Background/Objectives: DGAT1 p. K232A (rs109234250) is a well-established causal variant influencing milk fat and protein content in dairy cattle, but it is often absent from commercial genotyping arrays. PPP1R16A rs109146371 frequently appears as a top signal in genome-wide association studies (GWAS) for milk traits. This study aimed to evaluate the linkage disequilibrium (LD) between these two variants in Japanese Holsteins and assess whether rs109146371 exerts an independent effect on milk traits. Methods: A total of 256 Japanese Holstein cows were genotyped for DGAT1 p. K232A and PPP1R16A rs109146371 using TaqMan SNP assays. LD statistics (r², D′) were computed, and linear mixed-effects models were used to evaluate associations with 305-day milk yield, fat percentage, protein percentage, and solids-not-fat (SNF) percentage. Likelihood ratio tests were conducted to assess the independence of SNP effects. Results: Strong LD was observed between DGAT1 p. K232A and PPP1R16A rs109146371 (r² = 0.91, D′ = 0.9962). Both SNPs showed significant associations with all milk production traits; however, model comparisons indicated that rs109146371 did not improve model fit when K232A was included, suggesting no independent effect. Conclusions: PPP1R16A rs109146371 serves as a proxy for DGAT1 K232A rather than an independent determinant of milk traits.

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Canine hepatocellular carcinoma (HCC) requires further molecular characterization to identify diagnostic and therapeutic targets, and to establish whether dogs with this condition can model the human disease. Accordingly, we aimed to identify differentially expressed genes (DEGs) in canine HCC and evaluate cross‐species transcriptomic dysregulation in canine and human HCC. Liver tissue samples from three dogs with HCC and three healthy dogs were subjected to next‐generation sequencing, followed by RT‐qPCR validation. Identified DEGs were then targeted in bioinformatics analyses (pathway enrichment, protein‐protein interaction network, and hub gene analyses) for molecular characterization and comparison with human HCC datasets. We identified 975 DEGs (upregulated: 604; and downregulated: 371). Extracellular matrix‐receptor interaction, focal adhesion, cell adhesion molecule, PI3K/Akt signaling, and cytokine/chemokine‐related pathways were enriched. C1R, APOC3, C1QA, APOA1, C1QB, ACTG1, C1QC, CRP, ANXA5, and ANXA2 were identified as hub genes. Canine and human HCCs share 118 DEGs, highlighting conserved alterations in metabolic pathways, PI3K‐Akt signaling, focal adhesion, and PPAR signaling pathways. Based on human HCC data, SPP1, NQO1, RRM2, APOA1, APOC3, ALDOB, and IGF1 were identified as prognosticators indicating poor overall survival. This study presents the first cross‐species transcriptomic analysis of canine HCC, revealing significant molecular resemblances to human HCC, indicating it may be a promising comparative model for studying tumor biology, drug responses, and novel therapeutic interventions.

Canine hepatocellular carcinoma (HCC), the most common primary liver malignancy in dogs, shares many clinicopathological and molecular similarities with human HCC. However, its molecular characteristics remain insufficiently defined, and reliable diagnostic biomarkers are lacking. Elucidating dysregulated microRNAs (miRNAs) may aid in both disease characterization and comparative oncology research. Small RNA sequencing datasets from canine HCC were analyzed to identify significantly dysregulated miRNAs with high expression and biomarker potential. The top candidate was validated in clinical tissues, cell lines, patient’s plasma and plasma exosomes using RT-qPCR. Comparative analyses were conducted using human HCC datasets (TCGA and GEO), followed by target prediction and functional enrichment to identify conserved molecular pathways. Among the 59 differentially expressed miRNAs, cfa-miR-10a showed the highest average expression level and yet was significantly downregulated in canine HCC tissues. RT-qPCR confirmed reduced expression of cfa-miR-10a in canine HCC tissues, whereas plasma exosomes showed significant enrichment, demonstrating excellent diagnostic performance (AUC = 0.94). The mature sequence of cfa-miR-10a is highly conserved with hsa-miR-10a-5p. TCGA datasets confirmed downregulation of hsa-miR-10a-5p in HCC tissues, whereas a GEO dataset showed no significant change in serum exosome levels. Target prediction and functional annotation identified 59 overlapping genes, with the Proteoglycans in cancer pathways being conserved in both species, mediated by ACTG1, SDC1, FRS2, and WNT9B. Collectively, these findings demonstrate distinct intra-tumoral and exosomal expression pattern of miR-10a in canine HCC and support its potential as a non-invasive biomarker with translational relevance.

We evaluated four candidate SNPs (GC-NPFFR2 rs137147462, GC-NPFFR2 rs109452259, BRCA1 rs134817801, and DGAT1 p.K232A) previously reported in relation to mastitis or milk production traits, using 10,729 test-day phenotypic records collected over 10 years from 269 Japanese Holstein cows (Bos taurus) enrolled in the national Dairy Herd Improvement (DHI) program. Linear mixed models were used to estimate genotypic effects on somatic cell score (SCS) and to test multiple inheritance models. To assess clinical relevance, mastitis severity was further analyzed using categories defined by somatic cell counts (SCC). Among the SNPs tested, GC-NPFFR2 rs137147462 showed the clearest and most consistent association with SCS under a recessive model, with GG cows exhibiting higher SCS throughout lactation. Ordinal logistic regression confirmed a higher probability of progression to severe mastitis in GG cows. DGAT1 p.K232A showed additive effects, with the A allele increasing milk yield while lowering fat and protein percentages. AA cows also showed higher SCS under a modest recessive effect. BRCA1 rs134817801 and GC-NPFFR2 rs109452259 had minimal effects. These findings support GC-NPFFR2 rs137147462 as a promising marker for mastitis resistance and indicate the importance of considering not only additive but also recessive genetic models in genomic selection strategies.