
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00734-X
10.1016/j.psj.2024.104155
104155
PHYSIOLOGY AND REPRODUCTION
Prehatch thermal manipulation of embryos and posthatch baicalein supplementation increased liver metabolism, and muscle proliferation in broiler chickens
Al Amaz Sadid
Shahid Md Ahosanul Haque
Jha Rajesh
Mishra Birendra bmishra@hawaii.edu
1
Department of Human Nutrition, Food and Animal Sciences, College of Tropical Agriculture and Human Resources, University of Hawaiʻi at Manoa, Honolulu, HI 96822
1 Corresponding author: bmishra@hawaii.edu
03 8 2024
11 2024
03 8 2024
103 11 10415524 5 2024
29 7 2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The exposure of broiler chickens to high ambient temperatures causes heat stress (HS), negatively affecting their health and production performance. To mitigate heat stress in broilers, various strategies, including dietary, managerial, and genetic interventions, have been extensively tested with varying degrees of efficacy. For sustainable broiler production, it is imperative to develop an innovative approach that effectively mitigates the adverse effects of HS. Our previous studies have provided valuable insights into the effects of prehatch embryonic thermal manipulation (TM) and posthatch baicalein supplementation on embryonic thermotolerance, metabolism, and posthatch growth performance. This follow-up study investigated the effect of these interventions on gluconeogenesis and lipid metabolism in the liver, as well as muscle proliferation and regeneration capacity in heat-stressed broiler chickens. A total of six-hundred fertile Cobb 500 eggs were incubated for 21 d. After candling, 238 eggs were subjected to TM at 38.5°C with 55% relative humidity (RH) from embryonic day (ED) 12 to 18. These eggs were transferred to the hatcher and kept at a standard temperature (37.5°C) from ED 19 to 21, while 236 eggs were incubated at a controlled temperature (37.5°C) till hatch. After hatching, 180 day-old chicks from both groups were raised in 36 pens treatment (n = 10 birds/pen, 6 replicates per treatment). The treatments were: 1) Control, 2) TM, 3) Control heat stress (CHS), 4) Thermal manipulation heat stress (TMHS), 5) Control heat stress supplement (CHSS), and 6) Thermal manipulation heat stress supplement (TMHSS). Baicalein was added to the treatment group diets starting from d 1. All birds were raised under the standard environment for 21 d, followed by chronic heat stress from d 22 to 35 (32–33 ⁰C for 8 h) in the CHS, TMHS, CHSS, and TMHSS groups. A thermoneutral (22–24⁰C) environment was maintained in the Control and TM groups. RH was constant (50 ± 5%) throughout the trial. In the liver, TM significantly increased (P < 0.05) IGF2 expression. Baicalein supplementation significantly increased (P < 0.05) HSF3, HSP70, SOD1, SOD2, TXN, PRARα, and GHR expression. Moreover, the combination of TM and baicalein supplementation significantly increased (P < 0.05) the expression of HSPH1, HSPB1, HSP90, LPL, and GHR. In the muscle, TM significantly increased (P < 0.05) HSF3 and Myf5 gene expression. TM and baicalein supplementation significantly increased (P < 0.05) the expression of MyoG and significantly (P < 0.05) decreased mTOR and PAX7. In conclusion, the prehatch TM of embryos and posthatch baicalein supplementation mitigated the deleterious effects of HS on broiler chickens by upregulating genes related to liver gluconeogenesis, lipid metabolism, and muscle proliferation.

