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. 2024 Jun 26;103(9):104034. doi: 10.1016/j.psj.2024.104034

Embryo thermal manipulation enhances mitochondrial function in the skeletal muscle of heat-stressed broilers by regulating transient receptor potential V2 expression

Sheng Li 1, Xiaoqing Li 1, Kai Wang 1, Le Liu 1, Ketian Chen 1, Wenhan Shan 1, Luyao Liu 1, Mohamed Kahiel 1, Chunmei Li 1,1
PMCID: PMC11298950  PMID: 39003798

Abstract

Heat stress induces mitochondrial dysfunction, thereby impeding skeletal muscle development and significantly impacting the economic efficiency of poultry production. This study aimed to investigate the effects of embryo thermal manipulation (TM, 41.5°C, 65% RH, 3 h/d during 16–18th embryonic age) on the mitochondrial function of the pectoralis major (PM) in broiler chickens exposed to thermoneutral (24 ± 1°C, 60% RH) or cyclic heat stress (35 ± 1°C, 60% RH, 12 h/d) from day 22 to 28, and to explore potential mechanisms involving transient receptor potential V2 (TRPV2). Additionally, in vitro experiments were conducted to assess the regulatory effects of TRPV2 pharmacological activation and inhibition on mitochondrial function in primary myotubes. The results revealed that TM had no discernible effect on the body weight and feed intake of broiler chickens under heat stress conditions (P > 0.05). However, it did delay the increase in rectal temperature and accelerate the decrease in serum T3 levels (P < 0.05). Furthermore, TM promoted the development of PM muscle fibers, significantly increasing myofiber diameter and cross-sectional area (P < 0.05). Under heat stress conditions, TM significantly upregulated the expression of mitochondrial electron transport chain (ETC) genes and TRPV2 in broiler PM muscle (P < 0.05), with a clear positive correlation observed between the two (P < 0.05). In vitro, pharmacological activation of TRPV2 not only increased its own expression but also enhanced mitochondrial ETC genes expression and oxidative phosphorylation function by upregulating intracellular calcium ion levels (P < 0.05). Conversely, TRPV2 inhibition had the opposite effect. Overall, this study underscores the potential of prenatal thermal manipulation in regulating postnatal broiler skeletal muscle development and mitochondrial function through the modulation of TRPV2 expression.

Key words: thermal manipulation, skeletal muscle, mitochondrial function, TRPV2, broiler chicken

INTRODUCTION

With the escalating severity of global warming, heat stress induced by excessive ambient temperatures emerges as a primary environmental factor contributing to significant economic losses in poultry production. Broiler chickens exhibit vigorous metabolism, making them particularly vulnerable to heat stress when environmental temperatures exceed their thermoneutral range. Heat stress detrimentally impacts broiler production performance, notably manifesting in skeletal muscle development retardation and energy metabolism disorders (Zaboli et al., 2019; Ma et al., 2021). Mitochondria, pivotal organelles responsible for energy metabolism and redox balance in skeletal muscle, play a critical role in sustaining skeletal muscle development (Bottje, 2019). Heat stress diminishes the activity of the mitochondrial electron transport chain (ETC) and disrupts oxidative phosphorylation function, resulting in inadequate muscle energy supply and impaired muscle development (Huang et al., 2015; Lu et al., 2017). Furthermore, research indicates that the impairment of the ETC leads to the leakage of reactive oxygen species and superoxide (Mujahid et al., 2006; Kikusato et al., 2015), thereby initiating oxidative stress and exacerbating muscle oxidative damage (Azad et al., 2010a).

Thermal manipulation (TM) involves elevating the conventional incubation temperature during chick embryo development to enhance postnatal thermal adaptational capacity, encompassing aspects such as thermoregulation and energy metabolism. Numerous studies have demonstrated that appropriate TM can bolster broiler resistance to heat stress post-hatching (Al-Zhgoul et al., 2013; Goel et al., 2023). A previous study showed that TM fosters the proliferation of myoblasts and the hypertrophy of myofibers during the early post-hatch in broiler chickens (Piestun et al., 2015). Not only that, TM during the embryonic stage has long-term effects on energy metabolism and skeletal muscle development in broilers (Piestun et al., 2011; Piestun et al., 2013). Even in the later period of growth, it can also improve the loss of skeletal muscle mass caused by thermal challenge (Collin et al., 2007), which may involve the regulation of mitochondrial energy metabolism genes expression (Loyau et al., 2016). Nevertheless, the precise effects and underlying mechanisms of TM on skeletal muscle mitochondrial function remain unclear so far.

The transient receptor potential ion channels (TRP) are non-selective cation channels located on the cell membrane, exhibiting high permeability to calcium ions. Studies indicate that certain members of the TRP family are temperature-sensitive and play pivotal roles in thermoregulation (Lv et al., 2023), immune modulation (Zhang et al., 2022), sensory perception (Kashio and Tominaga, 2022), and homeostasis of energy metabolism (Uchida et al., 2017; Christie et al., 2018). A recent study has reported that TRPV family is involved in the mechanism of thermal manipulation regulating host thermotolerance (Xu et al., 2023). Among these, TRPV2 is notable for its high expression in skeletal muscle and its initial identification as a growth factor regulatory channel, with elevated levels found in dystrophic muscle (Iwata et al., 2003). TRPV2 activation can trigger the phosphorylation of muscle Ca2+/calmodulin-dependent protein kinase II (Iwata et al., 2008). Hence, TRPV2 activity in normal muscle might be calcium-dependent, and influencing critical processes such as muscle contraction, mitochondrial biogenesis, energy expenditure, and glucose uptake (Bishnoi et al., 2018; Jiang et al., 2024). To date, the function of TRPV2 in broiler chickens remains poorly understood. Whether TRPV2 can respond to thermal manipulation and play a role in regulating mitochondrial function in broiler skeletal muscle remains to be elucidated.

