NC+PPE (36.05±0.32oC at 2 PM, with PPE), Heat Stress (HS, 42.20±1oC at 2 PM, no PPE), HS+PPE (42.20±1oC at 2 PM, with PPE). Heat stress was induced using portable electric heater for the HS groups. After a 14-day acclimatization period, PPE was administered through drinking water. Feed was withdrawn from 12:00 hrs. and 16:00 hrs, while water was provided ad libitum for all the birds.

    Result: The NC+PPE and HS+PPE groups showed significantly higher (P<0.05) body weight and body weight gain compared to their respective control groups. The cumulative weekly mortality percentage was lowest in the NC+PPE group (1.36%, 6 birds). Feather HSP70 levels on day 41 were lowest (P<0.05) in the HS+PPE group. No significant differences were observed in the haematological parameters between the treatment groups. The NC+PPE and HS+PPE both groups performed comparatively better than other treatment groups. Both the groups had a comparatively better reduction of stress biomarkers, viz., feather HSP70, as compared to the respective control groups.

    INTRODUCTION

    India’s poultry industry is among the fastest-growing agricultural sectors, expanding at an annual rate of approximately 8% and currently valued at around USD 7.5 billion (Kumar et al., 2024). Global population is projected to reach 9.7 billion by 2050, driving a 60% increase in food demand and a 70% rise in animal-based product consumption (Ritchie et al., 2019). As a result, global meat production is expected to reach 347 million tonnes by 2030, with poultry accounting for approximately 52% (OECD-FAO Agricultural Outlook 2021-2030, 2021). Despite this growth, heat stress remains a major constraint, causing losses of up to USD 2.36 billion annually in the U.S. poultry sector (Abbas et al., 2025). Heat stress disrupts physiological balance by overwhelming thermoregulation, leading to oxidative damage and reduced performance (Greene et al., 2019). Broiler chickens are especially prone to heat stress due to dense feathers and absence of sweat glands, limiting heat loss (Song and King, 2015).
           
    Heat shock proteins (HSPs) function as molecular chaperones that play a crucial role in preserving cellular homeostasis by inhibiting apoptosis and enhancing cell survival mechanisms (Madkour et al., 2022). Under stressful conditions, HSPs are induced to synthesize specific proteins that maintain cell viability and stabilize the internal environment, with HSP70 playing a central role in thermotolerance and serving as a key biomarker for heat stress in animals (Song and King, 2015). Feather-expressed HSP70 serves as a reliable, non-invasive biomarker for assessing thermal stress in broilers (Greene et al., 2019).
           
    Phytogenic compounds from various plant parts act as bioactive agents that promote health, food safety and shelf-life. Mangifera indica provides mangiferin, a potent antioxidant (Okwu and Ezenagu, 2008), while Citrullus lanatus (watermelon) is rich in antioxidants like beta-carotene, vitamins C and E, arginine and citrulline, which help protect cells, DNA and tissues from oxidative damage (Collins et al., 2007). Lemongrass (Cymbopogon citratus) contains region-specific phytochemicals, including antimicrobial myrcene and flavonoids like luteolin, isoorientin 2'-O-rhamnoside, quercetin, kaempferol and apigenin (Shah et al., 2011). Onion (Allium cepa) naturally contains allicin and sulfur compounds such as S-methylcysteine sulfoxide and S-allylcysteine sulfoxide (Kothari et al., 2019).
           
    Rosemary (Rosmarinus officinalis) contains bioactives like camphor, ursolic acid, carnosol, carnosic acid and caffeine (Ulbricht et al., 2010). Garlic (Allium sativum) is rich in organosulfur compounds such as allicin and diallylsulfide, contributing to anti-inflammatory and immune-enhancing effects (Jang et al., 2018). However, evidence on the role of phytogenic in reducing heat stress in broilers remains limited (Chiwaya et al., 2025). Therefore, our current objective is to investigate the potential of phytogenic plant extracts (PPE), comprising blends of Mangifera indica, Citrullus lanatus, Cymbopogon citratus, Allium cepa, Rosmarinus officinalis and Allium sativum, in alleviating heat stress in broilers and to evaluate their synergistic effects.

    MATERIALS AND METHODS

    The research trial was conducted following ethical guidelines at Dr. B.V. Rao Institute of Poultry Management and Technology (IPMT), Pune, using 1,760 one-day-old Cobb 430Y broiler chicks (mixed sex) procured from Venkateshwara Hatcheries, Pune. The 42-day trial was conducted from 17 January to 27 February 2025. It involved four groups: NC (normal temperature, no PPE), NC+PPE (normal temperature with PPE), HS (heat stress at 42oC, no PPE) and HS+PPE (heat stress with PPE). Each group consisting of 440 birds, subdivided into four replicates of 110 birds each. Groups HS and HS+PPE were housed in shed C1, while NC and NC+PPE were in shed C2.
           
