Establishment and characterization of an equine skeletal muscle in vitro platform: gene expression validation and hypoxia-responsive signatures

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The study focused on generating a primary equine skeletal muscle cell line, confirming its myogenic identity through qPCR analysis of the markers MYOD1 , MYF5 , MYOG , and PAX7 , identifying and validating suitable endogenous reference genes for normalization, and assessing cellular responses to normoxia and hypoxia based on the expression profile of HIF1A , a master regulator reflecting oxygen availability in working muscle. Methods Primary equine skeletal muscle cells were isolated using collagenase and pronase treatment or the explant method and cultured under standard conditions. Gene expression was assessed by qPCR, including the evaluation of housekeeping genes for normalization. Hypoxic cultures were subsequently maintained at 3% O₂ in a HypoxyLab incubator, while normoxic controls were kept at 37°C with 5% CO₂. All cultures were handled in parallel under otherwise identical conditions. Results Collagenase digestion provided the most viable myogenic cells, whereas pronase and explant methods yielded less suitable populations. Among eight candidate reference genes evaluated with ΔCt, BestKeeper, NormFinder, and geNorm, ACTB emerged as the most stable, followed by RN18S , SDHA , and GAPDH . In contrast, B2M and TFRC showed the lowest stability and were deemed unsuitable for normalization. Conclusions Collagenase digestion is the most suitable method for establishing equine myogenic cell cultures, with ACTB as the optimal endogenous control. Furthermore, these findings suggest that hypoxia does not markedly affect myogenic progression, thus it may not be a favourable condition for modelling accelerated muscle regeneration or stress-induced differentiation in high-performance animals such as racing horses. Equine skeletal muscle derived cells Reference genes Hypoxia houskeeping genes Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The outstanding velocity, endurance, and muscular power observed in modern sport horses reflect both their evolutionary adaptation as grazing animals and the cumulative effects of selective breeding, which have produced a wide spectrum of specialized breeds, including those optimized for athletic performance. Moreover, the pronounced plasticity of equine physiological systems in response to training and exercise underlies their capacity to achieve elite performance across diverse equestrian disciplines [ 1 ]. Among the key traits influencing performance is muscle mass, which plays a central role in determining a horse’s speed, acceleration, and endurance [ 2 , 3 ]. In horses, the skeletal muscles comprise up to 60% of the entire body weight and generate the force required for locomotion, postural support, and athletic performance. Their ability to contract rapidly and repeatedly under varying loads is fundamental to performance in both high-speed and endurance-based disciplines. Healthy equine skeletal muscle can fully regenerate after exercise, even though physical workload induces a transient inflammatory and stress response [ 4 , 5 ]. This inflammation is a physiological adaptation to muscle growth; however, excessive muscle inflammation, such as overexertion, insufficient warm-up or cool-down, nutritional deficiencies, or muscle disorders, might lead to muscle pain, stiffness, and thus reduced performance [ 6 ]. Muscle function relies on a continuous supply of oxygen for respiratory purposes, enabling the generation of contractile force. Fluctuating oxygen levels, particularly the decrease in oxygen supply (hypoxia) during physical exertion, have a twofold effect on muscle physiology, promoting beneficial adaptations while also posing potential stress-related risks. Reduced oxygen levels in skeletal muscle during exercise are mainly a consequence of heightened metabolic demand. This shift in cellular oxygen concentration has a direct impact on metabolic processes [ 7 ]. In equine studies, traditional approaches such as muscle biopsies, although informative, are invasive, raise ethical concerns, and present limitations in terms of repeatability and large-scale application. Consequently, within the framework of sport translational medicine, there is increasing emphasis on developing innovative research strategies that minimize reliance on live animal experimentation. The establishment of immortalized and functionally characterized equine cell lines provides a valuable alternative, enabling detailed investigation of molecular pathways underlying exercise adaptation, muscle physiology, and metabolic performance in vitro [ 8 ]. Such cellular models not only align with the principles of the 3Rs (Replacement, Reduction, Refinement) but also offer a reproducible and ethically responsible research platform, ultimately facilitating the translation of fundamental discoveries into practical applications in equine sports medicine and performance optimization. Moreover, in human research, skeletal muscle cell models are gaining popularity due to their ability to reflect donor-specific metabolic traits, offer lower variability than tissue obtained through biopsies, and present greater translational relevance than rodent-derived cell lines [ 9 ]. Reverse transcription real-time quantitative PCR (RT-qPCR) remains the preferred technique for precise mRNA quantification due to its exceptional sensitivity and the availability of a wide array of commercial assays targeting diverse equine transcripts. However, high precision can complicate interpretation: small fluctuations in mRNA introduced during sample handling or preparation may be mistaken for genuine changes in gene expression. To mitigate such issues, rigorous and appropriate normalization of the data is essential [ 10 ]. In response to growing interest in equine regenerative medicine and translation-al/nutrigenomic applications, we are developing an in vitro platform. This study aimed to develop an in vitro platform for equine skeletal muscle by establishing a primary cell line, confirming its myogenic identity by qPCR of MYOD1 (Myogenic Differentiation 1), MYF5 (Myogenic Factor 5), MYOG (Myogenin), and PAX7 (Paired Box 7) genes [ 11 , 12 ], identifying and normalizing suitable endogenous reference genes, and examining cellular responses under normoxic and hypoxic conditions via HIF1A (hypoxia-inducible factor 1) gene expression pattern that mirrors oxygen levels in working muscle. 2. Materials and Methods 2.1. Sample collection Skeletal muscle cells were isolated from cadaveric tissue, which was considered the most ethically appropriate source for the purposes of this study. Tissue collection was performed with the explicit permission of the animal's owner. The material was obtained from a 10-month premature foal that could not be saved despite veterinary intervention. Muscle samples were collected immediately following the cessation of vital functions. Under the Polish Act of 15 January 2015 on the protection of animals used for scientific or educational purposes (Journal of Laws 2015, item 266), which implements Directive 2010/63/EU of the European Parliament and of the Council, ethical approval is required only for procedures performed on living animals. Consequently, the use of biological material collected post-mortem, with owner consent, does not constitute an animal experiment and is exempt from ethical review. Dissection was carried out using a scalpel to a depth of approximately 6 cm in the gluteus medius muscle, under aseptic conditions to preserve sample quality for further cellular analyses. Immediately post-extraction, samples of skeletal muscle were rinsed twice in ice-cold PBS supplemented with penicillin-streptomycin (300 U/ml and 300 µg/ml) and then placed directly into 50 ml Falcon tube with ice-cold cell culture medium (DMEM Gluta-Max; Gibco) supplemented with 20% fetal bovine serum (FBS; Gibco), 10% horse serum (HS; Gibco) and penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomy-cin). Tissue samples were kept at 4°C and delivered to the laboratory within 4 hours post-collection to initiate primary myoblast isolation. 2.2. Equine skeletal muscle cell isolation and culture Preparation of the culture took place under aseptic conditions in a biosafety cabinet, utilizing sterile equipment. The muscle sample was divided into three portions, each weighing approximately 1 g, and washed with PBS solution in a Petri dish. All visible non-muscle components, including connective tissue, fat, blood vessels, and tendons, were meticulously removed. Two portions of skeletal muscle tissue (approximately 1 g each) were finely minced and transferred into separate 50 ml Falcon tubes. After allowing the fragments to settle by gravity, the supernatant was carefully aspirated and discarded. The minced tissue was subsequently resuspended in either 0.1% pronase [E.1.1.] or collagenase (440 U/ml) [E.1.2.] solution in DMEM medium supplemented with penicillin-streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin) and incubated at 37°C for 1 hour with agitation every 15 min. After enzymatic digestion, the reaction was terminated by adding an equal volume of culture medium supplemented with 10% FBS. The remaining tissue was then mechanically dissociated by repeated pipetting through pi-pettes of decreasing volumes (10 mL followed by 5 mL), to facilitate cell release. The sample was then passed through a 70 µm and 40 µm cell strainer, rinsed with fresh medium, and centrifuged at 1000 × g for 10 min to collect the cells. The cell pellet was resuspended in a culture medium (DMEM GlutaMax, 20% FBS, 10% HS, 100 U/ml penicillin and 100 µg/ml streptomycin) and subjected to a 1-hour pre-plating step in a 2% gelatin-coated T25 flask to minimize fibroblast contamination. After this period, non-adherent cells were transferred to fresh 2% gelatin-coated T25 flasks. One remaining piece [E.2.] of not minced and not digested muscle tissue was individually placed into 2% gelatin-coated T25 flask with supplemented medium and incubated at 37°C under a controlled atmosphere containing 5% CO₂. Cultures were incubated undisturbed for at least 3 days, after which the medium was gently replaced. Once the cells reached optimal confluence, the first passage was performed using trypsin solution (Trypsin 0.25%, Gibco) and a pre-plating step to further enrich the myoblast population. 