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Full Research Article

Effect of Heat Stress on Biochemical Parameters and Anti-oxidant Enzyme Activity of Cobb Strain Chicks Supplemented with Lemongrass (Cymbopogon citratus) and Aloe vera (Aloe barbadensis)

S
Simranjeet Kour1
0009-0002-0061-6713
J
Jonali Devi1,*
0000-0001-6630-2659
K
Kamal Sarma2
0000-0002-6333-5516
K
Kawardeep Kour1
0009-0008-3150-6502
D
Dibyendu Chakraborty3
0000-0001-7411-0913
J
Jasvinder Singh Sasan2
0000-0002-7175-477X
1Division of Veterinary Physiology and Biochemistry, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.
2Division of Veterinary Anatomy, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.
3Division of Animal Genetics and Breeding, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.

ABSTRACT

Background: The study designed to investigate the impact of lemongrass and aloe vera on biochemical parameters and antioxidant activity in chicks under normal and heat stress conditions.

Methods: A total number of three hundred chicks were randomly distributed into different control and treatment groups, with three replicates having 20 chicks in each group. The chicks were kept for acclimatization for three days and then exposed to heat stress from day 4 to the termination of the experimental period. Blood samples were collected from nine birds (three birds from each replicate from each group) on days 3, 28 and 42 to analyze biochemical parameters and antioxidant enzyme activities.

Result: The results revealed that creatinine levels increased significantly (P<0.05) with age in C1 and C2 groups, with significantly higher (P<0.05) levels observed in the C2 group on days 28 and 42. Between the control groups, significantly higher (P<0.05) values of creatinine were recorded in C2 group and among treatment groups, higher values of creatinine were observed in T2 group. With the advancement of age, an increasing trend of SOD, GPx and Catalase enzyme activities were obtained in all the experimental groups. Between the control groups, higher antioxidant enzyme activities were recorded in C2 group. Among the treatment groups, lower activities of antioxidant parameters were recorded in T3.

INTRODUCTION

The poultry industry is vital to the Indian economy, with high demand for chicken meat. Cobb strain broilers are the most efficient breed due to low production cost, superior feed efficiency and fast growth. After the ban on antibiotic growth promoters, natural herbs like aloe vera, fenugreek, ashwagandha, moringa, cinnamon, tulsi, garlic and pepper are being used as alternatives (Lewis, 1986; Abd El-Hack et al., 2022). Lemongrass contains flavonoids, phenolic compounds and essential oils with antibacterial, antifungal and antioxidant properties that improve poultry growth and performance (Shah et al., 2011) and reproductive parameters (Aswathi et al., 2019) in broilers. Aloe vera improves growth, carcass quality, intestinal health, immunity and reduces production costs. Chickens maintain a core body temperature of ~41°C, so when they are exposed to thermal challenges, they cannot maintain a balance between the heat generation and heat removal from the body, resulting in incremented core body temperature that leads to mortality. Therefore, this study has been undertaken to evaluate the effect of lemongrass and aloe vera on biochemical parameters and antioxidant enzyme activity in chickens reared under normal and heat stress conditions.

MATERIALS AND METHODS

Location of work of experiment
 
The study was conducted during the year 2024-25 in the Division of Veterinary Physiology and Biochemistry, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology Jammu, R.S. Pura, J and K.
 
Experimental birds under study and housing
 
Total number of 300 one day old Cobb strain chicks obtained from Venky’s Private Limited, Satwari, Jammu were used in this study. The experiment was started in the month of mid-April and ended in the month of May in their growth phase for a period of 42 days with the birds reared under standard managemental practices. The rearing of birds was done in two phases viz. starter phase (day 1 to day 14) and grower phase (day 15 to day 42). Deep litter system with floor area @ 0.5 sq ft per chick with light intensity of 20 lux and duration of 24 hours of light was used. The birds were individually weighed before being randomly assigned to 15 pens with 20 birds in each pen. The chicks were randomly distributed in different groups of control and treatment viz. C1, C2, T1, T2 and T3 with 3 replicates each containing 20 chicks.
 
Dietary regime
 
The diet for chicks was formulated as per NRC recommendations (1994), the composition of which is depicted in Table 1.