    An acclimatization period (day 0-14) without heat stress or test treatments was provided. Venky’s Aminovit Gold was supplemented via drinking water for the first five days. Heat stress was induced from day 15 to 42 using a portable electric heater from 12:00 to 16:00 hrs daily. Recorded average temperatures in the HS shed were 26.71±0.50 oC at 08:00, 42.20±0.10 oC at 14:00 and 34.31 ±0.39 oC at 17:00.
           
    Vaccination was performed as follows: Newcastle disease (Super LaSota™) intranasally on day 7, booster orally on day 21; IBD vaccine on day 14 and day 28 (intermediate strain). Birds were fed in-house formulated mash diets: pre-starter (weeks 1-2), starter (weeks 3-4) and finisher (weeks 5-6) with ad libitum water. Proximate analysis was performed by Envirocare Labs Pvt. Ltd., Thane, as presented in Table 1. Feed was restricted daily from 12:00 to 16:00 hrs. Growth performance (body weight, weight gain, feed intake and FCR) was recorded weekly from 20 randomly selected birds per replicate.

    Table 1: Nutrient composition of pre-starter, starter and finisher broiler diets.


           
    The heat stress-relieving agent (PPE) was a blend of plant extracts: Rosmarinus officinalis, Cymbopogon citratus, Mangifera indica, Citrullus lanatus, Allium sativum and Allium cepa, administered via drinking water at 0.25  mL/L from day 15 to 42 for 12 hours daily (6 AM-6 PM).
     
    Sample collection and analysis
     
    On day 13 and 41, blood and feather samples were collected for cortisol and HSP70 estimation. Feather samples were collected twice daily morning (9:30-10:30 AM) and afternoon (2:30 PM), while blood was sampled only in the morning. Eight birds per replicate were marked; serum was collected from three birds/replicate without EDTA. Feather samples from all eight birds were pooled. Morning sampling: HS and HS+PPE (9:30-10:30 AM), then NC and NC+PPE (10:45–11:45 AM). On day 13, feather collection was done outside the shed; on day 41, inside. Sampling followed shed order: C1 then C2. Cortisol and HSP70 were quantified using chicken-specific sandwich ELISA kits (Cortisol: Cat. No. EA0017Ch; HSP70: Cat. No. E0326Ch; BT LAB, China). Tests were done according to the manufacturer’s protocol. For haematological analysis (day 41), 2 mL blood was collected from three birds per replicate (n=48) into 4% EDTA vacutainers. Hematological parameters were assessed using an automated veterinary hematology analyser (MicroCC Vet, High Technology Inc., USA). Differential leukocyte counts were performed on Field stain. Field Stains A (Molychem 31680) and B (Molychem 31720) processed blood smears.
     
    Statistical analysis
     
    One-way ANOVA (Quirk, 2012) was used to analyze treatment effects followed by Tukey’s multiple comparison test (Abdi and Williams, 2010). All analyses were performed using GraphPad Prism (Version 10.3.0). 

    RESULTS AND DISCUSSION

    Growth performance parameters and mortality
     
    The results of various growth performance parameters are presented in the Table 2,3,4,5 and 6, respectively. The average live body weight (g) and cumulative body weight gain (g) were significantly (p<0.05) higher in the NC+PPE and HS+PPE groups supplemented with PPE, compared to their respective control groups, NC and HS, which did not receive PPE. Throughout the research trial, the NC+PPE group exhibited a significantly (p<0.05) higher LBW and BWG compared to the other three groups. No significant (p>0.05) difference was observed in the 5th and 6th weeks between the NC and HS+PPE groups, indicating that both groups was relatively performed similarly in terms of LBW and BWG. At the end of the experimental period, all four groups showed no significant (p>0.05) difference in average cumulative feed consumption (g). After one week of PPE administration, in the 3rd week, both the NC+PPE and HS+PPE groups showed a significantly (p<0.05) better average cumulative feed conversion ratio compared to their respective control groups. In the 4th and 5th weeks, the HS+ PPE group continued to show a better FCR compared to the HS group. In the 6th week the average cumulative mortality was lowest in normal control group (Table 4) supplemented with PPE and second lowest seen in the heat stress group supplemented with PPE.

    Table 2: Average weekly live body weight (g) of experimental birds from different groups.



    Table 3: Average cumulative weekly live body weight gain (g) of experimental birds from different groups.



    Table 4: Average cumulative weekly feed consumption (g/bird) of experimental birds from different groups.



    Table 5: Average cumulative weekly feed conversion ratio (FCR) of experimental birds from different groups.



    Table 6: Average cumulative weekly mortality (%) of birds from different experimental groups.


