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Contents Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey ( Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys. [ABSTRACT FROM AUTHOR]

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 study is aimed at identifying selection footprints within the genome of Limousin cattle. With the use of Extended Haplotype Homozygosity test, supplemented with correction for variation in recombination rates across the genome, we created map of selection footprints and detected 173 significant (p < 0.01) core haplotypes being potentially under positive selection. Within these regions, a number of candidate genes associated inter alia with skeletal muscle growth ( GDF15, BMP7, BMP4 and TGFB3) or postmortem proteolysis and meat maturation ( CAPN1 and CAPN5) were annotated. Noticeable clusters of selection footprints were detected on chromosomes 1, 4, 8 and 14, which are known to carry several quantitative trait loci for growth traits and meat quality. The study provides information about the genes and metabolic pathways potentially modified under the influence of directional selection, aimed at improving beef production characteristics in Limousin cattle. [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.)