2.3. Induction of hypoxia Approximately 2 × 10⁵ cells were seeded into T25 flasks pre-coated with 2% gelatin and allowed to attach overnight under standard culture conditions. The following day, flasks assigned to the hypoxic condition were transferred to a HypoxyLab incubator (Ox-ford Optronix) set to 3% O₂, using a custom gas mixture consisting of 5% air, 5% CO₂, and 90% N₂, delivered at a flow rate of 25 psi/min. Normoxic cultures remained in standard humidified incubators at 37°C with 5% CO₂. Both groups were cultured in parallel under otherwise identical conditions and sampled at four time points: 3, 6, 18, and 24 hours, to assess oxygen-dependent changes in gene expression. For each condition and time point, three technical replicates were performed to ensure the reliability of the measurements. 2.4. Gene expression studies At each time point, cells from both normoxic and hypoxic groups were harvested for downstream analysis. Total RNA was extracted using the PureLink RNA Mini Kit with DNase treatment (Ambion, Life Technologies). At each time point, cells were harvested for downstream analysis in three technical replicates. The RNA concentration and purity were assessed on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and integrity was evaluated by TapeStation 2200 (RNA ScreenTapes, Agilent Technologies, Santa Clara, CA, USA). The samples' RNA concentrations range from 32 ng/µL to 412 ng/µL, with all samples showing RIN values above 7.5. Complementary DNA (cDNA) was synthesized from 0.5 µg of total RNA using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems), following the manufacturer's instructions. Quantitative PCR (qPCR) was performed on a QuantStudio 7 system (Applied Biosystems, Life Technologies) in a final reaction volume of 10 µl. Each reaction contained 0.5 µl of cDNA, 5 µl of 2× HS Mix EvaGreen (A&A Biotechnology), 0.3 µl of each gene-specific primer (10 µmol/µl), and 4.4 µl of nuclease-free water. The thermal cycling protocol included an initial denaturation at 95°C for 10 seconds, followed by 40 amplification cycles of 95°C for 15 seconds and 55°C for 34 seconds. Gene expression analysis was carried out using three technical replicates. Primer pairs for: MYOD1, MYF5, MYOG, PAX7, HIF1A, ACTB, RN18S, SDHA, GAPDH, HPRTF, RPL32, B2M and TFRC were designed with Primer3 (v4.0.0). Sequences are provided in Supplementary Table S1 . Primers were positioned across exon–exon junctions and targeted conserved regions of the genes to ensure stable expression measurements in the presence of multiple transcript variants. Amplicon lengths were optimized to 104–194 bp, and primer specificity was confirmed in silico using BLAST. 2.4. Estimation of housekeeping genes Housekeeping gene stability was evaluated using widely accepted and publicly available algorithms, including geNorm [ 13 ], NormFinder version 0.953 [ 14 ], BestKeeper, and the comparative delta Ct method [ 15 ]. 2.5. Statistical analysis Data were collected at five time points (0, 3, 6, 18, and 24 hours) under normoxic and hypoxic (3% O₂) conditions, with time point 0 representing the start of the experiment. Statistical analysis of the stability of putative reference genes and ranking was provided by geNorm, NormFinder and BestKeeper. Expression levels of the myogenesis-related genes were normalized to each of the evaluated housekeeping genes. Gene expression ratios were then calculated using the 2^−ΔΔCT method. Expression patterns were compared across the different reference (normalizing) genes to evaluate the impact of reference gene selection.. Data are expressed as mean ± S.E. Statistical analysis was performed in R (version 4.3.1). Group comparisons were assessed using one-way ANOVA followed by Tukey's HSD (Honestly Significant Difference) post hoc test. For multiple pairwise comparisons, Holm’s correction was applied to control for false positives. p-values indicating sta-tistical significance are marked as: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 3. Results 3.1. Cell culture experiment Two muscle samples were enzymatically digested prior to culture using either pronase [E.1.1.] or collagenase [E.1.2.]. In the pronase-digested culture, a substantial amount of tissue debris was observed, accompanied by a lower proliferation rate compared to the collagenase-digested culture. A piece of muscle tissue [E.2.] was placed in an individual culture dish containing growth medium and incubated at 37°C. Within 2–3 days of culture initiation, the first signs of cell outgrowth were observed surrounding the muscle explant. By day 5, a distinct halo of migrating cells had formed around the tissue, and the cell density was considered sufficient to remove the explant from the culture dish. Although the population derived from halo outgrowth was the most abundant, these cells displayed marked morphological heterogeneity and showed signs of possible contamination, limiting their utility for downstream applications (Figures S1 a-S1b) After the third passage and pre-plating steps to enrich for myogenic cells, gene expression analysis was performed in three technical replicates. The results should be interpreted as descriptive tendencies rather than statistically confirmed differences. All groups showed comparable HIF1A and PAX7 expression. Collagenase-digested cells exhibited the highest MYOD1 and increased MYOG , while cells digested with the use of pronase showed lower MYOD1 but higher MYF5 and MYOG expression. Cells from tissue explant displayed low myo genes expression (Fig. 1 ). Given its intermediate myogenic profile [E.1.2.], cells were selected as the most suitable population for further investigation. Based on these observations, collagenase digestion was determined to be the most effective and reliable method for isolating muscle-derived cells. Consequently, only collagenase-isolated cells were used in subsequent analyses under varying oxygen conditions. 3.2. Identification of the most stable reference gene and comprehensive ranking of the reference genes. The stability of eight candidate reference genes was evaluated using four commonly applied algorithms: ΔCt, BestKeeper, NormFinder, and geNorm. Based on these analyses, a comprehensive stability ranking was generated by calculating the geometric mean of the individual ranking values obtained from each method (Table 1) Method 1 2 3 4 5 6 7 8 Delta CT ACTB RN18S SDHA GAPDH HPRTF RPL32 B2M TFRC BestKeeper GAPDH SDHA ACTB RN18S B2M HPRTF RPL32 TFRC Normfinder ACTB SDHA RN18S GAPDH RPL32 HPRTF B2M TFRC Genorm ACTB RN18S HPRTF RPL32 GAPDH SDHA B2M TFRC Recommended comprehensive ranking ACTB RN18S SDHA GAPDH HPRTF RPL32 B2M TFRC The most stable gene was ACTB (geomean = 1.316), followed by RN18S , SDHA , and GAPDH , indicating low variability and high suitability as reference genes in equine skeletal muscle samples. In contrast, B2M and TFRC ranked lowest, with geometric means of 6.435 and 8.000, respectively, suggesting low stability across the tested conditions (Fig. 2 ). To further evaluate the impact of reference gene choice on expression analysis outcomes, we compared qPCR data normalized individually with each of the candidate reference genes. The statistical analysis of the Tukey post hoc test with HSD correction revealed considerable variation in normalized expression levels of target genes, depending on the reference gene used. The most significant differences (padj < 0.05) in RQ calculated according to the reference used were for MYOG gene, according to comparison of TFRC vs B2M , GAPDH , and HPRTF as references used (Fig. 3 ). 