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Expansion of the cumulus cells (CC) surrounding the oocyte is crucial for growth and maturation of a healthy oocyte, that can be fertilized and develop into an embryo. Factors that are released by CC into the follicular fluid (FF) induce paracrine signaling within the follicular environment, which is essential for proper folliculogenesis. FF is a rich source of proteins, nucleic acids or extracellular vesicles (EVs) which are considered as mediators of intercellular communication within the follicle. EVs influence target cells by interacting with membrane receptors or by internalizing and delivering molecules to cytoplasm or endosomes. Recent studies in cattle have shown an involvement of EVs in controlling cumulus expansion (Hung et al. Biol Reprod. 2015; 93(5):1-9), but this effect has to date not been observed in horses. The aim of this study was, therefore, to evaluate the effect of FF-derived EVs (ffEVs) on equine CC expansion, viability and transcriptome modulations. The material consisted of 43 slaughtered mares' ovaries, from which 304 cumulus-oocyte complexes (COCs) were obtained. EVs were isolated from small follicles (<20 mm) and characterized as described before (Gabryś et al. Theriogenology 2022; 188:116-124). COCs were matured in vitro (IVM) in the presence (200 µg protein/ml) or absence (control) of ffEVs. For the confirmation of ffEVs internalization, particles were labeled using ExoGlow-protein EV Labeling Kit, added to maturing COCs, which were afterwards imagedusing confocal microscopy. CC expansion was assessed by measuring the COCs diameter before and after IVM and viability test was performed after IVM using staining with ethidium bromide and fluorescein diacetate. Additionally, CC were isolated after 12 hrs of IVM, after which RNA was extracted and cDNA libraries were generated with QuantSeq 3' mRNA-Seq Library Prep Kit. Transcriptome alterations of CC were investigated with next generation sequencing and bioinformatic analysis. Confocal microscopy images show the internalization of ffEVs by both cumulus cells and oocytes. Supplementation with ffEVs had a significant impact on cumulus expansion in both compacted (Cp, p<0.0001) and expanded (Ex, p<0.05) COCs. There were no differences in diameters of COCs from the control groups measured before and after IVM. The results of viability test indicate a divergent effect of COCs supplementation, as in Cp group ffEVs highly significantly increased (p<0.01), while in Ex decreased (p<0.05) CC viability compared to the control group. Despite the observed differences in CC physiology, transcriptome analysis demonstrate that CC RNA profile is slightly affected in the supplemented group. Nevertheless, the differently expressed genes comprise ones with processes and functions (e.g. cellular and metabolic processes), which may be important for the properties of cumulus and hence the maturation of the oocyte. [ABSTRACT FROM AUTHOR]

The efficacy of in vitro embryo production (IVEP) in equines is relatively limited compared to other species due to the lack of a reliable superovulation technique, limited availability of cumulus oocyte complexes (COCs), low in vitro oocyte maturation (IVM) and fertilization rates. Extracellular vesicles (EVs), which are nanoparticles involved in intercellular signaling in the ovarian environment, have shown potential as supplements to improve oocyte development during IVM. This study tested the hypothesis that EVs from small (< 20 mm) ovarian follicles could enhance fertilization rates in mares. Follicular fluid was collected postmortem, and EVs were isolated and characterized. The IVM process was conducted with or without EVs (200 µg EV protein/ml). EV internalization during IVM was examined using fluorescent labeling and confocal microscopy. Following intracytoplasmic sperm injection (ICSI), presumptive zygotes were cultured in a time-lapse system. Confocal microscopy confirmed EV internalization by COCs. Nanoparticle tracking analysis showed that obtained EVs were submicron-sized, and flow cytometry identified surface markers CD81 and CD63 on a subpopulation of EVs. Transmission electron microscopy revealed the characteristic disk shape of EV isolates. After culture, 196 oocytes (36.84 %) exhibited a first polar body and were subjected to ICSI. The EV-treated group showed a significantly higher fertilization rate (34.7 % vs. 20.2 %; P < 0.05), reduced degeneration, and increased cleavage efficiency (P < 0.1). Despite early embryonic arrest in both groups, these results suggest that follicular fluid-derived EVs could play a supportive role in equine IVF procedures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sex chromosomal abnormalities are a well-established cause of reproductive failure in domestic horses. Because of its difficult diagnosis, the Pura Raza Español breeding program established a routine screening for chromosomal abnormalities in all the horses prior to enrolling in the studbook. This genomic procedure combines an initial assessment based on the results from Short Tandem Repeat (STR) parentage testing followed by a Single-Nucleotide Polymorphism (SNP) based copy number aberration (CNA) confirmative analysis in positive cases. Using this methodology, we identified five new individuals carrying a 65,XXY chromosomal number aberration (CNA) among 27,330 foals enrolled over the past two reproductive seasons. The animals were initially flagged as CNA candidates due to abnormal results in STR testing. Subsequent analysis genotyping using an STR sex-linked dedicated panel and a medium-density SNP array in ECAX and ECAY confirmed the diagnosis as 65,XXY carriers. Four cases (upon sample availability) underwent further analysis using in situ fluorescent hybridization with ECAX and ECAY probes, showing identical results. Phenotypic analysis revealed abnormal gonad development in one of the cases, showing that the remaining four had a normal reproductive morphology. To our knowledge, this represents the largest number of horses exhibiting the equine form of Klinefelter syndrome (65,XXY) reported to date. Our study highlights the importance of genomic screening in the accurate detection of chromosomal abnormalities in horses.