Table 1: Composition of the ration.


 
Grouping of the experimental birds
 
A total of 300 Cobb strain chicks were randomly distributed in five different groups of control (C1 and C2) and treatment (T1, T2 and T3) with 3 replicates of each containing 20 chicks viz. group-I: C1 (Control group)- in which the birds were given the basal diet and kept under normal temperature, group-II: C2 (Control group)- in which the birds were given the basal diet and were kept under heat stress condition, group-III: T1(Treatment)- in which the birds were supplemented with powdered form of lemongrass @ 15mg/kg feed under heat stress condition, group-IV: T2 (Treatment)- in which the birds were supplemented with powdered form of aloe vera @ 1.5 mg/kg feed under heat stress condition and group-V: T3 (Treatment)- in which the birds were supplemented with a combination of powdered form of lemongrass and aloe vera @ 15 mg/kg and 1.5 mg/kg feed, respectively under heat stress condition.
 
Application of heat stress
 
The chicks were at first kept for acclimatization for 3 days after arrival. Heat stress was induced in C2, T1, T2 and T3 groups from day 4th to the termination of the experimental period by increasing the temperature using tarpaulin sheets and room heaters from 280C (4th day) to 48°C on day 17, then maintained at 48°C up to 42 days of trial period.
 
Sample collection and processing of samples
 
Collection of blood was done from 9 birds (3 birds from each replicate from each group) i.e. control (C1 and C2) and supplemented (T1, T2 and T3) groups on 3, 28 and 42 days of the experiment (Table 2).

Table 2: Effect of lemongrass and aloe vera supplementation on biochemical parameters (Mean±SE) in Cobb strain broilers under normal and heat stress conditions.


       
Blood collection was done from experimental birds through the experimental period on day 3, 28 and 42. Blood glucose was measured using Contour plus monitor glucometer. The collected blood samples were divided into two parts, one part (2 ml) was collected in aliquots with anticoagulant heparin @10 IU/ml of blood and the samples were subjected to centrifuge at 3000 rpm for a period of 15 minutes. The obtained erythrocyte sediment was diluted by gently pouring normal saline solution at the ratio of 1:1 on v/v basis and then thoroughly mixed with erythrocyte sediment. Then the diluted erythrocytes were centrifuged for a period of 10 minutes in 3000 rpm. After centrifugation, the supernatant was discarded along with buffy coat and then normal saline was added to the RBC on v/v basis, with gentle mixing and centrifugation and the process was repeated for three times. After the final washing, phosphate buffer solution (pH 7.4) was taken as diluent to make 1 percentage hemolysate (100 µl washed RBC +9.9 ml 0.1 M PBS) for antioxidant enzymes (GPx, SOD and Catalase) activities. The second 3ml was transferred to clean, dry and sterilized glass tube and kept in slanted position at room temperature for 3 to 4 hours, serum was collected in sterile tube and centrifuged at 3000 rpm for 10 minutes. Subsequently, the serum sample was separated and stored at -20°C for the biochemical estimation.
 
Biochemical parameters studied
 
Estimation of blood glucose was done by using contour plus monitor and the unit was expressed mg/dl. Similarly, estimation of total cholesterol was done by CHOD-PAP method (Allain et al., 1974) and analyzed by UV kinetics (IFCC) method by using analytical kits manufactured by Erba Mannheim, Solan HP India and the unit was expressed as mg/dl. Total Protein was estimated by using method describe by Fredman et al., (1980) and analysed by analytical kits manufactured by Erba Mannheim, Solan HP, India and the unit was expressed as g/dl. Estimation of creatinine was done by Jaffes method by Bartel (1972) and analyzed by analytical kits manufactured by Erba Mannheim, Solan (H.P), India and expressed the unit as mg/dl.
       
Antioxidant enzymes studied
 
The activity of glutathione peroxidase (GPx) in erythrocytes was assayed by the method of Hafeman et al. (1974). SOD activity was determined by method of Magnani et al. (2000) and the activity of catalase (CAT) in erythrocyte lysate was determined as per the method described by Aebi (1983).
 
