Serological and molecular detection of Toxoplasma gondii infection in apparently healthy horses in eastern of Spain.

Toxoplasmosis is one of the most common parasitic zoonoses and represents a significant health risk for humans, especially for immunodeficient patients. The main transmission route is by oral uptake of oocysts and consumption of undercooked meat of infected animals. Different species have been evaluated as possible reservoirs of the parasite, but few studies have been carried out to examine the role of horses in transmission of the disease. Given the proximity of these animals to humans and the widespread consumption of their meat in many countries, including the Mediterranean basin, it is important to determine the prevalence of T. gondii infection in this species. In this study, blood samples from 105 horses were collected and the presence of T. gondii was evaluated by serological and molecular methods. Antibodies against T. gondii of 12 horses (11.43%) were detected by enzyme-linked immunosorbent assay (ELISA), whereas 29 horses (27.62%) showed positive for PCR. Seroprevalence was related to use of the animals, being higher in horses used for dressage than in others. Purebreds had higher seroprevalence than crossbred animals. No differences between breed, sex or age were found. The results of this study confirm the presence of T. gondii infection in horses, highlighting the need to analyse the meat of this species before human consumption and to control of this infection in horses, as they could be an important reservoir of this zoonotic parasite.

Acute myeloid leukemia-M1 in a horse with neurologic signs and necrotizing enterocolitis.

An 18-y-old American Saddlebred mare was admitted with fever and acute onset of neurologic signs including grade 3 of 5 ataxia, difficulty in prehension, and dull mentation. Because of financial restraints, desired testing could not be performed; the horse's condition declined despite supportive treatment, and euthanasia was elected. Postmortem examination revealed petechiae and ecchymoses in the meninges and neuroparenchyma of the encephalon. Blast-like neoplastic round cells were identified within the vasculature and areas of hemorrhage in the neuroparenchyma, the intestinal submucosa, and other organs, including the liver, kidney, lung, and mesenteric lymph node. Necrotizing enterocolitis and acute fibrinonecrotizing bacterial pneumonia were also noted. Of the atypical round cells in the encephalon, >70% expressed ionized calcium-binding adapter molecule 1 (Iba1), 10-20% expressed myeloperoxidase (MPO), and <10% expressed PAX5, CD3, CD20, CD79a, or MUM1. The bone marrow was diffusely effaced by neoplastic round cells expressing Iba1, and ~70% of these cells expressed MPO with no expression of CD3 or CD20. CD172a also immunolabeled a portion of the neoplastic cells. These findings were consistent with the diagnosis of acute myeloid leukemia-M1 with an unusual neurologic presentation.

Effect of balanced vs. standard protein on muscle mass development in exercising horses.

Twelve horses weighing 570 ±â€…35 kg were split into two groups (standard protein, STD, and balanced protein, BAL) to evaluate the effect of balanced vs. unbalanced protein on muscle mass. Groups were fed grass hay and a 12% CP feed (one formulated for balanced protein). Horses were out of work for 14 wk prior and participated in 1 to 2 h of light-to-moderate exercise 5 d/wk for 12 wk. A 4-d total urine and feces collection period was conducted at the start and end of the study. Urine and feces samples were analyzed for nitrogen (N) to calculate N balance. Muscle mass status was evaluated using histochemistry, muscle scoring, and body measurements at the start and end of the study. Muscle biopsies were analyzed for free amino acid concentrations. An ANOVA was done using the Proc MIXED function of SAS (2006). Nitrogen retention was greater and urine nitrogen as a percent of intake less for the STD group at the end of the study (P = 0.01). Muscle free concentrations of methionine, leucine, phenylalanine, and arginine were greater in the BAL group at the end of the study compared to the STD group (P < 0.04) while muscle free histidine, threonine, and valine were less for the STD group at the end of the study compared to the start of the study (P < 0.05). Muscle fiber diameters were greater at the start of the study for the STD group (P = 0.004) but greater for the BAL group at the end of the study (P = 0.001). There is some evidence of improved muscle mass for the BAL group with greater muscle free amino acids (arginine, leucine, methionine, phenylalanine, and valine) as well as greater type II muscle fiber areas and diameters. While changes did not appear evident between groups at the "whole body" level, changes at the tissue level appear more evident.