The present study evaluates the impact of embryo thermal manipulation on mitochondrial function in the skeletal muscle of heat-stressed broiler chickens. Additionally, the expression of TRPV2 was assessed. The role of TRPV2 in regulating mitochondrial function was further validated using chicken primary myotubes in vitro. This study aims to mitigate the detrimental effects of heat stress through prenatal thermal manipulation and elucidate the underlying mechanisms governing mitochondrial function.

MATERIALS AND METHODS

All research procedures were approved by the Nanjing Agricultural University Animal Care and Use Committee and complied with the Regulations on the Administration of Laboratory Animals promulgated by the National Science and Technology Commission of the People's Republic of China (Beijing).

Animals and Treatment

The fertile eggs (Arbor Acres, Gallus gallus domesticus) were purchased from a hatchery in Jiangsu, China, and incubated at standard temperature and humidity (37.5°C, 65% RH) until the 15th embryonic age. Eggs that did not exhibit normal development were identified and discarded using the egg candling method. At the 16th embryonic age, 198 eggs were randomly divided into 2 groups: control (CON) and thermal manipulation (TM) with 3 replicates per group and 33 eggs per replicate. During the 16th to 18th embryonic ages, the hatching temperature of eggs in TM group was increased from the standard state of incubation (37.5°C, 65% RH) to the high temperature state of incubation (41.5°C, 65% RH) for 3 h every day. During the remainder of the incubation period, the temperature and relative humidity for the TM group remained consistent with the CON group, which continued to be incubated under standard conditions (37.5°C, 65% RH) throughout.

After hatching, all chicks were transferred and kept in environmentally controlled rooms. These rooms are equipped with a temperature and humidity sensor, the Zl-th10TP (CIMC Technology Co., Ltd., Beijing, China), to monitor indoor conditions. An intelligent control system (iRVC-045, Kooland, Beijing, China) integrates these temperature and humidity parameters and adjusts the indoor heating and cooling air conditioners, as well as humidifiers, in real-time to maintain conditions within the specified range. The system features a temperature control accuracy of 1°C and a humidity control accuracy of 7%. The ambient temperature was 35°C for the first 2 d and then gradually reduced to 24°C until 21 d. At 21 d of age, 48 healthy broilers with similar body weight were selected and randomly assigned to 4 groups, and each group had 4 pens of 3 birds. Broilers in the CON+RT and TM+RT groups were kept at the standard temperature and humidity at 24 ± 1°C and in 60% RH 24 h/d from day 22 to 28. Broilers in the CON+HT and TM+HT groups were subjected to cyclic heat stress at 35 ± 1°C, 60% RH, 12 h/d (08:00-20:00) from day 22 to 28, while maintaining the same temperature and relative humidity during the rest time as in groups CON+RT and TM+RT (24 ± 1°C, 60% RH). Broilers have access to feed and water immediately after hatching. During the entire experiment, the chicks were provided with adequate diet and clean water, and feeding pens were arranged to ensure that all chicks had free access to both. The composition of the diet is detailed in Table S1. Manure was removed daily to maintain a hygienic environment. The health status of the poultry was assessed weekly to prevent the occurrence and spread of disease.

Sample Collection

At 21 and 28 d of age, feed intake and body weight gain were recorded per cage to calculate feed conversion efficiency (FCR, ADFI/ADG). After a 12 h overnight fast, 6 broilers in each treatment group were randomly selected and euthanized. Euthanasia was conducted through CO2 asphyxiation, followed by exsanguination. The breast muscle (both the pectoralis major and minor) was harvested and weighed, then expressed as a percentage of BW. The muscle sample was obtained from the right pectoralis major (PM) muscle, and immediately frozen in liquid nitrogen for subsequent analysis. Portions (around 200 mg) of the left PM muscle was excised and fixed in 4% paraformaldehyde solution for histomorphological observation.

Body Temperature Measurement

At 28 d of age, the rectal temperature was assessed using a Thermalert monitoring thermometer (TH-5, Physitemp, Clifton, NJ). The thermometer probe was inserted into the rectum to a depth of 2 to 3 cm, and data from the thermometer were recorded 2 seconds later. The overall accuracy of the measuring system was ± 0.1°C.

Serum Parameters

At 28 d of age, blood samples were obtained from the wing vein and allowed to clot naturally. Following clotting, serum was separated by centrifugation at 3,000 × g for 10 min and then stored at -20°C for subsequent analysis. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were quantified by radioimmunoassay using Iodine (125I) thyroxine radioimmunoassay kit and Iodine (125I) 3,3′,5-triiodothyronine radioimmunoassay kit (Beijing North Institute of Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions.

Myofibers Histomorphological Analysis

The measurement of myofiber histomorphology was based on that described by the previous research (Patael et al., 2019). Briefly, the tissue cross-sectional slices of PM muscle were stained with hematoxylin and eosin (G1120, Solarbio, Beijing, China), and examined under a microscope (BX51, Olympus, Tokyo, Japan). The analysis of myofibers diameter and cross-sectional area was performed using Image pro plus (Media Cybernetics, Bethesda, MD) software. Twelve visual fields of sections were examined for each group. Within each visual field, the cross-sectional area and average diameter of 50 to 80 myofibers were measured and ranked based on size. The cross-sectional area of myofibers was assessed to determine the distribution of fibers of varying sizes and calculate their relative proportions.

Antioxidative Capacity of PM Muscle

The contents of malondialdehyde (MDA; A003-1) and total-antioxidative capacity (T-AOC, A015-1) in the muscle were determined spectrophotometrically (Spark Absorbance Microplate Reader, Tecan, Männedorf, Switzerland) with commercial diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Also determined were the activities of superoxide dismutase (SOD; A001-1-2) and catalase (CAT; A007-1-1). Total protein concentration of the homogenate was measured by bicinchoninic acid (BCA) assay (P0009, Beyotime, Shanghai, China) using bovine serum albumin as the standard.

