Review
Genetic expression markers for assessing cellular respiration status under heat stress in cattle: A review Dorin Alexandru Vizitiu 1, Şerban Blaga 1, Daniel George Bratu 1, Bianca Cornelia Zanfira 1, Andrei Alexandru Ivan 1, Liliana Căprinişan 2, Oana Maria Boldura 2,* and Ioan Huţu 1 1 University of Life Sciences ”King Mihai I”, Horia Cernescu Research Unit - Faculty of Veterinary Medicine, 300645, No. 119 Calea Aradului, Timisoara, Romania * Correspondence: oanaboldura@usvt.ro
Abstract: Thermal stress significantly affects the metabolic efficiency and health of cattle, with cellular bioenergetics and mitochondrial function being key targets. The regulation of oxidative metabolism and thermotolerance is largely governed by specific genetic markers that reflect adaptive responses to heat exposure. This review discusses genes involved in mitochondrial respiration and stress response mechanisms, such as those encoding components of the electron transport chain (ND1–ND5, COX1–COX3, CYCS), heat shock proteins (HSP70, HSP90), and antioxidant enzymes (SOD1, NRF2, PGC-1α). Alterations in the expression of these genes provide valuable insights into mitochondrial efficiency and cellular adaptation to elevated temperatures, reflecting the dynamic processes that allow cattle to cope with heat stress. Furthermore, disruptions in these pathways may contribute to metabolic inefficiencies, negatively impacting overall health and productivity. Additionally, this review explores the potential of integrating transcriptomic, proteomic, and genomic data to identify molecular markers associated with heat tolerance. Such approaches provide valuable insights into the mechanisms underlying thermal resilience, which can guide genetic selection strategies aimed at improving cattle health and productivity in extreme temperature conditions. Keywords: oxidative stress, thermotolerance, mitochondrial function, cellular adaptation, heat-stress biomarkers
1. Introduction Cellular respiration lies at the heart of energy metabolism in cattle, serving as the biochemical linchpin that sustains growth, lactation, and physiological homeostasis. Within mitochondria, the oxidative phosphorylation (OXPHOS) system— comprising five electron transport chain (ETC) complexes—orchestrates the conversion of nutrients into ATP, a process so efficient that it generates over 90% of cellular energy under thermoneutral conditions [Baumgard and Rhoads, 2013]. This metabolic machinery is particularly vital in high-producing dairy cows, where lactation demands a 25-40% surge in mitochondrial oxygen consumption [Collier et al., 2017]. However, this finely tuned system falters under heat stress, triggering cascading failures in energy homeostasis that compromise productivity, immune function, and fertility [Wheelock et al., 2010]. Heat stress, increasingly prevalent due to global climate change, disrupts this critical process. Elevated ambient temperatures can impair mitochondrial integrity, leading to fragmentation of mitochondrial networks, diminished electron transport chain activity, and reduced ATP synthesis. Such perturbations result in an accumulation of reactive oxygen species (ROS), which further damage cellular components and exacerbate metabolic dysfunction. These effects not only compromise energy production but also impact essential processes such as milk synthesis and immune responses [Lacetera, 2019, Sejian et al., 2018]. Complex I (NADH dehydrogenase) and Complex IV (cytochrome c oxidase) are especially vulnerable, with studies showing 30-50% reductions in ND4 and COX1 expression in heat-stressed Holsteins [Deb et al., 2014]. These declines starkly contrast with the resilience observed in adapted Bos indicus breeds like the Sahiwal, which maintain mitochondrial function through upregulated HSP90 and SOD1 expression—a testament to evolutionary adaptations honed in tropical climates. Such disparities underscore mitochondria as both casualties and arbiters of thermal stress responses [Kishore et al., 2014]. In high-producing dairy cattle, the energetic demands of lactation substantially elevate endogenous heat production, intensifying susceptibility to thermal stress. Under thermoneutral conditions—defined between approximately 5 °C and 25
Romanian Journal of Veterinary Sciences 2026, 59, 1 200 of 212 °C for dairy cows—metabolic heat can be dissipated effectively; however, exposure to environmental temperatures above this range disrupts thermal equilibrium, leading to hyperthermia and systemic physiological strain. As thermal load accumulates, cattle exhibit reduced feed intake, altered nutrient partitioning, and compromised metabolic efficiency, all of which exacerbate the energy deficit imposed by lactation. Although thermoregulatory mechanisms such as increased sweating and respiratory rate are activated, they often prove insufficient during prolonged or extreme heat exposure, as seen in (Figure 1). The resulting negative energy balance places additional pressure on mitochondrial oxidative phosphorylation, further impairing ATP synthesis and aggravating cellular energy shortfalls. Mitochondrial dysfunction underpins much of the observed systemic decline, linking organelle-level bioenergetic failure to organismal-level productivity losses. These dynamics highlight the mitochondria not only as casualties of heat stress but as pivotal arbiters of adaptive capacity. Consequently, identifying genetic markers related to mitochondrial function and thermotolerance emerges as a critical objective for breeding cattle better adapted to the demands of a warming climate [Kadzere et al., 2002].
Figure 1. Impact of heat stress on mitochondrial bioenergetics in cattle, generated by OpenAI Sora.
