Mitochondrial Haplogroups Influence Mitochondrial Structure and Function, Oxidative Stress, Autophagy, and Lipid Metabolism of Chicken Hepatocytes in Response to Energy Stimulation

TL;DR

This study shows how different mitochondrial haplogroups in chickens affect liver cell function, stress response, and fat metabolism when exposed to energy changes.

Contribution

The study reveals how mitochondrial haplogroups influence maternal effects through changes in mitochondrial structure, autophagy, and lipid metabolism in chicken hepatocytes.

Findings

E-group mitochondria showed shorter perimeters and lengths during refeeding compared to A-group.

E-group cells had higher reactive oxygen species levels and lower autophagy markers after energy stimulation.

Mitochondrial haplogroups affect lipid accumulation and structural integrity in response to fasting and refeeding.

Abstract

This article reports the effects of mitochondrial haplogroups on the structure and function of mitochondria, the level of oxidative stress, and fat content in chicken hepatocytes. Mitochondrial haplogroups are classified based on differences in the base sequences of the mitochondrial genome, and since mitochondria are important carriers of maternal effects, mitochondrial haplogroups may have an impact on maternal effects. The aim of this study was to elucidate the mechanism by which mitochondrial haplogroups affect maternal effects. By comparing the differences in mitochondrial structure and function, oxidative stress levels and fat content between mitochondrial haplogroups in chicken hepatocytes stimulated by nutrient or energy factors, this study found that the capabilities of mitochondrial fusion, renewal and autophagy, the resistance to oxidative stress, the capacity for fat deposition, and the ability to cope with energy stress in the cell were affected by mitochondrial haplogroups. These findings contribute to the understanding of how maternal effects influence animal physiological function and how mitochondrial haplogroup-based selection can improve animal production performance.

Mitochondria are crucial carriers of maternal effects, and their function is closely related to energy metabolism and disease occurrence. Previous studies have shown that chickens with different mitochondrial haplogroups exhibit differences in production performance, but the underlying mechanism remains unclear. This study investigates the differences in mitochondrial structure and function-related indices between the A and E mitochondrial haplogroups (referred to as A-group and E-group) in recessive white-feathered chickens. It was achieved using in vivo fasting/refeeding models and an in vitro model of treating hepatocytes with nutritional factors (glucose and fatty acids). In vivo study indicated that compared to A-group chicken hepatocytes, E-group hepatocytes had shorter perimeters of mitochondria and shorter lengths of mitochondria associated with the endoplasmic reticulum membrane during refeeding (p < 0.05); mitochondria were more abundant (p = 0.05) but displayed compromised structural integrity during fasting; mitochondrial swelling was more severe during both refeeding and fasting (p < 0.01, p < 0.05); the protein level of mitofusin 2 (MFN2) was lower during fasting (p < 0.05); and there were more vacuoles and lipid accumulation in liver sections during refeeding (p < 0.05). In cultured hepatocytes, compared to A-group cells, E-group cells had higher reactive oxygen species (ROS) level after oleic acid treatments (p < 0.001); the protein level of microtubule-associated protein 1A/1B-light chain 3 beta (LC3) was lower after glucose treatment (p < 0.01), and the protein levels of MFN2 and LC3 were lower after oleic acid treatment (p < 0.01, p < 0.05). These findings suggest that mitochondrial haplogroups are associated with mitochondrial structure and function, oxidative stress, autophagy, and lipid metabolism of chicken hepatocytes in response to energy stimulation. The findings may explain how mitochondrial haplogroups affect chicken production performance.

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Full text

1. Introduction

Chicken is an economically important animal as it contributes to human nutritional needs by providing high-quality and cost-effective protein in the form of eggs and meat. Improving chicken production performance holds significant practical importance. Apart from being economically important, chickens also serve as vital model organisms, playing a crucial role in analyzing the formation mechanisms of animal biological traits, particularly those related to production.