RT-qPCR

Total RNA was extracted from PM muscle tissue using TRIzol reagent (Invitrogen, Carlsbad, CA), followed by quantification of RNA concentration via spectrophotometry (Thermo Fisher Scientific, Waltham, MA). Subsequently, reverse transcription was conducted using 1 μg of total RNA for first-strand cDNA synthesis employing the Transcriptor First Strand cDNA Synthesis Kit (ABclonal, Wuhan, China). The synthesized cDNA was then subjected to amplification in a 20 μL PCR system containing 0.2 μmol/L of each specific primer (Sangon, Shanghai, China) and SYBR Green master mix (ABclonal, Wuhan, China) according to manufacturer's protocol. Real-time PCR was carried out using an ABI QuantStudio 7 PCR machine (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA), with the primer sequences outlined in Table S2. The PCR products were verified by electrophoresis and DNA sequencing. The PCR data were analyzed using the 2−ΔΔCT method, and the mRNA levels of the target genes were normalized to β-actin (ΔCT).

Western Blotting

Frozen muscles were homogenized, lysed, and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined using a BCA kit. An equal amount (60 μg) of each protein sample was loaded onto 10% SDS-PAGE gels for electrophoresis, followed by transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA), and blocked with Western blocking solution. After 2 h of blocking, the membrane was then incubated with specific primary antibodies overnight at 4°C: PGC-1α (AF7736, rabbit polyclonal antibody from Beyotime), mitochondrial ATP synthase α-subunit (AG1187, mouse monoclonal antibody from Beyotime), uncouple protein 3 (A23285, rabbit monoclonal antibody from Abclonal) and β-Tubulin (AF2835, mouse monoclonal antibody from Beyotime). Subsequently, the corresponding secondary antibody (HRP-conjugated goat anti-rabbit or anti-mouse IgG, Beyotime) was added and incubated at room temperature for 4 h. Chemiluminescence was detected using the BeyoECL Plus kit (P0018S, Beyotime, Beijing, China). The results were quantified using Fusion FX software (Vilber, Paris, France), with β-Tubulin serving as the internal control for the target protein expression assay.

Cell Culture and Treatment

The chick primary myoblasts cell was isolated and cultured according to a previous study (Li et al., 2022). Briefly, the pectoralis major muscles were aseptically collected from 14 d of age chicken embryos and rinsed twice with D-HANKS solution (H1045, Solarbio, Beijing, China). They were then minced and digested with a mixture of 0.1% Pronase E (P8811, Sigma, St. Louis, MO) and DMEM-HG medium (SH30243, HyClone, Logan, UT) for 40 min at 37°C, followed by filtration to remove large fragments. The resulting cell suspension underwent 2 washes with D-HANKS and was then subjected to density gradient centrifugation (3,000 × g, 40 min) in discrete layers of 20, 60, and 90% Percoll (P8370, Solarbio, Beijing, China). Myoblasts were harvested, and washed twice with D-HANKS before being resuspended in DMEM-HG containing 10% fetal calf serum (TBD21HY, Haoyang Biotechnology Co., Ltd., Tianjin, China) and 1% penicillin-streptomycin mixture (P1400, Solarbio, Beijing, China). Subsequently, myoblasts were seeded in a 6-well plate at a density of 1 × 105 cells/cm2 and cultured at 37°C in a humidified atmosphere of 5% CO2 for 72 to 96 h until they differentiated into myotubes. The purity of myoblast cultures, estimated to be > 95%, was assessed by desmin staining using a mouse anti-pig monoclonal antibody (BM0036, Boster Biotechnology Co., Ltd., Wuhan, China).

The differentiated myotubes were pre-incubated for 2 h in serum-free DMEM-HG, followed by 1 h incubation in various concentrations (0, 1, 5, 10, and 20 μM) of ruthenium red (R817195, Macklin, Shanghai, China). After determining the optimal treatment conditions of ruthenium red to induce TRPV2 inhibition, the cells were exposed to normal DMEM-HG medium (CON), or medium adding 10 μM ruthenium red (RR). After 1 h of exposure, cells were washed with D-Hanks' solution, collected for further analysis.

As above, various concentrations (0, 0.1, 1, 5, and 10 mM) of probenecid (IP0380, Solarbio, Beijing, China) were administered to treat differentiated myotubes to ascertain the optimal conditions for inducing TRPV2 activity. Subsequently, cells were exposed to DMEM-HG medium containing 0.01% DMSO (CON) or medium supplemented with 5mM probenecid (PB). Following a 1 h exposure, cells were washed with D-Hanks' solution and collected for further analysis.

Determination of Intracellular ATP

The ATP content in myotube lysates was assessed using the Bioluminescent Luciferase Assay (S0027, Beyotime, Shanghai, China) following the manufacturer's instruction. Briefly, cells were scraped and lysed in a buffer. After measuring the protein concentration, the cell lysates were added to the microplate containing the detection solution. The emitted light was measured using a luminometer (EnSpire, PerkinElmer, Waltham, MA), and the results were compared with a standard curve ranging from 10 to 1,000 nM to calculate ATP concentrations.

Mitochondrial Membrane Potential

Mitochondrial membrane potential was detected fluorometrically with commercial kits (BL711A, Biosharp Biotechnology Co., Ltd., Hefei, China) according to manufacturer's instructions. Briefly, myotubes were stained with JC-1. After 30 min-incubation, cells were washed twice by staining buffer, and then the fluorescence intensity of the JC-1 aggregates was measured using a fluorescence microplate reader (Spark, Tecan, Männedorf, Switzerland) at 525-590 nm (excitation-emission) wavelength.

Mito-Tracker Red CMXRos Staining

Myotubes were incubated in a medium containing 100 nM of Mito-Tracker Red CMXRos (C1049B, Beyotime, Shanghai, China) for 30 min at 37°C. After incubation, cells were washed twice with PBS buffer, and then the fluorescence of Mito-Tracker Red CMXRos were observed using a laser confocal microscope (LSM900, Zeiss, Oberkochen, Germany) at 570-590 nm (excitation-emission) wavelength.