In dairy cattle, coordinated metabolic adaptations across multiple organ systems are essential to meet heightened energy demands, particularly during lactation when nutrient mobilization is critical for milk production. Heat stress disrupts these physiological processes by altering nutrient partitioning and impairing mitochondrial bioenergetics throughout the body. This systemic disruption reduces the overall efficiency of energy utilization, ultimately resulting in decreased milk yield and compromised animal performance [Wheelock et al., 2010]. Advances in functional genomics have revealed that genetic regulation plays a central role in the adaptive response to heat stress. Differential expression of nuclear-encoded mitochondrial genes, heat shock proteins, and antioxidant enzymes has been observed in heat-stressed cattle. Such findings suggest that specific genetic markers could serve as early indicators of mitochondrial dysfunction under thermal stress, providing valuable tools for genetic selection and breeding programs aimed at enhancing thermotolerance [Dikmen et al., 2014, Garner et al., 2016]. This review aims to explore current evidence regarding the role of genetic markers linked to cellular respiration in the context of heat stress. It examines how elevated temperatures disrupt mitochondrial function, leading to metabolic inefficiencies that compromise overall energy balance and productivity in dairy cattle. Additionally, the review assesses the potential of integrated multi-omics approaches—including genomics, transcriptomics, and proteomics—to identify robust molecular signatures of thermotolerance. These insights are pivotal for informing breeding strategies and developing management practices to mitigate the adverse impacts of global warming on dairy cattle.
2. Materials and Methods 2.1. Literature Search Strategy To explore genetic markers involved in mitochondrial function and the heat stress response in cattle, this review utilized academic databases including PubMed, Scopus, Web of Science, and Google Scholar. Emphasis was placed on studies investigating the expression of mitochondrial and heat shock-related genes in relation to thermotolerance. AI tools such as OpenAI (GPT-4o-mini) and DeepSeek facilitated the development of targeted Boolean search queries, enhancing
Romanian Journal of Veterinary Sciences 2026, 59, 1 201 of 212 the retrieval of niche articles. Key search terms included "bovine heat stress," "mitochondrial genes," "cellular respiration," "HSP70," "ATP5A1," and "NRF2," combined through Boolean operators (AND/OR) to maximize search specificity. Sample queries included: ("heat stress" AND "ND1" OR "ND5" OR "COX1") AND ("bovine" OR "cattle") or ("cellular respiration" AND "HSP70" AND "thermotolerance"). 2.2. AI Integration and Semantic Analysis Artificial intelligence platforms, including OpenAI and DeepSeek, were utilized to optimize literature search strategies and facilitate data organization. These tools enabled a more efficient identification of relevant studies and assisted in recognizing key themes related to gene expression dynamics under heat stress. Their integration supported a more systematic and comprehensive exploration of the available scientific evidence. Also another tool used was OpenAI's advanced text-to-image generative AI model, named “Sora”, which was used to create relevant images based on a given prompt. 2.3. Data Extraction A systematic extraction of data focused on key findings regarding differential gene expression, fold-changes, and correlations with phenotypic heat tolerance traits. AI-assisted text mining enabled efficient compilation of information regarding the upregulation or downregulation of target genes such as ND4, CYCS, HSP90, and PGC-1α under heat stress. Extracted data were synthesized into structured summaries, improving the clarity and integration of findings across multiple studies. 2.4. Inclusion and Exclusion Criteria Only peer-reviewed articles published between 2014 and 2024 that examined gene expression in cattle under heat stress conditions were included. Studies had to provide original data or detailed reviews focusing on genes related to mitochondrial respiration or stress response. Exclusion criteria comprised articles without English full-text availability, conference abstracts, studies lacking methodological details, and publications unrelated to livestock species or thermal stress phenomena. addressed unrelated topics, including those behind paywalls. Although more recent sources are generally preferred, there are exceptions where older studies have been included due to the exceptional quality and relevance of the information they provide on the discussed subject. These studies offer valuable insights and foundational data that remain relevant to this day. 2.5. Methodological Workflow The methodological approach for this review incorporated the use of Zotero for systematic reference management and Excel for structured data cataloging. This workflow ensured rigorous tracking of sources, consistent extraction of relevant information, and coherent organization of findings, thereby enhancing the overall quality and transparency of the research process. 2.6. Data Extraction and Cataloging Relevant data regarding gene expression profiles, tissue specificity, and experimental conditions were systematically extracted and organized into comparative summary tables. This structured synthesis facilitated clearer cross-study comparisons and contributed to identifying consistent patterns in mitochondrial function and cellular stress responses under elevated temperature conditions. 2.7. Manual Validation All automatically extracted data underwent manual cross-verification against primary sources to ensure data integrity and methodological rigor. Cross-referencing through Excel spreadsheets and metadata validation via Zotero minimized potential discrepancies and reinforced the reliability of the synthesized findings.