3.3. Myogenesis-related molecular markers expression To investigate the effect of oxygen concentration on the expression of myogenic regu-latory factors, mRNA levels of HIF1A (hypoxia-inducible factor 1), MYOD1 (Myogenic Differentiation 1), MYF5 (Myogenic Factor 5), MYOG (Myogenin), and PAX7 (Paired Box 7) were quantified in cells cultured under normoxic (21% O₂) and hypoxic (3% O₂) conditions for 3, 6, 18, and 24 hours. Differentiation qPCR data were normalized to ACTB , identified as the most stable reference gene in this study. The mean Ct values of the analyzed genes ranged from 8.795 ± 0.359 ( RN18S ) to 33.9 ± 0.401 ( GAPDH ). For the myogenic markers, MYOD1 exhibited the highest mean Ct value (31,115 ± 3.411), and MYOG the lowest (22.408 ± 1.439) (Table S2 ). HIF1A , a marker of hypoxia response, peaked at 3 hours in both conditions but remained higher in hypoxia up to 6h. MYOD1 , a gene associated with early myogenic activation, showed increased expression at 24 hours under normoxic conditions, and a similar induction was also observed in hypoxia. MYF5 , a marker of muscle progenitor cells, showed elevated expression at early time points (3–6 h) in both conditions and subsequently declined, consistent with the expected temporal activation pattern. However, normoxic conditions appeared to better support the maintenance of an undifferentiated state. MYOG , indicative of terminal differentiation, showed an increase under both conditions, particularly at 8 hours. The transient decrease in PAX7 expression at 6 hours followed by recovery and a gradual increase at 24 hours, suggests a temporary activation phase of myogenic progenitors. This pattern indicates that the cells retain the capacity to maintain a progenitor pool under both oxygen conditions. Data represent the mean ± SEM of three technical replicates. No statistical testing was performed because this panel includes only technical replicates and no biological replicates, which precludes meaningful statistical inference (Fig. 5). Cell shape changes during in vitro equine myogenesis. Primary cultures of preterm foal were grown for 3, 6, 18, and 24 hours in different oxygen conditions. The microscopic images show morphological changes in cells exposed to hypoxic conditions compared to normoxic conditions. After 24 hours of exposure, both normoxic and hypoxic cultures display a dense monolayer of elongated, spindle-shaped cells typical of proliferating myogenic precursors. However, the hypoxia-treated cells appear slightly more aligned and elongated, suggesting early signs of myogenic commitment and differentiation. In contrast, the normoxic culture shows a more random orientation and distribution, which may reflect a less synchronized differentiation process. No overt signs of cell death or detachment were observed in either condition, indicating that short-term hypoxia was well tolerated and may even enhance structural organization in muscle cell populations. 4. Discussion An increasing number of studies are focused on establishing in vitro models for equine skeletal muscle; however, there is a clear need to refine these approaches to ensure reproducible and biologically relevant outcomes. As such, it is essential to standardize culture conditions, cell sources, and characterization methods to enable meaningful cross-study comparisons and translational applications in veterinary medicine [ 16 – 18 ]. In the present study, we derived an equine primary cell line from a premature-born foal and initiated work on characterizing its properties, addressing the limited availability of well-defined commercial cell lines for translational applications in equine regenerative medicine. Given the critical role of oxygen availability in maintaining muscle cell homeo-stasis and recognizing that its relative influence varies with the physiological state, we investigated cellular responses under varying oxygen conditions by quantifying the ex-pression of myogenic regulatory factors, including HIF1A , MYOD1 , MYF5 , MYOG , and PAX7 , in cells cultured under normoxic (21% O₂) and hypoxic (3% O₂) conditions for 3, 6, 18, and 24 hours. qPCR is a widely adopted gold-standard method for accurately quantifying gene expression and plays a central role in experimental and diagnostic applications [ 19 ], where the use of stable reference genes is essential. For establishing myogenic cells from skeletal muscle ( gluteus medius ) in a premature foal, we used enzymatic digestion with pronase [ 18 ] and collagenase [ 20 ] treatments, as well as the explant method [ 21 ]. Enzymatic isolation of satellite cells (e.g., with collagenase) is a well-established method that enables precise genetic and biochemical manipulation in vitro. It enables transfection, delivery of genetic material, drug treatments, and live-cell imaging, providing researchers with complete control over the microenvironment and the effects of specific signals on cell behaviour [ 22 ]. However, enzymatic isolation has a critical drawback. Once removed from their native environment, satellite cells lose their intimate association with the myofiber sarcolemma and basal lamina. This immediately triggers activation, re-entry into the cell cycle, and progression toward myogenic differentiation [ 23 ]. In the current study, collagenase digestion yielded the most promising myogenic population, characterised by balanced expression of MYOD1 , MYF5 , and MYOG . In contrast, pronase-isolated cells showed reduced early markers but elevated MYOG , suggesting a more differentiated or heterogeneous population with limited regenerative potential. Thus, collagenase is more effective for isolating progenitor-rich cells, while pronase appears biased toward differentiated populations, underscoring the need to align the isolation method with specific experimental goals. In turn, the myofiber explant method preserves satellite cells in their natural niche, maintaining attachment to the fibre and basal lamina. This enables studies of quiescence, activation, and proliferation under conditions close to in vivo, with the added advantage of transient manipulations and real-time observation [ 24 , 25 ]. In our study, the E.2 population, obtained without enzymatic digestion by placing tissue fragments directly on the culture dish, showed comparable expression levels of all analysed myogenic genes relative to the other isolation methods. This uniform expression pattern indicates that the explant approach effectively preserves the native composition of early muscle progenitor cells, consistent with reports highlighting its reliability for pri-mary cell isolation from various tissues [ 26 ]. As we have demonstrated, different methods of isolating cell lines from the same tissue can yield distinct cell fractions. Consequently, gene expression analysis serves as a valuable tool for distinguishing these populations and assessing their functional state. To ensure reliable RT-qPCR results in our in vitro experiments, we selected an appropriate reference gene. This step is critical, as the expression levels of commonly used reference genes may vary considerably depending on tissue type, cell type, and experimental conditions, potentially compromising accuracy if not properly controlled [ 27 , 28 ]. In this study, the stability of eight potential reference genes was evaluated using four widely recognized algorithms: the ΔCt method, BestKeeper, NormFinder, and geNorm, since no consensus currently exists on which algorithm should be applied to assess reference gene stability [ 29 ]. The comprehensive ranking was generated by calculating the geometric mean of individual rankings from each algorithm. The results identified ACTB as the most stable reference gene (geomean = 1.316), followed by RN18S , SDHA , and GAPDH . These genes demonstrated the most consistent expression across the tested samples and experimental conditions, making them suitable candidates for normalization in gene expression studies related to equine skeletal muscle cells. Conversely, B2M and TFRC showed the highest variability in expression (geomean = 6.435 and 8.000, respectively) and are therefore considered the least reliable reference genes in this context. The ACTB gene encodes the β-actin protein, a member of the actin family and the most abundant protein in eukaryotic cells. β-actin plays a crucial role in cell motility and cytoskeletal maintenance in virtually all cell types [ 30 ]. Along with other genes such as GAPDH, SDHA , 18S rRNA, and others, ACTB is constitutively expressed and participates in essential housekeeping processes that sustain cellular function. Consequently, these genes are frequently used as endogenous reference controls for normalizing gene expression analyses [ 31 ]. Several studies in equines have identified ACTB as the most stable reference gene in endometrial, testicular, and conceptus tissue samples [ 32 ], cryopreserved stallion semen [ 33 ], as well as equine skin and sarcoids [ 34 ]. However, in bronchoalveolar lavage cells from horses with inflammatory airway disease, ACTB shows a certain degree of variation in stability [ 35 , 36 ]. Our findings are consistent with previous reports indicating that some commonly used reference genes may display variable expression depending on tissue type or physiological conditions. Hypoxic conditions consistently elevated HIF1A expression, particularly at the 3-hour timepoints, confirming activation of hypoxia-responsive pathways. MYOD1 , associated with early myogenic activation, reaches its maximum at 24 h in both condition, with higher expression under normoxia. MYF5 , a marker of muscle progenitor cells, was higher under normoxia at early time points, indicating that normoxic conditions may favour maintenance of the undifferentiated progenitor state. MYOG , indicative of terminal differentiation, increased under both conditions, particularly at later time points, reflecting progression towards differentiation. Finally, PAX7 expression transiently decreased at 6 hours, followed by recovery and gradual increase, suggesting a temporary activation of myogenic progenitors while maintaining the progenitor pool. By contrast, under normoxic conditions (e.g., 5%), the higher initial expression of MYF5 indicates the maintenance of progenitor cells, suggesting that normoxia favours a more precursor-like state [ 37 ]. In contrast, normoxia supports