Effect of Glucose Supplementation on Apoptosis in the Pectoralis major of Chickens Raised under Thermoneutral or Heat Stress Environment

Reduced feed intake during heat stress (HS) disrupts glucose homeostasis, thereby resulting in endoplasmic reticulum (ER) stress and triggering apoptosis in chickens. We hypothesize that glucose supplementation could reduce apoptosis in chickens raised under HS. This study comprised 456 28-day-old broiler chickens randomly assigned to four treatment combinations under glucose supplementation and HS. The treatments were TN0, TN6, HS0, and HS6 with two glucose levels (0% and 6%) and two temperature levels (25 °C (thermoneutral-TN) and 35 °C (8.00 AM to 8.00 PM, (HS)). After 7 days post-HS, the blood glucose level for the HS6 group was higher than for TN0, TN6, and HS0. We studied the mRNA expression of genes and caspase-3 activity in the four experimental groups. The expressions of GCN2, ATF4, CHOP, and FOXO3a increased during HS regardless of glucose supplementation, while PERK and MAFbx increased only under HS with glucose supplementation. We show that under TN conditions, glucose supplementation led to a significant increase in cellular apoptosis in the Pectoralis (P.) major. However, under HS with glucose, the level of apoptosis was similar to that of chickens raised under TN conditions with no glucose supplementation. The utility of glucose to curtail apoptosis under HS should be tested under other intense models of HS.


Introduction
Heat stress (HS) poses a significant challenge within the poultry industry, especially in regions characterized by high ambient temperatures.The ramifications of HS extend to various physiological and cellular changes in avian species, encompassing disturbances in glucose metabolism, oxidative stress, apoptosis, and the modulation of cellular signaling pathways [1].Apoptosis, a multifaceted physiological process, holds paramount importance in the preservation of tissue homeostasis, as it undertakes the pivotal role of eliminating compromised or malfunctioning cells, thereby upholding the integral function and structural integrity of tissues at large [2].
Notably, HS has been empirically observed to augment apoptotic occurrences across diverse avian tissue types, including the liver, spleen, and testes [2,3].Li [4] substantiated this phenomenon by demonstrating that HS in laying hens upregulated the expression of tumor necrosis factor-α (TNFα) and Fas ligand (FasL), thereby instigating apoptosis in the follicles of these layers.Moreover, the prolonged exposure of broilers to chronic HS led to muscular atrophy but the atrophy was attenuated when their dietary intake was supplemented with taurine, resulting in a significant reduction in muscle loss.This effect was attributed to the reversal of apoptosis induced by endoplasmic reticulum (ER) stress and the inhibition of protein breakdown [5].
Genes 2023, 14, 1922 2 of 11 Heat stress causes reduced feed intake, which leads to deficiencies in both nutrients and energy.This, in turn, triggers the activation of the general control nonderepressible-2 genes (GCN2) and adenosine monophosphate-activated protein kinase (AMPK) [6].The AMPK gene subsequently activates protein kinase RNA-like ER kinase (PERK), which plays a vital role in the cell's response to ER stress by triggering the unfolded protein response (UPR).Additionally, GCN2 detects amino acid deficiency and regulates cellular responses, including the inhibition of protein synthesis, promotion of autophagy, and modulation of gene expression, to restore cellular balance [7].Both GCN2 and PERK phosphorylate elongation transcription factor 2 α (EIF2α), triggering apoptosis in birds exposed to HS [8].Heat stress disrupts glucose homeostasis, leading to alterations in glucose utilization, transport, and signaling pathways in avian tissues due to reduced feed intake [9].
Glucose is a major source of energy for animals, and it has been shown to improve performance, reduce stress, and either enhance antioxidant capacity or accelerate oxidative stress [10,11].The interconnection between glucose metabolism and apoptosis is complex and multifaceted.Glucose does not only fuel metabolic activities but also serves as a signaling molecule that can modulate various cellular processes, including apoptosis.Glucose supplementation has been shown to defend against apoptosis induced by oxidative stress in hepatocytes [12] and cardiomyocytes [13].Also, glucose has been shown to reduce apoptosis in the skeletal muscle of mice subjected to hindlimb unloading [14].Glucose increases antioxidant capacity and reduces oxidative stress in the fatty livers of geese [11].However, high glucose concentrations have been shown to induce apoptosis in Schwann cells [15] and cardiac microvascular endothelial cells [16].
Despite the growing interest in the effects of glucose on apoptosis, the specific factors that modulate the apoptotic response to glucose supplementation in chickens under different thermal conditions remain to be fully elucidated.Understanding the role of glucose in regulating apoptosis under HS conditions is critical for developing effective management strategies to enhance bird welfare and productivity.Hence, this study investigated the effect of glucose supplementation on apoptosis in the P. major of chickens raised under HS or TN environment.