Development of an ultrasound-guided radiofrequency ablation technique in the equine cadaveric distal limb: histological findings and potential for treating chronic lameness.

Introduction: Radiofrequency (RF) relieves chronic pain in humans, but it is unexplored in horses affected by chronic lameness. This study aims to describe the technique and the histological effects of ultrasound (US)-guided radiofrequency ablation (RFA) of palmar digital nerves (PDNs) in horse's fetlock and pastern, ex vivo. Methods: After assessing the US anatomy of lateral and medial PDNs in fetlock and pastern in vivo (n = 10 horses; 20 forelimbs), US-guided RFA was performed on these sites in cadaveric forelimbs (n = 10) applying four different settings with increasing invasiveness (n = 40 total treatments): 60°C, 6 min (GROUP LOW); 70°C, 4 min (GROUP MEDIUM); 90°C, 2 min (GROUP HIGH); 80°C, 8 min (GROUP VERY HIGH). Needle-tip-to-nerve proximity was assessed with US and methylene blue, injected through the port of the RF needle. Nerves were collected for microscopical assessment. Results: Transverse palmaro-lateral and palmaro-medial US images of fetlock and pastern detected PDNs consistently, close to the palmar digital artery. With in-plane US technique, RFA was performed at target in 31/40 cases, with significantly higher number of failures in fetlock (p = 0.008). PDNs histology identified thermal injury/coagulation with axonal degeneration and collagen homogenation. Nuclear smearing of arterial leyomyocytes was also observed. Nerve coagulation was significantly associated with treatment (p = 0.03) and needle-tip-to-nerve proximity (US distance: p = 0.009; blue distance: p = 0.04). Discussion: The PDNs were easily visualized and reached with the RF needle by US in-plane-guided technique. RFA produced axonal thermal damage and intensity-related coagulation effectiveness. To ensure effective nerve coagulation, it is crucial that the needle is accurately positioned in close proximity to the target nerve. Based on the histopathological findings, HIGH and VERY HIGH RFA treatments might be worth of being tested in vivo in clinical studies aimed at treating chronic lameness of the distal forelimb in horses.

Serological survey on Hepatitis E virus in Namibian dogs, cats, horses, and donkeys.

The present study investigated the seropositivity rate of Hepatitis E virus (HEV) in domestic and working animals in Namibia, which included dogs, cats, horses, and donkeys. HEV poses a growing threat as a significant cause of human hepatitis globally and has several genotypes of varying zoonotic potential. As epidemiological data on the seroprevalence of HEV in Namibia is scarce, a serosurvey was conducted on archived serum samples of 374 dogs, 238 cats, 98 horses, and 60 donkeys collected between 2018 and 2022 from different regions, to assess the potential of these animals as sources of HEV infection. The findings revealed that 10.43% (n = 39/374) canine and 5.88% (n = 14/238) feline samples tested positive for HEV antibodies, whereas no seropositivity was detected in horses and donkeys. The study further examined the risk factors associated with HEV seropositivity, including animal sex, age, and geographical region, and noted a higher prevalence in dogs living in areas with intensive pig farming. Although there is no direct evidence indicating that these animals served as major reservoirs for HEV transmission to humans, the study underscores the importance of preventive measures to minimize contact exposure with pets considering the potential zoonotic risk, especially for susceptible risk groups. Further research is needed to explore the zoonotic potential of domestic animals and the epidemiological links between animal and human HEV transmissions in Namibia.

Comparison and characterization of the bacterial microbiota and SIgA production in different gastrointestinal segments in horses.