the preservation of stem cell populations. This has important implications for cell-based therapies, as hypoxic conditions may enhance regenerative efficacy by stimulating the activation and differentiation phases of muscle cells. Overall, hypoxic conditions enhanced both early activation (via HIF1A and MYOD1 ) and late differentiation (via MYOG ), whereas normoxia maintained progenitor characteristics (via MYF5 ) [ 38 ]. In this study, these gene expression profiles are primarily used to characterize the cell population employed in our experiments and to provide a situational overview of the myogenic status of the cultured cells, and should be regarded as descriptive and exploratory rather than definitive. In this study, these gene expression profiles are primarily used to characterize the cell population employed in our experiments and to provide a situational overview of the myogenic status of the cultured cells, and should be regarded as descriptive and exploratory rather than definitive. These findings indicate that hypoxic conditions (3% O₂) do not markedly affect myogenic progression in primary equine cell lines, suggesting that early activation and differentiation dynamics are largely maintained under both normoxic and hypoxic conditions. Conclusions Our results indicate that the choice of muscle cell isolation method should be tailored to the specific aim of the experiment. For experiments involving myogenic cell lines, ACTB proved to be the most stable endogenous control. Furthermore, these findings suggest that hypoxia does not markedly affect myogenic progression, thus it may not be a favorable condition for cells modeling in vitro. Declarations Funding statement This study was supported by a statutory grant from the National Research Institute of Animal Production (No. 501-182-821) Conflict of interest statement The authors declare no potential conflicts of interest Author Contribution M.S.S. and A.S. conceived and designed the study and developed the methodology and software. M.S.S. performed the formal analyses and investigation and prepared the original draft. M.P. provided resources and curated the data. K.R.M. contributed to validation and supervised the study together with M.S.S. K.R.M., M.P., and A.S. reviewed and edited the manuscript. M.S.S. and A.S. prepared the visualizations. M.S.S. managed project administration and acquired funding. 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Cell 12:767–781. https://doi.org/10.1016/0092-8674(77)90276-8 Stürzenbaum SR, Kille P (2001) Control genes in quantitative molecular biological techniques: The variability of invariance. Comp Biochem Physiol - B Biochem Mol Biol 130:281–289. https://doi.org/10.1016/S1096-4959(01)00440-7 Klein C, Rutllant J, Troedsson MH (2011) Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues. BMC Res Notes 4:1–9. https://doi.org/10.1186/1756-0500-4-120/FIGURES/8 Pérez-Rico A, Crespo F, Sanmartín ML et al (2014) Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen. Anim Reprod Sci 149:204–211. https://doi.org/10.1016/J.ANIREPROSCI.2014.08.007 Bogaert L, Van Poucke M, De Baere C et al (2006) Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids. BMC Biotechnol 6. https://doi.org/10.1186/1472-6750-6-24 Beekman L, Tohver T, Dardari R, Léguillette R (2011) Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol Biol 12:1–10. https://doi.org/10.1186/1471-2199-12-5;TYPE Ruan W, Lai M (2007) Actin, a reliable marker of internal control? Clin Chim Acta 385:1–5. https://doi.org/10.1016/J.CCA.2007.07.003 Elashry MI, Kinde M, Klymiuk MC et al (2022) The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice. Stem Cell Res Ther 13:1–17. https://doi.org/10.1186/S13287-022-02730-5/FIGURES/5 Pircher T, Wackerhage H, Aszodi A et al (2021) Hypoxic Signaling in Skeletal Muscle Maintenance and Regeneration: A Systematic Review. Front Physiol 12:684899. https://doi.org/10.3389/FPHYS.2021.684899/FULL Additional Declarations No competing interests reported. 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12:29:46","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117807,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/581a3baf475b5680c2261e93.html"},{"id":98763559,"identity":"6a306d7f-db22-4140-8d47-7bb6f674fc30","added_by":"auto","created_at":"2025-12-22 10:04:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22249,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of isolated cell populations after the third passage and pre-plating (three technical replicates).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/446b4bfa003414376c65c95d.png"},{"id":98763561,"identity":"b1e3233b-81e3-44c6-9cfd-e2f1485a1d94","added_by":"auto","created_at":"2025-12-22 10:04:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15792,"visible":true,"origin":"","legend":"\u003cp\u003eStability ranking of the eight candidate reference genes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eACTB\u003c/em\u003ewas identified as the most stable gene (geomean = 1.316), followed by \u003cem\u003eRN18S\u003c/em\u003e, \u003cem\u003eSDHA\u003c/em\u003e, and \u003cem\u003eGAPDH\u003c/em\u003e, demonstrating low expression variability and high suitability as reference genes in equine skeletal muscle. In contrast, \u003cem\u003eB2M\u003c/em\u003eand \u003cem\u003eTFRC\u003c/em\u003e showed the highest geometric means (6.435 and 8.000, respectively), indicating limited stability and reduced appropri-ateness for normalisation under the tested conditions.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/abf21ebc099b217a5a51796b.png"},{"id":98780731,"identity":"284ebea4-fb0b-4579-a9a9-996edce86af3","added_by":"auto","created_at":"2025-12-22 12:31:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326007,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap illustrating adjusted p-values for pairwise comparisons between reference genes and target transcripts. Significant differences (padj \u0026lt; 0.05) are marked accordingly.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/e9ced8cc7adff2b258cca900.png"},{"id":98763566,"identity":"bf5a3afb-4932-4366-b7c3-6d93aa1e7217","added_by":"auto","created_at":"2025-12-22 10:04:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43546,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression (RQ) of myogenic genes from passage 3 onward under two oxygen conditions.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/d3611167fe204c19fdcf315b.png"},{"id":98787403,"identity":"3297c5b4-ba6e-4cb5-a1d8-1eb0bdfb4658","added_by":"auto","created_at":"2025-12-22 12:43:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/7bea98bb-00cf-40a0-8b96-f3aac12552f7.pdf"},{"id":98763557,"identity":"dd1aa361-9db4-4e94-8723-49a74707f0da","added_by":"auto","created_at":"2025-12-22 10:04:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18707,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/108415b8f24cc0d547223bde.docx"},{"id":98780266,"identity":"398958cd-666e-4d00-85be-a7747f75e446","added_by":"auto","created_at":"2025-12-22 12:31:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19780,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/425d7fb522d2ea04cdc777c4.docx"},{"id":98763575,"identity":"56c1bf7a-6e74-4519-a35a-0ee1d9b95b0d","added_by":"auto","created_at":"2025-12-22 10:04:38","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37423904,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1c.tif","url":"https://assets-eu.researchsquare.com/files/rs-8374782/v1/bab15f28f07e9f3e7c35765e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment and characterization of an equine skeletal muscle in vitro platform: gene expression validation and hypoxia-responsive signatures","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe outstanding velocity, endurance, and muscular power observed in modern sport horses reflect both their evolutionary adaptation as grazing animals and the cumulative effects of selective breeding, which have produced a wide spectrum of specialized breeds, including those optimized for athletic performance. Moreover, the pronounced plasticity of equine physiological systems in response to training and exercise underlies their capacity to achieve elite performance across diverse equestrian disciplines [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the key traits influencing performance is muscle mass, which plays a central role in determining a horse\u0026rsquo;s speed, acceleration, and endurance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In horses, the skeletal muscles comprise up to 60% of the entire body weight and generate the force required for locomotion, postural support, and athletic performance. Their ability to contract rapidly and repeatedly under varying loads is fundamental to performance in both high-speed and endurance-based disciplines. Healthy equine skeletal muscle can fully regenerate after exercise, even though physical workload induces a transient inflammatory and stress response [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This inflammation is a physiological adaptation to muscle growth; however, excessive muscle inflammation, such as overexertion, insufficient warm-up or cool-down, nutritional deficiencies, or muscle disorders, might lead to muscle pain, stiffness, and thus reduced performance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMuscle function relies on a continuous supply of oxygen for respiratory purposes, enabling the generation of