2024, Cieślak, Jakub, Maćkowski, Mariusz, Skrzetuska, Weronika, Fidos-Tama, Ewa, Siwinska, Natalia, Szczerbal, Izabela

2025, Skrzetuska, Weronika, Maćkowski, Mariusz, Borowska, Alicja Helena, Kalski, Roman, Musiał, Adrianna, Bieniek, Agnieszka, Ropka-Molik, Katarzyna, Cieślak, Jakub

The protection and balanced development of all existing maternal lines is one of the primary goals of the Polish Primitive Horse (PPH or Konik) conservation programme. However, previous studies have indica- ted that managing PPH conservative breeding may encounter challenges because of the numerous existing pedigree errors. We therefore attempted to check whether mtDNA markers may prove useful in correcting PPH pedigrees and improving the breed conservation programme. A 510 bp mtDNA D-loop fragment was sequenced for 396 samples representing all sixteen officially recognised maternal lines. These samples were derived from different time points in the PPH breeding history. Our analysis confirmed the presence of nineteen mtDNA haplotypes. A comparison of the molecular and pedigree data showed that the frequency of particular haplotypes was highly uneven. Although for the majority of the maternal lines we were able to identify a potential 'founder haplotype', only four of them (Dzina I, Popielica, Geneza and Bona) turned out to be 'genetically pure'. Our study confirmed that an mtDNA analysis is a useful method for assessing PPH maternal genetic diversity and illustrating the breed's history. Our findings suggest that PPH conservation programmes would benefit from revising the official pedigrees using molecular data alongside breeding records.

2025, Nowacka-Woszuk, Joanna, Niemiec, Anna, Dzimira, Stanislaw, Racheniuk, Monika, Kaznowski, Arkadiusz, Maćkowski, Mariusz, Świtoński, Marek, Szczerbal, Izabela

ABSTRACTAn adult mare with ambiguous external genitalia, observed at a slaughterhouse, was subjected to detailed examination. The mare exhibited fused labia and an enlarged clitoris located at the ventral commissure of the vulva. Anatomical and histopathological studies revealed the presence of two testicles, vas deferens, fallopian tubes, a small uterus with blindly ending horns and a normally developed cervix. Cytogenetic analysis, using fluorescent in situ hybridisation (FISH) of in vitro cultured lymphocytes, showed a female karyotype—64,XX. Molecular detection of X‐ and Y‐linked genes (SRY and ZFX/ZFY) in blood cells confirmed the presence of X‐linked genes only. In contrast, in hair follicles and gonadal tissue, the presence of genes originating from the Y chromosome was also detected. The use of digital droplet PCR (ddPCR) revealed the presence of a SRY‐positive cell line; however, at a very low level (< 5%). Analysis of polymorphic short tandem repeats (STRs), recommended for parentage testing, did not detect chimerism, which would be indicated by the presence of three or four variants at some STR loci. In conclusion, the studied case was classified as a sex chromosome disorder of sex development (DSD) due to gonadal XX/XY mosaicism. To the best of our knowledge, this is the first reported case of such an abnormality in a DSD horse.

2025, Cullen, Jonah N., Cieślak, Jakub, Petersen, Jessica L., Bellone, Rebecca R., Finno, Carrie J., Kalbfleisch, Ted S., Calloe, Kirstine, Capomaccio, Stefano, Cappelli, Katia, Coleman, Stephen J., Distl, Ottmar, Durward-Akhurst, Sian A., Giulotto, Elena, Hamilton, Natasha A., Hill, Emmeline W., Katz, Lisa M., Klaerke, Dan A., Lindgren, Gabriella, MacHugh, David E., Maćkowski, Mariusz, MacLeod, James N., Metzger, Julia, Murphy, Barbara A., Orlando, Ludovic, Raudsepp, Terje, Silvestrelli, Maurizio, Strand, Eric, Tozaki, Teruaki, Trachsel, Dagmar S., Valderrama Figueroa, Laura S., Velie, Brandon D., Wade, Claire M., Waud, Bianca, Mickelson, James R., McCue, Molly E.

MicroRNAs (miRNAs) are essential regulators of gene expression, yet few comprehensive databases exist for miRNA expression in non-model species, limiting our ability to characterize their roles in gene regulation, development, and disease. Similarly, isomiRs - length and sequence isoforms of canonical miRNAs with potentially altered regulatory targets and functions - have received even less attention in non-model species, including the horse, leaving a critical gap in our understanding of their biological significance. To address these challenges, we developed an open-source, containerized pipeline for identifying and quantifying miRNAs and isomiRs (FARmiR: Framework for Analysis and Refinement of miRNAs), and an associated interactive browser (AIMEE: Animal IsomiR and MiRNA Expression Explorer). AIMEE was developed to make miRNA expression data more accessible and user-friendly, a feature often lacking from other expression atlases. These tools were developed using equine data but can be readily extended to other species. Using these tools, we aggregated 461 small RNA-seq datasets, spanning 61 distinct tissues, integrating data from public repositories, an American Quarter Horse cohort, and the Functional Annotation of ANimal Genome (FAANG) consortium Thoroughbred samples, predicting 5,781 miRNAs and isomiRs. This work represents the largest systematically curated atlas of equine miRNA expression to date, providing a valuable resource that will enhance our understanding of miRNA and isomiR functions in tissue-specific regulation and ultimately improve biomarker discovery, functional genomics, and precision veterinary medicine.