The genetic effects of nuclear genome components are crucial in determining animal biological traits; however, maternal genetic influences are equally essential. Mitochondria, with their own genomic DNA (mitochondrial DNA, mtDNA), are maternally inherited [1,2], and thus, alterations in mtDNA composition significantly impact maternal genetic effects on biological traits. However, research in this area is very limited. Since mtDNA lacks homologous recombination, changes in its nucleotide sequences are cumulative, causing mtDNA from the same maternal lineage to cluster together. This clustered DNA is known as a mitochondrial haplogroup [3]. The classification of chicken mitochondrial haplogroups primarily originates from Liu et al. [4]. Research on the mitochondrial control region (displacement loop, D-loop) sequences of Asian domestic chickens and red junglefowl initially categorized chickens into nine haplogroups (A-I). Subsequently, Zhang et al. [5] reconstructed the haplogroup phylogenetic tree of domestic chickens and red junglefowl using a substantial amount of published and newly sequenced mitochondrial genome data from domestic chickens, further dividing chickens into 13 haplogroups (A-I and W-Z). The study revealed that 94.84% of domestic chickens belong to haplogroups A, B, C, E, F, G, and I, with A and B being widely distributed in East and Southeast Asia, C being unique to East Asia, and E being prevalent in South Asia and Europe and America [6].

Mitochondria are essential organelles within eukaryotic cells that sustain cellular homeostasis. They not only serve as the cell’s powerhouses, facilitating cellular material and energy metabolism, but also play a vital role in biological processes such as the production of reactive oxygen species (ROS), regulation of calcium ions, and signal transduction [7]. Consequently, mitochondria can impact animal growth, development, and production performance by engaging in material and energy metabolism and other biological processes. Oxidative stress can lead to mitochondrial dysfunction and damage to mtDNA, thereby impairing mitochondrial biosynthesis and function [8]. Research has been conducted on the correlation between mitochondrial haplogroups and mitochondrial function as well as production performance [9,10,11], but mechanistic insights are very limited. Studies have indicated that there are significant variations between Tibetan chickens and Camellia chickens in the effects of mtDNA haplogroups on cellular adenosine triphosphate (ATP) synthesis capacity, mtDNA copy number, and the expression of genes associated with mitochondrial biosynthesis [12]. Tang et al. [6] confirmed that the E-haplogroup (E-group) was advantageous for the growth performance of broiler chickens, resulting in higher birth weights, a younger age to reach market weight (1.8 kg), and a higher feed conversion ratio.

The liver of animals is rich in mitochondria, which are primarily responsible for metabolism, glycogen storage, protein synthesis, detoxification, and lipid metabolism (including the absorption, synthesis, breakdown, and secretion of fats). These functions are crucial for maintaining metabolic homeostasis. For example, the majority of fatty acids synthesized from scratch in poultry come from the liver [13]. Many metabolic disorders, such as metabolic dysfunction-associated fatty liver disease (MAFLD), diabetes, and metabolic syndrome, are closely associated with mitochondrial dysfunction and disrupted metabolic function in the liver [14,15].

Among broiler lines, the recessive white-feathered chicken is frequently utilized as a key parent for the production of yellow-feathered broilers in China, due to its rapid growth rate and higher egg production. The hybrid offspring of these recessive white-feathered chickens exhibit not only a significantly enhanced growth rate but also maintain the phenotypic traits of the local chickens from which they are bred. Consequently, the majority of fast-growing yellow-feathered broiler lines in China are derived from the recessive white-feathered chickens. Previous research indicated that the mitochondrial haplogroups of these chickens are predominantly the A-haplogroup (A-group) and the E-group [6], yet the distinctions in mitochondrial structure and function between these are still unclear. In this study, the differences in mitochondrial structure and function between the A-group and the E-group were examined using recessive white-feathered chickens with the haplogroups. The aim of this study was to investigate how mitochondrial haplogroups influence specific maternal effect-related phenotypes—cellular fat deposition and stress resilience—by regulating mitochondrial dynamics, functional activity, and redox homeostasis in chicken hepatocytes.

2. Materials and Methods

2.1. Experimental Animals

For this experiment, forty 320-day-old healthy hens (20 from A-group and 20 from E-group) with similar body weights were selected from a recessive white-feathered chicken line with the same genetic background. The Jiangsu Institute of Poultry Science provided these hens, and Yangzhou University’s Animal Care and Use Committee (SYXK checked and approved all animal-related procedures (SYXK (Su) 2022-0044).