contractile force. Fluctuating oxygen levels, particularly the decrease in oxygen supply (hypoxia) during physical exertion, have a twofold effect on muscle physiology, promoting beneficial adaptations while also posing potential stress-related risks. Reduced oxygen levels in skeletal muscle during exercise are mainly a consequence of heightened metabolic demand. This shift in cellular oxygen concentration has a direct impact on metabolic processes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn equine studies, traditional approaches such as muscle biopsies, although informative, are invasive, raise ethical concerns, and present limitations in terms of repeatability and large-scale application. Consequently, within the framework of sport translational medicine, there is increasing emphasis on developing innovative research strategies that minimize reliance on live animal experimentation. The establishment of immortalized and functionally characterized equine cell lines provides a valuable alternative, enabling detailed investigation of molecular pathways underlying exercise adaptation, muscle physiology, and metabolic performance in vitro [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Such cellular models not only align with the principles of the 3Rs (Replacement, Reduction, Refinement) but also offer a reproducible and ethically responsible research platform, ultimately facilitating the translation of fundamental discoveries into practical applications in equine sports medicine and performance optimization. Moreover, in human research, skeletal muscle cell models are gaining popularity due to their ability to reflect donor-specific metabolic traits, offer lower variability than tissue obtained through biopsies, and present greater translational relevance than rodent-derived cell lines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReverse transcription real-time quantitative PCR (RT-qPCR) remains the preferred technique for precise mRNA quantification due to its exceptional sensitivity and the availability of a wide array of commercial assays targeting diverse equine transcripts. However, high precision can complicate interpretation: small fluctuations in mRNA introduced during sample handling or preparation may be mistaken for genuine changes in gene expression. To mitigate such issues, rigorous and appropriate normalization of the data is essential [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn response to growing interest in equine regenerative medicine and translation-al/nutrigenomic applications, we are developing an in vitro platform. This study aimed to develop an in vitro platform for equine skeletal muscle by establishing a primary cell line, confirming its myogenic identity by qPCR of \u003cem\u003eMYOD1\u003c/em\u003e (Myogenic Differentiation 1), \u003cem\u003eMYF5\u003c/em\u003e (Myogenic Factor 5), \u003cem\u003eMYOG\u003c/em\u003e (Myogenin), and \u003cem\u003ePAX7\u003c/em\u003e (Paired Box 7) genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], identifying and normalizing suitable endogenous reference genes, and examining cellular responses under normoxic and hypoxic conditions via \u003cem\u003eHIF1A\u003c/em\u003e (hypoxia-inducible factor 1) gene expression pattern that mirrors oxygen levels in working muscle.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample collection\u003c/h2\u003e \u003cp\u003eSkeletal muscle cells were isolated from cadaveric tissue, which was considered the most ethically appropriate source for the purposes of this study. Tissue collection was performed with the explicit permission of the animal's owner. The material was obtained from a 10-month premature foal that could not be saved despite veterinary intervention. Muscle samples were collected immediately following the cessation of vital functions. Under the Polish Act of 15 January 2015 on the protection of animals used for scientific or educational purposes (Journal of Laws 2015, item 266), which implements Directive 2010/63/EU of the European Parliament and of the Council, ethical approval is required only for procedures performed on living animals. Consequently, the use of biological material collected post-mortem, with owner consent, does not constitute an animal experiment and is exempt from ethical review. Dissection was carried out using a scalpel to a depth of approximately 6 cm in the gluteus medius muscle, under aseptic conditions to preserve sample quality for further cellular analyses.\u003c/p\u003e \u003cp\u003eImmediately post-extraction, samples of skeletal muscle were rinsed twice in ice-cold PBS supplemented with penicillin-streptomycin (300 U/ml and 300 \u0026micro;g/ml) and then placed directly into 50 ml Falcon tube with ice-cold cell culture medium (DMEM Gluta-Max; Gibco) supplemented with 20% fetal bovine serum (FBS; Gibco), 10% horse serum (HS; Gibco) and penicillin-streptomycin (100 U/ml penicillin and 100 \u0026micro;g/ml streptomy-cin). Tissue samples were kept at 4\u0026deg;C and delivered to the laboratory within 4 hours post-collection to initiate primary myoblast isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Equine skeletal muscle cell isolation and culture\u003c/h2\u003e \u003cp\u003ePreparation of the culture took place under aseptic conditions in a biosafety cabinet, utilizing sterile equipment. The muscle sample was divided into three portions, each weighing approximately 1 g, and washed with PBS solution in a Petri dish. All visible non-muscle components, including connective tissue, fat, blood vessels, and tendons, were meticulously removed. Two portions of skeletal muscle tissue (approximately 1 g each) were finely minced and transferred into separate 50 ml Falcon tubes. After allowing the fragments to settle by gravity, the supernatant was carefully aspirated and discarded. The minced tissue was subsequently resuspended in either 0.1% pronase [E.1.1.] or collagenase (440 U/ml) [E.1.2.] solution in DMEM medium supplemented with penicillin-streptomycin (100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin) and incubated at 37\u0026deg;C for 1 hour with agitation every 15 min. After enzymatic digestion, the reaction was terminated by adding an equal volume of culture medium supplemented with 10% FBS. The remaining tissue was then mechanically dissociated by repeated pipetting through pi-pettes of decreasing volumes (10 mL followed by 5 mL), to facilitate cell release. The sample was then passed through a 70 \u0026micro;m and 40 \u0026micro;m cell strainer, rinsed with fresh medium, and centrifuged at 1000 \u0026times; g for 10 min to collect the cells. The cell pellet was resuspended in a culture medium (DMEM GlutaMax, 20% FBS, 10% HS, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin) and subjected to a 1-hour pre-plating step in a 2% gelatin-coated T25 flask to minimize fibroblast contamination. After this period, non-adherent cells were transferred to fresh 2% gelatin-coated T25 flasks. One remaining piece [E.2.] of not minced and not digested muscle tissue was individually placed into 2% gelatin-coated T25 flask with supplemented medium and incubated at 37\u0026deg;C under a controlled atmosphere containing 5% CO₂.\u003c/p\u003e \u003cp\u003eCultures were incubated undisturbed for at least 3 days, after which the medium was gently replaced. Once the cells reached optimal confluence, the first passage was performed using trypsin solution (Trypsin 0.25%, Gibco) and a pre-plating step to further enrich the myoblast population.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Induction of hypoxia\u003c/h2\u003e \u003cp\u003eApproximately 2 \u0026times; 10⁵ cells were seeded into T25 flasks pre-coated with 2% gelatin and allowed to attach overnight under standard culture conditions. The following day, flasks assigned to the hypoxic condition were transferred to a HypoxyLab incubator (Ox-ford Optronix) set to 3% O₂, using a custom gas mixture consisting of 5% air, 5% CO₂, and 90% N₂, delivered at a flow rate of 25 psi/min. Normoxic cultures remained in standard humidified incubators at 37\u0026deg;C with 5% CO₂. Both groups were cultured in parallel under otherwise identical conditions and sampled at four time points: 3, 6, 18, and 24 hours, to assess oxygen-dependent changes in gene expression. For each condition and time point, three technical replicates were performed to ensure the reliability of the measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Gene expression studies\u003c/h2\u003e \u003cp\u003eAt each time point, cells from both normoxic and hypoxic groups were harvested for downstream analysis. Total RNA was extracted using the PureLink RNA Mini Kit with DNase treatment (Ambion, Life Technologies). At each time point, cells were harvested for downstream analysis in three technical replicates. The RNA concentration and purity were assessed on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and integrity was evaluated by TapeStation 2200 (RNA ScreenTapes, Agilent Technologies, Santa Clara, CA, USA). The samples' RNA concentrations range from 32 ng/\u0026micro;L to 412 ng/\u0026micro;L, with all samples showing RIN values above 7.5. Complementary DNA (cDNA) was synthesized from 0.5 \u0026micro;g of total RNA using the High-Capacity RNA-to-cDNA\u0026trade; Kit (Applied Biosystems), following the manufacturer's instructions. Quantitative PCR (qPCR) was performed on a QuantStudio 7 system (Applied Biosystems, Life Technologies) in a final reaction volume of 10 \u0026micro;l. Each reaction contained 0.5 \u0026micro;l of cDNA, 5 \u0026micro;l of 2\u0026times; HS Mix EvaGreen (A\u0026amp;A Biotechnology), 0.3 \u0026micro;l of each gene-specific primer (10 \u0026micro;mol/\u0026micro;l), and 4.4 \u0026micro;l of nuclease-free water. The thermal cycling protocol included an initial denaturation at 95\u0026deg;C for 10 seconds, followed by 40 amplification cycles of 95\u0026deg;C for 15 seconds and 55\u0026deg;C for 34 seconds. Gene expression analysis was carried out using three technical replicates.