           
    Lemongrass essential oil may improve broiler growth under heat stress through its antioxidant, digestive and antimicrobial properties (Alagawany et al., 2021). Thymol enhances trypsin and pancreatic enzyme activity (Lee et al., 2003). Supplementation with garlic, lemon and their combination (200 mg/kg) significantly increased body weight gain, improved FCR and mortality, without affecting feed intake (Elbaz et al., 2022). Similarly, rosemary extract at 50 mg/kg improved ABW and BWG without affecting feed intake, likely due to carnosic acid, a potent antioxidant derived from ferruginol, scavenging ROS and reducing lipid peroxidation. Conversely, 100 mg/kg under heat stress significantly improved FCR, while 50 mg/kg showed no such effect (Madkour et al., 2024). Similarly, the supplementation of a mixture of 1.5 g/kg lemongrass leaf and wormwood powders significantly improved live weight gain in heat-stressed birds compared to the control group (Al-Gburi, 2025). In contrast, Rokade et al. (2017) reported improved FCR (p<0.001) in broilers under hot summer conditions with mannan oligosaccharide (3% and 5%) supplementation. The mitigation of heat-induced reductions in broiler performance with PPE supplementation may be due to lemongrass essential oils’ antioxidant, digestive enzyme-stimulating and antimicrobial effects (Alagawany et al., 2021). The results are consistent with our findings, supplementation with 40% watermelon juice (WJ) resulted in 0% mortality (100% survival rate) compared to the control group (Jimoh et al., 2018). Similarly, provision of Amla juice, Aloe vera gel and rough lemon juice (2% each) through drinking water reduced mortality in broilers during summer (Gowri et al., 2022).
     
    Hematological parameters
     
    PPE significantly (p<0.05) increased red blood cell counts under heat stress and improved (P<0.05) the heterophil-to-lymphocyte ratio (H:L) in the NC+PPE group compared to the NC group. While all other hematological parameters were showed no significant (p>0.05) difference (Table 7). The higher H:L ratio in the NC group may be due to differences in blood collection timing, as noted in the materials and methods. Similarly, Rabadan (2022) reported no significant (p>0.05) changes in total leukocyte counts, hemoglobin, packed cell volume, or mean corpuscular volume in broilers given various concentrations of lemongrass hydrosol extract. Likewise, El-Sahn et al., (2024) observed that 200-300 mL/L lemongrass extract significantly (P<0.05) improved RBC count. On contrary, they also reported significantly (P<0.05) improved haemoglobin, lymphocytes, PCV, MCV and MCH in broilers.

    Table 7: Average haematological parameters of birds from different experimental groups.


     
    Stress biomarkers
     
    HSP70 (ng/mL) and COR (ng/mL) levels in feathers and serum are presented in Table 8. No significant (P>0.05) difference was observed in blood serum HSP70 (ng/mL) and COR (ng/mL) levels in different experimental groups, on both 13th and 41st days. On day 41 (morning and afternoon), significantly lower (P<0.05) feather HSP70 levels (ng/mL) were observed in the NC+PPE and HS+ PPE groups compared to their respective controls. Additionally, the HS+PPE group recorded significantly (P<0.05) lower levels than the other three treatment groups.

    Table 8: Average HSP70 and COR testing (ng/ml) of birds of various experimental groups.


     
    Serum HSP70 and COR
     
    Heat stress was applied from 12:00 PM to 4:00 PM, allowing birds overnight recovery, which may have helped maintain physiological stability and contributed to the non-significant differences in serum HSP70 and COR levels. Sarma et al., (2022) reported higher HSP70 in 13-day-old broilers compared to 41-day-olds, suggesting age-related regulation, while developed feathers in older birds may stabilize HSP70 during winter. Their study also reported no significant (p>0.05) difference in COR levels across agroclimatic zones in Assam during winter, corroborated with our findings. In contrast, Jimoh et al., (2024) observed a significant (p<0.05) reduction in HSP70 levels when garlic essential oil (30% v/v) was administered via drinking water for 49 days. In divergence, supplementation with antioxidants such as vitamin E and selenium significantly (p<0.001) lowered serum HSP70 levels (ng/mL) in broilers during heat stress (Bora et al., 2024).
     
    Feather HSP70 and COR
     
    In this study, ELISA was used to quantify HSP70 and cortisol (COR) levels (ng/mL). Heat stress increased HSP70 expression. On day 41, both HSP70 and COR levels were lower in the afternoon than in the morning. This may be due to sampling conditions as explained in materials and methods: morning samples were collected outdoors at ~37oC, while afternoon samples were taken indoors at ~42.5oC. Both temperatures exceeded the thermoneutral range. On the contrary, Greene et al., (2019) reported significantly (p<0.01) higher HSP70 levels, both in mRNA expression and protein content, in growing feathers collected in the afternoon compared to the morning from the same birds. Feather HSP70 expression reflects systemic and cellular stress, serving as a reliable, non-invasive biomarker for longitudinal stress monitoring. Sampling is quick, low-stress and suitable for repeated measures (Greene et al., 2019).

    CONCLUSION

    Based on the findings of the present study, it is concluded that inclusion of phytogenic plant extracts under both normal and heat-stressed conditions improved growth performance and reduced mortality in broilers. The NC+PPE and HS+ PPE groups showed greater reductions in stress biomarkers, such as feather HSP70, compared to their respective control groups. PPE supplementation shows potential in mitigating the adverse effects of heat stress in current fast growing commercial broilers raised under high-temperature conditions.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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