Intracellular Calcium Ion Measurement

The intracellular calcium ion level was measured by fluorescence imaging using Ca2+ fluorescence probe Fluo-3 AM according to the previous study (Han et al., 2022). Briefly, after the experimental treatment, cells were incubated with the dye Fluo-3 AM (S1056, Beyotime, Shanghai, China) at a concentration of 5 μM for 30 min at 37°C, and then the fluorescence of Ca2+ probes were observed using a laser confocal microscope at 488-520 nm (excitation-emission) wavelength. The analysis of fluorescence intensity was performed using Image pro plus software.

Statistical Analysis

Data are presented as the mean ± SEM. Statistical analysis was conducted using Student's t-test or two-way ANOVA with Statistical Analysis System software (version 8e; SAS Institute, Cary, NC). G*Power software (version 3.1; Düsseldorf University, Düsseldorf, Germany) was utilized to conduct a post hoc power analysis, ensuring the detection of meaningful differences between groups. Two-way ANOVA was utilized to evaluate the main effects (HT and TM) and their interactions. In case of significant main effects or interactions, one-way ANOVA was employed. Mean separation was carried out using Duncan's multiple comparisons, and treatment effects were deemed statistically significant at a probability level of P < 0.05.

RESULTS

Effect of Thermal Manipulation on Growth Performance in Broilers Under Different Ambient Temperatures

There was no significant difference in hatchability between the control and TM groups (Figure S1). During the growth period (1–21 d), TM had no negative effect on ADFI, ADG and FCR in broilers (P > 0.05). During the temperature challenge (22–28 d), HT exposure decreased ADFI (PHT < 0.001) and ADG (PHT < 0.001) of broilers compared with RT conditions (Figure 1). Specifically, ADG and ADFI of broilers in control and TM groups were all reduced by HT challenge (P < 0.05). TM treatment significantly increased ADFI (PTM < 0.01) and ADG (PTM < 0.01). Under RT conditions, broilers in the TM group exhibited higher ADFI (P < 0.05) than the control group, while there was no significant difference in ADFI, ADG, and FCR between the control and TM groups under HT conditions (P > 0.05).

Figure 1.

Figure 1

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Effect of embryo thermal manipulation on performance of broilers under different temperature conditions. (A) Average daily feed intake. (B) Average daily gain. (C) Feed conversion ratio. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 4.

Effect of Thermal Manipulation on Thermoregulation in Broilers Under Different Ambient Temperatures

As shown in Figure 2, HT exposure significantly increased the rectal temperature of broilers (PHT < 0.001), while TM treatment decreased it (PTM < 0.01). In detail, the rectal temperature of broilers in the control and TM groups were all up-regulated by HT challenge (P < 0.001), but broilers in the TM group exhibited lower rectal temperatures than those in the control group under HT conditions (P < 0.05). Hormone levels related to thermoregulation were further measured in serum. TM treatment had no effect on serum T3 and T4 concentrations or the T3/T4 ratio (PTM > 0.05). However, HT exposure resulted in a decrease in serum T3 levels and T3/T4 ratio (PHT < 0.001), and this decrease was only observed in broilers in the TM group (P < 0.01).

Figure 2.

Figure 2

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Effect of embryo thermal manipulation on thermoregulation of broilers under different temperature conditions. (A) Rectal temperature (n = 10). (B) T3 concentration in serum. (C) T4 concentration in serum. (D) The ratio of T3 and T4. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6. T3: Triiodothyronine; T4: Thyroid hormone.

Effect of Thermal Manipulation on Myofiber Development in PM Muscle of Broilers Under Different Ambient Temperatures

There were no significant differences in the breast muscle index among all groups (Figure 3). However, heat stress led to a reduction in the absolute weight of the breast muscle (PHT < 0.01), and particularly noticeable in the control broilers (P < 0.05). Conversely, compared with the control group, broiler chickens in the TM group exhibited a tendency towards increased breast muscle weight under HT conditions (P = 0.071). HE staining was employed to further evaluate myofiber development in the PM muscle. As shown in Figure 3C, heat stress led to a looser arrangement of broiler PM muscle fibers, increased muscle fascicle gaps, and induced a small amount of congestion, while TM ameliorated the muscle damage caused by heat stress. Additionally, statistical analysis revealed that TM significantly increased the cross-sectional area (PTM < 0.001) and average diameter (PTM < 0.001) of myofibers under both RT and HT conditions, with a significant interaction observed under HT (PInteraction < 0.001). Overall, heat stress increased the proportion of smaller PM muscle fibers (< 1,000 μm2) in broiler chickens, while TM augmented the proportion of larger myofibers (> 4,000 μm2).

Figure 3.

Figure 3

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Effect of embryo thermal manipulation on myofiber development in PM muscle of broilers under different temperature conditions. (A) The breast muscle (pectoralis major and minor muscle) index. (B) The breast muscle weight. (C) Representative micrograph of hematoxylin and eosin-stained myofiber cross-sections of broilers (top: magnification 20×; bottom: magnification 100×). (D) Statistical analysis of myofiber diameter. (E) Statistical analysis of myofiber cross-sectional area. (F) Proportion and distribution of different sized myofibers. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6.

Effect of Thermal Manipulation on Antioxidant Capacity in PM Muscle of Broilers Under Different Ambient Temperatures

Compared with RT (Figure 4), HT exposure significantly reduced SOD and CAT levels in the PM muscle (PHT < 0.001), with no effect on MDA and T-AOC levels (PHT > 0.05). Specifically, SOD levels declined in broilers from both control and TM groups during HT challenge (P < 0.05), whereas CAT levels decreased only in the control group (P < 0.001). Moreover, TM treatment notably decreased MDA concentration (PTM < 0.01) and increased SOD (PTM < 0.05), CAT (PTM < 0.01), and T-AOC (PTM < 0.05) levels. Under RT conditions, no differences were observed between control and TM groups (P > 0.05). Under HT conditions, the CAT levels in the TM group were higher than that in the control group (P < 0.05).