Key words

Antioxidant
broiler
embryo
gene expression
heat stress
==== Body
pmcINTRODUCTION

The natural environment is changing at an unprecedented rate due to human activity and continuous rises in temperature worldwide, making life physiologically more stressful. Nearly all the hottest years since the mid-19th century have occurred in the past decade (Stillman, 2019). With the intensity of global warming, heat stress (HS) has become a threat to animal health and production performance. HS is a significant challenge in the poultry industry, adversely affecting bird well-being, productivity, and carcass quality. Heat stress results in various physiological changes, including oxidative stress, acid-base imbalance, and suppressed immunocompetence. These changes lead to higher mortality, decrease feed efficiency, body weight, and feed intake, and affect the meat quality (Wasti et al., 2020). Modern broilers are particularly susceptible to HS due to intense genetic selection, hypoplasia of the sweat gland, high metabolic rates, restricted heat loss capacity, and high heat production (Nawab et al., 2018). HS can potentially modify various biological functions, likely resulting in behavioral adaptations in poultry to enhance their survival under stressful conditions. The body's inability to withstand elevated temperatures may lead to detrimental pathological alterations in various organs on macroscopic and microscopic levels (Rebez et al., 2023). The liver is one of the most vital organs impacted by high ambient temperature (Chen et al., 2017). Studies have demonstrated that chronic HS leads to the development of necrosis in the central lobular regions of the hypoxic liver in broiler chickens (Aengwanich and Simaraks, 2004). Furthermore, the hepatic cells of broilers exhibited fatty degeneration characterized by vacuolation, sinusoidal dilation, and necrosis with leukocyte granulation tissue, particularly in the centritubular region. Muscle is also greatly affected by the HS. The primary function of muscle tissue is to facilitate metabolic adaptation to negative energy balance and HS injury (Koch et al., 2016). Fat infiltration is a common pathological change in muscles during HS. When exposed to HS, the meat quality of chickens is significantly altered with increased fat content and reduced protein content (Zhang et al., 2012). Several strategies have been implemented to alleviate the adverse effects of HS on poultry production. These approaches include genetic, management, and nutritional strategies (Nawab et al., 2018). The genetic approach involved developing poultry lines with some genes such as the naked neck (Meteyake et al., 2023), frizzle (Yunis and Cahaner, 1999), and dwarf genes that help to reduce HS (Wasti et al., 2020). The management strategies encompass the utilization of suitable housing design, provision of shade, utilization of sprinklers, implementation of cooling devices, and utilization of fans and ventilation systems (Wasti et al., 2020).

Studies have been conducted to enhance the ability of embryonic thermal manipulation (TM) to withstand heat in poultry (Yahav et al., 2004; Loyau et al., 2013). In TM, during crucial stages of embryonic development, embryos are exposed to a comparatively higher incubation temperature to improve the thermotolerant capacity of the embryos (Yalçin et al., 2008; Al Amaz and Mishra, 2024), and higher thermotolerance can be attained through prompt thermal stress response, acclimation, and epigenetic temperature adaptation (Yahav, 2009). Also, it is well-established that nutritional programming can reduce the negative effects of HS in broiler chickens (Abdel-Moneim et al., 2021). The nutritional interventions involve the optimization of feed composition and the supplementation of vital micronutrients to enhance the productivity capabilities of poultry birds. In the poultry industry, the most commonly implemented nutritional strategies to promote optimal production entail the application of varied supplements such as fat, antioxidants, yeast, and electrolytes (Vandana et al., 2021). A primary flavonoid called baicalein (5,6,7 trihydroxy flavone) is obtained from the root of Scutellaria baicalensis Georgi, also known as Huang Qin or Chinese skullcap. It has been demonstrated that supplementing broiler diets with baicalein improved the animals' growth, immune response, antioxidant profile, and serum lipid metabolism (Zhou et al., 2019). Our study revealed that embryonic TM improved hatchability, thermotolerance capacity, and liver metabolism while decreasing hatch time (Al Amaz et al., 2024b). Furthermore, prehatch embryonic TM and posthatch baicalein supplementation enhanced body weight, average daily gain, average daily feed intake, feed conversion ratio, cecal microbial diversity, and volatile fatty acids of heat-stressed broilers (Al Amaz et al., 2024a).

Based on the efficiency of prehatch TM and posthatch baicalein supplementation, we hypothesized that prehatch TM and posthatch baicalein supplementation may alleviate oxidative stress and improve liver gluconeogenesis and lipid metabolism in the liver and promote muscle proliferation. In this follow-up study, we aimed to investigate the effects of TM and baicalein supplementation on the markers of liver oxidative stress, gluconeogenesis, lipid metabolism, muscle proliferation, and regeneration capacity in broiler chickens under heat stress.

MATERIALS AND METHODS

Animal Ethics Statement

All the animal experimentation was carried out following the guidelines approved by the Institutional Animal Care and Use Committee of the University of Hawaii (Approval No. 17-2605-6).

Experimental Design

This study used animal experimentation and samples from our previous study (Al Amaz et al., 2024a). Briefly, 600 fertilized eggs of the Cobb 500 broiler breed were sourced from a local hatchery (Asagi Hatchery Inc, Honolulu, HI). The eggs were randomly incubated into 3 incubators (GQF incubator, Savannah, GA; 200 eggs each) at standard temperature (37.5°C at 55% RH, 24h/d) for the first embryonic day (ED) 11. The eggs were candled to separate the live embryos (n = 474) to be used in the study. On ED 12, eggs were divided into 2 incubation groups: 1) Control (n = 236) (standard temperature until the hatch day, ED 21), and 2) TM group (n = 238) (38.5°C at 55% RH, 12 h/d, from ED 12 to ED 18 and standard temperature from ED 19 to ED 21) in 2 incubators for each treatment with automatic temperature control, 55% RH, and egg turning every 2 h.