\u003c/p\u003e \u003cp\u003ePrimer pairs for: \u003cem\u003eMYOD1, MYF5, MYOG, PAX7, HIF1A, ACTB, RN18S, SDHA, GAPDH, HPRTF, RPL32, B2M\u003c/em\u003e and \u003cem\u003eTFRC\u003c/em\u003e were designed with Primer3 (v4.0.0). Sequences are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Primers were positioned across exon\u0026ndash;exon junctions and targeted conserved regions of the genes to ensure stable expression measurements in the presence of multiple transcript variants. Amplicon lengths were optimized to 104\u0026ndash;194 bp, and primer specificity was confirmed in silico using BLAST.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Estimation of housekeeping genes\u003c/h2\u003e \u003cp\u003eHousekeeping gene stability was evaluated using widely accepted and publicly available algorithms, including geNorm [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], NormFinder version 0.953 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], BestKeeper, and the comparative delta Ct method [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were collected at five time points (0, 3, 6, 18, and 24 hours) under normoxic and hypoxic (3% O₂) conditions, with time point 0 representing the start of the experiment. Statistical analysis of the stability of putative reference genes and ranking was provided by geNorm, NormFinder and BestKeeper. Expression levels of the myogenesis-related genes were normalized to each of the evaluated housekeeping genes. Gene expression ratios were then calculated using the 2^\u0026minus;ΔΔCT method. Expression patterns were compared across the different reference (normalizing) genes to evaluate the impact of reference gene selection.. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. Statistical analysis was performed in R (version 4.3.1). Group comparisons were assessed using one-way ANOVA followed by Tukey's HSD (Honestly Significant Difference) post hoc test. For multiple pairwise comparisons, Holm\u0026rsquo;s correction was applied to control for false positives. p-values indicating sta-tistical significance are marked as: * p\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cell culture experiment\u003c/h2\u003e \u003cp\u003eTwo muscle samples were enzymatically digested prior to culture using either pronase [E.1.1.] or collagenase [E.1.2.]. In the pronase-digested culture, a substantial amount of tissue debris was observed, accompanied by a lower proliferation rate compared to the collagenase-digested culture.\u003c/p\u003e \u003cp\u003eA piece of muscle tissue [E.2.] was placed in an individual culture dish containing growth medium and incubated at 37\u0026deg;C. Within 2\u0026ndash;3 days of culture initiation, the first signs of cell outgrowth were observed surrounding the muscle explant. By day 5, a distinct halo of migrating cells had formed around the tissue, and the cell density was considered sufficient to remove the explant from the culture dish.\u003c/p\u003e \u003cp\u003eAlthough the population derived from halo outgrowth was the most abundant, these cells displayed marked morphological heterogeneity and showed signs of possible contamination, limiting their utility for downstream applications (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-S1b)\u003c/p\u003e \u003cp\u003eAfter the third passage and pre-plating steps to enrich for myogenic cells, gene expression analysis was performed in three technical replicates. The results should be interpreted as descriptive tendencies rather than statistically confirmed differences. All groups showed comparable \u003cem\u003eHIF1A\u003c/em\u003e and \u003cem\u003ePAX7\u003c/em\u003e expression. Collagenase-digested cells exhibited the highest \u003cem\u003eMYOD1\u003c/em\u003e and increased \u003cem\u003eMYOG\u003c/em\u003e, while cells digested with the use of pronase showed lower \u003cem\u003eMYOD1\u003c/em\u003e but higher \u003cem\u003eMYF5\u003c/em\u003e and \u003cem\u003eMYOG\u003c/em\u003e expression. Cells from tissue explant displayed low myo genes expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven its intermediate myogenic profile [E.1.2.], cells were selected as the most suitable population for further investigation. Based on these observations, collagenase digestion was determined to be the most effective and reliable method for isolating muscle-derived cells. Consequently, only collagenase-isolated cells were used in subsequent analyses under varying oxygen conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Identification of the most stable reference gene and comprehensive ranking of the reference genes.\u003c/h2\u003e \u003cp\u003eThe stability of eight candidate reference genes was evaluated using four commonly applied algorithms: ΔCt, BestKeeper, NormFinder, and geNorm. Based on these analyses, a comprehensive stability ranking was generated by calculating the geometric mean of the individual ranking values obtained from each method (Table\u0026nbsp;1)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelta CT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eACTB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRN18S\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSDHA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eHPRTF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eRPL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eTFRC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBestKeeper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSDHA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eACTB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRN18S\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eHPRTF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eRPL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eTFRC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormfinder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eACTB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSDHA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRN18S\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRPL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eHPRTF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eTFRC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenorm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eACTB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRN18S\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHPRTF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRPL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eSDHA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eTFRC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecommended comprehensive ranking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eACTB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eRN18S\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSDHA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eHPRTF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eRPL32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eB2M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eTFRC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe most stable gene was \u003cem\u003eACTB\u003c/em\u003e (geomean\u0026thinsp;=\u0026thinsp;1.316), followed by \u003cem\u003eRN18S\u003c/em\u003e, \u003cem\u003eSDHA\u003c/em\u003e, and \u003cem\u003eGAPDH\u003c/em\u003e, indicating low variability and high suitability as reference genes in equine skeletal muscle samples. In contrast, \u003cem\u003eB2M\u003c/em\u003e and \u003cem\u003eTFRC\u003c/em\u003e ranked lowest, with geometric means of 6.435 and 8.000, respectively, suggesting low stability across the tested conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the impact of reference gene choice on expression analysis outcomes, we compared qPCR data normalized individually with each of the candidate reference genes. The statistical analysis of the Tukey post hoc test with HSD correction revealed considerable variation in normalized expression levels of target genes, depending on the reference gene used. The most significant differences (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in RQ calculated according to the reference used were for \u003cem\u003eMYOG\u003c/em\u003e gene, according to comparison of \u003cem\u003eTFRC\u003c/em\u003e vs \u003cem\u003eB2M\u003c/em\u003e, \u003cem\u003eGAPDH\u003c/em\u003e, and \u003cem\u003eHPRTF\u003c/em\u003e as references used (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Myogenesis-related molecular markers expression\u003c/h2\u003e \u003cp\u003eTo investigate the effect of oxygen concentration on the expression of myogenic regu-latory factors, mRNA levels of \u003cem\u003eHIF1A\u003c/em\u003e (hypoxia-inducible factor 1), \u003cem\u003eMYOD1\u003c/em\u003e (Myogenic Differentiation 1), \u003cem\u003eMYF5\u003c/em\u003e (Myogenic Factor 5), \u003cem\u003eMYOG\u003c/em\u003e (Myogenin), and \u003cem\u003ePAX7\u003c/em\u003e (Paired Box 7) were quantified in cells cultured under normoxic (21% O₂) and hypoxic (3% O₂) conditions for 3, 6, 18, and 24 hours. Differentiation qPCR data were normalized to \u003cem\u003eACTB\u003c/em\u003e, identified as the most stable reference gene in this study. The mean Ct values of the analyzed genes ranged from 8.795\u0026thinsp;\u0026plusmn;\u0026thinsp;0.359 (\u003cem\u003eRN18S\u003c/em\u003e) to 33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.401 (\u003cem\u003eGAPDH\u003c/em\u003e). For the myogenic markers, \u003cem\u003eMYOD1\u003c/em\u003e exhibited the highest mean Ct value (31,115\u0026thinsp;\u0026plusmn;\u0026thinsp;3.411), and \u003cem\u003eMYOG\u003c/em\u003e the lowest (22.408\u0026thinsp;\u0026plusmn;\u0026thinsp;1.439) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). \u003cem\u003eHIF1A\u003c/em\u003e, a marker of hypoxia response, peaked at 3 hours in both conditions but remained higher in hypoxia up to 6h. \u003cem\u003eMYOD1\u003c/em\u003e, a gene associated with early myogenic activation, showed increased expression at 24 hours under normoxic conditions, and a similar induction was also observed in hypoxia. \u003cem\u003eMYF5\u003c/em\u003e, a marker of muscle progenitor cells, showed elevated expression at early time points (3\u0026ndash;6 h) in both conditions and subsequently declined, consistent with the expected temporal activation pattern. However, normoxic conditions appeared to better support the maintenance of an undifferentiated state. \u003cem\u003eMYOG\u003c/em\u003e, indicative of terminal differentiation, showed an increase under both conditions, particularly at 8 hours. The transient decrease in \u003cem\u003ePAX7\u003c/em\u003e expression at 6 hours followed by recovery and a gradual increase at 24 hours, suggests a temporary activation phase of myogenic progenitors. This pattern indicates that the cells retain the capacity to maintain a progenitor pool under both oxygen conditions. Data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three technical replicates. No statistical testing was performed because this panel includes only technical replicates and no biological replicates, which precludes meaningful statistical inference (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell shape changes during in vitro equine myogenesis. Primary cultures of preterm foal were grown for 3, 6, 18, and 24 hours in different oxygen conditions. The microscopic images show morphological changes in cells exposed to hypoxic conditions compared to normoxic conditions. After 24 hours of exposure, both normoxic and hypoxic cultures display a dense monolayer of elongated, spindle-shaped cells typical of proliferating myogenic precursors. However, the hypoxia-treated cells appear slightly more aligned and elongated, suggesting early signs of myogenic commitment and differentiation. In contrast, the normoxic culture shows a more random orientation and distribution, which may reflect a less synchronized differentiation process. No overt signs of cell death or detachment were observed in either condition, indicating that short-term hypoxia was well tolerated and may even enhance structural organization in muscle cell populations.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAn increasing number of studies are focused on establishing in vitro models for equine skeletal muscle; however, there is a clear need to refine these approaches to ensure reproducible and biologically relevant outcomes. As such, it is essential to standardize culture conditions, cell sources, and characterization methods to enable meaningful cross-study comparisons and translational applications in veterinary medicine [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the present study, we derived an equine primary cell line from a premature-born foal and initiated work on characterizing its properties, addressing the limited availability of well-defined commercial cell lines for translational applications in equine regenerative medicine. Given the critical role of oxygen availability in maintaining muscle cell homeo-stasis and recognizing that its relative influence varies with the physiological state, we investigated cellular responses under varying oxygen conditions by quantifying the ex-pression of myogenic regulatory factors, including \u003cem\u003eHIF1A\u003c/em\u003e, \u003cem\u003eMYOD1\u003c/em\u003e, \u003cem\u003eMYF5\u003c/em\u003e, \u003cem\u003eMYOG\u003c/em\u003e, and \u003cem\u003ePAX7\u003c/em\u003e, in cells cultured under normoxic (21% O₂) and hypoxic (3% O₂) conditions for 3, 6, 18, and 24 hours. qPCR is a widely adopted gold-standard method for accurately quantifying gene expression and plays a central role in experimental and diagnostic applications [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], where the use of stable reference genes is essential. For establishing myogenic cells from skeletal muscle (\u003cem\u003egluteus medius\u003c/em\u003e) in a premature foal, we used enzymatic digestion with pronase [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and collagenase [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] treatments, as well as the explant method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Enzymatic isolation of satellite cells (e.g., with collagenase) is a well-established method that enables precise genetic and biochemical manipulation in vitro. It enables transfection, delivery of genetic material, drug treatments, and live-cell imaging, providing researchers with complete control over the microenvironment and the effects of specific signals on cell behaviour [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, enzymatic isolation has a critical drawback. Once removed from their native environment, satellite cells lose their intimate association with the myofiber sarcolemma and basal lamina. This immediately triggers activation, re-entry into the cell cycle, and progression toward myogenic differentiation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the current study, collagenase digestion yielded the most promising myogenic population, characterised by balanced expression of \u003cem\u003eMYOD1\u003c/em\u003e, \u003cem\u003eMYF5\u003c/em\u003e, and \u003cem\u003eMYOG\u003c/em\u003e. In contrast, pronase-isolated cells showed reduced early markers but elevated \u003cem\u003eMYOG\u003c/em\u003e, suggesting a more differentiated or heterogeneous population with limited regenerative potential. Thus, collagenase is more effective for isolating progenitor-rich cells, while pronase appears biased toward differentiated populations, underscoring the need to align the isolation method with specific experimental goals. In turn, the myofiber explant method preserves satellite cells in their natural niche, maintaining attachment to the fibre and basal lamina. This enables studies of quiescence, activation, and proliferation under conditions close to in vivo, with the added advantage of transient manipulations and real-time observation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our study, the E.2 population, obtained without enzymatic digestion by placing tissue fragments directly on the culture dish, showed comparable expression levels of all analysed myogenic genes relative to the other isolation methods. This uniform expression pattern indicates that the explant approach effectively preserves the native composition of early muscle progenitor cells, consistent with reports highlighting its reliability for pri-mary cell isolation from various tissues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As we have demonstrated, different methods of isolating cell lines from the same tissue can yield distinct cell fractions. Consequently, gene expression analysis serves as a valuable tool for distinguishing these populations and assessing their functional state. To ensure reliable RT-qPCR results in our in vitro experiments, we selected an appropriate reference gene. This step is critical, as the expression levels of commonly used reference genes may vary considerably depending on tissue type, cell type, and experimental conditions, potentially compromising accuracy if not properly controlled [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, the stability of eight potential reference genes was evaluated using four widely recognized algorithms: the ΔCt method, BestKeeper, NormFinder, and geNorm, since no consensus currently exists on which algorithm should be applied to assess reference gene stability [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The comprehensive ranking was generated by calculating the geometric mean of individual rankings from each algorithm. The results identified \u003cem\u003eACTB\u003c/em\u003e as the most stable reference gene (geomean\u0026thinsp;=\u0026thinsp;1.316), followed by \u003cem\u003eRN18S\u003c/em\u003e, \u003cem\u003eSDHA\u003c/em\u003e, and \u003cem\u003eGAPDH\u003c/em\u003e. These genes demonstrated the most consistent expression across the tested samples and experimental conditions, making them suitable candidates for normalization in gene expression studies related to equine skeletal muscle cells. Conversely, \u003cem\u003eB2M\u003c/em\u003e and \u003cem\u003eTFRC\u003c/em\u003e showed the highest variability in expression (geomean\u0026thinsp;=\u0026thinsp;6.435 and 8.000, respectively) and are therefore considered the least reliable reference genes in this context. The \u003cem\u003eACTB\u003c/em\u003e gene encodes the β-actin protein, a member of the actin family and the most abundant protein in eukaryotic cells. β-actin plays a crucial role in cell motility and cytoskeletal maintenance in virtually all cell types [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Along with other genes such as \u003cem\u003eGAPDH, SDHA\u003c/em\u003e, 18S rRNA, and others, \u003cem\u003eACTB\u003c/em\u003e is constitutively expressed and participates in essential housekeeping processes that sustain cellular function. Consequently, these genes are frequently used as endogenous reference controls for normalizing gene expression analyses [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Several studies in equines have identified \u003cem\u003eACTB\u003c/em\u003e as the most stable reference gene in endometrial, testicular, and conceptus tissue samples [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], cryopreserved stallion semen [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], as well as equine skin and sarcoids [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, in bronchoalveolar lavage cells from horses with inflammatory airway disease, \u003cem\u003eACTB\u003c/em\u003e shows a certain degree of variation in stability [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our findings are consistent with previous reports indicating that some commonly used reference genes may display variable expression depending on tissue type or physiological conditions.\u003c/p\u003e \u003cp\u003eHypoxic conditions consistently elevated \u003cem\u003eHIF1A\u003c/em\u003e expression, particularly at the 3-hour timepoints, confirming activation of hypoxia-responsive pathways. \u003cem\u003eMYOD1\u003c/em\u003e, associated with early myogenic activation, reaches its maximum at 24 h in both condition, with higher expression under normoxia. \u003cem\u003eMYF5\u003c/em\u003e, a marker of muscle progenitor cells, was higher under normoxia at early time points, indicating that normoxic conditions may favour maintenance of the undifferentiated progenitor state. \u003cem\u003eMYOG\u003c/em\u003e, indicative of terminal differentiation, increased under both conditions, particularly at later time points, reflecting progression towards differentiation. Finally, \u003cem\u003ePAX7\u003c/em\u003e expression transiently decreased at 6 hours, followed by recovery and gradual increase, suggesting a temporary activation of myogenic progenitors while maintaining the progenitor pool. By contrast, under normoxic conditions (e.g., 5%), the higher initial expression of \u003cem\u003eMYF5\u003c/em\u003e indicates the maintenance of progenitor cells, suggesting that normoxia favours a more precursor-like state [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, normoxia supports the preservation of stem cell populations. This has important implications for cell-based therapies, as hypoxic conditions may enhance regenerative efficacy by stimulating the activation and differentiation phases of muscle cells. Overall, hypoxic conditions enhanced both early activation (via \u003cem\u003eHIF1A\u003c/em\u003e and \u003cem\u003eMYOD1\u003c/em\u003e) and late differentiation (via \u003cem\u003eMYOG\u003c/em\u003e), whereas normoxia maintained progenitor characteristics (via \u003cem\u003eMYF5\u003c/em\u003e) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, these gene expression profiles are primarily used to characterize the cell population employed in our experiments and to provide a situational overview of the myogenic status of the cultured cells, and should be regarded as descriptive and exploratory rather than definitive. In this study, these gene expression profiles are primarily used to characterize the cell population employed in our experiments and to provide a situational overview of the myogenic status of the cultured cells, and should be regarded as descriptive and exploratory rather than definitive. These findings indicate that hypoxic conditions (3% O₂) do not markedly affect myogenic progression in primary equine cell lines, suggesting that early activation and differentiation dynamics are largely maintained under both normoxic and hypoxic conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results indicate that the choice of muscle cell isolation method should be tailored to the specific aim of the experiment. For experiments involving myogenic cell lines, ACTB proved to be the most stable endogenous control. Furthermore, these findings suggest that hypoxia does not markedly affect myogenic progression, thus it may not be a favorable condition for cells modeling in vitro.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding statement\u003c/h2\u003e \u003cp\u003eThis study was supported by a statutory grant from the National Research Institute of Animal Production (No. 501-182-821)\u003c/p\u003e \u003cp\u003eConflict of interest statement\u003c/p\u003e \u003cp\u003eThe authors declare no potential conflicts of interest\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.S.S. and A.S. conceived and designed the study and developed the methodology and software. M.S.S. performed the formal analyses and investigation and prepared the original draft. M.P. provided resources and curated the data. K.R.M. contributed to validation and supervised the study together with M.S.S. K.R.M., M.P., and A.S. reviewed and edited the manuscript. M.S.S. and A.S. prepared the visualizations. M.S.S. managed project administration and acquired funding. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRivero JLL, Hill EW (2016) Skeletal muscle adaptations and muscle genomics of performance horses. Vet J 209:5\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKearns CF, McKeever KH, John-Alder H et al (2002) Relationship between body composition, blood volume and maximal oxygen uptake. 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Front Physiol 12:684899. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/FPHYS.2021.684899/FULL\u003c/span\u003e\u003cspan address=\"10.3389/FPHYS.2021.684899/FULL\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Equine skeletal muscle derived cells, Reference genes, Hypoxia, houskeeping genes","lastPublishedDoi":"10.21203/rs.3.rs-8374782/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8374782/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn response to the increasing interest in equine regenerative medicine and translational nutrigenomics, we sought to establish an in vitro platform for studying skeletal muscle biology in horses. The study focused on generating a primary equine skeletal muscle cell line, confirming its myogenic identity through qPCR analysis of the markers \u003cem\u003eMYOD1\u003c/em\u003e, \u003cem\u003eMYF5\u003c/em\u003e, \u003cem\u003eMYOG\u003c/em\u003e, and \u003cem\u003ePAX7\u003c/em\u003e, identifying and validating suitable endogenous reference genes for normalization, and assessing cellular responses to normoxia and hypoxia based on the expression profile of \u003cem\u003eHIF1A\u003c/em\u003e, a master regulator reflecting oxygen availability in working muscle.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePrimary equine skeletal muscle cells were isolated using collagenase and pronase treatment or the explant method and cultured under standard conditions. Gene expression was assessed by qPCR, including the evaluation of housekeeping genes for normalization. Hypoxic cultures were subsequently maintained at 3% O₂ in a HypoxyLab incubator, while normoxic controls were kept at 37\u0026deg;C with 5% CO₂. All cultures were handled in parallel under otherwise identical conditions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCollagenase digestion provided the most viable myogenic cells, whereas pronase and explant methods yielded less suitable populations. Among eight candidate reference genes evaluated with ΔCt, BestKeeper, NormFinder, and geNorm, \u003cem\u003eACTB\u003c/em\u003e emerged as the most stable, followed by \u003cem\u003eRN18S\u003c/em\u003e, \u003cem\u003eSDHA\u003c/em\u003e, and \u003cem\u003eGAPDH\u003c/em\u003e. In contrast, \u003cem\u003eB2M\u003c/em\u003e and \u003cem\u003eTFRC\u003c/em\u003e showed the lowest stability and were deemed unsuitable for normalization.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollagenase digestion is the most suitable method for establishing equine myogenic cell cultures, with \u003cem\u003eACTB\u003c/em\u003e as the optimal endogenous control. Furthermore, these findings suggest that hypoxia does not markedly affect myogenic progression, thus it may not be a favourable condition for modelling accelerated muscle regeneration or stress-induced differentiation in high-performance animals such as racing horses.\u003c/p\u003e","manuscriptTitle":"Establishment and characterization of an equine skeletal muscle in vitro platform: gene expression validation and hypoxia-responsive signatures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 10:04:31","doi":"10.21203/rs.3.rs-8374782/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad5245b0-5021-4b3f-a650-46b698275eca","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T10:54:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 10:04:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8374782","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8374782","identity":"rs-8374782","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}