Figure 4.

Figure 4

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Effect of embryo thermal manipulation on antioxidative capacity of broilers PM muscle under different temperature conditions. (A) Malondialdehyde contents. (B) Superoxide dismutase activity. (C) Catalase activity. (D) Total-antioxidative capacity. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6.

Effect of Thermal Manipulation on Mitochondrial Function in PM Muscle of Broilers Under Different Ambient Temperatures

Compared with RT (Figure 5), HT exposure significantly increased the expression of PGC-1α (PHT < 0.001), Cyt c (PHT < 0.001), COX IV (PHT < 0.001), ATP5A (PHT < 0.01), ATP5B (PHT < 0.01), IDH3a (PHT < 0.001), and SERCA1 (PHT < 0.001) in the PM muscle. Among these genes, upregulation occurred exclusively in the TM group under HT conditions, except for SERCA1, which exhibited increased expression in both TM and control groups during HT challenge. Furthermore, TM treatment notably increased the mRNA expression levels of PGC-1α (PTM < 0.05), Cyt c (PTM < 0.001), COX IV (PTM < 0.001), ATP5A (PTM < 0.05), ATP5B (PTM < 0.01), and SERCA1 (PTM < 0.05). Under RT conditions, there were no significant differences in the expression of genes relative to mitochondrial function between the two groups (P > 0.05). However, under HT conditions, broilers in the TM group exhibited higher expression levels of PGC-1α (P < 0.05), Cyt c (P < 0.001), COX IV (P < 0.001), ATP5A (P < 0.05), ATP5B (P < 0.05), and IDH3a (P < 0.05) compared to the control group.

Figure 5.

Figure 5

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Effect of embryo thermal manipulation on mitochondrial function of broilers PM muscle under different temperature conditions. (A–I) The mRNA expression of genes related to mitochondrial function. (J) Representative images of Western blotting. (K–M) The protein expression of PGC-1α, avUCP, and ATP5A1. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6. avUCP: Avian uncoupling protein; ATP5A: Mitochondrial ATP synthase 5 alpha-subunit; ATP5B: Mitochondrial ATP synthase 5 beta-subunit; COX IV: Cytochrome c oxidase subunit IV; Cyt c: Cytochrome c; IDH3α: Isocitrate dehydrogenase 3 (NAD+) α; PGC-1α: Peroxisome proliferator-activated receptor γ coactivator 1α; SERCA1: Sarcoplasmic/endoplasmic reticulum calcium ATPase 1; Tfam: Mitochondrial transcription factor A.

As illustrated in Figure 5J–5M, HT exposure significantly elevated ATP5A1 protein abundance in the PM muscle compared to RT (PHT< 0.01). Additionally, TM treatment led to increased abundance of PGC-1α (PTM < 0.01) and ATP5A1 (PTM < 0.05) proteins, with a tendency for avUCP (PTM = 0.054) protein levels to increase as well. Specifically, TM increased PGC-1α protein levels in broiler chickens under RT conditions (P < 0.01), while it enhanced avUCP (P < 0.01) and ATP5A1 (P < 0.05) protein abundance under HT conditions.

Effect of Thermal Manipulation on TRPV2 Expression in PM Muscle of Broilers Under Different Ambient Temperatures

TRPV family mRNA expression profiles in the broiler PM muscle were characterized (Figure 6). The results showed that the expression of TRPV2 in PM muscle was significantly higher than that of other members (P < 0.001). Furthermore, HT exposure increased TRPV2 mRNA expression compared with RT (PHT < 0.001), and this change was observed only in the TM group (P < 0.01). Additionally, TM treatment also upregulated TRPV2 expression levels (PTM < 0.001). Under HT conditions, the expression of TRPV2 in the TM group was significantly increased compared with the control group (P < 0.01). The relationship between TRPV2 and mitochondrial function was further assessed through Pearson correlation analysis, demonstrating a significant positive correlation between TRPV2 and the expression of mitochondria-related genes, including PGC-1α (P < 0.01), Cyt c (P < 0.001), COX IV (P < 0.01), ATP5A (P < 0.01), ATP5B (P < 0.01), and IDH3a (P < 0.001).

Figure 6.

Figure 6

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Effect of embryo thermal manipulation on TRPV2 expression of broilers PM muscle under different temperature conditions. (A) TRPV family expression profile in PM muscle (n = 12). (B) The mRNA expression of TRPV2 (n = 6). (C–H) Correlation analysis between TRPV2 and mitochondrial genes expression (n = 24). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. TRPV1-4: Transient receptor potential vanilloid 1-4 ion channels.

Effects of TRPV2 Agonists and Inhibitors on Mitochondrial Function of Myotubes

The expression of TRPV2 in myotube was significantly higher (P < 0.001) than that of other members (Figure S2). To further validate the role of TRPV2 in regulating mitochondrial function, the potent and specific TRPV2 antagonist ruthenium red and the agonist probenecid were used to treat myotubes (Figures S2B and S2C). Compared with the control group, TRPV2 expression was downregulated after treatment with 10 μM or 20 μM ruthenium red for 1 h (P < 0.001), while it was upregulated after treatment with 5 mM and 10 mM probenecid (P < 0.01). Based on these results, 10 μM ruthenium red and 5 mM probenecid were determined as the optimal treatment concentration and used in subsequent experiments.

Compared to the control group (Figure 7), probenecid treatment enhanced the expression of genes related to mitochondrial function and the calcium ion pathway in myotubes, such as ATP5A (P < 0.05), ATP5B (P < 0.05), Tfam (P < 0.01), COX IV (P < 0.05), PPARα (P < 0.01) and CaMK II (P < 0.05). It also increased MitoTracker Red, JC-1, and calcium ion fluorescence signals, and significantly elevated ATP content and intracellular Ca2+ levels (P < 0.05).

Figure 7.