Hatching and Rearing Management

The hatching rate in the control group was 91%, while in the TM group, it was 94.5% (Al Amaz et al., 2024b). After hatching, unsexed day-old chicks (n = 360) were divided equally into 2 primary cohorts: the Control (n = 180 from the Control group at hatch) and the TM (n = 180 from the TM group at hatch). Subsequently, 3 treatment groups were formed from each of the Control and TM cohorts, resulting in a total of 6 treatment groups. The posthatch treatments comprised: 1) Control, 2) Control heat stress (CHS), 3) Control heat stress baiclein supplement (CHSS), 4) Thermal manipulation (TM), 5) Thermal manipulation heat stress (TMHS), and 6) Thermal heat stress with baiclein supplement (TMHSS). The chicks were weighed individually, winged tagged, and randomly assigned in 36 pens (10 birds per pen), making 6 replicates for each treatment group (n = 60 birds per treatment). The Control and HS birds were raised in different rooms. The chicks were raised on a litter floor pen following standard Cobb-500 broiler rearing and management guidelines. From d 21 to d 35, birds in the CHS, CHSS, TMHS, and TMHSS groups were exposed to cyclic heat stress (33–35°C) from 8:00 to 18:00 h (to mimic the environmental temperature) and 22 to 24°C during the night with 55% RH. Meanwhile, the Control and TM groups were raised at standard room temperature (22–24°C) with 55% RH throughout the study. The birds were monitored thrice daily (morning, afternoon, and evening) to ensure proper management and health conditions. The pens were completely randomized in this study. The pen's size was 1 m × 0.61 m, and the stocking density was 1,500 cm2/bird. Wood shavings were used as litter. Birds were raised with a standard lighting system (23 h light: 1 h dark).

Diets

The corn-soybean meal-based basal diets were formulated in 2 phases; starter (d 1–21) and finisher (d22–35), to meet the nutritional requirements of the Cobb 500 broilers (Cobb Broiler Management Guide, 2021). Feed and water were supplied ad libitum throughout the study. Control, CHS, TM, and TMHS groups were fed the basal diet throughout the study. CHSS and TMHSS groups were fed baicalein supplemented (250 mg/kg) in basal diets throughout the study. Baicalein was purchased in powder form and mixed into the diets. The dose of the baicalein supplement was chosen based on its antioxidant properties and the dose rate used in rodents, cattle, and human studies. The diet's composition and nutrient profile are presented in Table 1.Table 1 Composition of experimental diets and their nutrient profile.

Table 1Ingredients, %	Starter diet (1–21 d)	Finisher diet (22–35 d)	
Control	Test	Control	Test	
Corn	53.67	53.67	60.84	60.84	
SBM	38.00	38.00	31.00	31.00	
Soybean oil	5.00	5.00	5.50	5.50	
Limestone	1.35	1.35	1.20	1.20	
Monocalcium phosphate	0.75	0.75	0.44	0.44	
Lysine	0.18	0.18	0.10	0.10	
Met	0.18	0.18	0.13	0.13	
Thr	0.04	0.04	0.00	0.00	
Tryptophan	0.00	0.00	0.00	0.00	
Choline Cl	0.00	0.00	0.00	0.00	
Nacl	0.20	0.20	0.18	0.18	
Sodium bicarbonate	0.12	0.12	0.10	0.10	
Vitamin+mineral mix	0.50	0.50	0.50	0.50	
Baicalein	0.00	0.00020	0.00	0.00020	
Phytase	0.01	0.01	0.01	0.01	
Total	100.00	100.00	100.00	100.00	
Nutrients Contents in the diet, %	
AMEn, kcal/kg	3040	3040	3165	3165	
CP	21.47	21.47	18.54	18.54	
Ca	0.91	0.91	0.77	0.77	
Total P	0.71	0.71	0.61	0.61	
AvP	0.45	0.45	0.37	0.37	
Lys	1.32	1.32	1.09	1.09	
Met	0.52	0.52	0.44	0.44	
Cys	0.42	0.42	0.40	0.40	
Thr	0.87	0.87	0.73	0.73	
Trp	0.31	0.31	0.27	0.27	
Met+Cys	0.92	0.92	0.82	0.82	
Arg	1.55	1.55	1.35	1.35	
Val	1.18	1.18	1.05	1.05	
Ile	0.90	0.90	0.78	0.78	
Leu	1.82	1.82	1.66	1.66	
NDF	8.86	8.86	8.73	8.73	
CF	3.84	3.84	3.51	3.51	
Na	0.16	0.16	0.14	0.14	
Cl	0.16	0.16	0.15	0.15	
Choline (mg/kg)	1371	1371	1224	1224	
dig Lys	1.17	1.17	0.95	0.95	
dig Met	0.48	0.48	0.40	0.40	
dig Thr	0.67	0.67	0.55	0.55	
Provides following nutrients (per kg of diet): vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E (all-rac-tocopherol-acetate), 30 mg; vitamin B1, 2 mg; vitamin B2, 8 mg; vitamin B6, 4 mg; vitamin B12 (cyanocobalamin), 0.025 mg; vitamin K3 (bisulfate menadione complex), 3mg; choline (choline chloride), 250 mg; nicotinic acid, 60 mg; pantothenic acid (D-calcium pantothenate), 15 mg; folic acid, 1.5 mg; betaíne anhydrous, 80 mg; D-biotin, 0.15 mg; zinc (ZnO), 80 mg; manganese (MnO), 70 mg iron (FeCO3), 60 mg; copper (CuSO4·5H2O), 8 mg; iodine (KI), 2 mg; selenium (Na2SeO3), 0.2 mg.