The hens were randomly assigned to two groups, the fasting group (including 10 from A-group with an average body weight of 2.664 ± 0.0867 kg and 10 from E-group with an average body weight of 2.676 ± 0.0906 kg) and the refeeding group (including 10 from A-group with an average body weight of 2.690 ± 0.0590 kg and 10 from E-group with an average body weight of 2.700 ± 0.0702 kg), and housed individually in cages. The fasting group was deprived of food for 12 h, whereas the refeeding group underwent a 12 h fast followed by a 2 h feeding period. The feed intake per hen per day was 130 g. The feed for the peak phase of laying hens was purchased from Yancheng Yuanyao Feed Co., Ltd. (Yancheng, China).All experimental hens had unrestricted access to water. At the end of the experiment, the hens were humanely euthanized by CO_2_ inhalation. Liver tissue samples were promptly collected from the central region of the largest lobe, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.

2.2. Classification of Mitochondrial Haplogroups

Based on the control region or displacement loop of the chicken mitochondrial reference sequence (GenBank accession number: NC_007235), a pair of specific primers, including a forward primer (5′-AAACACCCAAACTCACTAAC-3′) and a reverse primer (5′-CACTGGGATGCGGATACTTGC-3′), was designed using Primer Premier 5.0 and synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China). The primers were used in PCR with DNA samples isolated from chicken blood according to previously described procedures [6]. The PCR products were then sequenced by Shenggong Bioengineering Co., Ltd. The mitochondrial haplogroups were classified according to the methods previously described [6].

2.3. Isolation and Culture of Primary Hepatocytes

Primary hepatocytes were isolated from the livers of 15 d post-hatching embryos from the same batch of recessive white-feathered breeding hens according to a previous publication [16]. These cells were then cultured in complete medium, including high-glucose (25 mmol/L) Dulbecco’s Modified Eagle Medium (DMEM) (KeyGEN BioTECH, Nanjing, China), 10% Fetal Bovine Serum (FBS) (GIBCO, Waltham, MA, USA) and 0.02% Epidermal Growth Factor (EGF) (Pepro Tech, Cranbury, NJ, USA). The cells were cultured at 37 °C and 5% CO_2_.

2.4. Treatment of Primary Chicken Hepatocytes with Glucose and Oleic Acid

After culturing chicken primary hepatocytes for 12 h, the cells were rinsed three times with Phosphate-Buffered Saline (PBS) (Solarbio, Beijing, China) and subsequently treated with oleic acid or glucose. For fatty acid treatment, oleic acid (Sigma, Burlington, MA, USA) was initially prepared as a 100 mmol/L stock solution using ethanol as the solvent. This 100 mmol/L oleic acid stock solution was then thoroughly mixed with a complete medium containing 2% Bovine Serum Albumin (BSA) (Solarbio, Beijing, China) to create a medium with a final oleic acid concentration of 0.25 mmol/L. The control cells received complete medium supplemented with 2% BSA and the equivalent volume of ethanol. For glucose treatment, glucose (Sigma, Burlington, MA, USA) was added to the complete medium to make a culture medium with a final concentration of 50 mmol/L glucose, which was then used to treat the primary hepatocytes. The control cells were treated with complete medium alone. Following 14 h of treatment, the cells were harvested for protein extraction.

2.5. Western Blot Assay

Approximately 100 mg of liver tissue was lysed with 100 μL of Radioimmunoprecipitation Assay Buffer (RIPA) (Applygen, Beijing, China) and Phenylmethylsulfonyl Fluoride (PMSF) (Solarbio, Beijing, China). The protein concentration of the samples was determined using the Bicinchoninic Acid (BCA) Protein Assay kit (Vazyme, Nanjing, China). Protein samples were separated by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to a Polyvinylidene Fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). The PVDF membranes were blocked with a rapid blocking solution (NCM Biotech, Suzhou, China). The blocked membranes were incubated with the primary antibody at 4 °C overnight, followed by incubation with the secondary antibody at room temperature for 1.5 h. Images were taken after development using an Enhanced Chemiluminescence (ECL) kit (Vazyme, Nanjing, China). Primary antibodies used in this study include Mitofusin 2 (MFN2) (sc-100560, Santa Cruz Biotechnology, Dallas, TX, USA), Beta-Actin (β-actin) (AC026, ABclonal, Wuhan, China), Superoxide Dismutase 2 (SOD2) (24127-1-AP, Proteintech, Wuhan, China), Microtubule-Associated Protein 1A/1B-Light Chain 3 Beta (LC3B) (3868T, Cell Signaling Technology, Danvers, MA, USA), Mitochondrially Encoded NADH Dehydrogenase 1 (MT-ND1) (YA2089, MedChemExpress, Monmouth Junction, NJ, USA), and Cytochrome B (CYTB) (55090-1-AP, Proteintech, Wuhan, China). Secondary antibodies used were goat anti-rabbit Immunoglobulin G (IgG) (CW0103S, Cwbiotech, Taizhou, China) or goat anti-mouse IgG (CW01025, Cwbiotech, Taizhou, China).