Romanian Journal of Veterinary Sciences 2026, 59, 1 202 of 212
3. Theoretical basis 3.1. Genes involved in mitochondrial metabolism and cellular respiration 3.1.1. Role of HSP90 in cellular stress response and thermotolerance HSP90 is one of the most abundant molecular chaperones in eukaryotic cells, constituting approximately 1–2% of total cellular protein under normal conditions. It plays a fundamental role in maintaining protein homeostasis by stabilizing, folding, and refolding denatured proteins, particularly during heat stress. Two major cytoplasmic isoforms exist: HSP90α, the inducible form, and HSP90β, the constitutively expressed form [Sreedhar et al., 2004]. HSP90α is rapidly upregulated during thermal stress through heat shock elements (HSEs) in its promoter region, supporting immediate cytoprotection and cellular repair mechanisms. In contrast, HSP90β maintains general cellular stability and can also be moderately induced through intronic HSEs, contributing to longer-term adaptation. These isoforms differ in dimerization efficiency and client protein interactions, with HSP90α showing higher activity under acute stress conditions. The differential regulation and functional specificity of HSP90 isoforms underscore their essential role in cellular resilience to elevated temperatures. In cattle, upregulation of HSP90 under heat stress likely prevents protein aggregation and preserves mitochondrial and cytoskeletal function, enhancing thermotolerance. Given its central role in the cellular stress response, HSP90 represents a critical genetic marker for assessing thermal resilience in livestock populations [Sreedhar et al., 2004]. The HSP90AA1 gene encodes a major heat shock protein involved in maintaining cellular protein homeostasis under stress conditions. In dairy cattle, exposure to elevated ambient temperatures activates heat shock responses, leading to increased expression of molecular chaperones such as HSP90, which assist in stabilizing and refolding denatured proteins. Recent findings have demonstrated that genetic polymorphisms within the HSP90AA1 gene are significantly associated with thermotolerance traits in cattle [Badri et al., 2018]. Structurally, HSP90 proteins share three conserved regions interspersed with four variable regions and exhibit molecular masses close to 90 kDa. Their functional domains mediate ATP binding, ATPase activity, and interaction with cochaperones. Evolutionary analysis indicates that HSP90 genes underwent multiple duplication and loss events, reflecting their central role in adaptation and cellular stress responses. Given its broad functional repertoire and evolutionary conservation, HSP90 is a critical player in both normal cellular physiology and the organism’s response to environmental stressors [Chen, B et al., 2006]. Badri et al. (2018) identified five novel single nucleotide polymorphisms (SNPs) within the HSP90AA1 gene in Chinese Holstein cows. Two of these polymorphisms, located in the promoter region (g.-87G>C) and the 3'-untranslated region (g.4172A>G), were found to influence gene expression during heat stress. Functional assays revealed that the mutant C allele at position g.-87 substantially increased promoter activity, resulting in a 297% higher luciferase reporter signal compared to the G allele under heat shock conditions. This suggests that animals carrying the C allele possess an enhanced transcriptional response to thermal stress [Badri et al., 2018]. Moreover, microRNA analysis showed that miR-2279 binds to the 3'-UTR containing the G allele, inhibiting HSP90AA1 expression post-transcriptionally. The G to A substitution at g.4172 reduces this miRNA binding, allowing higher HSP90 expression levels under stress conditions. This finding highlights the importance of post-transcriptional regulation in finetuning cellular heat shock responses [Badri et al., 2018]. Phenotypically, cattle harboring the C allele in the promoter region or the A allele in the 3'-UTR exhibited improved physiological parameters under thermal stress, including lower somatic cell counts (SCC), which reflect better mammary gland health during adverse environmental conditions. These results strongly support the role of HSP90AA1 polymorphisms in modulating heat stress resilience in dairy cattle [Badri et al., 2018].
Romanian Journal of Veterinary Sciences 2026, 59, 1 203 of 212
The identification of regulatory variants in HSP90AA1 that enhance transcriptional and post-transcriptional control mechanisms provides a promising avenue for selecting thermotolerant cattle. The upregulation of HSP90AA1 in response to environmental heat may help protect critical proteins from denaturation, thus preserving cellular function and maintaining animal productivity under rising temperature challenges [Badri et al., 2018]. Garner et al. (2020) reported that HSP90AB1 expression was significantly upregulated in both peripheral white blood cells and milk somatic cells of Holstein cows exposed to short-term moderate heat stress. This sustained increase suggests that HSP90 participates in maintaining proteostasis by stabilizing regulatory proteins, including kinases, transcription factors, and hormone receptors, which are essential for cellular survival under prolonged thermal challenges. Unlike heat shock protein 70 (HSP70), which is characterized by a rapid and transient induction following heat exposure, HSP90 expression appears to follow a more gradual and enduring pattern. This behavior reflects its function in preserving cellular signaling pathways and preventing apoptosis during the recovery phase. The coordinated upregulation of HSP90 under heat stress conditions emphasizes its role in safeguarding systemic physiological functions and supporting long-term cellular adaptation in dairy cattle [Garner et al., 2020]. HSP90 plays an indispensable role in the cellular defense system against heat-induced proteotoxicity, extending beyond acute protection to ensure the long-term stability of essential regulatory proteins. By safeguarding kinases, hormone receptors, and transcription factors under thermal stress, HSP90 maintains cellular signaling integrity and supports systemic adaptation to environmental challenges. Its robust induction during heat stress, influenced by both transcriptional and posttranscriptional mechanisms, underscores its potential as a biomarker for thermotolerance and a target for genetic improvement programs in dairy cattle. Multi-omics approaches, particularly transcriptomics and protein–protein interaction mapping, are essential for uncovering the regulatory networks involving HSP90 in heat-stressed cattle. Collectively, these properties position HSP90 as a central molecular component in orchestrating the resilience of livestock under climateinduced stress conditions [Morán Luengo et al., 2019]. 