Genetic resources constitute the cornerstone of seed-industry revitalization and sustainable agriculture. Their conservation and judicious utilization are imperative for safeguarding national seed security, enhancing livestock competitiveness, and maintaining biodiversity. The Huchuan Mountain pigs, a representative indigenous pig cluster in Southwest China, possess a unique genetic background and valuable local traits. It is a crucial source of income for local farmers and underpins regional agricultural development. However, frequent outbreaks of African swine fever and intensifying market competition have severely eroded its genetic diversity. Several populations—most notably Luopanshan, Hechuan Black, and Quxi pigs—are now critically endangered, demanding urgent conservation measures. Systematically dissecting the genetic structure, population divergence, and putative adaptive variants of the Huchuan Mountain pigs is therefore of both theoretical and practical importance for informed conservation and genetic improvement.

This study aimed to comprehensively characterize the population structure and genetic differentiation of the Huchuan Mountain pigs cluster, to identify selection signatures under contrasting conservation schemes, and to uncover candidate adaptive genes, thereby providing a robust scientific basis for the conservation and genetic enhancement of local pig resources.

Taking these six Huchuan Mountain pig populations as study subjects, whole-genome resequencing was performed. Based on high-quality SNP data, principal component analysis (PCA) and Admixture analysis were employed to evaluate genetic differences and admixture levels among populations, thereby revealing the genetic structure of each population. By constructing a phylogenetic tree, the kinship relationships and introgression events among populations were analyzed to further clarify their genetic connections. Combining the Fst (population genetic differentiation index) and π (nucleotide diversity) ratio, those selection signatures were detected to identify regions and candidate genes under selection during adaptive evolution in populations conserved under different conservation models. GO and KEGG functional enrichment analyses of these candidate genes were conducted to uncover their potential functions and biological characteristics related to adaptation, thus systematically dissecting the genetic structure and the genetic mechanisms underlying adaptive phenotypes in Huchuan Mountain pig populations.

PCA and ADMIXTURE analysis revealed pronounced genetic differentiation among populations. Yacha and Enshi Black pigs exhibited distinct genetic backgrounds, whereas Quxi and Penzhou Mountain pigs displayed extensive genetic admixture. A subset of Hechuan Black individuals clustered closely with Enshi Black pigs, and introgression signals were detected in multiple directions, especially involving Luopanshan pigs. The phylogeny tree corroborated these findings, underscoring the complex reticulate evolution within the cluster. Selection signal analysis based on Fst and θπ ratio revealed 998 highly differentiated loci corresponding to 7088 candidate genes in the nature-reserve conservation model and 616 highly differentiated loci corresponding to 3360 candidate genes in the preserving farms model. Functional enrichment indicated that genes under natural selection in the nature-reserve conservation model were predominantly associated with immune response, energy metabolism, and environmental adaptation, whereas those under artificial selection in the preserving farms model were enriched for reproductive regulation, hormone signaling, and metabolic pathways, reflecting distinct genetic adaptations under different rearing and management conditions.

The Huchuan Mountain pig cluster was characterized by intricate population structure and marked genetic differentiation. Nature-reserve conservation effectively preserved genetic diversity and adaptive potential, whereas preserving farms conservation enhanced production and reproductive performance. These findings provided the critical genomic evidence for designing complementary conservation strategies tailored to the sustainable utilization of this invaluable genetic resource.

Germplasm resources are the foundation of the revitalization of the swine breeding industry, and their protection and utilization are critical. However, the outbreak of African swine fever and increasing market competition have caused a significant decline in the number of local Chinese pig breeds, therefore threatening the genetic diversity of pig germplasm resources, with many breeds now facing endangerment. Somatic cell nuclear transfer (SCNT) is a promising strategy for restoring endangered pig breeds, but the aging of donor cells severely limits the efficiency of pig SCNT. Cycloastragenol (CAG) has demonstrated anti-aging effects in earlier studies.

This study aimed to evaluate the impact of CAG on aging-related phenotypes of pig ear marginal fibroblasts (PEMFs) and to explore the molecular mechanisms by which CAG regulates cellular senescence using RNA sequencing. Additionally, the potential of CAG as a pre-treatment for SCNT donor cells was further investigated.

PEMFs exhibiting aging phenotypes were obtained through continuous passaging. Cell proliferation assays and flow cytometry were performed to detect Senescence-associated β-galactosidase (SA-β-Gal) activity, in order to analyze the optimal concentrations and treatment times of CAG for PEMFs at different stages of aging. The anti-aging effects of CAG were further assessed by measuring p53 protein levels and the expression of Senescence-associated secretory phenotype (SASP) markers, coupled with immunofluorescence staining of the cytoskeleton. By integrating phenotypic analysis with transcriptomic data, the potential mechanisms by which CAG slowed down PEMF senescence were elucidated. Additionally, SCNT embryo cleavage and blastocyst rates were statistically analyzed, followed by staining and quantification of the total cell number and trophectoderm cell number in SCNT blastocysts to investigate the effects of CAG-pretreated PEMFs on SCNT embryo developmental potential.

SA-β-Gal assay and CCK-8 cell proliferation assay demonstrated that the anti-aging and proliferation-inhibitory effects of CAG were concentration- and time-dependent. Based on dose-effect balance analysis, the recommended treatment conditions were 100 μmol·L^-1 CAG for 24 hours in P3/P6 PEMFs and 12 hours in P9 PEMFs. Under these conditions, CAG significantly reduced SA-β-Gal activity, p53 protein expression, and the secretion of certain SASP factors in PEMFs. Additionally, CAG treatment led to a significant reduction in cell surface area, focal adhesion number, and stress fiber thickness and quantity, while also reorganizing the cytoskeleton. Transcriptomic analysis and subsequent validation results revealed that CAG modulates pathways such as focal adhesion and extracellular matrix (ECM) signaling, thereby affecting the stability of the cytoskeleton. In embryo culture experiments, PEMFs treated with CAG exhibited higher cleavage and blastocyst formation rates, as well as greater blastocyst total cell numbers and trophoblast cell counts, indicating a significant improvement in the quality and developmental potential of early embryos.

CAG effectively delayed donor cell senescence, reorganized the cytoskeleton, and significantly enhanced SCNT efficiency and embryo developmental potential. This discovery offered a novel approach to addressing the challenges of pig germplasm conservation and low cloning efficiency.