Sample Collection

At the end of the animal trial (d35), 1 bird from each pen (n = 6 per treatment) was euthanized using carbon dioxide asphyxiation for sampling. Liver tissues were collected from the middle portion of the lobe, and breast muscle tissues were collected from the central portion for all the birds. After sampling, tissue pieces were immediately snap-frozen, and stored at −80 °C until RNA extraction.

Quantitative real-time PCR

TRIzol reagent (Invitrogen, Carlsbad, CA) was used to isolate total RNAs from frozen tissues (50–100 mg) following the manufacturer's instructions. Total RNA concentration was determined using NanoDrop One (Thermo Fisher Scientific, Madison, WI). RNA quality was determined by running samples on 2% agarose. The RNA samples were stored at -80°C until further analysis. The expressions of candidate genes were analyzed using quantitative real-time PCR (qPCR) (Quant Studio 3, Applied Biosystems, Foster City, CA), as described previously (Chaudhary et al., 2023). NCBI Primer-Blast tool was used to design specific primer pairs for detecting each gene. 1 μg of total RNA (20 μL reaction of RT mixture) was reverse-transcribed into complementary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and then diluted with nuclease-free water (1:25). Using Real-time PCR system (Applied Biosystems, Foster City, CA), qPCR was performed with PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA). The qPCR reaction mixture contained 3 μL of cDNA, 5 μL of PowerUp SYBR Green Master Mix, and 1 μL of each forward and reversed primer at a concentration of 5 μmol to yield a final reaction volume of 10 μL. Standard cycling mode was utilized for the qPCR reaction. A melting curve was constructed to validate the SYBR Green-based objective amplicon. In addition, the specificity of each primer pair was determined by running the qPCR products through 1% gel electrophoresis. Three housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (ß-actin), and TATA-box binding protein (TBP), were analyzed in triplicate. The most stable housekeeping genes in the liver and muscle were GAPDH and β-actin, respectively, which were used to normalize target gene expression. The target genes were analyzed in duplicate, and the average value of each experimental replicate was calculated. The expression levels of target genes were determined using cycle threshold (Ct) values normalized with GAPDH in the liver and β-actin in the muscle. The fold change of each gene was determined using the 2−ΔΔCt method. The gene primers list is presented in Supplementary Table 1.

Statistical Analyses

The gene expression data were analyzed using GraphPad (GraphPad Software, San Diego, CA). All data are presented as mean ± SEM. Following a 1-way analysis of variance (ANOVA), the Tukey-HSD test was used to compare the means of the various treatment groups. The statistical significance threshold was set at P < 0.05.