2.6. Isolation of Mitochondria

Mitochondria were isolated using the Mitochondria and Cytosol Extraction Kit (Applygen, Beijing, China) following the manufacturer’s instructions. Approximately 150 mg of liver tissue was ground in 1 mL of pre-cooled mitochondrial extraction reagent using a glass homogenizer. Then, the homogenate was transferred to a centrifuge tube and centrifuged at 800× g and 4 °C for 5 min. The precipitate was discarded, and the supernatant was retained. This procedure was repeated twice, after which the mixture was centrifuged at 10,000× g and 4 °C for 10 min. After centrifugation, the precipitate was discarded, and the supernatant was collected. Subsequently, 1 mL of pre-cooled mitochondrial extraction reagent was added, and the mixture was resuspended. Finally, the mixture was centrifuged at 12,000× g and 4 °C for 10 min, resulting in the precipitation of mitochondria.

2.7. Measurement of Mitochondrial Membrane Potential

The mitochondrial membrane potential assay kit for JC-1 (Beyotime, Shanghai, China) was used. According to the manufacturer’s instructions for the kit, the cells isolated from liver tissue or the cultured cells were added to 0.5 mL of JC-1 staining working solution, thoroughly mixed, and subsequently incubated in a cell culture incubator at 37 °C for 20 min. After incubation, the cells were centrifuged at 600× g and 4 °C for 3–4 min, and the supernatant was discarded. The cells were then washed twice with 1 mL of JC-1 staining buffer, with the supernatant removed by centrifugation after each resuspension. Lastly, the cells were resuspended in 1 mL of buffer, followed by analysis with flow cytometry.

2.8. ROS Measurement

According to the manufacturer’s protocol, intracellular ROS levels were determined with a Reactive Oxygen Species Assay Kit (Solarbio, Beijing, China). DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate) was initially diluted with the Optimized Minimum Essential Medium (Opti-MEM) (GIBCO, Waltham, MA, USA) to a concentration of 10 μmol/L at a ratio of 1:1000, and subsequently, 1 mL of this dilution was added to the cells. The cells were incubated at 37 °C in a cell culture incubator for 20 min, with gentle shaking every 4 min. After incubation, the cells were centrifuged at 600× g and 4 °C for 5 min, and the supernatant was removed. Next, the cells were resuspended in 1 mL of Opti-MEM, centrifuged again at 600× g and 4 °C for 5 min, and the supernatant was discarded. This process was repeated three times. Finally, the cells were resuspended in 200 μL of Opti-MEM and analyzed using flow cytometry.

2.9. Mitochondrial Swelling Measurement

Mitochondrial swelling was detected using the Mitochondrial Swelling Assay Kit (HalingBio, Shanghai, China) following the manufacturer’s instructions. Twenty μL of mitochondria (200 μg per sample) isolated from liver tissue were added to a 96-well plate, followed by the addition of 170 μL of buffer (Reagent A) to each well. After mixing thoroughly, the initial reading of each well was acquired at a wavelength of 520 nm using a microplate reader. After sitting for 1 min at room temperature, 10 μL of inflation solution (Reagent B) was added to each well, followed by thorough mixing and sitting for 30 min at room temperature. The absorbance value of each well was then measured at a wavelength of 520 nm using the microplate reader. The degree of mitochondrial swelling was calculated by subtracting the initial readings from the readings acquired at 30 min.