Ethical Clearance
The broiler chickens used in the current study were specifically reared at the Poultry Research Center of the University of Georgia, following the guidelines and regulations set by the Institutional Animal Care and Use Committee, University of Georgia.

Experimental Approach and Sample Collection
The present study used 456-day-old Cobb 500 chicks housed in floor pens.These broilers were fed different starter, grower, and finisher diets based on their respective growth phases.The experiment followed a full factorial design, where the birds were randomly assigned to four treatments with six replicates each, with an average starting weight of about 42 g at day 0.
The experimental design of this study included two factors, a heat stress (HS) and thermoneutral (TN) environment, each with two levels of glucose treatment (0% and 6%).Birds in the TN group were maintained at a constant environmental temperature of 25 • C (77 • F), whilst HS birds were kept under a temperature of 35 • C (95 • F) between the hours of 8.00 am and 8.00 pm from day 28 to 35.The birds had access to water and feed ad libitum, following standard animal care practices [17].In the treatment group, the drinking water of the birds was supplemented with D-glucose at either a 0% or 6% concentration from day 28 to 35.
Birds were then selected at random from each replicate group at the end of the experiment.Birds were euthanized humanely via cervical dislocation and the P. major samples were collected and stored at −86 • C.

Determining Blood Glucose Levels
Blood glucose levels were determined using the VetScan i-STAT ® 1 handheld blood analyzer, software version OSi15(A-500.3.36-1)manufactured by Abbott Laboratories, Abbott Park, IL, USA.We followed the manufacturer's instructions carefully to ensure proper calibration and the accurate measurement of blood glucose levels.Blood samples of 75 µL (two drops) were collected from the jugular veins of birds and loaded onto the i-STAT cartridge.The loaded cartridge was inserted into the i-STAT analyzer, and the analysis was initiated.The i-STAT system utilizes an electrochemical detection method to measure glucose levels in the blood sample.

Gene Expression Analysis via RT-qPCR
Total mRNA was extracted from the P. major samples using Trizol reagent according to the established protocol of the manufacturer.Subsequently, complementary DNA (cDNA) synthesis was carried out using the total mRNA as a template with a high-capacity reverse transcriptase kit.Real-time quantitative PCR analysis (RT-qPCR) was conducted to measure the relative expression levels of the target genes.The cDNA samples obtained from the previous step were used as templates for the RT-qPCR reaction in triplicate.The reaction mixture, containing specific primers (Appendix A) with 2× SYBER ® (Applied Biosystems, Carlsbad, CA, USA), was prepared according to the manufacturer's instructions.Amplification was carried out under the following cycling conditions: 95 • C for 15 s and 60 • C for 30 s, for 35 cycles.The threshold cycle (CT) values were determined at the end of each cycle.The relative gene expression levels for general control nonderepessible-2 (GCN2), protein kinase-like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF4), C/EBP homologous protein (CHOP), eukaryotic translation initiation factor 2a (EIF2α), muscle atrophy forkhead box (MAFbx), and forkhead box 3a (FOXO3a) were then calculated using the 2 −∆∆CT method [18] and analyzed statistically to determine any significant differences between TN 0 , TN 6 , HS 0 , and HS 6 groups.