Background: Chromosomal abnormalities occur in the equine population at a rate of approximately 2%. The use of molecular cytogenetic techniques allows a more accurate identification of chromosomal abnormalities, especially those with a low rate of abnormal metaphases, demonstrating that the actual incidence in equine populations is higher., Objectives: Estimation of the number of carriers of karyotypic abnormalities in a sample from a population of young horses of various breeds, using molecular cytogenetic techniques., Study Design: Cross-sectional., Methods: Venous blood samples were collected from 500 young horses representing 5 breeds (Purebred Arabian, Hucul, Polish primitive horse [Konik], Małopolska, Coldblood, Silesian). Chromosomes and DNA were obtained from blood lymphocytes and evaluated by fluorescence in situ hybridisation (FISH) and PCR, using probes and markers for the sex chromosomes and select autosomes., Results: Nineteen horses, 18 mares and 1 stallion, were diagnosed with different chromosomal abnormalities: 17 cases of mosaic forms of sex chromosome aneuploidies with a very low incidence (0.6%-4.7%), one case of a SRY-negative 64,XY sex reversal mare, and one mare with X-autosome translocation. The percentage of sex chromosomal aberrations was established as 3.8% in the whole population, 6.08% in females and 0.49% in males., Main Limitations: Limited sample size, confined to horses from Poland., Conclusions: The rate of sex chromosomal abnormalities we identified was almost double that reported in previous population studies that used classical chromosome staining techniques. FISH allowed the detection of aneuploid cell lines which had a very low incidence. The FISH technique is a faster and more precise method for karyotype examination; however, it is usually focused on only one or two chromosomes while banding karyotyping includes the entire chromosome set., (© 2024 EVJ Ltd.)

Mastitis is a multifactorial inflammatory disease. The increase in antibiotic resistance of bacteria that cause mastitis means that cattle breeders would prefer to reduce the use of antibiotics. Recently, therapies using mesenchymal stem cells (MSCs) from various sources have gained significant interest in the development of regenerative medicine in humans and animals, due to their extraordinary range of properties and functions. The aim of this study was to analyze the effectiveness of an allogeneic stem cells derived from bone marrow (BMSC) and adipose tissue (ADSC) in treating mastitis in dairy cattle. The research material consisted of milk and blood samples collected from 39 Polish Holstein-Friesian cows, 36 of which were classified as having mastitis, based on cytological evaluation of their milk. The experimental group was divided into subgroups according to the method of MSC administration: intravenous, intramammary, and intravenous + intramammary, and according to the allogeneic stem cells administered: BMSC and ADSC. The research material was collected at several time intervals: before the administration of stem cells, after 24 and 72 h, and after 7 days. Blood samples were collected to assess hematological parameters and the level of pro-inflammatory cytokines, while the milk samples were used for microbiological assessment and to determine the somatic cells count (SCC). The administration of allogeneic MSCs resulted in a reduction in the total number of bacterial cells, Staphylococcus aureus, bacteria from the Enterobacteriaceae group, and a systematic decrease in SCC in milk. The therapeutic effect was achieved via intravenous + intramammary or intramammary administration., (© 2024. The Author(s).)

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups ( p < 0.01), but decreased in Ex groups ( p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-β1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (P adjusted  < 0.05, log2FC ≥ 1.0/log2FC ≤  - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-β1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA)., (© 2023. Springer Nature Limited.)