3.1.2. Role of HSP70 in cellular stress response and thermotolerance Beyond the role of HSP90 in sustaining proteome stability during prolonged thermal stress, HSP70 emerges as a crucial early responder, orchestrating rapid cytoprotection by preventing protein aggregation and facilitating the recovery of heatdamaged proteins. HSPs are a critical component of the cellular defense machinery activated under environmental stress, particularly heat stress [Bharati et al., 2017]. Under thermal stress conditions, HSP70 is rapidly upregulated to stabilize unfolded proteins, assist in refolding denatured proteins, and prevent irreversible aggregation. Among them, HSP70 is one of the most conserved and functionally important, acting as a molecular chaperone that ensures proper protein folding, refolding of denatured proteins, and degradation of damaged proteins. Its role in maintaining cellular homeostasis makes it central to the development of thermotolerance in livestock species [Lindquist and Craig, 1988]. Recent studies, including the work of Rakib et al. (2024), have highlighted the practical relevance of HSP70 detection in the context of livestock management. HSP70 expression levels are positively correlated with traditional physiological markers of heat load such as rectal temperature, respiration rate, and heart rate, reinforcing its validity as a functional indicator of systemic stress. Moreover, HSP70 can be assessed in multiple biological matrices, including blood, peripheral blood mononuclear cells (PBMCs), saliva, and milk, offering various options for non-invasive or minimally invasive stress monitoring Among these, milk-based HSP70 detection holds particular promise for field applications due to ease of sample collection without additional animal handling. Functionally, HSP70 also modulates apoptosis by inhibiting apoptosome formation and caspase activation, supporting cell survival during acute and prolonged heat exposure Its chaperone activity extends into the modulation of the immune system, where extracellular HSP70 can act as a danger-associated molecular pattern (DAMP), activating innate and adaptive immune responses during stress episodes [Rakib et al., 2024]. In a study conducted by Bharati et al. (2017) on Tharparkar cattle (Bos indicus), HSP70 was shown to play a pivotal role in adaptive responses to chronic heat stress. Cattle exposed to 42 °C for 23 days exhibited a biphasic HSP70 expression pattern, with peaks at Day 17 and Day 32 of thermal challenge. The initial peak reflected an acute cytoprotective response, Rom. J. Vet. Sci. 2026, 59, 1
Romanian Journal of Veterinary Sciences 2026, 59, 1 204 of 212 while the second peak suggested the activation of a delayed, secondary adaptive mechanism, potentially providing a "second window" of protection during prolonged exposure [Bharati et al., 2017]. Both intracellular HSP70 levels in PBMCs and extracellular serum HSP70 concentrations mirrored this biphasic trend, highlighting the potential of eHSP70 as a non-invasive biomarker for assessing heat stress. In vitro experiments with cultured PBMCs further demonstrated that HSP70 expression increased in a temperature- and time-dependent manner, with the highest induction observed after 6 hours at 42 °C. Immunocytochemical analysis revealed HSP70 localization within the cytoplasm, nucleus, and cell membrane, underscoring its widespread protective role across cellular compartments [Bharati et al., 2017]. Molecularly, HSP70 functions by binding to hydrophobic regions of non-native proteins, using ATP hydrolysis to assist in proper folding or targeting irreversibly damaged proteins for degradation. In heat stress contexts, this action is critical to maintaining mitochondrial and cytosolic proteostasis and preventing cellular apoptosis [Mayer and Bukau, 2005]. Compared to HSP70, the heat shock protein HSP90 also participates in cellular defense mechanisms but follows a slightly different expression pattern and functional focus. HSP90 generally exhibits a more gradual induction during heat stress and plays a predominant role in stabilizing signal transduction proteins, steroid receptors, and kinases [Sreedhar et al., 2004]. Although Badri et al. (2018) demonstrated that certain HSP90AA1 promoter polymorphisms lead to increased gene expression under seasonal heat stress, specific time-course dynamics (e.g., expression peaks at 12h, 24h) were not characterized in that study. Thus, while HSP70 ensures rapid buffering against acute proteotoxic stress, HSP90 supports sustained recovery and regulatory stability. This complementary action suggests that monitoring both HSP70 and HSP90 expression may provide a more complete picture of thermotolerance potential [Badri et al., 2018]. Recent in vivo studies have provided valuable insights into the molecular adaptations of dairy cattle to thermal stress. Garner et al. (2020) demonstrated that heat stress leads to significant upregulation of heat shock proteins, notably members of the HSP70 family. In Holstein cows subjected to short-term heat challenges, HSPA6, a gene encoding an HSP70 family member, showed a 2.1-fold increase in expression in peripheral white blood cells and a 2.3-fold increase in milk somatic cells compared to thermoneutral controls [Garner et al., 2020]. This pronounced upregulation of HSP70 suggests a critical role in the acute cellular recovery phase by facilitating protein refolding, preventing aggregation of damaged proteins, and enhancing cell survival under proteotoxic conditions. Moreover, the persistence of HSP70 expression during the four-day heat exposure period aligns with previous findings indicating that thermotolerance mechanisms, once activated, can sustain cellular protection for several days post-stress. These observations reinforce the potential of HSP70 as an early and sensitive molecular biomarker for heat stress resilience in dairy cattle [Garner et al., 2020]. Given the dynamic, stress-inducible nature of HSP70 and its association with improved heat resilience in cattle, HSP70 represents a promising biomarker for assessing thermal stress responses [Sreedhar et al., 2004]. These findings, supported by concurrent physiological responses such as increased rectal temperature and respiration rate during heat exposure, establish a strong theoretical basis for considering HSP70 not only as a molecular indicator of thermal stress but also as an active mediator of cellular thermotolerance. High inducibility of HSP70 could serve as an early molecular indicator of an animal’s capacity to survive and maintain performance under heat load, offering valuable tools for selection and management in climate-adapted livestock systems [Bharati et al., 2017]. To illustrate the distinct roles of HSP70 and HSP90 during thermal stress adaptation, a comparative schematic is presented in Figure 2. HSP70 is rapidly induced during the acute phase of heat stress to prevent protein misfolding and aggregation, while HSP90 exhibits a slower, sustained response, primarily stabilizing regulatory proteins critical for cellular homeostasis. Integrating transcriptomic and proteomic data has proven valuable in assessing HSP70 gene expression levels and protein abundance under thermal stress, offering a comprehensive view of its role as a thermotolerance biomarker. Their complementary functions underscore the importance of evaluating both chaperones when assessing thermotolerance potential in cattle [Bharati et al., 2017, Sreedhar et al., 2004].