Statistical analysis
 
All the generated data in the present experiment were subjected to standard statistical procedures (Snedecor and Cochran 2004). The data were mentioned with their mean and standard and were assessed by two-way analysis of variance. The data were analysed by polynomial contrast and Duncans post hoc multiple comparison at the significance of 0.05 level.

RESULTS AND DISCUSSION

Biochemical parameters
 
Blood glucose
 
In the study, it was observed that the blood glucose level (mg/dl) showed an increasing tendency with advancing age of the birds in all the experimental groups (Table 2). The results are in agreement with the findings of Sturkie (2000). However, Silva et al., (2007) in broilers and Nihad (2018) in Ross 308 of broiler found no interrelationship of age with the blood glucose levels. In contrast, Lakehal et al., (2015) found that the blood glucose significantly (P<0.05) decreased with the age in broilers.
       
Comparing the experimental groups in present study, significantly higher (P<0.05) glucose level was recorded in T1 group as compared to heat stress control group (C2) and T2 group on day 3 of experiment (Table 2). No significant variations were seen among different groups on day 28 and 42 of trial period, however, the levels were higher in C2 group on day 28 (287.33±6.58 mg/dl) and day 42 (298.89±2.41 mg/dl) when compared to normal control and lemongrass and aloe vera supplemented groups. Heat stress raises glucose via glucocorticoids (Silva et al., 2011). Aloe vera lowers glucose by improving insulin response (Parade et al., 2017; Rajasekaran et al., 2001), while Tekce and Gul (2017) and Safamehr et al., (2012) found no effect of lemongrass or aloe vera on glucose. Parade et al., (2017) reported increased glucose with higher lemongrass supplementation.
 
Total cholesterol
 
As reflected in the current study (Table 2), the total cholesterol level incremented significantly (P<0.05) as age advanced in all the experimental groups similar to the findings of Oloyo et al., (2003) and Elsabbagh (2011) in broilers.
       
The present findings showed higher value of cholesterol in C2 group on day 28 and 42 as compared to normal control (C1) and supplemented (T1, T2, T3) groups; however, there was no significant difference observed among the treatment groups (Table 2). Heat stress increases cholesterol in broilers at 21 days (Antonio et al., 2017). Lemongrass lowers serum cholesterol due to flavonoids and alkaloids that inhibit HMG-CoA reductase, the key enzyme in cholesterol synthesis (Ganjewala, 2009; Krishan and Narang, 2014; Crowell, 1999). Our results align with Mukhtar et al., (2012) and Olorunnisola et al., (2014), who reported significant cholesterol reduction with lemongrass oil supplementation in broilers. However, Tekeli et al., (2011) found no effect of lemongrass oil on cholesterol levels.
       
Naghi Shokri et al. (2017) showed that aloe vera gel reduced total blood cholesterol in poultry. Acemannan, its main polysaccharide contained in aloe vera, regulates liver fat metabolism. Birds fed with 1.5% aloe vera gel in drinking water had significantly lower cholesterol. Taraneh (2016) also found decreased cholesterol with 3% aloe vera solution in drinking water.
 
Total protein
 
The total protein levels showed an increasing trend in control and supplemented groups with the advancement of age from day 3 to 42 (Table 2), as also reported by Filipovic et al., (2007) and Piotrowska et al. (2011) in Ross broiler chickens and Huang et al. (2018) in broilers. Again, the value of total protein was higher in C2 group on day 28 and 42 of experiment as compared to C1 and supplemented (T1, T2 and T3) groups; however, no significant difference was observed among the treatment groups as also mentioned by Khattak et al., (2014). Sharma and Singh (2018) and Xie et al. (2015) stated no significant differences in total protein level among the birds fed dietary aloe vera. In contrast, Singh et al., (2013) recorded that the total protein values were recorded to be the highest in the broilers fed with lemongrass essential oil at the levels of 150, 300 and 450 mg/kg when compared with that of the non-supplemented (control) group. Alzawqari et al. (2016) also illustrated similar trend as compared to birds supplemented with antibiotic growth promoters.
 