2.10. Hematoxylin–Eosin Staining and Oil Red O Staining

The liver tissues were dissected into approximately 1 cm × 1 cm × 0.5 cm pieces and fixed in 4% paraformaldehyde (Yuanye, Shanghai, China) overnight at room temperature. Subsequently, hematoxylin–eosin staining and Oil Red O staining were performed according to standard protocols [17].

2.11. Determination of Triglyceride Content

The triglyceride contents in liver tissues or the cultured cells were measured using a triglyceride determination kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions. For tissue samples, approximately 100 g of liver tissue was put into 900 μL of anhydrous ethanol, followed by manually homogenizing the tissue 30–35 times in an ice-water bath. After centrifugation for 10 min, the homogenate was collected for triglyceride content determination.

For the cell samples, the cultured cells were washed with PBS 1–2 times, centrifuged at 600× g and 4 °C for 10 min, and the supernatant was discarded. Subsequently, the cells were manually homogenized in 200 μL of PBS, and the resulting homogenate was used to determine triglyceride content. The triglyceride content was calculated using the formula provided in the manual of the triglyceride determination kit.

2.12. Transmission Electron Microscopy (TEM) Analysis

The livers were sliced into small pieces and placed in centrifuge tubes containing glutaraldehyde, then stored at 4 °C in the dark. Tissue sections for TEM were prepared as follows: A phosphate buffer (0.1 mol/L, pH 7.4) with 1% osmium tetroxide was prepared and kept in the dark at room temperature to fix the tissue samples for 2 h. The tissue sections were rinsed with the phosphate buffer three times, with each rinse lasting 15 min. The tissue samples underwent a graded series of alcohol dehydrations: 30%, 50%, 70%, 80%, 95%, and 100%, with each step lasting 20 min. The samples were then treated with 100% acetone twice, for 15 min each time, and subsequently immersed in a 1:1 mixture of acetone and 812 embedding medium at 37 °C for 2–4 h. Next, the tissue samples were permeated in a 1:2 mixture of acetone and 812 embedding medium overnight. The samples were then transferred to an embedding plate filled with pure 812 embedding medium and placed in an oven at 37 °C overnight. The embedding plate and tissue samples were polymerized in the oven at 60 °C for 48 h to create a resin block, which was then removed and set aside. The resin block was sliced into ultra-thin sections with thickness of 60–80 nm using a slicer, and the sections were placed on a 150-mesh square copper grid coated with a carbon film. The grid was then placed in a 2% uranyl acetate solution in ethanol, kept in the dark, for staining for 8 min. It was then washed three times with 70% ethanol and three times with ultrapure water, followed by staining with a 2.6% lead citrate solution in the absence of carbon dioxide for 8 min. The grid was washed three times with ultrapure water and dried with filter paper. The copper grid sections were placed in a copper grid storage box and left to dry at room temperature overnight. Finally, the sections were observed under a transmission electron microscope, and images were collected and analyzed.

2.13. Statistical Analysis

The data were normalized relative to the control group. Use the Student’s t-test to assess the statistical significance of the differences in means between the control and treatment groups. * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered acceptable levels of significance. The results are presented as the mean ± standard deviation.

3. Results

3.1. Effects of Mitochondrial Haplogroups on Mitochondrial Number, Structure, and Morphology in the Fed or Fasted States

Transmission electron microscopy was used to observe the liver cells of chickens in the A-group and the E-group. The findings indicated that, in response to refeeding, the E-group mitochondria had significantly shorter circumferences (p < 0.05, Figure 1A,B) and reduced lengths of the mitochondria-associated endoplasmic reticulum membrane (MAM) compared to the A-group mitochondria (p < 0.05, Figure 1C,D). Additionally, there were more aggregated ribosomal particles (Figure 1E,F), an increased number of mitochondria (Figure 1G), and a smaller mitochondrial area in the E-group cells (Figure 1H), although these differences were not statistically significant. In response to fasting, the E-group cells had more mitochondria (p = 0.05, Figure 2A,B), and the structural integrity of the mitochondria was compromised (Figure 2C,D). Furthermore, there were more aggregated ribosomal particles (Figure 2E,F), a reduced mitochondrial area (Figure 2G), and a shorter mitochondrial circumference in the E-group cells (Figure 2H), but none of these differences were statistically significant.