In the gastrointestinal mucosa, there is a close cooperation between secretory immunoglobulin A (SIgA) and the composition of the microbiota, which aims to maintain homeostasis as well as act as a protective barrier. The purpose of this study was to determine the composition of microbiota and SIgA production in different parts of the digestive tract (small intestine, cecum, colon and rectum) of nine healthy horses and its reflection in the feces. For this purpose, we determined: the composition of the microbiome (by next-generation Sequencing of Hypervariable Regions V3-V4 and V7-V9 of the 16 S rRNA gene analysis), the amount of SIgA in the intestinal content samples (by ELISA), as well as the number of IgA-producing cells (IgA+) in the tissue samples (by immohistochemical analysis). Significant differences were observed between the small intestine and the large colon in the composition and diversity of the microbiome, as well as the number of IgA + cells in the mucosal lamina propria and the abundance of SIgA in the intestinal lumen. The small intestine in relation to the large colon is characterised by fewer IgA + cells, more SIgA in the intestinal contents and a less diverse microbiome. However, the cecum appears to be the third separate ecosystem, with a high number of IgA + cells and a diverse microbiome. The fecal sample reflects the current state of the large colon, both in terms of the microbiome and SIgA content; however, it is not known to what extent it may be influenced by dysbiosis in other parts of the digestive tract.

Molecular detection of Chlamydia abortus in endometrial biopsies of mares from western Canada.

Chlamydia abortus is a reported cause of infertility and endometritis in sheep, cattle, and pigs; however, the association between uterine disease and C. abortus is poorly understood in horses. Recently, a high prevalence of C. abortus in equine aborted chorioallantoises was reported in horses in western Canada. Based on this high prevalence, investigation into the effects of C. abortus on infertility and endometritis in western Canadian mares is prudent. We examined 98 formalin-fixed, paraffin-embedded endometrial biopsies from western Canada submitted between 2014 and 2022 using a Chlamydia-specific 16S rRNA PCR test; 40 samples tested positive for Chlamydia on PCR, and 28 were sequenced as C. abortus. The C. abortus-positive cases were primarily associated with a history of failure to conceive, early embryonic loss, or abortion. Our findings suggest that C. abortus may be a cause of conception failure and abortion in horses in western Canada.

Effects of weaning-related stress on the emotional health of horses-A scoping review.

BACKGROUND: Weaning represents one of the most stressful events in the life of a horse, and may have long-term impacts on behaviour. There are a range of approaches used to wean foals from mares, including abrupt and progressive separation methods. There is currently a lack of consensus on how stress and impact are measured. OBJECTIVES: To conduct a scoping review to identify and chart measures of weaning-related stress on the mare and foal. STUDY DESIGN: Scoping review. METHODS: The scoping review was conducted using the PRISMA extension for scoping reviews. Systematic searches were conducted in three scientific databases (CAB abstracts, Medline and Embase). The title, abstract and full text of retrieved studies were reviewed against inclusion and exclusion criteria. Publications that remained after full text review were analysed. Data on study design, population characteristics, weaning method and health and behavioural outcomes were extracted and charted. RESULTS: A total of 366 publications were identified; 22 were retained for inclusion and charting. Eighteen studies reported behavioural measures, four only reported physiological measures and 12/22 studies used a combination of both. Fifteen studies analysed foal behaviour only (15/22), six analysed foals with their dams and one analysed the mare only. Duration of most studies (19/22) was a maximum of 12 months post weaning; three studies continued measurements until the foal age was 3-4 years old. MAIN LIMITATIONS: Limited number of studies available for analysis. CONCLUSIONS: There is significant variation in the current evidence around weaning-related impacts in the mare and foal. This includes variation in how weaning methods and management of mares and foals are described, and very limited research evaluating long-term impacts, or the impact on the mare. This scoping review makes recommendations on how to improve the future evidence base.

Equine Sarcocystosis in the Northern Region of the Republic of Kazakhstan.

BACKGROUND: A total of 396 samples were taken from the hearts, oesophagi, and diaphragms of 132 horses slaughtered at slaughterhouses in 2023 for subsequent examination. METHODS: The histological method revealed pathomorphological changes in the muscle tissue. The molecular method identified the pathogen species. RESULTS: Histological examination revealed thick-walled cysts with internal septa and numerous bradyzoites, and mononuclear inflammatory cells with pericyst infiltrates. Microcyst samples were amplified by polymerase chain reaction. Molecular genetic analysis allowed for the identification of 18 sarcocysts. Phylogenetic analysis of Sarcocystis isolates revealed three separate clades of Sarcocystis bertrami and two separate clades of Sarcocystis fayeri. Comparison and phylogenetic analysis revealed a very close relationship between the identified Sarcocystis species and other equine Sarcocystis DNA sequences from China and Japan. Based on the results obtained, the epizootic situation and the parasitic level of sarcocystosis contamination of horses in the northern Kazakhstan were determined. CONCLUSION: This is the first histological and molecular study to identify Sarcocystis spp. isolated from microscopic forms of equine sarcocysts in the northern Kazakhstan. This research will contribute to the fight against the spread of sarcocystosis in the Republic of Kazakhstan and will allow us to develop proposals for improving the mechanisms of sarcocystosis control.