Romanian Journal of Veterinary Sciences 2026, 59, 1 205 of 212
Figure 2. Comparative Roles of HSP70 and HSP90 in the Cellular Response to Heat Stress, generated by OpenAI Sora.
3.1.3. Role of PGC-1α as a Master Regulator of energy homeostasis and thermotolerance PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a cold-inducible transcriptional coactivator critically involved in the regulation of mitochondrial and peroxisomal biogenesis, oxidative metabolism, and cellular energy balance [pg]. Initially discovered in brown adipose tissue as a key player in non-shivering thermogenesis, PGC-1α enhances mitochondrial uncoupling and heat production through the co-activation of PPARα/γ and RXRα receptors, particularly inducing UCP-1 expression. In skeletal muscle and liver, it coordinates fatty acid oxidation and gluconeogenesis, respectively, ensuring that energy demands during environmental or physiological stress are efficiently met [Mihaylov et al., 2023]. Beyond its classical roles, PGC-1α also directly modulates oxidative stress responses. It stimulates the expression of reactive oxygen species (ROS)-detoxifying enzymes, thus minimizing oxidative damage. Evidence from knockout mouse models demonstrates that PGC-1α deficiency leads to elevated oxidative stress, dopaminergic cell death, and impaired thermogenic responses, underlining its protective role during cellular stress [Xu et al., 2016]. A striking feature of PGC-1α is its upregulation during environmental challenges such as cold exposure, fasting, and exercise, which correlates with increased mitochondrial biogenesis and OXPHOS gene expression. This response involves direct interactions with nuclear respiratory factors (NRF-1 and NRF-2) and the mitochondrial transcription factor A (TFAM), ultimately enhancing mitochondrial DNA replication and transcription [Mihaylov et al., 2023]. Interestingly, PGC-1α not only promotes mitochondrial adaptations but also interacts with the cellular heat shock response machinery. Recent findings reveal that PGC-1α physically associates with heat shock factor 1 (HSF1) at heat shock elements (HSEs) in promoter regions of HSP genes like HSP70. This interaction suggests that PGC-1α can directly regulate stress-responsive genes, providing a dual mechanism for coping with thermal insults: augmenting mitochondrial resilience and enhancing protein homeostasis [Xu et al., 2016]. Functionally, experimental data show that PGC-1α ectopic expression leads to a significant upregulation of genes involved in heat shock responses, while its deficiency impairs HSP production and increases susceptibility to apoptosis after heat exposure. Thus, PGC-1α emerges as a pivotal orchestrator of thermotolerance through both mitochondrial and proteostatic pathways [Xu et al., 2016]. Furthermore, nuclear functions of PGC-1α extend to the export of mitochondrial mRNA. The serine/arginine-rich (RS) domain of PGC-1α interacts with the nuclear RNA export receptor NXF1, ensuring proper trafficking of transcripts encoding mitochondrial proteins. This mechanism supports mitochondrial biogenesis and functionality under stressful conditions, adding a novel layer to PGC-1α’s multifaceted role in cellular defense [Mihaylov et al., 2023]. Overall, the evidence highlights PGC-1α not merely as a metabolic coactivator but as a master integrator of mitochondrial adaptation, oxidative stress mitigation, and heat stress resilience. Genomic studies combined with transcriptomics can help elucidate the regulation of PGC-1α pathways during mitochondrial biogenesis under heat stress, providing insight into cattle energy metabolism adaptation. Its regulation of energy metabolism and stress response
Romanian Journal of Veterinary Sciences 2026, 59, 1 206 of 212 pathways positions it as a potential biomarker and therapeutic target for improving thermotolerance and metabolic stability in livestock and possibly in broader biological systems [Mihaylov et al., 2023, Xu et al., 2016]. 3.1.4. NRF2 activation and its implications for cellular thermotolerance Nuclear factor erythroid 2–related factor 2 (NRF2) is a pivotal transcription factor responsible for orchestrating cellular antioxidant defenses and maintaining redox homeostasis during environmental stress, including heat stress. Under basal conditions, NRF2 is retained in the cytoplasm by Kelch-like ECH-associated protein 1 (KEAP1), which targets it for ubiquitin-mediated degradation. Upon exposure to oxidative or thermal insults, conformational changes in KEAP1 liberate NRF2, enabling its nuclear translocation and binding to antioxidant response elements (AREs) in the promoter regions of target genes [Bellezza et al., 2018]. Through this mechanism, NRF2 drives the transcription of a broad array of cytoprotective genes, including heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD1), and glutathione peroxidase (GPX). This comprehensive antioxidant response mitigates the accumulation of reactive oxygen species (ROS), thus preserving mitochondrial function and protecting cells against heat-induced oxidative damage [Bellezza et al., 2018, Loboda et al., 2016]. Experimental evidence demonstrates that NRF2 activation supports mitochondrial biogenesis and quality control. NRF2 enhances the expression of mitochondrial protective factors, including transcription factors like NRF1 and TFAM, thereby sustaining mitochondrial DNA integrity and promoting efficient oxidative phosphorylation during periods of environmental challenge [Bellezza et al., 2018]. Conversely, deficiencies in NRF2 signaling have been linked to increased mitochondrial dysfunction, heightened ROS generation, and exacerbated cellular injury following heat exposure [Loboda et al., 2016]. Although NRF2 is not the primary regulator of HSPs, studies suggest that NRF2 signaling interacts with the heat shock response. The upregulation of antioxidant defenses by NRF2 complements the chaperone-mediated refolding functions of HSPs, jointly enhancing cellular resilience under thermal stress. This functional interplay highlights the importance of NRF2 not only in redox regulation but also in preserving proteostasis during heat challenges [Bellezza et al., 2018]. Furthermore, genetic polymorphisms in components