RESULT

Effect of TM and Baicalein Supplementation on the Liver Gene Expression

The expression pattern of the heat shock protein-related genes (HSF3, HSPH1, HSPB1, HSP70, and HSP90) among the treatment is presented in Figure 1. The expressions of HSF3 and HSP70 were significantly higher (P < 0.05) in the CHSS group than in the CHS group. HSPH1 and HSPB1 expression was significantly higher (P < 0.05) in the TMHSS group than in other groups. HSP90 expression was significantly higher (P < 0.05) in TMHSS than in the CHS treatment group.Figure 1 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of heat shock protein-related genes on the liver. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 1

The expression of the antioxidant genes (SOD1, SOD2, GPX1, GPX3, and TXN) among the treatment is presented in Figure 2. SOD1 and TXN mRNA expression was significantly higher (P < 0.05) in the CHSS group than in the TM group. SOD2 mRNA expression was significantly higher (P < 0.05) in the CHSS group than in the CHS group.Figure 2 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of antioxidant genes in the liver. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 2

The expression pattern of the metabolism-related genes (PC, PCKc, PCKm, FOXO1, CPT1, FBP1, PRARα, SREBP-1c, ME, Leptin, LPL, and 4EBP1) among the treatments is presented in Figure 3. The expression of the PRARα was significantly higher (P < 0.05) in the CHSS group than in the CHS group. The LPL expression was significantly higher (P < 0.05) in the TMHSS than in the Control, TM, CHS, and TMHS groups.Figure 3 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of metabolism-related genes in the liver. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 3

The expression pattern of the growth factor-related genes (IGF1, IGF2, and GHR) among the treatment is presented in Figure 4. IGF2 gene expression was significantly higher (P < 0.05) in the TMHS group than in the Control, TM, and CHS groups. GHR gene expression was significantly higher (P < 0.05) in the CHSS and TMHSS groups than in the other treatment groups.Figure 4 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of growth factor-related genes in the liver. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 4

Effect of TM and Baicalein Supplementation on Muscle Gene Expression

The expression pattern of the heat shock protein-related genes (HSF3, HSPH1, HSPB1, HSP70, and HSP90) among the treatment is presented in Figure 5. HSF3 mRNA expression was significantly higher (P < 0.05) in the TMHS group compared to the Control, TM, CHSS, and TMHSS groups.Figure 5 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of heat shock protein-related genes on the muscle. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 5

The expression of the antioxidant genes (Keap1, Nrf2, SOD1, SOD2, GPX1, GPX3, and TXN) among the treatments is presented in Figure 6.Figure 6 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of antioxidant genes on the muscle. Data showed as mean ± SEM.

Figure 6

The expression of the proliferation-related genes (MSTN, MyoG, MyoD1, Myf5, mTOR, MuRF1 and MAPbx1, PCNA, and PAX7) among the treatments are presented in Figure 7. MyoG expression was significantly higher (P < 0.05) in the TMHSS group than in the TM and CHS groups. Myf5 expression was significantly higher (P < 0.05) in TMHS than in the CHSS and TMHSS groups. The expression of the mTOR was significantly lower (P < 0.05) in TMHSS than in the CHS group.Figure 7 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of proliferation-related genes on the muscle. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 7

The expression of the proliferation-related genes (PCNA and PAX7) among the treatments is presented in Figure 8. PAX7 gene expression was significantly lower (P < 0.05) in the TMHSS group than in the Control, CHS, and TMHS groups.Figure 8 Effects of embryonic TM and posthatch baicalein supplement on the mRNA expression of satellite cell-related genes on the muscle. Data showed as mean ± SEM. Different letters indicate a significant difference (P < 0.05) among the treatment groups.