Creatinine
 
In this study, the values of creatinine in treatment groups (T1, T2 and T3) increased on 28 day and then declined on day 42; However, in C1 (normal temperature) and C2 (heat stress) groups creatinine level increased significantly (P<0.05) with age (Table 2). A significant enhancement (P<0.05) in the values of creatinine level with the advancement of age was also reported by Silva et al., (2007) in Hybro PG broilers, Lakehal et al., (2015) in Arbor Acres strain of broiler and Kawasaki et al., (2016) in broilers.
       
As compared to C1 and supplemented (T1, T2 and T3) groups, the level of creatinine was found to be significantly higher (P<0.05) in C2 group on day 28 and 42 of the experiment. Stress has been linked to elevated creatinine levels in broiler chicken (Huang et al., 2020; Sugiharto, 2021; Reddy et al., 2018 and Ghanima et al., 2021). Zhang et al. (2018) indicated that the lemongrass can be a beneficial natural-base resource for decreasing the oxidative stress in growing poultry. In contrast, Sharma and Singh (2018) observed no significant differences in creatinine level of broilers supplemented with aloe vera when compared with non-supplemented group.
 
Antioxidant enzyme activities
       
Increasing trend of Superoxide dismutase (SOD), Glutathionine peroxide (GPx) and Catalase enzyme activities were observed with advancing of age of the birds in all the experimental groups (Table 3). While comparing the different enzyme activities on day 42 with day 3 and 28 of experiment, significantly higher (P<0.05) GPx activities were observed in C2 and supplemented groups whereas; significantly higher (P<0.05) SOD activities were observed in C1, C2 and supplemented groups and significantly higher (P<0.05) catalase activities were found only in C1 and C2 groups. Increased oxidative enzyme activities with increase of age in broilers was also reported by Torres et al., (2013) and Stahl et al., (2003).

Table 3: Effect of lemongrass and aloe vera on antioxidant enzymes activity (Mean±SE) in Cobb strain broilers under normal and heat stress condition.


       
In this study, significantly elevated (P<0.05) activities of SOD, GPx and catalase were recorded in C2 group while compared to other experimental groups of chicken on day 42 of trial period. Pamok et al., (2009) found that broilers usually suffer from oxidative stress induced by heat stress and in intensive farming system and infectious disease processes that resulted from the overproduction of free radicals and the disfunction of antioxidative defense mechanism to degenerate these radicals. Extracts of lemongrass and aloe vera inhibited the oxidative stress (Ojo et al., 2006). Citral is the well-known key antioxidant constituent of lemongrass essential oil which can also anchor the endogenous antioxidant defense system in alveolar macrophages cells through augmenting superoxide dismutase and glutathione activity (Nambiar, 2012). Matela (2012) observed that the dietary supplementation of lemongrass essential oil has significantly augmented the Superoxide dismutase, Glutathionine peroxide and catalase activities as compared to the antibiotic growth promoters as also observed by Mishra and Jha (2019) in birds that received 1 or 1.5% of aloe vera gel. Birds treated with aloe vera gel in drinking water had enhanced GPx activity (Maini et al., 2007). Ghanima et al., (2021) and Li et al., (2019) reported significant reduction of SOD and GPx activities during heat stress condition. Daader et al. (2018)  dietary lemongrass essential oil supplementation reduced the glutathione peroxidase and catalase activity of growing rabbits.
       
The mortality rate of the birds in the treatment groups was recorded as nil.

CONCLUSION

The present study enlightened that the incorporation of lemongrass and aloe vera in feed of chicken significantly elevated antioxidant parameters of blood as compared to heat stressed group. Significantly higher (P<0.05) activities of oxidative enzymes under study were recorded in all the groups at the final stages of the experiment as compared to earlier periods. Higher levels of cholesterol and creatinine were found in heat stressed birds (C2 group) on day 28 and 42 as compared to supplemented groups. However, no significant variations were observed in glucose and total protein levels among different groups. Again, the birds provided with combination of lemongrass and aloe vera @ 15 mg + 1.5 mg/kg i.e. T3 group exhibited the best results followed by birds fed aloe vera alone @ 1.5 mg/kg (T2 group) and then the birds provided with lemongrass only @ 15 mg/kg (T1 group).