Figure 7

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Effect of TRPV2 activation (exposure probenecid for 1 h) on mitochondrial function in myotubes. (A) The expression of genes related to mitochondrial function. (B) Quantitative analysis of JC-1 fluorescence intensity. (C) Representative fluorescence image of mitochondria stained with MitoTracker Red CMXRos. (D) ATP content in myotubes. (E) Fluo-3AM fluorescence imaging with laser confocal microscopy. (F) Quantitative analysis of intracellular Ca2+. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6. ATP: Adenosine triphosphate; MMP: Mitochondrial membrane potential.

Compared to the control group (Figure 8), ruthenium red treatment markedly suppressed the expression of genes associated with mitochondrial function, including avUCP (P < 0.001), PGC-1α (P < 0.001), ATP5A (P < 0.05), ATP5B (P < 0.001), IDH3a (P < 0.01), Tfam (P < 0.01), and PPARα (P < 0.05). Additionally, the fluorescence signals of MitoTracker Red and JC-1 were significantly diminished following ruthenium red treatment (P < 0.05). Furthermore, ATP contents (P < 0.05) and intracellular Ca2+ levels (P < 0.001) in myotubes were also reduced upon ruthenium red treatment.

Figure 8.

Figure 8

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Effect of TRPV2 inhibition (exposure ruthenium red for 1 h) on mitochondrial function in myotubes. (A) The expression of genes related to mitochondrial function. (B) Quantitative analysis of JC-1 fluorescence intensity. (C) Representative fluorescence image of mitochondria stained with MitoTracker Red CMXRos. (D) ATP content in myotubes. (E) Fluo-3AM fluorescence imaging with laser confocal microscopy. (F) Quantitative analysis of intracellular Ca2+. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6.

DISCUSSION

TM Improves Heat Tolerance of Broiler Chickens During Heat Stress

Heat stress presents a significant challenge to the growth and development of broiler chickens. Elevated ambient temperatures disrupt the broilers' ability to maintain a stable body temperature, resulting in a cascade of heat stress reactions, including reduced growth performance. Interestingly, there is some controversy regarding the impact of thermal manipulation (TM) on body weight. Certain studies suggest that TM does not impact post-hatching body weight when eggs are incubated at 39.5°C for 3 h daily during the 8 to 10th or 16 to 18th embryonic age (Collin et al., 2007). Similarly, our study did not observe significant changes in growth performance with TM, suggesting that short-term, moderate temperature increases might not adversely affect body weight. Conversely, eggs subjected to incubation at temperatures of 38.5°C and 40.0°C for 18 h daily during the 12 to 18th embryonic age exhibited higher body weights after hatching (Al-Zghoul and El-Bahr, 2019). The disparities observed in these studies may be attributed to variations in the temperature and duration of TM exposure. Besides, TM at 39.5°C for 3 h during embryonic day 16 to 18 was found to increase body weight and feed intake in broilers under heat stress conditions (Xu et al., 2023). Although our results do not support this view, the complexity of embryonic development results in varied responses to specific temperatures and durations, as well as diverse environmental conditions. This suggests that the specific temperature conditions of TM may be the key factor influencing its effectiveness.

Changes in body temperature serve as the initiating factor for heat stress and are therefore considered a crucial indicator of heat tolerance. Pre-hatch thermal manipulation may enhance the heat tolerance of chickens by reducing rectal temperature. Previous studies have shown that TM leads to a decrease in basal body temperature post-hatching and delays the rise in rectal temperature during heat stress episodes (Xu et al., 2023). Consistent with these findings, our study revealed a reduction in rectal temperature in broilers subjected to TM under high-temperature conditions. Body temperature changes are regulated by the Hypothalamus-Pituitary-Thyroid (HPT) axis. TM not only decreases plasma T3 concentration post-hatching but also exerts significant long-term effects on thyroid hormone metabolism by modulating the mRNA expression of deiodinase DIO3 (Loyau et al., 2014; Cong et al., 2023). Our findings demonstrate that T3 concentration and the T3/T4 ratio in broiler chickens of the TM group were notably reduced under heat stress conditions. As the ultimate effector of the HPT axis, the decline in T3 concentration signifies reduced thermogenesis and metabolic efficiency, potentially serving as the direct cause of the decrease in body temperature (Mullur et al., 2014). These results indicate that thermal manipulation during the embryonic stage can enhance the heat tolerance of broilers post-hatching.

In the present study, we used broilers from a single commercial strain to minimize genetic variability. Although selecting a homogeneous genetic background helped reduce the potential confounding effects of genetic differences on our results, it is important to note that these results may not be directly applicable to other broiler strains. Furthermore, despite our efforts to control for maternal factors, we recognize that some variability may still exist, such as egg size, yolk composition, and initial health status of the embryos. Future research could further control for maternal effects by using eggs from multiple flocks or by including maternal characteristics as covariates in the analysis.

TM Enhances Mitochondrial Function and Promotes Myofiber Development in PM Muscle of Heat-Stressed Broiler Chickens

The application of various thermal manipulation procedures during the incubation period has been shown to positively impact post-hatching muscle growth, leading to an increase in the relative weight and diameter of PM muscle fibers (Collin et al., 2007; Piestun et al., 2015). Although no significant change in the relative weight of PM muscle was observed in this study, we noted a trend towards increased absolute weight of PM muscle in TM-treated broiler chickens. This trend may be obscured by changes in body weight, resulting in no discernible difference in relative weight. Furthermore, TM notably augmented the diameter and cross-sectional area of muscle fibers and mitigated skeletal muscle damage induced by heat stress, whether under thermoneutral or heat stress conditions. Previous research has suggested that the mechanism underlying TM-induced muscle fiber hypertrophy may involve enhanced proliferation and differentiation of myoblasts during or after hatching, leading to an expanded myogenic cell pool in embryonic and post-hatching broiler chickens and an increase in muscle fiber count (Piestun et al., 2015). Conversely, our findings indicate that TM primarily enhances the proportion of larger muscle fibers rather than altering the total number of muscle fibers. Hence, the increase in PM muscle weight appears to depend more on the developmental process of PM muscle fibers.

