Current knowledge about the evolutionary history of donkeys is still incomplete due to the lack of archeological and whole-genome diversity data. To fill this gap, we have de novo assembled a chromosome-level reference genome of one male Dezhou donkey and analyzed the genomes of 126 domestic donkeys and seven wild asses. Population genomics analyses indicate that donkeys were domesticated in Africa and conclusively show reduced levels of Y chromosome variability and discordant paternal and maternal histories, possibly reflecting the consequences of reproductive management. We also investigate the genetic basis of coat color. While wild asses show diluted gray pigmentation (Dun phenotype), domestic donkeys display non-diluted black or chestnut coat colors (non-Dun) that were probably established during domestication. Here, we show that the non-Dun phenotype is caused by a 1 bp deletion downstream of the TBX3 gene, which decreases the expression of this gene and its inhibitory effect on pigment deposition.

Circular RNAs (circRNAs) are a class of noncoding RNAs with a covalently closed loop. Studies have demonstrated that circRNA can function as microRNA (miRNA) sponges or competing endogenous RNAs. Although circRNA has been explored in some species and tissues, the genetic basis of testis development and spermatogenesis in donkeys remain unknown. We performed RNA-seq to detect circRNA expression profiles of adult donkey testes. Length distribution and other characteristics were shown a total of 1971 circRNAs were differentially expressed and 12,648 and 6261 circRNAs were detected from the testis and caput epididymis, respectively. Among these circRNAs, 1472 circRNAs were downregulated and 499 circRNAs were upregulated in the testis. Moreover, KEGG pathway analyses and Gene Ontology were performed for host genes of circRNAs. A total of 39 upregulated circRNA host genes were annotated in spermatogenesis terms, including PIWIL2, CATSPERD, CATSPERB, SPATA6, and SYCP1. Other host genes were annotated in the focal adhesion, Rap1 signaling pathway. Downregulated expressed circRNA host genes participated in the TGF-β signaling pathway, GnRH signaling pathway, estrogen signaling pathway, and calcium signaling pathway. Our discoveries provide a solid foundation for identifying and characterizing critical circRNAs involved in testis development or spermatogenesis.

Controlling low ambient temperatures and ammonia levels is critical for effective environmental management in poultry houses during winter, as both represent persistent stressors affecting bird health and productivity. However, evidence regarding their combined long-term effects on the physiological responses of laying hens remains limited. In this study, 576 eighteen-week-old Hy-Line Brown hens were randomly assigned to six treatments (8 replicates with 12 birds per replicate each treatment) and housed in environmentally controlled chambers for 20 weeks: T1 (8 °C, ≤5 ppm ammonia), T2 (8 °C, 20 ppm ammonia), T3 (8 °C, 45 ppm ammonia), T4 (20 °C, ≤5 ppm ammonia; control), T5 (20 °C, 20 ppm ammonia), and T6 (20 °C, 45 ppm ammonia). Plasma samples were collected at 22, 26, 30, 34, and 38 weeks to evaluate physiological stress biomarkers (corticosterone, CORT; total antioxidant capacity, T-AOC), immunoglobulins (IgG, IgM, and IgA), and reproductive hormones (luteinizing hormone, LH; follicle-stimulating hormone, FSH; estradiol, E2). At 38 weeks, hypothalamus, pituitary, and spleen tissues were collected to assess the relative mRNA expression of gonadotropin-releasing hormone (GnRH), FSH, tumor necrosis factor-α (TNF-α), and interleukins (IL-1β, IL-6, and IL-10). Results showed that both cold and ammonia stress reduced antioxidant capacity, disrupted immune homeostasis, and altered reproductive hormone profiles. Cold exposure induced acute immunoendocrine alterations with partial physiological adaptation over time, whereas ammonia exerted progressive and cumulative damage, including elevated immunoglobulins (IgG and IgM) and downregulation of GnRH and FSH expression. Combined exposure significantly upregulated TNF-α and IL-1β expression, suggesting a synergistic inflammatory response. These results highlight complex, parameter-specific interactions between cold and ammonia stressors, emphasizing the need for targeted environmental strategies. Stage-specific interventions—thermal regulation in early laying and ammonia control in later phases—are recommended to safeguard hen health and optimize productivity under winter conditions.

Producing translucent eggs has been found to reduce the quality and safety of the eggs, as well as the demand from consumers. However, the intestinal function and the molecular mechanism for the production of translucent eggs remain uncertain. A total of 120 eggs from 276-day-old Jining Bairi were divided into two groups based on eggshell translucence: the translucent egg group (group T) and the normal group (group C). Group T exhibited thicker eggshells and a lower egg yolk color. Subsequently, we divided the chickens into translucent and normal groups based on their egg quality. We then assessed the plasma biochemical index, intestinal morphology and structure, enzyme activity, and antioxidant capacity of the hens producing translucent eggs compared to those producing normal eggs. The results showed that the ratio of duodenal villus length to crypt depth, succinate dehydrogenase (SDH) activity, chymotrypsin, total ATPase (T-ATPase), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-Px) activities were decreased in the hens produced translucent eggs (p < 0.05), but malondialdehyde (MDA) content was increased (p < 0.05); jejunal lipase activity, Na⁺K⁺-ATPase activity, total antioxidant capacity (T-AOC), and GSH-Px activities were decreased (p < 0.05) in group T; ileal amylase and Ca²⁺Mg²⁺-ATPase activities were also decreased (p < 0.05) in group T. In addition, we identified a total of 471 differentially expressed genes (DEGs) in duodenal tissue, with 327 up-regulated genes and 144 down-regulated genes (|log₂FC| ≥ 1 and p < 0.05). Enrichment analysis showed that the up-regulated genes, such as GSTT1, GSTO2, and GSTA3, were mostly enriched in metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, and oxidative phosphorylation pathways. The results of our study indicate that plasma lipid metabolism disorder, decreased intestinal antioxidant capacity, and altered intestinal metabolism capabilities may influence the formation of translucent eggs.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21014-9

Eggshell quality is a major concern in the poultry industry. The speckle phenotype in eggshell affects eggshell color, thickness, and Haugh Unit (HU) and significantly affects the hatchability rate. The rate of speckled eggs increases during the late-laying period, which can lead to notable economic losses. Eggshell speckles have medium heritability; however, the genetic architecture of speckled eggshells remains unclear. In this study, the chickens were divided into two groups based on the rate of egg speckling. Uterine tissues were collected and analyzed using 10X Genomics single-cell RNA sequencing to delineate the molecular signatures of the various cell types. A total of 10 cell types were identified: immune (B, T, monocyte macrophages, and plasma cells), endothelial, epithelial, stromal, neuroendocrine, serous, and protoporphyrin IX pigment cells. The trends of cell type distributions were consistent in two groups. Functional enrichment analysis showed that unique biological processes, such as IgA production and Toll-like C-type lectin receptor signaling pathways, were enriched in immune cells, whereas TGF-beta, Wnt, and NOD-like receptor signaling pathways were enriched in epithelial cells. Subsequently, we identified differentially expressed genes in the two groups, among which SPP1 (secreted phosphoprotein 1) was down-regulated in all cell types in the speckled group. The cellular interaction signals of speckled group were significantly enhanced compared to the control group. The cell proliferation and differentiation pathways mediated by WNT, SPP1, and BMP were inhibited and the interaction related to cell migration was activated in the speckled group. This study explored cell heterogeneity, extended prior findings by adding single-cell resolution atlas, and identified cell type specific patterns that can guide future functional studies of speckled eggs.