ACKNOWLEDGEMENT

The present study was supported by Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J and K).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are 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.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were duly approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Effect of Heat Stress on Biochemical Parameters and Anti-oxidant Enzyme Activity of Cobb Strain Chicks Supplemented with Lemongrass (Cymbopogon citratus) and Aloe vera (Aloe barbadensis)

    S
    Simranjeet Kour1
    0009-0002-0061-6713
    J
    Jonali Devi1,*
    0000-0001-6630-2659
    K
    Kamal Sarma2
    0000-0002-6333-5516
    K
    Kawardeep Kour1
    0009-0008-3150-6502
    D
    Dibyendu Chakraborty3
    0000-0001-7411-0913
    J
    Jasvinder Singh Sasan2
    0000-0002-7175-477X
    1Division of Veterinary Physiology and Biochemistry, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.
    2Division of Veterinary Anatomy, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.
    3Division of Animal Genetics and Breeding, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, Jammu-181 102, Jammu and Kashmir, India.

    ABSTRACT

    Background: The study designed to investigate the impact of lemongrass and aloe vera on biochemical parameters and antioxidant activity in chicks under normal and heat stress conditions.

    Methods: A total number of three hundred chicks were randomly distributed into different control and treatment groups, with three replicates having 20 chicks in each group. The chicks were kept for acclimatization for three days and then exposed to heat stress from day 4 to the termination of the experimental period. Blood samples were collected from nine birds (three birds from each replicate from each group) on days 3, 28 and 42 to analyze biochemical parameters and antioxidant enzyme activities.

    Result: The results revealed that creatinine levels increased significantly (P<0.05) with age in C1 and C2 groups, with significantly higher (P<0.05) levels observed in the C2 group on days 28 and 42. Between the control groups, significantly higher (P<0.05) values of creatinine were recorded in C2 group and among treatment groups, higher values of creatinine were observed in T2 group. With the advancement of age, an increasing trend of SOD, GPx and Catalase enzyme activities were obtained in all the experimental groups. Between the control groups, higher antioxidant enzyme activities were recorded in C2 group. Among the treatment groups, lower activities of antioxidant parameters were recorded in T3.

    INTRODUCTION

    The poultry industry is vital to the Indian economy, with high demand for chicken meat. Cobb strain broilers are the most efficient breed due to low production cost, superior feed efficiency and fast growth. After the ban on antibiotic growth promoters, natural herbs like aloe vera, fenugreek, ashwagandha, moringa, cinnamon, tulsi, garlic and pepper are being used as alternatives (Lewis, 1986; Abd El-Hack et al., 2022). Lemongrass contains flavonoids, phenolic compounds and essential oils with antibacterial, antifungal and antioxidant properties that improve poultry growth and performance (Shah et al., 2011) and reproductive parameters (Aswathi et al., 2019) in broilers. Aloe vera improves growth, carcass quality, intestinal health, immunity and reduces production costs. Chickens maintain a core body temperature of ~41°C, so when they are exposed to thermal challenges, they cannot maintain a balance between the heat generation and heat removal from the body, resulting in incremented core body temperature that leads to mortality. Therefore, this study has been undertaken to evaluate the effect of lemongrass and aloe vera on biochemical parameters and antioxidant enzyme activity in chickens reared under normal and heat stress conditions.

    MATERIALS AND METHODS

    Location of work of experiment
     
    The study was conducted during the year 2024-25 in the Division of Veterinary Physiology and Biochemistry, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology Jammu, R.S. Pura, J and K.
     
    Experimental birds under study and housing
     
    Total number of 300 one day old Cobb strain chicks obtained from Venky’s Private Limited, Satwari, Jammu were used in this study. The experiment was started in the month of mid-April and ended in the month of May in their growth phase for a period of 42 days with the birds reared under standard managemental practices. The rearing of birds was done in two phases viz. starter phase (day 1 to day 14) and grower phase (day 15 to day 42). Deep litter system with floor area @ 0.5 sq ft per chick with light intensity of 20 lux and duration of 24 hours of light was used. The birds were individually weighed before being randomly assigned to 15 pens with 20 birds in each pen. The chicks were randomly distributed in different groups of control and treatment viz. C1, C2, T1, T2 and T3 with 3 replicates each containing 20 chicks.
     
    Dietary regime
     
    The diet for chicks was formulated as per NRC recommendations (1994), the composition of which is depicted in Table 1.