Colorimetric Caspase-3 Assay
The activity of caspase-3 was measured using a colorimetric caspase-3 assay kit following the manufacturer's protocol (ab39401 Abcam, Waltham, MA, USA).Caspase-3 is an executioner caspase that results in apoptosis by breaking down proteins.A total of 24,100 mg P. major tissue samples were homogenized in 800 µL of chilled lysis buffer at a pH of 7.5.The resultant mixture was centrifuged at 10,000× g for 10 min (4 • C).The supernatant was then kept on ice for 10 min and the accurate concentration of protein was determined immediately using the Pierce BCA Protein Assay Kit according to the manufacturer's guidelines (Thermo Scientific, Waltham, MA, USA, catalog number: 23227).The absorbance was measured at 562 nm using a Spectra Max 5 microplate reader.In each sample (200 µL), a 2× Reaction Buffer (50 µL) that contained 10 mM dithiothreitol was added in duplicate.This was followed by the addition of 4 mM DEVD-p-NA (5 µL) substrate to each duplicated sample with the exception of the negative control wells.Subsequently, the mixture was incubated in the dark at 37 • C for 90 min.The resulting output was then measured at an optical density (OD) of 400 nm using a microplate reader.

Statistical Analysis
The current study investigated the levels of blood glucose, gene expression changes in the apoptotic pathway, and the activities of caspase-3 in the TN 0 , TN 6 , HS 0 , and HS 6 groups.The data obtained were analyzed independently for the expression of different genes, blood glucose levels, and caspase-3 activity in all treatment groups (TN 0 , TN 6 , HS 0 , and HS 6 ) using two-way analysis of variance (ANOVA) [19].The generalized linear model (GLM) approach was used.The model used was as follows: where y ijK is either the blood glucose composition, relative mRNA expression level, or the caspase-3 activity; µ is the overall mean of the response; a i is the effect of the temperature; b j is the effect of glucose; (ab) ij is the interaction effect between temperature and glucose; and e ijk is the random error.The means were separated using Tukey-HSD [19] and significance among treatments was declared at p < 0.05.

Blood Glucose Levels
The blood glucose levels for TN 0 , TN 6 , HS 0 , and HS 6 are summarized in Figure 1.The blood glucose levels for the TN groups (TN 0 and TN 6 ) were lower (p < 0.05) than those of the HS 6 group.The HS groups (HS 0 and HS 6 ) had higher (p < 0.05) blood glucose levels than the TN 0 group.Also, the TN 0 group had the lowest (p < 0.05) levels of blood glucose compared with TN 6 , HS 0 , and HS 6 .Additionally, the blood glucose level for TN 6 was higher (p < 0.05) than for TN 0 .However, there were no differences (p > 0.05) in blood glucose levels between HS 0 and HS 6 , or between HS 0 and TN 6 .
(GLM) approach was used.The model used was as follows: where   is either the blood glucose composition, relative mRNA expression level, or t caspase-3 activity; μ is the overall mean of the response; ai is the effect of the temperatu bj is the effect of glucose; (ab)ij is the interaction effect between temperature and gluco and eijk is the random error.The means were separated using Tukey-HSD [19] and sign icance among treatments was declared at p < 0.05.

Blood Glucose Levels
The blood glucose levels for TN0, TN6, HS0, and HS6 are summarized in Figure 1.T blood glucose levels for the TN groups (TN0 and TN6) were lower (p < 0.05) than those the HS6 group.The HS groups (HS0 and HS6) had higher (p < 0.05) blood glucose leve than the TN0 group.Also, the TN0 group had the lowest (p < 0.05) levels of blood gluco compared with TN6, HS0, and HS6.Additionally, the blood glucose level for TN6 w higher (p < 0.05) than for TN0.However, there were no differences (p > 0.05) in blood gl cose levels between HS0 and HS6, or between HS0 and TN6.The mean blood glucose levels (±SEM) of birds raised under a thermoneutral or heat stress environment with or without glucose supplementation (n = 24).Treatment groups with di ferent connecting letters are considered significant (p < 0.05).TN0: thermoneutral with no glucose supplementation; TN6: thermoneutral with glucose supplementation; HS0: heat-stressed birds w no glucose supplementation; HS6: heat-stressed birds with glucose supplementation.