Figure 8

DISCUSSION

The HS response in chicken is characterized by a series of systematic and sequential reactions. The initial phase involves the activation of the central nervous system to transmit signals to various areas of the body through neurons. In addition, the antioxidant enzyme system and the immune system play a part (Goel et al., 2021). All living organisms produce heat-shock proteins (HSP) in response to stress. HSPs prevent the toxic effects of heat-related stressors on cells. It is also strongly correlated with the thermotolerance induction in the animals (Lindquist and Craig, 1988). To understand the beneficial mechanism of prehatch TM and posthatch dietary baicalein supplementation, we analyzed important genes associated with HS in liver tissue samples. The liver is a very sensitive organ that HS can greatly affect. Transcription factors called heat shock factors (HSF) regulate heat shock proteins. It is known that 4 heat shock variables (HSF1, HSF2, HSF3, and HSF4) control how HSPs are expressed (Morimoto, 1998). HSF3 is the master regulator of heat shock genes in avian cells (Fujimoto and Nakai, 2010). Our study showed a significantly higher HSF3 expression in the CHSS group than in the CHS and TMHSS group. HSPB1 (HSP27) is a vital mediator in the development of cancer (Garrido et al., 1999) and functions as an IGF-I signaling downstream activator that promotes cell survival pathways (Zoubeidi et al., 2010). HSP70 functions as a cellular thermometer, controlling the expression of all heat shock proteins (Craig et al., 1991). HSP70 has an inhibitory mechanism that prevents the expression of cytokines that promote inflammation (Stocki and Dickinson, 2012), and glutathione reductase (GR) and glutathione peroxidase (GPX) can be stimulated by HSP70 (Gu et al., 2012). In the liver, HSP90 controlled the expression of genes linked to adipocyte differentiation and PPARƔ, which controlled the accumulation of triglycerides and adipocyte differentiation (Kang et al., 2023). HSPH1 (HSP110) primarily serves as a molecular chaperone and maintains protein homeostasis in physiological and stressful environments (Zuo et al., 2016). Our results showed a significant upregulation of the HSF3 gene in the liver in the CHSS group compared to the CHS and TMHSS groups, which might have upregulated the HSP70 in the CHSS group. Previous studies revealed that the hepatic heat shock proteins (HSP70, HSP90A, and HSP90B) exhibited a significant decrease in expression in the thermally conditioned group that was exposed to thermal stress conditions (Ncho et al., 2022; Hemida et al., 2023). In our study, we did not analyze HSP90A and HSP90B, but we found a significant increase in the expression of HSP90, which contradicts the previous findings. The significant increase in HSPB1 and HSPH1 mRNA expression in the TMHSS group among the treatment group and the significant increase in HSP90 mRNA expression in the TMHSS group compared to the CHS group may suggest that the combination of TM and baicalein supplementation had a positive effect on thermal acquisition and protective cellular function in molecular level. Additionally, we observed that there were no effects of baicalein in the CHSS group compared to the others, except for the TMHSS group, which may be due to the birds' own adaptive mechanism. The HS period lasted from d 22 to d 35. By d 35, the birds had already become accustomed to the heat stress.

Reactive oxygen species (ROS)/ reactive nitrogen species (RNS) serve as crucial signaling molecules, and the antioxidant defense system meticulously regulates the concentration of numerous transcription factors that mediate their biological effects. Under normal conditions, the ROS to RNS ratio is balanced. Birds under HS have higher than normal concentrations of ROS and RNS in their bodies (He et al., 2017). Nrf2 is the first transcription factor that activates vitagenes, synthesizing a greater range of protective molecules to counteract increased ROS/RNS production (Yamamoto et al., 2018). Subsequently, it initiates the process of radical detoxification by activating the 3 primary antioxidant enzymes, namely superoxide dismutase (SOD), GPX, and catalase, at the outset of their formation (Surai et al., 2019). An adaptive mechanism for lowering ROS formation, preventing oxidative stress, and maintaining adaptive homeostasis is increased synthesis of SOD1 and SOD2 in response to stress. SODs are a catalyst that converts superoxide to hydrogen peroxide (Azadmanesh and Borgstahl, 2018). GPX1 and GPX3 are glutathione forms dependent on selenium and are present in the primary and secondary levels of the antioxidant network in avian species. Whereas GPX3 is more commonly found in plasma, GPX1 is mainly found in the cytoplasm and mitochondria. GPXs catalyze the reduction of hydroperoxides and H2O2 by glutathione (Surai et al., 2018). The TXN and GPX systems can rely on one another in mammalian cells. Because of its ability to regulate the protein dithiol/disulfide equilibrium through disulfide reductase activity, TXN is an essential antioxidant system in the body's defense against oxidative stress (Lu and Holmgren, 2014). A previous study found that SOD2 was significantly downregulated in the TM birds under HS (Hemida et al., 2023). In this study, SOD1 and SOD2 were significantly upregulated in the CHSS group compared to the TM in SOD1 and CHS in SOD2. More interestingly, TXN mRNA expression was also significantly upregulated in the CHSS group than in the TM group. The TMHSS group did not show a similar effect, possibly because the TM birds were more thermotolerant at the embryonic stage, as discussed in our previous study (Al Amaz et al., 2024b). There were no heat stress effects on the antioxidant-related genes between the CHS and Control groups, likely because the birds were already habituated to heat stress at that time. Additionally, there were no significant TM effects between the Control and TM, as well as TMHS and TMHSS groups; this difference may be explained by examining the expression of antioxidant-related genes in the embryonic stage of these birds. In our previous study (Al Amaz et al., 2024), TM birds showed a higher antioxidant capacity in the liver at the embryonic stage, and this higher antioxidant capacity may persist throughout the postnatal period of these birds, resulting in a lower antioxidant response in the liver for all TM birds. It is important to note that the combination of baicalein and TM also did not work in that case. However, a thorough molecular investigation is needed to conclude this topic