    Table 1: Composition of the ration.


     
    Grouping of the experimental birds
     
    A total of 300 Cobb strain chicks were randomly distributed in five different groups of control (C1 and C2) and treatment (T1, T2 and T3) with 3 replicates of each containing 20 chicks viz. group-I: C1 (Control group)- in which the birds were given the basal diet and kept under normal temperature, group-II: C2 (Control group)- in which the birds were given the basal diet and were kept under heat stress condition, group-III: T1(Treatment)- in which the birds were supplemented with powdered form of lemongrass @ 15mg/kg feed under heat stress condition, group-IV: T2 (Treatment)- in which the birds were supplemented with powdered form of aloe vera @ 1.5 mg/kg feed under heat stress condition and group-V: T3 (Treatment)- in which the birds were supplemented with a combination of powdered form of lemongrass and aloe vera @ 15 mg/kg and 1.5 mg/kg feed, respectively under heat stress condition.
     
    Application of heat stress
     
    The chicks were at first kept for acclimatization for 3 days after arrival. Heat stress was induced in C2, T1, T2 and T3 groups from day 4th to the termination of the experimental period by increasing the temperature using tarpaulin sheets and room heaters from 280C (4th day) to 48°C on day 17, then maintained at 48°C up to 42 days of trial period.
     
    Sample collection and processing of samples
     
    Collection of blood was done from 9 birds (3 birds from each replicate from each group) i.e. control (C1 and C2) and supplemented (T1, T2 and T3) groups on 3, 28 and 42 days of the experiment (Table 2).

    Table 2: Effect of lemongrass and aloe vera supplementation on biochemical parameters (Mean±SE) in Cobb strain broilers under normal and heat stress conditions.


           
    Blood collection was done from experimental birds through the experimental period on day 3, 28 and 42. Blood glucose was measured using Contour plus monitor glucometer. The collected blood samples were divided into two parts, one part (2 ml) was collected in aliquots with anticoagulant heparin @10 IU/ml of blood and the samples were subjected to centrifuge at 3000 rpm for a period of 15 minutes. The obtained erythrocyte sediment was diluted by gently pouring normal saline solution at the ratio of 1:1 on v/v basis and then thoroughly mixed with erythrocyte sediment. Then the diluted erythrocytes were centrifuged for a period of 10 minutes in 3000 rpm. After centrifugation, the supernatant was discarded along with buffy coat and then normal saline was added to the RBC on v/v basis, with gentle mixing and centrifugation and the process was repeated for three times. After the final washing, phosphate buffer solution (pH 7.4) was taken as diluent to make 1 percentage hemolysate (100 µl washed RBC +9.9 ml 0.1 M PBS) for antioxidant enzymes (GPx, SOD and Catalase) activities. The second 3ml was transferred to clean, dry and sterilized glass tube and kept in slanted position at room temperature for 3 to 4 hours, serum was collected in sterile tube and centrifuged at 3000 rpm for 10 minutes. Subsequently, the serum sample was separated and stored at -20°C for the biochemical estimation.
     
    Biochemical parameters studied
     
    Estimation of blood glucose was done by using contour plus monitor and the unit was expressed mg/dl. Similarly, estimation of total cholesterol was done by CHOD-PAP method (Allain et al., 1974) and analyzed by UV kinetics (IFCC) method by using analytical kits manufactured by Erba Mannheim, Solan HP India and the unit was expressed as mg/dl. Total Protein was estimated by using method describe by Fredman et al., (1980) and analysed by analytical kits manufactured by Erba Mannheim, Solan HP, India and the unit was expressed as g/dl. Estimation of creatinine was done by Jaffes method by Bartel (1972) and analyzed by analytical kits manufactured by Erba Mannheim, Solan (H.P), India and expressed the unit as mg/dl.
           
    Antioxidant enzymes studied
     
    The activity of glutathione peroxidase (GPx) in erythrocytes was assayed by the method of Hafeman et al. (1974). SOD activity was determined by method of Magnani et al. (2000) and the activity of catalase (CAT) in erythrocyte lysate was determined as per the method described by Aebi (1983).
     