Antimicrobial Resistance in Equines: A Growing Threat to Horse Health and Beyond-A Comprehensive Review.

The equine industry holds substantial economic importance not only in the USA but worldwide. The occurrence of various infectious bacterial diseases in horses can lead to severe health issues, economic losses, and restrictions on horse movement and trade. Effective management and control of these diseases are therefore crucial for the growth and sustainability of the equine industry. While antibiotics constitute the primary treatment strategy for any bacterial infections in horses, developing resistance to clinically important antibiotics poses significant challenges to equine health and welfare. The adverse effects of antimicrobial overuse and the escalating threat of resistance underscore the critical importance of antimicrobial stewardship within the equine industry. There is limited information on the epidemiology of antimicrobial-resistant bacterial infections in horses. In this comprehensive review, we focus on the history and types of antimicrobials used in horses and provide recommendations for combating drug-resistant bacterial infections in horses. This review also highlights the epidemiology of antimicrobial resistance (AMR) in horses, emphasizing the public health significance and transmission dynamics between horses and other animals within a One Health framework. By fostering responsible practices and innovative control measures, we can better help the equine industry combat the pressing threat of AMR and thus safeguard equine as well as public health.

Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with Sepsis.

Cell-free DNA (cfDNA) is fragmented extracellular DNA that is released from cells into various body fluids. Previously published data from adult horses supports cfDNA as a potential disease biomarker, but also shows that direct measurement in plasma is inaccurate due to matrix effect. It is currently unknown whether a similar matrix effect exists in foal plasma. Given this, the objectives of the current study were to investigate foal plasma for potential matrix effect during fluorescence measurement of cfDNA using a Qubit fluorometer, and to determine whether neat and/or extracted plasma cfDNA concentrations are significantly different in healthy, sick non-septic (SNS) or septic foals. We hypothesized that matrix effect would interfere with accurate fluorescent measurement of cfDNA in foal plasma. Further, we hypothesized that mean extracted cfDNA concentrations, and/or extracted cfDNA:neutrophil ratio, would be elevated in plasma of septic foals compared to healthy or SNS foals. Cell-free DNA was measured in neat plasma, and following DNA extraction with a commercial kit, from 60 foals. Foal plasma exhibited both autofluorescence and non-specific dye binding, confirming matrix effect. However, even with extraction, no significant difference was found in cfDNA concentrations, or cfDNA:neutrophil ratios, between healthy (sepsis score ≤ 5), SNS (sepsis score 6-11 and negative blood culture), or septic (sepsis score ≥ 12 ± positive blood culture) foals. Our data show that matrix effect interferes with accurate Qubit measurement of cfDNA in foal plasma and supports previous findings that plasma cfDNA concentrations are not associated with sepsis diagnosis in foals. Further research is needed to better understand neutrophil function and dysfunction in foal sepsis.

Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland.

Equine rhinitis A (ERAV) and B (ERBV) viruses are respiratory pathogens with worldwide distribution. The current study aimed to determine the frequency of infection of ERAV and ERBV among horses and foals at Polish national studs, and to determine genetic variability within the viruses obtained. Virus-specific quantitative RT-PCR assays targeting a 5' untranslated region were used to screen nasal swabs collected from 621 horses at 16 national horse studs from throughout Poland, including 553 healthy horses and 68 horses with respiratory disease. A partial DNA polymerase gene was amplified and sequenced from the qRT-PCR-positive samples. The obtained sequences were analysed using phylogeny and genetic network analysis. None of the nasal swabs were positive for ERAV, whereas ERBV was found in 11/621 (1.78%) samples collected from 10 healthy horses and one foal affected by respiratory disease. Partial DNA polymerase gene sequence variability was correlated with individual horses and studs from which samples were collected when only Polish sequences were analysed, but there was no correlation between country of origin and ERBV sequence when Polish and international sequences were included in the network. The report presents the first detection of ERBV in Poland.