Gene Expression Analysis
The relative fold expression levels of GCN2, PERK, ATF4, CHOP, EIF2α, FOXO3a, and MAFbx are shown in Figure 2. The mRNA expressions of GCN2, ATF4, CHOP, and FOXO3a in the HS groups (HS 0 and HS 6 ) were significantly higher (p < 0.05) than those in the TN groups (TN 0 and TN 6 ).However, there were no differences (p > 0.05) between the TN 0 and TN 6 or the HS 0 and HS 6 groups for GCN2, ATF4, CHOP, and FOXO3a expression levels.Also, the mRNA expression level of PERK and MAFbx in the HS 6 group was significantly higher (p < 0.05) than in the TN 0 , TN 6 , and HS 0 groups.Nevertheless, HS 0 and TN 6 were Genes 2023, 14, 1922 5 of 11 comparable (p > 0.05) to the control group (TN 0 ) in the expression of PERK, but for MAFbx, only HS 0 was similar (p > 0.05) to TN 0 .Furthermore, ElF2α showed upward (p < 0.05) expressions for HS 6 when compared with TN 0 and TN 6 .The mRNA expression of EIF2α in the TN 6 group was significantly lower (p < 0.05) than that in the HS groups (HS 0 and HS 6 ) but comparable (p > 0.05) to TN 0 .aFOXO3a in the HS groups (HS0 and HS6) were significantly higher (p < 0.05) than those the TN groups (TN0 and TN6).However, there were no differences (p > 0.05) between TN0 and TN6 or the HS0 and HS6 groups for GCN2, ATF4, CHOP, and FOXO3a express levels.Also, the mRNA expression level of PERK and MAFbx in the HS6 group was sig icantly higher (p < 0.05) in the TN0, TN6, and HS0 groups.Nevertheless, HS0 and T were comparable (p > 0.05) to the control group (TN0) in the expression of PERK, but MAFbx, only HS0 was similar (p > 0.05) to TN0.Furthermore, ElF2α showed upward ( 0.05) expressions for HS6 when compared with TN0 and TN6.The mRNA expression EIF2α in the TN6 group was significantly lower (p < 0.05) than that in the HS groups (H and HS6) but comparable (p > 0.05) to TN0.a .The relative mRNA expressions of genes related to apoptosis in chickens either under heat stress or thermoneutral environment, regardless of their glucose levels (TN0, TN6, HS0, HS6 For each sample (n = 18), the RT-qPCR analysis was carried out three times.The output is expressed as 2 −∆∆CT (±SEM).Significant groups in the treatment are those with distinct letters (p < 0.05).TN0: thermoneutral with no glucose supplementation; TN6: thermoneutral with glucose s plementation; HS0: heat-stressed birds with no glucose supplementation; HS6: heat-stressed bird with glucose supplementation.

Caspase-3 Assay Analysis
The caspase-3 activities of TN0, TN6, HS0, and HS6 groups are presented in Figur After 7 d post-HS, the OD measured in the TN6 group for caspase-3 activity was sign cantly higher (p < 0.05) than in the TN0 and HS6 groups, but TN6 was similar (p > 0.05 that of the HS0 group.Also, there was no significant difference (p > 0.05) between the groups (HS0 and HS6) and TN0 group for the fold increase in caspase-3 activity.
Figure 2. The relative mRNA expressions of genes related to apoptosis in chickens either under a heat stress or thermoneutral environment, regardless of their glucose levels (TN 0 , TN 6 , HS 0 , HS 6 ).For each sample (n = 18), the RT-qPCR analysis was carried out three times.The output is expressed as 2 −∆∆CT (±SEM).Significant groups in the treatment are those with distinct letters (p < 0.05).TN 0 : thermoneutral with no glucose supplementation; TN 6 : thermoneutral with glucose supplementation; HS 0 : heat-stressed birds with no glucose supplementation; HS 6 : heat-stressed birds with glucose supplementation.

Caspase-3 Assay Analysis
The caspase-3 activities of TN 0 , TN 6 , HS 0 , and HS 6 groups are presented in Figure 3.After 7 d post-HS, the OD measured in the TN 6 group for caspase-3 activity was significantly higher (p < 0.05) than in the TN 0 and HS 6 groups, but TN 6 was similar (p > 0.05) to that of the HS 0 group.Also, there was no significant difference (p > 0.05) between the HS groups (HS 0 and HS 6 ) and TN 0 group for the fold increase in caspase-3 activity.