    Statistical analysis
     
    All the generated data in the present experiment were subjected to standard statistical procedures (Snedecor and Cochran 2004). The data were mentioned with their mean and standard and were assessed by two-way analysis of variance. The data were analysed by polynomial contrast and Duncans post hoc multiple comparison at the significance of 0.05 level.

    RESULTS AND DISCUSSION

    Biochemical parameters
     
    Blood glucose
     
    In the study, it was observed that the blood glucose level (mg/dl) showed an increasing tendency with advancing age of the birds in all the experimental groups (Table 2). The results are in agreement with the findings of Sturkie (2000). However, Silva et al., (2007) in broilers and Nihad (2018) in Ross 308 of broiler found no interrelationship of age with the blood glucose levels. In contrast, Lakehal et al., (2015) found that the blood glucose significantly (P<0.05) decreased with the age in broilers.
           
    Comparing the experimental groups in present study, significantly higher (P<0.05) glucose level was recorded in T1 group as compared to heat stress control group (C2) and T2 group on day 3 of experiment (Table 2). No significant variations were seen among different groups on day 28 and 42 of trial period, however, the levels were higher in C2 group on day 28 (287.33±6.58 mg/dl) and day 42 (298.89±2.41 mg/dl) when compared to normal control and lemongrass and aloe vera supplemented groups. Heat stress raises glucose via glucocorticoids (Silva et al., 2011). Aloe vera lowers glucose by improving insulin response (Parade et al., 2017; Rajasekaran et al., 2001), while Tekce and Gul (2017) and Safamehr et al., (2012) found no effect of lemongrass or aloe vera on glucose. Parade et al., (2017) reported increased glucose with higher lemongrass supplementation.
     
    Total cholesterol
     
    As reflected in the current study (Table 2), the total cholesterol level incremented significantly (P<0.05) as age advanced in all the experimental groups similar to the findings of Oloyo et al., (2003) and Elsabbagh (2011) in broilers.
           
    The present findings showed higher value of cholesterol in C2 group on day 28 and 42 as compared to normal control (C1) and supplemented (T1, T2, T3) groups; however, there was no significant difference observed among the treatment groups (Table 2). Heat stress increases cholesterol in broilers at 21 days (Antonio et al., 2017). Lemongrass lowers serum cholesterol due to flavonoids and alkaloids that inhibit HMG-CoA reductase, the key enzyme in cholesterol synthesis (Ganjewala, 2009; Krishan and Narang, 2014; Crowell, 1999). Our results align with Mukhtar et al., (2012) and Olorunnisola et al., (2014), who reported significant cholesterol reduction with lemongrass oil supplementation in broilers. However, Tekeli et al., (2011) found no effect of lemongrass oil on cholesterol levels.
           
    Naghi Shokri et al. (2017) showed that aloe vera gel reduced total blood cholesterol in poultry. Acemannan, its main polysaccharide contained in aloe vera, regulates liver fat metabolism. Birds fed with 1.5% aloe vera gel in drinking water had significantly lower cholesterol. Taraneh (2016) also found decreased cholesterol with 3% aloe vera solution in drinking water.
     
    Total protein
     
    The total protein levels showed an increasing trend in control and supplemented groups with the advancement of age from day 3 to 42 (Table 2), as also reported by Filipovic et al., (2007) and Piotrowska et al. (2011) in Ross broiler chickens and Huang et al. (2018) in broilers. Again, the value of total protein was higher in C2 group on day 28 and 42 of experiment as compared to C1 and supplemented (T1, T2 and T3) groups; however, no significant difference was observed among the treatment groups as also mentioned by Khattak et al., (2014). Sharma and Singh (2018) and Xie et al. (2015) stated no significant differences in total protein level among the birds fed dietary aloe vera. In contrast, Singh et al., (2013) recorded that the total protein values were recorded to be the highest in the broilers fed with lemongrass essential oil at the levels of 150, 300 and 450 mg/kg when compared with that of the non-supplemented (control) group. Alzawqari et al. (2016) also illustrated similar trend as compared to birds supplemented with antibiotic growth promoters.
     
