Maintaining maximum genetic diversity and preserving breed viability in conserved populations necessitates the rigorous evaluation of conservation schemes. Three chicken breeds (Baier Yellow Chicken (BEC), Beijing You Chicken (BYC) and Langshan Chicken (LSC)) are currently in conservation programs in China. Changes in genetic diversity were measured by heterozygosity, genomic inbreeding coefficients, and autozygosity, using estimates derived from runs of homozygosity (ROH) that were identified using SNPs. Ninety DNA samples were collected from three generations for each breed. In the most recent generation, the highest genetic diversity was observed in LSC, followed by BEC and BYC. Inbreeding coefficients based on ROH for the three breeds declined slightly between the first and middle generations, and then rapidly increased. No inbreeding coefficients exceeded 0.1. Population structure assessments using neighbor-joining tree analysis, principal components analysis, and STRUCTURE analysis indicated that no genetic differentiation existed within breeds. LD decay and ROH at different cut-off lengths were used to identify traces left by recent or ancient inbreeding. Few long ROH were identified, indicating that inbreeding has been largely avoided with the current conservation strategy. The observed losses in genetic diversity and occurrences of inbreeding might be consequences of sub-optimal effective population sizes. The conserved Chinese chicken populations have high genomic diversity under the current conservation program (R: F). Furthermore, this study highlights the need to monitor dynamic changes in genetic diversity in conserved breeds over successive generations. Our research provides new insights into genetic diversity dynamics in conserved populations, and lays a solid foundation for improving conservation schemes.

Conservation of natural resources is a vital and challenging task. Numerous animal genetic resources have been effectively conserved worldwide. However, the effectiveness of conservation programmes and the variation information of species have rarely been evaluated. Here, we performed whole-genome and whole-genome bisulfite sequencing of 90 Chinese indigenous chickens, which belonged to the Tibetan, Wenchang and Bian chicken breeds, and have been conserved under different conservation programmes. We observed that low genetic diversity and high DNA methylation variation occurs during ex situ in vivo conservation, while higher genetic diversity and differentiation occurs during in situ conservation. Further analyses revealed that most DNA methylation signatures are unique within ex situ in vivo conservation. Moreover, a high proportion of differentially methylated regions is found in genomic selection regions, suggesting a link between the effects of genomic variation and DNA methylation. Altogether our findings provide valuable information about genetic and DNA methylation variations during different conservation programmes, and hold practical relevance for species conservation.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 μg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 μg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 μg/mL and 100 μg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 μg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

Objective: Inbreeding depression of reproduction is a major concern in the conservation of native chicken genetic resources. Here, based on the successful development of strongly inbred (Sinb) and weakly inbred (Winb) Langshan chickens, we aimed to evaluate inbreeding effects on reproductive traits and identify candidate genes involved in inbreeding depression of reproduction in Langshan chickens.Methods: A two-sample t-test was performed to estimate the differences in phenotypic values of reproductive traits between Sinb and Winb chicken groups. Three healthy chickens with reproductive trait values around the group mean values were selected from each of the groups. Differences in ovarian and hypothalamus transcriptomes between the two groups of chickens were analyzed by RNA sequencing (RNA-Seq).Results: The Sinb chicken group showed an obvious inbreeding depression in reproduction, especially for traits of age at the first egg and egg number at 300 days (p<0.01). Furthermore, 68 and 618 differentially expressed genes (DEGs) were obtained in the hypothalamus and ovary between the two chicken groups, respectively. In the hypothalamus, DEGs were mainly enriched in the pathways related to vitamin metabolism, signal transduction and development of the reproductive system, such as the riboflavin metabolism, Wnt signaling pathway, extracellular matrix-receptor interaction and focal adhesion pathways, including stimulated by retinoic acid 6, serpin family F member 1, secreted frizzled related protein 2, Wnt family member 6, and frizzled class receptor 4 genes. In the ovary, DEGs were significantly enriched in pathways associated with basic metabolism, including amino acid metabolism, oxidative phosphorylation, and glycosaminoglycan degradation. A series of key DEGs involved in folate biosynthesis (gamma-glutamyl hydrolase, guanosine triphosphate cyclohydrolase 1), oocyte meiosis and ovarian function (cytoplasmic polyadenylation element binding protein 1, structural maintenance of chromosomes 1B, and speedy/RINGO cell cycle regulator family member A), spermatogenesis and male fertility (prostaglandin D2 synthase 21 kDa), Mov10 RISC complex RNA helicase like 1, and deuterosome assembly protein 1) were identified, and these may play important roles in inbreeding depression in reproduction.Conclusion: The results improve our understanding of the regulatory mechanisms underlying inbreeding depression in chicken reproduction and provide a theoretical basis for the conservation of species resources.