Effect of Exogenous Glucose on Blood Glucose Level
The current study aimed to investigate the effect of HS on blood glucose levels in meat-type chickens, and whether glucose supplementation could modulate blood glucose levels.There was a significant difference in blood glucose levels for the TN0, TN6, HS0, and HS6 groups, indicating the impact of HS and glucose supplementation on glucose homeostasis in broiler chickens.Consistent with previous research [20], HS birds exhibited elevated blood glucose levels compared to birds under TN conditions.Heat stress triggers corticosterone that can elevate blood glucose levels by stimulating gluconeogenesis and glycogenolysis in the liver [20].
Broilers receiving glucose supplementation displayed higher blood glucose levels compared to those without supplementation, regardless of their environmental conditions.This suggests that glucose supplementation directly influenced blood glucose concentrations and further contributed to glucose metabolism in broiler chickens.Glucose is the primary fuel source for cellular respiration and serves as a substrate for ATP production through glycolysis and the tricarboxylic acid (TCA) cycle [21].In the current study, the observed increase in blood glucose levels with glucose supplementation could be attributed to the availability of an exogenous source of glucose, which bypasses the endogenous glucose production pathways in both TN and HS birds.Exogenous glucose is readily absorbed and enters the bloodstream, leading to increased blood glucose concentrations [22,23].
Furthermore, glucose is transported into cells via glucose transporters, which are regulated by insulin signaling.Once inside the cell, glucose can undergo glycolysis to produce ATP or be converted into glycogen for storage in the liver and muscles [24].Elevated blood glucose concentrations influence hormonal regulation, particularly insulin secretion.Insulin is a key hormone involved in glucose metabolism, as it promotes glucose uptake by cells and inhibits glucose production in the liver [25].In general, elevated blood

Effect of Exogenous Glucose on Blood Glucose Level
The current study aimed to investigate the effect of HS on blood glucose levels in meat-type chickens, and whether glucose supplementation could modulate blood glucose levels.There was a significant difference in blood glucose levels for the TN 0 , TN 6 , HS 0 , and HS 6 groups, indicating the impact of HS and glucose supplementation on glucose homeostasis in broiler chickens.Consistent with previous research [20], HS birds exhibited elevated blood glucose levels compared to birds under TN conditions.Heat stress triggers corticosterone that can elevate blood glucose levels by stimulating gluconeogenesis and glycogenolysis in the liver [20].
Broilers receiving glucose supplementation displayed higher blood glucose levels compared to those without supplementation, regardless of their environmental conditions.This suggests that glucose supplementation directly influenced blood glucose concentrations and further contributed to glucose metabolism in broiler chickens.Glucose is the primary fuel source for cellular respiration and serves as a substrate for ATP production through glycolysis and the tricarboxylic acid (TCA) cycle [21].In the current study, the observed increase in blood glucose levels with glucose supplementation could be attributed to the availability of an exogenous source of glucose, which bypasses the endogenous glucose production pathways in both TN and HS birds.Exogenous glucose is readily absorbed and enters the bloodstream, leading to increased blood glucose concentrations [22,23].
Furthermore, glucose is transported into cells via glucose transporters, which are regulated by insulin signaling.Once inside the cell, glucose can undergo glycolysis to produce ATP or be converted into glycogen for storage in the liver and muscles [24].Elevated blood glucose concentrations influence hormonal regulation, particularly insulin secretion.Insulin is a key hormone involved in glucose metabolism, as it promotes glucose uptake by cells and inhibits glucose production in the liver [25].In general, elevated blood glucose concentrations contribute to glucose metabolism in birds by providing a readily available energy source and supporting anabolic processes, as observed in the HS 6 group of the current study.