Mitochondria are abundant in skeletal muscle and play a pivotal role in energy production and redox balance within muscle fibers. Heat stress has been shown to induce mitochondrial dysfunction, impair mitochondrial ATP synthesis, disrupt the electron transport chain, and impede muscle formation and development (Huang et al., 2015). Significant reductions in the activity or expression of mitochondrial respiratory chain complexes in response to heat stress can lead to inefficient electron transport, heightened reactive oxygen species (ROS) production, and consequent oxidative damage (Jiao et al., 2018). While the present study did not report alterations in expression of ETC-related genes, this discrepancy with other studies may be attributed to variations in the severity and duration of heat stress (Huang et al., 2015; Uyanga et al., 2022). Such discrepancies might diminish over time as animals adapt to environmental heat stress (Azad et al., 2010b). Nevertheless, it is evident that heat stress diminishes antioxidant capacity, notably SOD and CAT. Consisting with our findings, it has been reported that blunted antioxidant capacity in serum under heat stress (Ouyang et al., 2023), thereby exacerbating oxidative damage induced by mitochondrial dysfunction.

The mitochondrial electron transport chain comprises 4 complexes (I–IV) integrated into the inner mitochondrial membrane, regulated by both mitochondrial DNA and the nuclear genome (Vercellino and Sazanov, 2021). These complexes facilitate oxidative phosphorylation and establish a proton gradient essential for ATP production. Avian uncoupling protein (avUCP) mitigates proton gradients by uncoupling, reducing ROS leakage, and preserving the integrity of the mitochondrial ETC (Criscuolo et al., 2006; Ruuskanen et al., 2021). Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) coactivates ETC genes expression with nuclear transcription factors, promoting mitochondrial biogenesis and energy metabolism (Wu et al., 1999; Rowe et al., 2013). In a previous study, TM altered markers of energy utilization and growth in the PM muscle and liver, potentially limiting energy metabolism as a long-term adaptation (Loyau et al., 2014). However, we did not observe similar changes. Instead, TM significantly upregulates PGC-1α and avUCP levels in PM muscle under heat stress conditions, alongside enhanced expression of genes linked to the mitochondrial ETC, suggesting that TM enhances mitochondrial energy metabolism. Notably, consistent with our results, the study also reported that TM increased markers of energy utilization and growth during heat challenges, which may underscore the temperature-selective effect (Loyau et al., 2014). Additionally, it has been reported that augmented avUCP levels and elevated expression of mitochondrial ETC genes effectively mitigate ROS production (Kikusato and Toyomizu, 2013; Mujahid et al., 2007), potentially elucidating the enhanced antioxidant capacity and reduced oxidative damage observed in the TM group of broilers. Concurrently, the increased expression of mitochondrial ETC genes ensures efficient oxidative phosphorylation, supplying ample energy for muscle fiber development (Brito et al., 2017; Smith et al., 2023). In summary, our study confirms that TM enhances mitochondrial function in the PM muscle of heat-stressed broilers, thereby promoting the development of muscle fibers.

The changes in the feed efficiency phenotype may be related to differences or inefficiencies in mitochondrial function, as it affects energy consumption. In previous research, compared with high feed efficiency broilers, low feed efficiency broilers exhibited defects in the mitochondrial ETC at specific points, resulting in reduced ETC coupling efficiency and enzyme complex activity (Ojano-Dirain et al., 2005; Bottje et al., 2022). Considering the reduction in feed conversion ratio (FCR), the failure of the aerobic or oxidative energy system is related to heat stress-induced muscle atrophy. Feed efficiency, being a heritable and commercially valuable trait, has not been conclusively linked to the physiological mechanisms underlying its phenotypic variation through TM. We found that although feed intake had a mild to marked increases after embryonic TM, FCR remained unchanged under both thermoneutral and heat stress conditions. This contradicts another study, which pointed out that genotypes interact with TM in terms of chick performance and FCR (Meteyake et al., 2023), indicating that the differences in TM effects may be related to genetic factors. However, at least in this study, feed intake was not the primary reason for changes in mitochondrial function. It can be speculated that under stress, there is a mitochondrial renewal mechanism in TM broilers that is not related to genetic background or feed conversion rate.

TRPV2 is a Potential Mechanism Mediating TM to Regulate Mitochondrial Function in Heat-Stressed Broiler Chickens

Notably, TM exhibits significant differences in the regulation of mitochondrial function under varying environmental temperatures, implying a clear temperature specificity. We speculate that this process may involve a temperature-selective mitochondrial regulatory mechanism. The TRPV subfamily (TRPV1-V4), a temperature-sensitive group within the TRP family, can be activated in response to temperature changes and participates in processes such as thermal sensation formation, cell metabolism signaling, and hormone release in a calcium ion-dependent manner (Nazıroğlu and Braidy, 2017). Recent research has demonstrated that TM can mitigate heat stress-induced intestinal inflammation in broiler chickens by modulating TRPV4 (Xu et al., 2023). However, our examination of the TRPV family expression profile in PM muscle tissue and myotubes revealed that TRPV2 exhibited the highest expression levels both in vivo and in vitro. Despite limited literature on whether TRPV2 responds to TM, our findings indicate that TM treatment significantly upregulates TRPV2 expression in the PM muscle of heat-stressed broiler chickens. This suggests that TRPV2 may play a crucial role in skeletal muscle development as a major regulatory factor.