Lipofuscin accumulates in ganglionic neurons in chronic equine dysautonomia.

Lipofuscin is a complex mixture of highly oxidized, cross-linked macromolecules that accumulates in neurons with age and some neurodegenerative diseases. Equine dysautonomia (ED) is a polyneuropathy that mainly affects autonomic and enteric nervous systems, resulting in alimentary tract dysfunction. Our main aim was to determine whether neuronal lipofuscin increased with increasing duration of ED. We investigated the prevalence of lipofuscin in cranial cervical ganglia of horses with acute (AED), subacute (SED), and chronic ED (CED), young controls (of similar age to ED cases), and aged controls (n = 8 per group). We used Schmorl stain for histologic detection of lipofuscin and assessed its accumulation in neurons using image analysis software. The percentage of neurons positive for lipofuscin increased with age in individual groups and all groups combined (p < 0.001). There were fewer positive neurons in AED and SED compared to aged controls (p < 0.001) and more in CED than AED cases (p = 0.042) and young controls (p = 0.012). We found a strong positive correlation between percentage positive neurons and percentage positive area of the neuron containing lipofuscin for combined groups (p < 0.001). Although neuronal lipofuscin increased in cranial cervical ganglion in CED cases, it remains to be determined whether this is a cause or consequence of neuronal degeneration.

Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Polyclonal Peptide Antisera.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages.The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare.Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

Changes in saliva analytes in equine gastric ulcer syndrome (EGUS) after treatment: a pilot study.

Equine gastric ulcer syndrome (EGUS) is a prevalent condition in horses, affecting up to 93% of racehorses. Comprising the equine squamous gastric disease (ESGD) and the equine glandular gastric disease (EGGD), EGUS poses significant health challenges. Saliva, a non-invasive and easily obtainable sample, is increasingly recognized for its potential as a source of biomarkers in horses. This study investigates changes in saliva analytes using automated assays before and after EGUS treatment, aiming to identify biomarkers indicative of treatment success or failure. A total of 28 horses diagnosed with EGUS were treatment with omeprazole for six weeks and further divided into successful (n = 15) or unsuccessful (n = 13) treatment group. Saliva samples were collected before and after treatment, and analytes related to enzymes, metabolites, proteins, redox biomarkers, and minerals were measured using an automated chemistry analyzer. Results revealed that horses with successful treatment, indicated by reduced EGGD and ESGD scores, showed significant increases in bicarbonate and urea, and decreases in adenosine deaminase (ADA), and creatine kinase (CK). Conversely, horses with non-successful treatment showed no significant changes in salivary analytes. These analytes have the advantages of an easy and fast measurement and the possibility of being applied in routine. Further studies with larger populations should be performed to establish the possible practical application of these analytes as biomarkers of treatment.

Timing of feeding a protein supplement on nitrogen balance and plasma amino acids during exercise recovery in horses.

Eight geldings weighing 544 ± 16 kg were used to evaluate feeding a postexercise protein meal on plasma amino acids during recovery. Horses were fed sweet feed, corn, grass hay and equal amounts of a protein pellet (32% CP) with meals (MP group) or postexercise (EP group). Horses exercised 1-2 h/day, 5 days/week, for 12 weeks. A pre and poststudy 4 days total urine and feces collection was conducted. Urine and fecal samples were analyzed for nitrogen (N) to calculate N balance. Blood samples were drawn immediately after and at 1 and 3 h postexercise at the start and end of the study for plasma amino acid concentrations. Absorbed N and N retention were greater for the MP group compared to the EP group (p = 0.038, 0.033 respectively). An interaction revealed an increase in fecal N (p = 0.01) and decreased N digestibility for the MP group compared to the EP group at the end of the study. Plasma concentrations for 8 out of 14 amino acids were less for the EP group immediately after exercise compared to the MP group (p < 0.02). Plasma concentrations of lysine and arginine were greater for the EP group compared to the MP group at 1 and 3 h after exercise (p < 0.05 and 0.04 respectively). Changes were different for 8 out of the 14 amino acids immediately post exercise, 7 out of 14 amino acids at 1 h postexercise and 10 out of 14 amino acids at 3 h postexercise with positive changes for the EP group and negative changes for the MP group. The EP group had improved supply of plasma amino acids in the recovery period that sustained for 3 h postexercise and are indicative of better amino acid supply supporting muscle development.