Effect of Ambient Temperature and Glucose Supplementation on Apoptosis
The gene expression analysis provided valuable insights into the variations in the mRNA expression levels of GCN2, PERK, ATF4, CHOP, EIF2α, FOXO3a, and MAFbx in the HS and TN birds with or without glucose supplementation.The GCN2 gene is an important regulatory protein involved in cellular stress responses, including apoptosis.The current study revealed the expression of GCN2 and its downstream effect in relation to apoptosis in birds under different thermal conditions.In the current study, we observed a significant upward expression of GCN2 in birds subjected to HS regardless of their glucose supplementation levels as compared to the TN groups.The GCN2 gene is a protein kinase activated in response to cellular stress, particularly amino acid (AA) deprivation.The activation of GCN2 occurs when there is an imbalance or insufficiency of a specific AA, leading to the accumulation of uncharged transfer RNA (tRNA) molecules [26].Under normal physiological conditions, when AAs are abundant, tRNAs are charged with their respective AA and translation initiation proceeds.Amino acids are crucial for charging tRNA molecules, which are responsible for delivering AAs to the ribosomes during translation [27].However, during reduced feed intake, the decreased supply of dietary AAs results in the insufficient charging of tRNA molecules.Consequently, the accumulation of uncharged tRNAs serves as a signal for cellular stress and triggers the activation of GCN2, allowing it to phosphorylate its downstream target ElF2α, which initiates apoptosis.
Nevertheless, glucose availability plays a role in modulating the cellular energy status, while energy depletion affects activation of stress-responsive kinases like GCN2.It is worth noting that glucose metabolism can indirectly influence GCN2 activity [28].In situations where glucose levels are low or energy reserves are depleted, GCN2 activity may be upregulated as part of a broader cellular stress response and vice versa [7].Furthermore, glucose metabolism influences the regulation of AA transporters and AA homeostasis [29,30], potentially affecting GCN2 activation indirectly through alterations in AA availability.In the current study, GCN2 expression was not affected by the exogenous supply of glucose, but HS increased GCN2 expression.The expression of GCN2 may be more sensitive to nutrients than energy levels as there was no difference between the two HS groups.The mRNA expression of GCN2 across the treatments suggests that its expression changes was due to HS and not the exogenous glucose supply.
In contrast, PERK is a stress sensor activated in response to stress.Heat stress is known to induce cellular stress and trigger the unfolded protein response (UPR), a cellular signaling pathway that aims to restore protein homeostasis in the ER [31].Under prolonged HS, the UPR fails to restore ER homeostasis, leading to the induction of apoptosis through the activation of PERK.Also, immunoglobulin binding protein (BiP), an ER chaperone, normally binds and maintains PERK in an inactive state [32].However, during ER stress, BiP is released from PERK due to its binding to misfolded proteins.This dissociation exposes the activation domain of PERK.Once released from BiP, PERK undergoes autophosphorylation, which involves the phosphorylation of its own residues.This autophosphorylation leads to the activation of PERK and its downstream targets, including EIF2α, ATF4, and CHOP, to initiate apoptosis in tissues [32].
In the current study, the PERK expression level for HS birds without glucose supplementation (HS 0 ) was comparable to that of the control group (TN 0 ).However, a study by Seremelis [33] showed an increase in PERK expression in HS broiler chickens compared to in TN birds, indicating the activation of UPR.Another study by Ma [34] showed elevated PERK expression in HS broilers, also suggesting the induction of the ER stress response.In the current study, PERK expression for HS 0 was slightly higher than for TN 0 but the difference was not statistically significant.It was expected that glucose would modulate the mRNA expression of PERK in HS birds, but that was not the case.Birds under HS conditions supplemented with glucose (HS 6 ) showed a significant increase in the expression of PERK compared to TN 0 , TN 6 , and HS 0 .It should be pointed out that glucose could act as a substrate for glycosylation, a post-translational modification process that can influence protein folding and stability, thereby leading to protein dysfunction [35].The current study suggests that the exogenous supply of glucose may have contributed to ER stress, which subsequently induced the mRNA expression of PERK in HS 6 birds.