Egg production plays a pivotal role in the economic viability of hens. To analyze the genetic rules of egg production, a total of 3151 Luhua chickens were selected, the egg production traits including egg weight at first laying (Start-EW), egg weight at 43 weeks (EW-43), egg number at 43 weeks (EN-43), and total egg number (EN-All) were recorded. Then, the effects of related factors on egg production traits were explored, using a multi-trait animal model for genetic parameter estimation and a genome-wide association study (GWAS). The results showed that body weight at first egg (BWFE), body weight at 43 weeks (BW-43), age at first egg (AFE), and seasons had significant effects on the egg production traits. Start-EW and EW-43 had moderate heritability of 0.30 and 0.21, while EN-43 and EN-All had low heritability of 0.13 and 0.16, respectively. Start-EW exhibited a robust positive correlation with EW-43, while Start-EW was negatively correlated with EN-43 and EN-All. Furthermore, gene ontology (GO) results indicated that Annexin A2 (ANXA2) and Frizzled family receptor 7 (FZD7) related to EW-43, Cyclin D1 (CCND1) and A2B adenosine receptor (ADORA2B) related to EN-All, and have been found to be mainly involved in metabolism and growth processes, and deserve more attention and further study. This study contributes to accelerating genetic progress in improving low heritability egg production traits in layers, especially in Luhua chickens.

Tibetan animals have several unique advantages owing to the harsh ecological conditions under which they live. However, compared to Tibetan mammals, understanding of the advantages and underlying mechanisms of the representative high-latitude bird, the Tibetan chicken ( Gallus gallus , TC), remains limited. The gut microbiota of animals has been conclusively shown to be closely related to both host health and host environmental adaptation. This study aimed to explore the relationships between the cecal microbiome and the advantages of TCs based on comparisons among three populations: native TCs residing on the plateau, domestic TCs living in the plain, and one native plain species. Metatranscriptomic sequencing revealed a significant enrichment of active Bacteroidetes but a loss of active Firmicutes in native TCs. Additionally, the upregulated expression of genes in the cecal microbiome of native TCs showed enriched pathways related to energy metabolism, glycan metabolism, and the immune response. Furthermore, the expression of genes involved in the biosynthesis of short-chain fatty acids (SCFAs) and secondary bile acids (SBAs) was upregulated in the cecal microbiome of native TCs. Data from targeted metabolomics further confirmed elevated levels of certain SCFAs and SBAs in the cecum of native TCs. Based on the multi-omics association analysis, we proposed that the higher ratio of active Bacteroidetes/Firmicutes may be attributed to the efficient energy metabolism and stronger immunological activity of native TCs. Our findings provide a better understanding of the interactions between gut microbiota and highland adaptation, and novel insights into the mechanisms by which Tibetan chickens adapt to the plateau hypoxic environment. IMPORTANCE: The composition and function of the active cecal microbiome were significantly different between the plateau Tibetan chicken population and the plain chicken population. Higher expression genes related to energy metabolism and immune response were found in the cecal microbiome of the plateau Tibetan chicken population. The cecal microbiome in the plateau Tibetan chicken population exhibited higher biosynthesis of short-chain fatty and secondary bile acids, resulting in higher cecal content of these metabolites. The active Bacteroidetes/Firmicutes ratio in the cecal microbiome may contribute to the high-altitude adaptive advantage of the plateau Tibetan chicken population.