of the NRF2 signaling pathway have been associated with differences in thermotolerance among individuals, emphasizing its potential as a biomarker for resilience to oxidative and thermal stress. Strategies aimed at enhancing NRF2 activation through genetic selection or nutritional interventions could thus offer promising avenues for improving heat tolerance in livestock species. Overall, NRF2 serves as a central mediator of oxidative defense and mitochondrial preservation, contributing significantly to cellular thermotolerance mechanisms and offering a valuable target for future breeding and management strategies [Loboda et al., 2016]. The potential of NRF2 as a biomarker for heat stress resilience in cattle lies in its central role in orchestrating antioxidant defenses and maintaining mitochondrial integrity under thermal challenges. By upregulating cytoprotective genes such as HO-1, NQO1, and SOD1, NRF2 mitigates oxidative damage that typically intensifies during heat exposure, preserving cellular homeostasis and energy metabolism [Bellezza et al., 2018, Loboda et al., 2016]. Also, genetic variability in NRF2-related pathways has been associated with differential thermotolerance across cattle breeds, suggesting that NRF2 expression levels or activity patterns could serve as early molecular indicators of resilience to heat stress. Integrated omics analyses, including transcriptome and proteome profiling, allow detailed evaluation of NRF2-driven antioxidant responses and their modulation during thermal stress conditions [Loboda et al., 2016]. 3.1.5. Role of SOD1 in oxidative stress defense and thermotolerance Superoxide dismutase 1 (SOD1) plays a pivotal role in the antioxidant defense system of bovine cells, particularly under heat stress conditions. As one of the primary enzymes responsible for catalyzing the dismutation of superoxide radicals into hydrogen peroxide and oxygen, SOD1 protects cellular structures from oxidative damage. Heat stress leads to an overproduction of reactive oxygen species (ROS), challenging cellular homeostasis. Khan et al. (2021) demonstrated that silencing SOD1 expression in bovine granulosa cells under thermal stress significantly increased ROS levels, promoted apoptosis, disrupted mitochondrial membrane potential, and impaired steroid hormone synthesis. These findings highlight the critical cytoprotective function of SOD1 during heat-induced oxidative stress, especially in reproductive tissues [Khan et al., 2021].
Romanian Journal of Veterinary Sciences 2026, 59, 1 207 of 212
Genetic variations in the SOD1 gene have also been associated with differing levels of heat tolerance among cattle breeds. Zeng et al. (2018) identified specific SNPs within the SOD1 gene that correlated with improved thermal adaptability in Chinese cattle populations. Animals carrying favorable alleles exhibited enhanced antioxidant responses, suggesting that SOD1 genetic variability could serve as a valuable marker for selecting thermotolerant individuals in breeding programs. This genetic association reinforces the role of SOD1 not only as a functional antioxidant but also as a potential molecular biomarker for improving resilience to heat stress at the population level [Zeng et al., 2018]. Taken together, the available evidence underscores the dual importance of SOD1 in cattle exposed to thermal stress: first, through its essential antioxidant action protecting cells from oxidative damage, and second, through its potential use as a genetic marker in thermotolerance selection programs. Incorporating SOD1 expression profiles or polymorphism screening into cattle breeding strategies could provide significant advantages for sustaining livestock productivity and health under the increasingly challenging conditions imposed by global warming [Zeng et al., 2018]. Several studies suggest that SOD1 can serve as a reliable biomarker for evaluating oxidative stress and thermotolerance in cattle. Heat stress conditions elevate oxidative damage, and animals capable of upregulating antioxidant enzymes like SOD1 exhibit greater cellular resilience. Zeng et al. (2018) reported that specific polymorphisms in the bovine SOD1 gene were associated with enhanced thermotolerance traits, supporting its use in genetic selection. Similarly, Khan et al. (2021) demonstrated that reduced SOD1 expression in heat-stressed bovine granulosa cells led to higher oxidative stress and apoptosis, reinforcing the idea that SOD1 levels reflect the degree of thermal adaptation. Thus, both expression studies and genetic association analyses validate the potential of SOD1 as a biomarker for identifying heat-resilient cattle [Khan et al., 2021, Zeng et al., 2018]. 3.2. Genes involved in mitochondrial metabolism and cellular respiration 3.2.1. Role of Cytochrome C in mitochondrial function and stress adaptation Cytochrome c (CYCS) is a highly conserved mitochondrial protein essential for cellular energy metabolism and apoptosis regulation. Within the electron transport chain, CYCS shuttles electrons between Complex III and Complex IV, sustaining OXPHOS and ATP production. Proper regulation of CYCS activity minimizes electron leakage and ROS generation, preserving mitochondrial integrity under stress conditions [Morse et al., 2024]. Under heat and oxidative stress, CYCS assumes a dual role. While supporting mitochondrial respiration under homeostatic conditions, its release into the cytosol acts as a potent pro-apoptotic signal. Heat stress-induced mitochondrial membrane depolarization facilitates CYCS leakage, promoting apoptosome formation and caspase activation, leading to programmed cell death. Maintaining mitochondrial membrane potential is therefore crucial to prevent CYCS-mediated apoptosis during thermal challenges [Morse et al., 2024]. Post-translational modifications (PTMs) of CYCS further modulate its function during stress adaptation. Phosphorylation at residues such as threonine 28, serine 47, and tyrosine 48 reduces electron transport efficiency but lowers ROS production and apoptotic susceptibility, acting as a protective adaptation. Conversely, acetylation at lysine 39 enhances mitochondrial respiration and inhibits CYCS release, promoting survival during ischemic or