The activation of both GCN2 and PERK kinases phosphorylate EIF2α and ATF4 [36,37].In the current study, birds under HS with or without glucose supplementation exhibited an increase in ATF4 mRNA expression compared to their counterparts reared under TN conditions.The phosphorylation of EIF2α activates the integrated stress response pathway (ISR), which modulates apoptotic processes by regulating the expression of apoptotic regulators, caspases, and Bcl-2 family members [33].One of the key downstream targets of EIF2α phosphorylation is the transcription factor ATF4, which regulates the expression of a variety of genes involved in stress responses, including those associated with apoptosis.The ATF4 gene promotes apoptosis under persistent stress conditions by suppressing the expression of anti-apoptotic genes and promoting the transcription of pro-apoptotic genes such as CHOP, which plays a significant role in inducing apoptosis during the stress response [38].The mRNA expressions of ATF4, CHOP, and EIF2α were similar between the two HS groups, suggesting that the exogenous supply of glucose during HS had an insignificant effect on their mRNA abundances.
CHOP functions together with FOXO3a to induce apoptosis under stressful conditions.The activation of FOXO3a via dephosphorylation triggers a series of events that enhance the expression of genes responsible for apoptosis and cell cycle arrest [39].However, high glucose levels can induce the expression of FOXO3a, which, in turn, leads to apoptosis [16].In the current study, the HS group (HS 0 and HS 6 ) showed an increased level of FOXO3a as compared to the TN group (TN 0 and TN 6 ), indicating that FOXO3a expressions were mainly due to the effect of HS and not the exogenous supply of glucose.FOXO3a influences the mRNA expression of MAFbx [40] subsequently leading to atrophy.In the current study, the supplementation of glucose had opposing effects in the TN and HS birds.In the TN birds, glucose supplementation led to a decrease in MAFbx mRNA expression when compared with the TN 0 birds.However, in the HS birds, the glucose-supplemented birds had higher MAFbx expression compared with the TN 0 and HS 0 birds.Thus, HS does not appear to affect the mRNA expression of MAFbx.In a study reported by Zuo [41] from a 21-day continuous HS experiment (28-56 days of age), the expression of MAFbx in the P. major of broilers was not altered; however, there was a significant increase in the mRNA of MAFbx in the thigh muscles of HS birds compared to their control counterparts.In another study, Ma [34] reported an increase in MAFbx expression in the P. major of broiler chickens subjected to continuous HS for 35 days when compared with their TN controls.Changes in mRNA expression during HS may be affected by the experimental model and the tissues investigated.The duration of the current study was 7 days, and the HS model was cyclical and not continuous as in the studies of Zuo [41] and Ma [34].
We further proceeded to measure the activities of caspase-3 in the P. major of broilers under both TN and HS conditions, with or without glucose supplementation.Caspase-3 is a protease enzyme that plays a crucial role in the execution phase of apoptosis [42].In the caspase-3 assay, the DEVD-pNA sequence was used as a substrate to mimic the specific cleavage site recognized by caspase-3 [42].Intracellular caspase-3 could be activated by stress signaling molecules, and upon activation, cleaves the peptide bond between aspartic acid (D) and glutamic acid (E) residues.This cleavage event releases the pNA moiety.p-nitroanilide usually absorbs light at a specific wavelength (405 nm).Caspase-3 activity is directly proportional to the level of substrate to be cleaved.Under TN conditions, the exogenous supply of glucose led to an increase in blood glucose level.There was a significant increase in caspase-3 activity in the P. major of chickens supplied with exogenous glucose.There are reports that show that apoptosis is among the many cellular responses to high glucose levels [15,16].High glucose levels cause oxidative and nitrosative stress, leading to apoptosis and necrosis in various cell types [43].High glucose levels lead to the production of more ATP in the mitochondria.Increased ATP production triggers proapoptotic proteins such as cytochrome c to be released from the intramembrane space of the mitochondria into the cytosol [44].Cytochrome c then facilitates the allosteric activation of apoptosis protease-activating factor 1 (Apaf-1), which forms an apoptosome complex [45].Each apoptosome has the capacity to assemble apoptotic proteases, subsequently resulting in the catalytic development of caspase-3 and other caspases [46].Ariyo [47] showed that under TN conditions, supplementary glucose improves feed efficiency by significantly reducing feed intake and marginally improving growth.Despite the improvement in performance, it also leads to a significant increase in apoptosis.
There was a marginal increase in caspase-3 activity in the HS 0 birds compared with the TN 0 birds.The mRNA changes in the ATF4, CHOP, and FOXO3a genes in the HS 0 birds compared with the TN 0 birds did not translate into significant changes in cellular apoptotic levels.This may be due to the cyclical model and duration of HS.Interestingly, under HS, the supplementation of glucose maintained the apoptosis level, which was similar to birds in the TN 0 group.The HS 6 birds had reduced glucose and energy from their feed.Supplementary glucose putatively provided some of the extracellular glucose and energy required to mitigate some of the negative physiological effects of HS and reduced losses in protein biosynthesis.The effect of glucose supplementation on apoptosis should be studied further under a continuous HS model to ascertain its utility in the production system.