Nanopore sequencing is a third-generation biopolymer sequencing technique that relies on monitoring the changes in an electrical current that occur as nucleic acids are passed through a protein nanopore. Increasing quality of reads generated by nanopore sequencing systems encourages their application in genome-wide polymorphism detection and genotyping. In this study, we employed nanopore sequencing to identify genome-wide polymorphisms in the horse genome. To reduce the size and complexity of genome fragments for sequencing in a simple and cost-efficient manner, we amplified random DNA fragments using a modified DOP-PCR and sequenced the resulting products using the MinION system. After initial filtering, this generated 28,426 polymorphisms, which were validated at a 3% error rate. Upon further filtering for polymorphism and reproducibility, we identified 9495 SNPs that reflected the horse population structure. To conclude, the use of nanopore sequencing, in conjunction with a genome enrichment step, is a promising tool that can be practical in a variety of applications, including genotyping, population genomics, association studies, linkage mapping, and potentially genomic selection.

The present study reports runs of homozygosity (ROH) distribution in the genomes of six horse breeds (571 horses in total) representing three horse types (primitive, light, and draft horses) based on the 65k Equine BeadChip assay. Of major interest was the length, quantity, and frequency of ROH characteristics, as well as differences between horse breeds and types. Noticeable differences in the number, length and distribution of ROH between breeds were observed, as well as in genomic inbreeding coefficients. We also identified regions of the genome characterized by high ROH coverage, known as ROH islands, which may be signals of recent selection events. Eight to fourteen ROH islands were identified per breed, which spanned multiple genes. Many were involved in important horse breed characteristics, including WFIKNN2 , CACNA1G , STXBP4 , NOG , FAM184B , QDPR , LCORL , and the zinc finger protein family. Regions of the genome with zero ROH occurrences were also of major interest in specific populations. Depending on the breed, we detected between 2 to 57 no-ROH regions and identified 27 genes in these regions that were common for five breeds. These genes were involved in, e.g., muscle contractility ( CACNA1A ) and muscle development ( miR-23 , miR-24 , miR-27 ). To sum up, the obtained results can be furthered analyzed in the topic of identification of markers unique for specific horse breed characteristics.

In recent years, a vast amount of sequencing data has been generated and large improvements have been made to reference genome sequences. Despite these advances, significant portions of reads still do not map to reference genomes and these reads have been considered as junk or artificial sequences. Recent studies have shown that these reads can be useful, e.g., for refining reference genomes or detecting contaminating microorganisms present in the analyzed biological samples. A special case of this is RNA sequencing (RNA-Seq) reads that come from tissue transcriptomes. Unmapped reads from RNA-Seq have received much less attention than those from whole-genome sequencing. In particular, in the horse, an analysis of unmapped RNA reads has not been performed yet. Thus, in this study, we analyzed the unmapped reads originating from the RNA-Seq performed through the Functional Annotation of Animal Genomes (FAANG) project in the horse, using eight different tissues from two mares. We demonstrated that unmapped reads from RNA-Seq could be easily assembled into transcripts relating to many important genes present in the sequences of other mammals. Large portions of these transcripts did not have coding potential and, thus, can be considered as non-coding RNA. Moreover, reads that were not mapped to the reference genome but aligned to the entries in NCBI database of horse proteins were enriched for biological processes that largely correspond to the functions of organ from which RNA was isolated and thus are presumably true transcripts of genes associated with cell metabolism in those tissues. In addition, a portion of reads aligned to the common pathogenic or neutral microbiota, of which the most common was Brucella spp. These data suggest that unmapped reads can be an important target for in-depth analysis that may substantially enrich results of initial RNA-Seq experiments for various tissues and organs., (© 2022. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.)

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24-38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The Y chromosome is a valuable genetic marker for studying the origin and influence of paternal lineages in populations. In this study, we conducted Y-chromosomal lineage-tracing in Arabian horses. First, we resolved a Y haplotype phylogeny based on the next generation sequencing data of 157 males from several breeds. Y-chromosomal haplotypes specific for Arabian horses were inferred by genotyping a collection of 145 males representing most Arabian sire lines that are active around the globe. These lines formed three discrete haplogroups, and the same haplogroups were detected in Arabian populations native to the Middle East. The Arabian haplotypes were clearly distinct from the ones detected in Akhal Tekes, Turkoman horses, and the progeny of two Thoroughbred foundation sires. However, a haplotype introduced into the English Thoroughbred by the stallion Byerley Turk (1680), was shared among Arabians, Turkomans, and Akhal Tekes, which opens a discussion about the historic connections between Oriental horse types. Furthermore, we genetically traced Arabian sire line breeding in the Western World over the past 200 years. This confirmed a strong selection for relatively few male lineages and uncovered incongruences to written pedigree records. Overall, we demonstrate how fine-scaled Y-analysis contributes to a better understanding of the historical development of horse breeds.