3.2. Effects of Mitochondrial Haplogroups on Mitochondrial Membrane Potential, the Degree of Swelling, and ROS Levels in the Fed or Fasted States

Under the refeeding conditions, both mitochondrial membrane potential and ROS levels in the E-group chicken hepatocytes were similar to those in the A-group chicken hepatocytes (Figure 3A–D). However, the E-group mitochondria displayed significantly greater swelling (p < 0.01, Figure 3E). Under the fasting conditions, the E-group mitochondria showed a lower membrane potential and a lower ROS level (Figure 4A–D), again with no significant differences. Nevertheless, the E-group mitochondria exhibited greater swelling (p < 0.05, Figure 4E).

3.3. Effects of Mitochondrial Haplogroups on the Abundance of Mitochondria-Related Proteins in the Fed or Fasting States

The results from the immunoblotting analysis indicated that, compared to the A-group cells, there were no significant differences in the protein levels of MFN2, CYTB, MT-ND1, LC3B II, and SOD2 in the E-group cells under the refeeding conditions (Figure 5A–F). However, the protein level of MFN2 was lower in the E-group cells under the fasting conditions (p < 0.05, Figure 6A,B), and no significant trends were detected for the remaining proteins (Figure 6C–F).

3.4. Effects of Mitochondrial Haplogroups on Intracellular Fat Content in the Fed and Fasted States

The HE and Oil Red O staining analyses indicated that, compared to the A-group chicken liver sections, the E-group chicken liver sections exhibited a greater number of vacuoles (Figure 7A) and a higher fat content (p < 0.05, Figure 7B,C) under the refeeding conditions. The E-group chicken liver sections also showed a greater number of vacuoles (Figure 8A) and a higher fat content under the fasting conditions (Figure 8B,C), although these differences were not statistically significant.

3.5. Effects of Mitochondrial Haplogroups on Mitochondrial Membrane Potential and ROS Level Following Glucose or Oleic Acid Treatments

In cultured hepatocytes, compared to the A-group cells, treatments with oleic acid led to a significantly higher level of ROS in the E-group cells (p < 0.001, Figure 9A,B and Figure 10A,B). However, there was no notable difference in mitochondrial membrane potential between the A-group and E-group cells (Figure 9C,D and Figure 10C,D).

3.6. Effects of Mitochondrial Haplogroups on the Content of Mitochondria-Related Proteins Following Glucose or Oleic Acid Treatments

The immunoblotting analysis indicated that, compared to the A-group cells, the E-group cells exhibited a lower protein level of LC3 following glucose treatment (p < 0.01, Figure 11A–F). However, the differences in the protein levels of MFN2 and CYTB were not statistically significant between the A-group and E-group cells. Both MFN2 and LC3 protein levels had decreased in the E-group cells compared to the A-group cells (p < 0.01, p < 0.05, Figure 12A–F).

3.7. Effects of Mitochondrial Haplogroups on Intracellular Fat Content Following Glucose or Oleic Acid Treatments

The results of the triglyceride content determination indicated that there was no significant difference in triglyceride content between the A-group and E-group cells following glucose treatment (Figure 13A). However, following oleic acid treatment, the triglyceride content increased in the E-group cells compared to the A-group cells (Figure 13B), yet this difference was also not significant.

4. Discussion

A substantial volume of evidence suggests that maternal effects significantly influence animal physiology, production performance, and health [18]. Given the role of mitochondria in various biological functions, including material and energy metabolism, oxidative stress, signal transduction, organelle communication, and apoptosis, mitochondria are crucial to maternal effects. Mitochondria have their own genomes, which include genes for proteins integral to their structural and functional integrity, as well as transfer RNA (tRNA) and ribosomal RNA (rRNA) necessary for protein synthesis [19]. Consequently, distinct mitochondrial haplogroups may affect mitochondrial structure and function. Furthermore, proteins encoded by the mitochondrial genome can interact with those encoded by the nuclear genome, also influencing mitochondrial structure and function [20]. This interaction could be another mechanism by which different mitochondrial haplogroups impact mitochondrial structure and function. Nevertheless, the precise mechanisms through which mitochondrial haplogroups influence mitochondrial structure and function are not yet fully understood.