Conclusions
The current study showed that glucose could serve as an extra source of energy for broiler chickens reared under HS conditions.The extra glucose serves as extra energy that can be used to mitigate some of the negative effects of HS.Heat stress with or without glucose supplementation led to an increase in the mRNA expressions of the GCN2, ATF4, CHOP, EIF2α, and FOXO3a genes compared with birds reared under TN conditions without glucose supplementation.The mRNA expressions of PERK and MAFbx increased only in the HS group that was supplemented with glucose when compared with the TN nonglucose-supplemented group.The mRNA expression in the selected apoptotic genes in the current study did not translate to apoptosis according to the caspase-3 cellular assay.Under TN conditions, supplementation with glucose led to a significant increase in apoptosis.However, under HS conditions, supplementation with glucose did not change the level of apoptosis when compared with the TN non-glucose-supplemented group.

Figure 1 .
Figure 1.The mean blood glucose levels (±SEM) of birds raised under a thermoneutral or heat stress environment with or without glucose supplementation (n = 24).Treatment groups with di ferent connecting letters are considered significant (p < 0.05).TN0: thermoneutral with no glucose supplementation; TN6: thermoneutral with glucose supplementation; HS0: heat-stressed birds w no glucose supplementation; HS6: heat-stressed birds with glucose supplementation.

Figure 1 .
Figure 1.The mean blood glucose levels (±SEM) of birds raised under a thermoneutral or heat stress environment with or without glucose supplementation (n = 24).Treatment groups with different connecting letters are considered significant (p < 0.05).TN 0 : thermoneutral with no glucose supplementation; TN 6 : thermoneutral with glucose supplementation; HS 0 : heat-stressed birds with no glucose supplementation; HS 6 : heat-stressed birds with glucose supplementation.

Figure 2
Figure2.The relative mRNA expressions of genes related to apoptosis in chickens either under heat stress or thermoneutral environment, regardless of their glucose levels (TN0, TN6, HS0, HS6 For each sample (n = 18), the RT-qPCR analysis was carried out three times.The output is expressed as 2 −∆∆CT (±SEM).Significant groups in the treatment are those with distinct letters (p < 0.05).TN0: thermoneutral with no glucose supplementation; TN6: thermoneutral with glucose s plementation; HS0: heat-stressed birds with no glucose supplementation; HS6: heat-stressed bird with glucose supplementation.

Figure 3 .
Figure 3.The optical density measurement (±SEM) of caspase-3 activity in the P. major of broilers (n = 12) raised under a heat stress or thermoneutral environment, with or without glucose supplementation.The treatment groups with different connecting letters are considered significant (p < 0.05).TN0: thermoneutral with no glucose supplementation; TN6: thermoneutral with glucose supplementation; HS0: heat-stressed birds with no glucose supplementation; HS6: heat-stressed birds with glucose supplementation.

Figure 3 .
Figure 3.The optical density measurement (±SEM) of caspase-3 activity in the P. major of broilers (n = 12) raised under a heat stress or thermoneutral environment, with or without glucose supplementation.The treatment groups with different connecting letters are considered significant (p < 0.05).TN 0 : thermoneutral with no glucose supplementation; TN 6 : thermoneutral with glucose supplementation; HS 0 : heat-stressed birds with no glucose supplementation; HS 6 : heat-stressed birds with glucose supplementation.