Comparative analysis of the differences in ear cartilage tissues between Duroc and Erhualian pigs

【Objective】The comparison of the differences in ear cartilage tissues between Duroc and Erhualian pigs was conducted to provide insights into the molecular mechanisms underlying the variation in outer ear area in pigs.【Method】 Outer ear cartilage tissues from male sibling pigs of Duroc(n=5)and Erhualian(n=5),aged 100 days,were collected for paraffin sectioning. Hematoxylin-eosin(HE)staining and Masson trichrome staining were performed to observe and compare the structural differences between these two groups. Duroc and Erhualian ear chondrocytes were then isolated and cultured in vitro to detect cell proliferation ability using CCK8. Finally,transcriptome sequencing was combined with a comparison of gene expression differences in ear cartilage tissues of Erhualian(n=3)and Duroc(n=3). Genes related to cartilage growth were screened and verified by real-time fluorescence quantitative PCR(qRT-PCR).【Result】 HE and Masson trichrome stainings revealed that compared to Duroc,Erhualian ear chondrocytes formed more isogenous groups consisting of 2-8 cells,and the cartilage tissues contained more elastic fibers. The cell proliferation ability testing demonstrated that the proliferation rate of Erhualian ear chondrocytes was significantly higher than that of Duroc after 36,48, and 60 h of in vitro culture(P<0.05). A total of 251 differentially expressed genes(DEGs)were identified through transcriptome sequencing,including 165 up-regulated genes and 86 down-regulated genes of Duroc comparing with Erhualian. GO functional annotation revealed that the up-regulated genes were mainly enriched in pathways related to skeletal system development and extracellular matrix,while the down-regulated genes are mainly enriched in pathways associated with cellular response to retinoic acid and Wnt signaling. Gene network and functional analysis showed that MMP3, BGLAP and GDF5 genes with high expression in Duroc ear cartilage tissues promoted the degradation and mineralization of extracellular matrix in cartilage tissues. The Wnt signaling pathway inhibitory gene DKK1 was highly expressed in Erhualian ear cartilage,suggesting negative regulation of the Wnt signaling pathway. Highly-expressed HMGA2,WNT10B and AXIN2 genes promoted the proliferation of Erhualian outer ear chondrocytes. qRT-PCR verified above results.【Conclusion】The ear chondrocytes of Erhualian pigs exhibit a more robust proliferation and division ability compared to that of Duroc pigs. In Duroc ear cartilage,genes such as MMP3,BGLAP,and GDF5 induce extracellular matrix degradation and osteogenic differentiation processes. In Erhualian ear cartilage tissues,the Wnt signaling pathway is negatively regulated,and genes such as HMGA2,WNT10B and AXIN2 promote the proliferation of ear chondrocytes and the growth of ear cartilage.