The liver plays a crucial role in maintaining energy and glucose homeostasis. PC, PCK, and FBP1 are the primary regulatory gluconeogenesis enzymes (Parsanejad et al., 2003). Pyruvate carboxylase (PC), a protein highly expressed in the liver, is involved in gluconeogenesis, lipogenesis, insulin signaling, and other metabolic processes. It catalyzes the conversion of pyruvate into oxaloacetate (Valle, 2017). There are 2 genotypes of phosphoenolpyruvate carboxykinase (PCK): PCKm, which is found in the mitochondrial matrix, and PCKc, which is found in the cytosol (Parsanejad et al., 2003). Glucocorticoid receptor binding sites are present in the PCK gene promoter region, and corticosterone may increase hepatic gluconeogenesis by upregulating the PCK gene expression (Cawley, 2012). Transfer of long-chain fatty acids from acyl-CoA to acylcarnitine is catalyzed, and then acylcarnitine is transported across mitochondrial membranes by carnitine palmitoyl transferase 1 (CPT1) (McGarry and Brown, 1997). The peroxisome proliferator-activated receptor α (PPARα) acts as a lipid receptor and lipid-activated transcription factors that regulate energy homeostasis and metabolism (Brown et al., 2018). It was discovered that Forkhead box O1 (FOXO1) caused the liver to produce more very low-density lipoprotein, or particles rich in triglycerides, which led to hypertriglyceridemia (Hyun Kim et al., 2011). The liver is one of the primary sites of action for FOXO1, which is a key player in controlling the insulin response. The liver adjusts to food by upregulating glycogen synthesis, inhibiting gluconeogenesis and glycogenolysis, and increasing glucose uptake into hepatocytes, all of which are mediated by insulin (Tikhanovich et al., 2013). Sterol regulator element binding proteins 1c (SREBP-1c) is essential for mediating the expression of genes linked to lipogenic growth triggered by glucose and insulin (Foretz et al., 1999). For this reason, the SREBP-1c gene might influence the upregulation of lipogenic gene ME in this study, which is critical for regulating lipid metabolism in HS conditions. Numerous physiological processes, such as the metabolism of fats and carbohydrates, immunological response, and reproduction, are significantly influenced by Leptin (Martínez-Uña et al., 2020). LPL diverts circulating triglycerides to the liver, sparing glucose and starting an ineffective cycle of increased ketone and low-density lipoprotein production. This might be crucial to maintaining muscle and brain function during metabolic stress when glucose is scarce (Merkel et al., 1998). In this study, the upregulation of LPL mRNA expression in the TMHSS group showed disrupted lipid metabolism even though they were under HS. The expression of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) is increased by amino acid deprivation and other cellular stressors (Sikalidis et al., 2013). Thus, upon evaluating all metabolism-related genes, the TMHSS group exhibits minimal impact on lipid metabolism under HS conditions.

Proper growth and weight gain are crucial during the finisher phase of broilers as they impact the profit margin at the marketing stage. IGF1 and IGF2 majorly impact cellular growth by controlling growth hormone pathways. In chickens, they can affect growth, differentiation, and other biological processes (McMurtry et al., 1997). Numerous studies have shown that IGF1 and IGF2 are related to carcass features and body weight (Zhou et al., 2005; Tang et al., 2010). The significant upregulation of IGF2 in TMHS groups compared to Control, TM, and CHS groups suggests heat acclimatization and a higher growth rate in the thermally manipulated birds in HS conditions according to our previously published data (Al Amaz et al., 2024a). The growth hormone receptor (GHR) is linked to several physiological and phenotypic changes, such as decreased muscle and body mass. For the mitochondrial function of chickens, GHR expression is necessary (Hu et al., 2019). Thus, a significant upregulation of GHR indicates a higher growth rate, supporting our previously published higher body weight data in CHSS and TMHSS groups under HS (Al Amaz et al., 2024a).