The findings of this study suggest that under nutritional stimulation, mitochondria in the E-group hepatocytes are smaller, more abundant, less intact, and exhibit a higher number of clustered ribosomes compared to those in the A-group hepatocytes. They also have reduced fusion and autophagy capabilities, increased swelling, elevated ROS levels, and a greater accumulation of fat content, along with more vacuoles in the liver tissue. MFN2, a key protein involved in mitochondrial fusion, has downregulated expression associated with impaired mitochondrial fusion [21]. Mitochondria in the E-group hepatocytes display a weaker fusion capacity under nutrient stimulation compared to those in the A-group hepatocytes, potentially resulting in a greater number of smaller mitochondria with shorter circumferences and MAM lengths. This diminishes their ability to interact with each other and with other organelles in response to nutrient stimulation. This weakened interaction leads to mitochondrial overload, heightened ROS levels, and mitochondrial damage. Moreover, with decreased mitochondrial autophagy capacity, damaged mitochondria are not efficiently cleared, further exacerbating ROS levels and oxidative stress. Under these circumstances, a substantial number of mitochondria with incomplete internal structures accumulate within the cell, causing increased mitochondrial membrane permeability and swelling. Consequently, fat oxidation capacity diminishes in the cell, potentially resulting in an increase in the number of fat droplets and vacuoles within the hepatocytes and liver tissue.

Previous studies have indicated that the structure of mitochondria is in a state of constant dynamic change, encompassing both fusion and fission [22]. Mitochondrial fusion and fission serve not only to regulate the exchange of mitochondrial contents but also to segregate damaged mtDNA, preserve normal mitochondrial DNA, and ensure the proper functioning of mitochondria [23]. Upon energy stimulation, mitochondria enhance fusion, transitioning from small spherical structures to elongated or branched, network-like forms [24]. Research has demonstrated that the spatial distribution of mitochondria is closely linked to their metabolic functions [25]. It is well-established that cellular energy synthesis primarily originates from glycolysis in the cytoplasm and oxidative phosphorylation (OXPHOS) in mitochondria. Changes in mitochondrial morphology can reprogram cellular metabolism by directly impacting the activity and efficiency of OXPHOS. Reports suggest that metabolic reprogramming can occur in various proliferating cells, with aerobic glycolysis (the Warburg effect) being a prime example of such reprogramming. This phenomenon is observed not only in cancer cells but also in proliferating cells such as pluripotent stem cells and antigen-activated immune cells [26]. This implies that metabolic patterns are essential for cells to sustain normal differentiation, proliferation, and function. When mitochondrial morphology is disrupted, energy metabolism is also disrupted, leading to compromised cell proliferation and fate determination [27]. As a result, mitochondrial fusion and fission influence not only cellular energy metabolism but also cell proliferation or apoptosis [28]. In this study, the E-group exhibited downregulated expression of MFN2 compared to the A-group in response to oleic acid treatment. The changes in the fusion capacity between the A-group and E-group mitochondria might reflect differences in their abilities for material metabolism, energy synthesis, and response to energy stress, as well as disparities in basal metabolic rate and cell proliferation rate. The mechanism underlying the effects of mitochondrial haplogroups on the expression of MFN2 likely involves the following process: subtle sequence variations in mtDNA-encoded oxidative phosphorylation subunits could alter electron transport chain efficiency, leading to differential production of mitochondrial signals (e.g., ROS, ATP). Subsequently, these signals may be sensed by some transcription factors, which further influence the transcription of nuclear-encoded genes such as MFN2.