    Creatinine
     
    In this study, the values of creatinine in treatment groups (T1, T2 and T3) increased on 28 day and then declined on day 42; However, in C1 (normal temperature) and C2 (heat stress) groups creatinine level increased significantly (P<0.05) with age (Table 2). A significant enhancement (P<0.05) in the values of creatinine level with the advancement of age was also reported by Silva et al., (2007) in Hybro PG broilers, Lakehal et al., (2015) in Arbor Acres strain of broiler and Kawasaki et al., (2016) in broilers.
           
    As compared to C1 and supplemented (T1, T2 and T3) groups, the level of creatinine was found to be significantly higher (P<0.05) in C2 group on day 28 and 42 of the experiment. Stress has been linked to elevated creatinine levels in broiler chicken (Huang et al., 2020; Sugiharto, 2021; Reddy et al., 2018 and Ghanima et al., 2021). Zhang et al. (2018) indicated that the lemongrass can be a beneficial natural-base resource for decreasing the oxidative stress in growing poultry. In contrast, Sharma and Singh (2018) observed no significant differences in creatinine level of broilers supplemented with aloe vera when compared with non-supplemented group.
     
    Antioxidant enzyme activities
           
    Increasing trend of Superoxide dismutase (SOD), Glutathionine peroxide (GPx) and Catalase enzyme activities were observed with advancing of age of the birds in all the experimental groups (Table 3). While comparing the different enzyme activities on day 42 with day 3 and 28 of experiment, significantly higher (P<0.05) GPx activities were observed in C2 and supplemented groups whereas; significantly higher (P<0.05) SOD activities were observed in C1, C2 and supplemented groups and significantly higher (P<0.05) catalase activities were found only in C1 and C2 groups. Increased oxidative enzyme activities with increase of age in broilers was also reported by Torres et al., (2013) and Stahl et al., (2003).

    Table 3: Effect of lemongrass and aloe vera on antioxidant enzymes activity (Mean±SE) in Cobb strain broilers under normal and heat stress condition.


           
    In this study, significantly elevated (P<0.05) activities of SOD, GPx and catalase were recorded in C2 group while compared to other experimental groups of chicken on day 42 of trial period. Pamok et al., (2009) found that broilers usually suffer from oxidative stress induced by heat stress and in intensive farming system and infectious disease processes that resulted from the overproduction of free radicals and the disfunction of antioxidative defense mechanism to degenerate these radicals. Extracts of lemongrass and aloe vera inhibited the oxidative stress (Ojo et al., 2006). Citral is the well-known key antioxidant constituent of lemongrass essential oil which can also anchor the endogenous antioxidant defense system in alveolar macrophages cells through augmenting superoxide dismutase and glutathione activity (Nambiar, 2012). Matela (2012) observed that the dietary supplementation of lemongrass essential oil has significantly augmented the Superoxide dismutase, Glutathionine peroxide and catalase activities as compared to the antibiotic growth promoters as also observed by Mishra and Jha (2019) in birds that received 1 or 1.5% of aloe vera gel. Birds treated with aloe vera gel in drinking water had enhanced GPx activity (Maini et al., 2007). Ghanima et al., (2021) and Li et al., (2019) reported significant reduction of SOD and GPx activities during heat stress condition. Daader et al. (2018)  dietary lemongrass essential oil supplementation reduced the glutathione peroxidase and catalase activity of growing rabbits.
           
    The mortality rate of the birds in the treatment groups was recorded as nil.

    CONCLUSION

    The present study enlightened that the incorporation of lemongrass and aloe vera in feed of chicken significantly elevated antioxidant parameters of blood as compared to heat stressed group. Significantly higher (P<0.05) activities of oxidative enzymes under study were recorded in all the groups at the final stages of the experiment as compared to earlier periods. Higher levels of cholesterol and creatinine were found in heat stressed birds (C2 group) on day 28 and 42 as compared to supplemented groups. However, no significant variations were observed in glucose and total protein levels among different groups. Again, the birds provided with combination of lemongrass and aloe vera @ 15 mg + 1.5 mg/kg i.e. T3 group exhibited the best results followed by birds fed aloe vera alone @ 1.5 mg/kg (T2 group) and then the birds provided with lemongrass only @ 15 mg/kg (T1 group).

    ACKNOWLEDGEMENT

    The present study was supported by Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J and K).
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are 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.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were duly approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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