Like the liver, we analyzed the same set of HSP-related genes in the breast muscle. An important protein involved in the contraction and migration of smooth muscle is HSPB1. It modulates the structure of the actin cytoskeleton and possibly the actin-myosin interaction, which is important for cell division (Salinthone et al., 2008). In skeletal muscle, HSP70 is an essential protein that controls muscle size and function in healthy and diseased conditions. HSP70 expression decreases with age and muscle inactivity, and research suggests that this loss of HSP70 may be a major factor in the atrophy, contractile dysfunction, and decreased capacity for regeneration of muscles that are linked to these diseases (Senf, 2013). Like HSP70 overexpression, HSP90 overexpression is believed to be involved in the recovery process activation of muscle repair and regeneration as well as the restoration of protein homeostasis (Krüger et al., 2019). Overexpression of HSPH1 (HSP110) can stop denatured proteins from aggregating and give cells a thermal tolerance (Oh et al., 1997). Taken together, thermally manipulated birds had a minimal effect on thermotolerance muscle gain and regeneration capacity with or without HS conditions.

Like the liver, we also analyzed the same set of antioxidant genes in the muscle. The main inducible protection against oxidative and electrophilic stresses is the Keap1-Nrf2 stress response pathway. Nrf2, a transcription factor, robustly generates chemical signals to regulate cytoprotective genes, while Keap1 functions as a cysteine thiol-rich sensor of redox insults. When Nrf2 is not under stress, Keap1 represses its activity; however, when Nrf2 is exposed to stress, Keap1-mediated repression is released (Yamamoto et al., 2018). Thus, the expression of antioxidant-related genes suggests a lower impact of TM and baicalein supplementation in the muscle antioxidant capacity.

Myostatin (MSTN) negatively regulates skeletal muscle mass, a member of the transforming growth factor-β family (Lee and McPherron, 2001). In animals, myostatin inhibits the development of muscles by cellular differentiation of developing somites during the embryonic stage and the growth of myofibrillar cells during the adult stage (Hickford et al., 2010). In chickens, silencing only the MSTN receptor, specifically ACVR2B, may promote the highest growth during the juvenile stage (Bhattacharya et al., 2019). Interestingly, the expression of myogenic regulators like MyoD, MyoG, and Myf5 was downregulated by MSTN, which inhibits myogenic differentiation (Langley et al., 2002). In mammalian cells, the rapamycin (mTOR) signaling pathway's mechanistic target is the regulator of autophagy (Jung et al., 2010). Significant downregulation of mTOR gene expression suggests a lower autophagy status in the TMHSS group, which is impressive under HS. MuRF1 and MAFbx belong to the original group of genes known as "atrophy-related genes" (atrogenes) (Bodine and Baehr, 2014). MuRF1 is a major contributor to skeletal muscle atrophy during catabolic states, making it a top choice for pharmacological treatments that prevent muscle atrophy (Peris-Moreno et al., 2020). Myogenic precursor cells are essential for muscle regeneration and hypertrophy in skeletal muscle (Campion, 1984). In mature muscle, these cells–also known as satellite cells–are typically found in a quiescent state. However, satellite cells multiply and eventually differentiate after activation, forming new fibers or fusing into existing myofibers. These processes are essential for work-induced hypertrophy and muscle regeneration (Johnson and Allen, 1993). Proliferating cell nuclear antigen (PCNA) is expressed as cells enter the S phase of the cell cycle to activate the satellite cells (Baserga, 1991). PAX7 limits alternative developmental programs in muscle-derived stem cells to induce satellite cell specification (Seale et al., 2000). In chicken, PAX7 expression mainly identifies satellite cells and their proliferating status (Halevy et al., 2004). Significant downregulation of PAX7 mRNA expression in the TMHSS group compared to the Control, CHS, and TMHS group suggests a lower affinity of satellite cell proliferation, meaning the birds were not prone to any injury or emergency that needed a muscle regeneration mechanism even though they were in elevated temperature condition.

In conclusion, prehatch TM and posthatch baicalein supplementation improved the expression of heat shock proteins, antioxidant markers, and gluconeogenesis in the liver, alongside improvements in muscle growth and a reduction in satellite cell proliferation under heat stress conditions. This combined approach demonstrates considerable potential for alleviating heat stress, as it simultaneously enhances thermotolerance acquisition in the liver and amplifies muscle mass. Nonetheless, further investigation is imperative to elucidate the mechanisms through which prehatch treatment and posthatch baicalein supplementation fortify liver and muscle functions at the cellular and molecular levels in heat-stressed broilers.

DISCLOSURES

The authors declare no conflict of interest in this manuscript.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

The research was funded by a USDA Multistate (2059R ) grant from the CTAHR University of Hawaii at Manoa to B.M. We thank Socorro Tauyan for her assistance with animal experiments. We also thank Ajay Chaudhary, Prem Lal Mohoto, Pravin Mishra, and Razib Das for their help in the sample collection.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104155.
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