In this study, in addition to having weaker fusion capabilities than the A-group mitochondria, the E-group mitochondria also exhibit fewer contact points with the endoplasmic reticulum (shorter MAM length), indicating that the E-group mitochondria have weaker response to energy stress. Given the critical role of the endoplasmic reticulum in protein synthesis, calcium ion regulation, and lipid synthesis, the reduced number of contact points between the endoplasmic reticulum and mitochondria may weaken the ability of mitochondria to collaborate with the endoplasmic reticulum via the MAM to address the demands of protein synthesis, calcium ion regulation, and lipid synthesis during energy stress [29]. When animals consume an energy-rich diet, the load on the mitochondrial electron transport chain increases. Due to the weaker ability of the E-group mitochondria to respond to energy stress, this leads to mitochondrial overload, resulting in increased leakage of electrons and the generation of ROS, ultimately elevating the level of oxidative stress in the cell. Furthermore, CYTB is the only cytochrome encoded by the animal mitochondrial genome and is a component of the respiratory chain complex III [30]. A decrease in its expression indicates reduced mitochondrial biogenesis and respiratory chain activity. LC3 is a key protein involved in autophagy, and a reduction in its protein content indicates decreased autophagy, including mitochondrial autophagy [31]. During energy intake, the protein levels of CYTB and LC3 in the E-group cells are lower than those in the A-group cells, reflecting weaker mitochondrial biogenesis and autophagy capabilities in the E-group cells, resulting in reduced mitochondrial renewal and damaged mitochondrial clearance capabilities. As damaged mitochondria accumulate, oxidative stress further intensifies, leading to increased oxidative damage to lipids and proteins in the mitochondrial membrane [32]. Previous studies have shown that the accumulation of ROS leads to increased mitochondrial membrane permeability [33], mitochondrial swelling [34], reduced mitochondrial membrane potential [35], and uncoupling of oxidative phosphorylation [15]. The mitochondrial membrane contains permeability-switching pores, whose structural and functional changes directly affect mitochondrial stability, regulate mitochondrial membrane permeability, and are crucial for maintaining mitochondrial membrane potential and the Ca^2+^, pH, and electrochemical balance in the matrix [36]. Consistent with this, our study also found that as ROS levels increased in the E-group cells, mitochondrial swelling increased and mitochondrial membrane potential decreased. Since maintaining mitochondrial membrane potential is a prerequisite for normal OXPHOS and ATP synthesis, the lower mitochondrial membrane potential in the E-group cells reflects impaired mitochondrial metabolic capacity, which may be one of the reasons for the increased fat content in the E-group cells.

It is noteworthy that the in vivo refeeding model and the in vitro model in this study are mutually consistent in some findings, such as the significant increase in fat content in the E-group liver cells under the state of nutritional or energy intake. The findings from the in vitro study also help interpret the findings from the in vivo study. Therefore, the in vivo study and the in vitro study are complementary to each other. As for the in vitro study, primary hepatocytes from chicken embryos were used instead of primary hepatocytes from adult chickens. Our main consideration is to understand whether the effects of mitochondrial haplogroups are affected by developmental stages. If some findings during embryonic and adult stages are consistent, then the findings are more universal and reliable. In addition, another factor to consider is that we observed that embryonic cells have better proliferation ability than adult cells.

In this study, the findings may have the following implications. First, it is necessary to select proper mitochondrial haplogroups during breeding based on the purpose of use (egg or meat), nutritional level, and rearing environment (tropical area or plateau). Secondly, different husbandry methods (e.g., restricted feeding, different nutritional levels and feed formulas) may be adopted for different mitochondrial haplogroups. For example, compared to the A-group mitochondria, the E-group mitochondria are weaker in fusion and autophagy and are more easily damaged under stress, which leads to more fat deposition, rapid growth, and a greater susceptibility to metabolic diseases such as fatty liver. Therefore, restricted feeding or feed with a low nutritional level may be applied to chickens with the E-group mitochondria at a specific rearing stage. As the E-group was more prone to oxidative stress during feeding, it suggests that chickens with the E haplogroup may need more antioxidants as feed additives.

5. Conclusions

Mitochondrial haplogroups are associated with the size, number, structural integrity, membrane potential, fusion, and renewal of mitochondria, as well as oxidative stress level, autophagy level, and fat content in chicken hepatocytes under energy factor stimulation. In the recessive white-feathered hens, mitochondrial fusion, renewal and autophagy capabilities, as well as antioxidant stress resistance and the ability to cope with energy stress in the A-group cells, are stronger than those in the E-group cells. However, the fat deposition capacity of the A-group cells is weaker than that of the E-group cells. These findings provide an important scientific reference for elucidating the relationship between mitochondrial haplogroups and production performance.