TRPV2 activity in normal muscle may be linked to Ca2+ dynamics and influences crucial processes such as muscle contraction, mitochondrial biogenesis, energy expenditure, and glucose uptake (Gailly, 2012; Iwata et al., 2016). Studies in mammals have elucidated the elevated expression of TRPV2 in muscular dystrophy mice (Iwata et al., 2008), and pharmacological inhibition of TRPV2 has been shown to ameliorate rat myocardial mitochondrial dysfunction by maintaining calcium homeostasis (Jiang et al., 2024). Contrary to these findings, in vitro experiments revealed that specific TRPV2 activation not only upregulates TRPV2 expression itself but also enhances the expression of mitochondria-related genes, leading to increased mitochondrial membrane potential and ATP production. Additionally, treatment with a TRPV2 inhibitor produced the opposite effect. These findings, consistent with the results from in vivo experiment but differing from those in mammals, demonstrate a clear positive correlation between TRPV2 and the expression of genes related to mitochondrial function. A major explanation is that the TRP family functions differently across species. For instance, TRPV1 is blunted in chickens, rendering it unresponsive to the painful effects of capsaicin (Jordt and Julius, 2002). Chicken lack TRPM4 and are less sensitive to cold than mammals (Saito and Shingai, 2006). Given the significant interspecies variability within the TRP family, activation thresholds and functions of TRPV2 may differ across species, and its unique role in chicken warrants further investigation. However, it is important to acknowledge that in vitro experiments cannot fully replicate the in vivo environment, including hormonal influences, inter-organ signaling, and complex physiological compensation mechanisms, which introduces certain limitations to this study. We cannot definitively determine whether the increase in TRPV2 expression in vivo is a result of its own activation or a compensatory mechanism triggered by changes in body temperature, which may impact our understanding of how it responds. However, it is clear that TRPV2 is a key regulator of mitochondrial function.

TRPV2 functions as a calcium ion channel, and its physiological role in skeletal muscle likely involves the calcium ion signaling cascade (Iwata et al., 2016). TRPV2 activity has been shown to induce Ca2+/calmodulin-dependent protein kinase (CaMK) phosphorylation (Iwata et al., 2008). The latter further maintains mitochondrial quality and biogenesis through the CaMK/PGC-1α pathway (Wu et al., 2002). Mitochondria play a crucial role in modulating cytoplasmic calcium concentration. They integrate the cellular metabolic state with the Ca2+ transport, thereby regulating mitochondrial ATP production in response to calcium signals and influencing cellular processes such as energy metabolism, growth, proliferation, and apoptosis (Marchi et al., 2018; Basse et al., 2021). Activation of TRPV2 not only enhances intracellular calcium ion flux but also upregulates the expression of CaMKII, which may contribute to the observed enhancement in mitochondrial function. Based on these findings, we propose that TRPV2 emerges as a potential mechanism through which TM regulates mitochondrial function in heat-stressed broiler chickens.

The commercial poultry industry requires high-yield, rapidly growing broiler chickens. Consequently, the market age is continuously shortened, with 30% to 40% of a broiler's life expectancy spent during embryo incubation (Hulet et al., 2007). This highlights the significant importance of the embryonic period. Therefore, implementing thermal manipulation or in ovo feeding with targeted TRPV2 during the embryonic stage may be a promising strategy to reduce the harmful effects of heat stress.

CONCLUSIONS

Chicken embryo thermal manipulation (41.5°C and 65% RH for 3 h/d during 16–18th embryonic age) can enhance mitochondrial energy metabolism in the pectoralis major muscle of heat-stressed broiler chickens and promote the development of muscle fibers. TRPV2 appears to regulate mitochondrial energy metabolism, potentially mediating the mechanism by which thermal manipulation influences skeletal muscle development in heat-stressed broilers. These findings offer promising strategies for enhancing broiler performance in high-temperature environments.

Acknowledgments

ACKNOWLEDGMENTS

This research was funded by the National Key Research and Development Program of China (2023YFD1300802-4); the National Natural Science Foundation of China, grant number 32372935; Jiangsu Agricultural Industry Technology System, grant number JATS (2023)437.

Author Contributions: SL: conceptualization, writing—original draft, writing—review and editing; XQL: data curation, formal analysis; KW: writing—review and editing; LL: methodology; KTC: methodology; WHS: methodology; LYL: methodology; MK: methodology; CML: conceptualization, supervision, writing—review and editing. All authors contributed to this article and approved the submission.

Ethics Approval and Consent to Participate: The study was reviewed and approved by the Institutional Animal Care and Use Committee of the College of Animal Science of Nanjing Agriculture University and carried out following the "Guidelines for Experimental Animal" of the Ministry of Science and Technology (Beijing, P. R. China).

We express our gratitude to all the staff members at the Research Centre for Livestock Environmental Control and Smart Production, Nanjing Agricultural University for their valuable assistance in conducting the experiments and providing valuable feedback during the revision of this article.

DISCLOSURES

The authors declare no conflicts of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104034.

Appendix. Supplementary materials

Figure S1. Effect of embryo thermal manipulation on hatchability and performance of broilers from 1 to 21 d. (A) Hatchability. (B) Average daily feed intake. (C) Average daily gain. (D) Feed conversion ratio. Data are expressed as the mean ± SEM. * P < 0.05. n = 4.

mmc1.jpg (411.4KB, jpg)

Figure S2. Effects of TRPV2 activator (B) and inhibitor (C) treatment on TRPV2 expression in myotubes. (A) TRPV family expression profile in myotubes. (B) Myotubes were treated with different concentrations of probenecid for 1 h. (C) Myotubes were treated with different concentrations of ruthenium red for 1 h. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6.

mmc2.jpg (280.3KB, jpg)
mmc3.docx (17KB, docx)
mmc4.docx (20KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Effect of embryo thermal manipulation on hatchability and performance of broilers from 1 to 21 d. (A) Hatchability. (B) Average daily feed intake. (C) Average daily gain. (D) Feed conversion ratio. Data are expressed as the mean ± SEM. * P < 0.05. n = 4.

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Figure S2. Effects of TRPV2 activator (B) and inhibitor (C) treatment on TRPV2 expression in myotubes. (A) TRPV family expression profile in myotubes. (B) Myotubes were treated with different concentrations of probenecid for 1 h. (C) Myotubes were treated with different concentrations of ruthenium red for 1 h. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6.

mmc2.jpg (280.3KB, jpg)
mmc3.docx (17KB, docx)
mmc4.docx (20KB, docx)

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