oxidative insults [Morse et al., 2024]. In bovine models, mitochondrial fragmentation under heat stress has been shown to trigger CYCS release, linking mitochondrial network integrity directly to cell survival. Zhang et al. (2020) reported that heat stress disrupts mitochondrial dynamics in bovine mammary epithelial cells, facilitating CYCS-mediated apoptosis. These findings emphasize the sensitivity of CYCS regulation to thermal insults and its relevance in maintaining cellular and tissue homeostasis under stress [Chen, K-L et al., 2020]. Thus, CYCS emerges as a critical regulator of mitochondrial function, shifting between energy metabolism and apoptotic signaling depending on cellular stress levels. Monitoring CYCS expression patterns, subcellular localization, or post-translational modifications may provide valuable biomarkers for assessing mitochondrial health and thermotolerance in cattle populations subjected to increasing environmental heat load [Chen, K-L et al., 2020, Morse et al., 2024]. 3.2.2. ND1, ND2, ND4, and ND5: Key mitochondrial genes of Complex I (NADH Dehydrogenase) Mitochondrial metabolism and cellular respiration are fundamental processes underpinning energy homeostasis in all eukaryotic cells. Complex I (NADH:ubiquinone oxidoreductase), the largest and most intricate component of the
Romanian Journal of Veterinary Sciences 2026, 59, 1 208 of 212 mitochondrial respiratory chain, plays a central role in these processes. It consists of over 45 subunits, seven of which are encoded by the mitochondrial genome: ND1, ND2, ND3, ND4, ND4L, ND5, and ND6. Among these, ND1, ND2, ND4, and ND5 are critical membrane-associated subunits responsible for electron transfer and proton translocation across the inner mitochondrial membrane—processes essential for maintaining mitochondrial membrane potential and driving ATP synthesis [Hirst, 2013]. Emerging experimental studies suggest that mitochondrial genes, including those encoding Complex I subunits, are sensitive to environmental and physiological stressors. Oxidative imbalance and metabolic overload can influence the expression and functional capacity of ND1, ND2, ND4, and ND5, thereby modulating the bioenergetic capacity of mitochondria. This adaptive regulation highlights the importance of mitochondrial plasticity in cellular responses to environmental stress, such as heat exposure, and in the maintenance of energy [Hirst, 2013]. Dorji et al. (2020) performed a comprehensive transcriptomic analysis across 17 tissues from Jersey crossbred cows, revealing that mitochondrial protein-coding genes, including ND1, ND2, ND4, and ND5, show tissue-specific expression patterns closely associated with metabolic demand. Specifically, these genes were highly expressed in energy-intensive tissues such as the heart, skeletal muscle, and mammary gland, highlighting their critical role in sustaining ATP production for high metabolic activity. Conversely, tissues with lower energy requirements, such as blood and lymph nodes, exhibited comparatively reduced ND gene expression. This differential expression underlines the adaptability of mitochondrial bioenergetics to tissue-specific physiological functions in cattle [Dorji et al., 2020]. Moreover, Dorji et al. (2020) noted that mitochondrial gene expression was tightly co-regulated, suggesting a coordinated control of the entire oxidative phosphorylation pathway to meet cellular energy needs. Although their study was conducted under normal physiological conditions, the findings imply that any external stressors, such as heat stress, could significantly disrupt this balance, especially in tissues reliant on continuous high energy output. Given that mitochondrial integrity is essential for thermotolerance, and ND genes form the core of Complex I, the expression dynamics described by Dorji et al. (2020) provide a crucial baseline for understanding how mitochondrial function might be impaired under thermal stress conditions in cattle. Future studies could leverage these insights to evaluate mitochondrial gene responsiveness under heat stress and identify potential biomarkers for selecting more resilient animals [Dorji et al., 2020]. Overall, the ND1, ND2, ND4, and ND5 genes represent crucial determinants of mitochondrial respiratory efficiency and cellular energy balance. Their vulnerability to mutations and their regulatory responsiveness to environmental stressors position them as important candidates for further investigation in the context of thermotolerance, metabolic resilience, and mitochondrial health in cattle. A deeper understanding of these genes’ functional dynamics is essential for developing strategies aimed at mitigating mitochondrial dysfunction and enhancing livestock adaptation to climateinduced thermal stress [Fassone and Rahman, 2012, Hirst, 2013]. 3.2.3. COX1, COX2, and COX3 Expression and Complex IV Function in Bovine Heat Stress Mitochondrial Complex IV, also known as cytochrome c oxidase (CcO), plays a fundamental role in the mitochondrial respiratory chain by catalyzing the transfer of electrons from cytochrome c to molecular oxygen, ultimately sustaining ATP synthesis. In mammals, CcO is composed of 13 subunits, of which COX1, COX2, and COX3 are encoded by the mitochondrial genome and form the catalytic core. COX1 harbors the heme a and heme a3/CuB centers essential for oxygen reduction, while COX2 contains the CuA center responsible for receiving electrons from cytochrome c. These mitochondrial-encoded subunits are highly conserved and crucial for the electron transfer activity and proton translocation that maintain the mitochondrial membrane potential, essential for energy production in bovine tissues [Kadenbach et al., 2004]. Under heat stress conditions, the regulation of CcO activity becomes critical for cellular survival. Vogt et al. (2011) demonstrated that HSP induction can protect mitochondrial function during stress by stabilizing CcO subunit composition and preventing its degradation. Heat-induced HSPs, particularly HSP70 and HSP60, assist in preserving the structure and