Equine Corneal Wound Healing Using Mesenchymal Stem Cell Secretome: Case Report.

Corneal ulcers are a common and potentially vision-threatening condition in horses that can be challenging to treat with conventional therapies alone. This case report describes the successful treatment of a non-healing corneal ulcer in a 28-year-old Hispano-Bretón mare using the secretome derived from adipose tissue-derived mesenchymal stem cells (ASCs). Despite initial treatment with antibiotics, anti-inflammatory drugs, and surgical debridement, the corneal ulcer failed to heal properly, exhibiting persistent epithelial defects and stromal complications. As an alternative regenerative approach, the ASC secretome, a rich source of trophic factors, cytokines, and extracellular vesicles, was topically administered to the affected eye. Remarkably, within one week of secretome treatment, the clinical signs of blepharospasm and epiphora resolved, and the corneal ulcer exhibited complete re-epithelialization, regained transparency, and reduced neovascularization. No recurrence was observed during the 1.5-year follow-up period. This case highlights the potential therapeutic benefits of the ASC secretome in promoting corneal wound healing and suggests its promise as a novel cell-free therapy for treating refractory corneal ulcers in horses.

Are Users Good Assessors of Social Dominance in Domestic Horses?

Horse users and caretakers must be aware of the risks of mixing social groups. The current study investigated whether eight equine practitioners can assess the social dominance rank of 20 horses. The horses' feeding time and agonistic/aggressive and submissive behaviours were observed during the feed confrontation test, and the dominance index (DI) was calculated. Kendal's W, Spearman correlations and factor analysis were applied to test the raters' agreement, the relationship between dominance ranks and the behavioural variables, and to determine the clustered behaviours. The agreement between all raters in the classification of dominance order ranged from moderate to perfect. The ranking by every rater was strongly and negatively correlated with the time of eating in feed confrontation tests and with the DI, evidencing shorter feeding times for more submissive horses. The withdrawal of the horse when threatened was the behavioural variable that was most often correlated with raters' ranking. The current study confirmed the abilities of practitioners to categorise the horses under their care according to their social interactions. Additionally, rolling when denied access to feed was proposed as frustration-releasing (redirected) behaviour.

Comparative transcriptome analysis of slow-twitch and fast-twitch muscles in Kazakh horses.

This study conducted a thorough analysis of the myofiber type composition in the extensor digitorum longus muscle (EDL) and soleus muscle (SOL) of Kazakh horses, across different genders (male and female). The results showed significant differences in myofiber type composition between EDL and SOL, with a higher proportion of Type I fibers in SOL muscles and a greater prevalence of Type II fibers in EDL muscles. Additionally, the myofiber diameter in Kazakh horses was relatively small, potentially related to the tenderness and edible quality of their muscles. Using high-throughput sequencing technology, we constructed 32 cDNA sequencing libraries and obtained high-quality read data. Gene expression analysis revealed 278 and 372 differentially expressed genes (DEGs) in EDL and SOL muscles, respectively, including genes related to muscle contraction, metabolism, and development. Intersection analysis of DEGs between genders showed that 60 DEGs were significantly different in both male and female horses. GO annotation and KEGG analysis further elucidated the roles of these DEGs in muscle structure, function, and cellular signaling. Protein-protein interaction (PPI) network analysis and identification of hub genes provided new insights into the molecular mechanisms underlying muscle growth and development. Finally, the reliability of the DEGs data was validated through quantitative real-time PCR (qRT-PCR). This study not only enhances our understanding of the biological characteristics of horse muscles but also provides potential molecular targets for improving horse muscle performance and health.