Romanian Journal of Veterinary Sciences 2026, 59, 1 209 of 212 assembly of CcO, safeguarding the catalytic activities of COX1, COX2, and COX3 subunits. Without this protection, heat stress could impair the transcription and processing of these mitochondrial genes, thereby compromising oxidative phosphorylation and exacerbating ROS production [Vogt et al., 2011]. Mitochondrial energy metabolism is finely regulated through mechanisms including the allosteric ATP-inhibition of CcO, where high ATP/ADP ratios inhibit CcO activity to maintain a lower mitochondrial membrane potential and reduce ROS generation. Under stress conditions such as heat exposure, this inhibition is often switched off through dephosphorylation or signaling pathways, leading to an increase in membrane potential and elevated ROS levels. This phenomenon highlights how failure to regulate COX1–COX3 function under heat stress could initiate mitochondrial dysfunction, contributing to cellular damage in cattle exposed to thermal extremes [Kadenbach et al., 2004]. Further evidence from ischemia-reperfusion models parallels heat stress responses, showing that hypoxia and subsequent reoxygenation can lead to phosphorylation changes in CcO subunits I (COX1), IV, and Vb, causing decreased enzyme activity. Such post-translational modifications impair the structural and functional integrity of Complex IV, underlining the vulnerability of mitochondrial-encoded subunits during oxygen flux disturbances. In cattle subjected to heat stress, similar dysregulation of mitochondrial dynamics could lead to impaired energy recovery and enhanced susceptibility to oxidative injury if CcO stability is not adequately maintained [Vogt et al., 2011]. Altogether, COX1, COX2, and COX3 are essential for sustaining bovine mitochondrial respiration and energy balance, particularly under thermal stress conditions. Heat-induced expression of stress proteins appears to be a critical adaptive response that stabilizes these subunits, maintaining Complex IV functionality. Understanding how bovine mitochondria regulate the expression, assembly, and activity of mitochondrial-encoded CcO subunits during heat stress could offer new avenues for improving cattle thermotolerance and resilience to climate challenges [Kadenbach et al., 2004, Vogt et al., 2011].
4. Final remarks and conclusions This review underscores the critical role of mitochondrial respiration genes, particularly those encoding subunits of Complex I (ND1, ND2, ND4, ND5) and Complex IV (COX1, COX2, COX3), in shaping the heat stress response in dairy cattle. Elevated temperatures disrupt the expression and function of these genes, impairing electron transport, reducing ATP synthesis, and increasing oxidative stress, ultimately compromising cellular energy balance and productivity. Additionally, stress-responsive genes such as HSP70, HSP90, NRF2 , SOD1, and CYCS play pivotal roles in protecting mitochondrial integrity, regulating antioxidant defenses, and controlling apoptosis under thermal challenges. Identifying and understanding the genetic architecture underpinning mitochondrial function and cytoprotection is therefore essential for mitigating the adverse metabolic consequences of heat stress in livestock. Integrated multi-omics approaches—including genomics, transcriptomics, and proteomics—offer powerful tools for deciphering the complex biological networks involved in thermotolerance. Recent studies highlight the value of combining gene expression profiling, mitochondrial DNA variant analysis, and protein abundance measurements to discover molecular signatures associated with heat resilience. These multi-layered datasets enable a more comprehensive understanding of how mitochondrial efficiency, oxidative stress responses, proteostasis, and cell survival mechanisms interact under thermal challenges, thereby providing a robust foundation for the identification of candidate markers for breeding selection programs. Harnessing these insights is pivotal for the development of precision breeding strategies and adaptive management practices aimed at enhancing cattle resilience to global warming. Future efforts should prioritize the validation of mitochondrial and nuclear genetic markers of thermotolerance—including those associated with respiratory chain stability, oxidative stress mitigation, and apoptosis regulation—across breeds and production systems. By targeting mitochondrial health, cellular respiration efficiency, and stress-adaptive pathways, the dairy industry can improve animal welfare, sustain productivity, and better prepare for the escalating environmental pressures imposed by climate change. To facilitate the integration of the evidence presented, a summary table is provided (Table 1), outlining key genetic markers linked to mitochondrial respiration, their main biological functions, the effects of heat stress on their activity, and their potential applications in breeding and management strategies. This synthesis highlights how multi-omics approaches can systematically identify and validate molecular signatures of thermotolerance in dairy cattle.
Romanian Journal of Veterinary Sciences 2026, 59, 1 210 of 212
Table 1. Key genetic markers of mitochondrial respiration associated with heat stress resilience in dairy cattle. Gene/Marker HSP70 HSP90 PGC-1α NRF2 SOD1 CYCS ND1, ND2, ND4, ND5 (Complex I) COX1, COX2, COX3 (Complex IV)
Main Function Molecular chaperone, protein folding Protein stabilization, signal transduction Mitochondrial biogenesis and energy metabolism Antioxidant defense regulator ROS scavenging (superoxide dismutase) Electron transport, apoptosis regulation NADH oxidation, proton pumping Terminal electron transfer to oxygen
Effect of Heat Stress Rapid upregulation, cytoprotection Modulates mitochondrial integrity, HSF1 activation Impaired activation under chronic stress Disrupted signaling increases ROS Decreased expression, oxidative damage Increased release, mitochondrial dysfunction Downregulated, ATP deficiency Downregulated, impaired respiration
Application Biomarker for heat stress detection Target for thermotolerance breeding Indicator of mitochondrial adaptation Candidate for antioxidant capacity enhancement Genetic marker for oxidative stress resistance Indicator of apoptosis and mitochondrial stability Target for mitochondrial efficiency breeding Marker for mitochondrial energy resilience