Automatically identifying abnormal behaviors of caged laying hens in a thermal environment improves manual management efficiency. It also provides reference indicators for breeding heat-tolerant hens. In this study, we propose a deep learning-based method for automatic recognition and evaluation of typical heat stress behaviors in hens. We developed a lightweight object detection algorithm, YOLO-HGP, based on the YOLOv8n as the baseline model. YOLO-HGP achieves Precision (P), Recall (R), and mean average precision (mAP) of 95.952%, 94.127%, and 97.667%, respectively, effectively detecting typical heat stress behaviors in hens. Compared to the original YOLO v8n, YOLO-HGP improves R, and mAP by 6.257%, and 1.963%, respectively. The FLOPs (floating point operations) and parameter count of YOLO-HGP are 4.3G and 1.729M, reducing by 47.56% and 42.58% compared to the original model. Additionally, we introduce the "ORC-ratio" (The ratio of the combined frequency of open-beak breathing and retching behaviors to the frequency of closed-beak behaviors.) as an evaluation indicator for the frequency of typical heat stress behaviors in hens and combine it with the Hybrid-SORT multiobject tracking algorithm to achieve tracking detection of individual hens. The study demonstrates that the proposed model effectively identifies and quantitatively evaluates typical behaviors of hens in a thermal environment, providing an effective approach for the automated recognition of heat stress behaviors in hens.

Excessive aromatase can reduce reproductive performance in aged roosters. Aromatase inhibitors (AI) can inhibit the aromatase activity and improve the semen quality of aged roosters. However, relevant molecular mechanism is still unclear. The purpose of this study was to explore the regulatory mechanism of AI letrozole improving semen quality in aged roosters by transcriptomic and proteomic sequencing. In this study, 56-week-old roosters were reared in separate cages on a standard basice diet and oral letrozole 42 days (D) at a daily dose 0.25 mg/kg. Semen quality and serum hormone were measured before (0 D) and after (42 D) letrozole administration. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected, respectively. The results indicated that semen volume, sperm motility, sperm density, MMP, testosterone (T) and gonadotropin releasing hormone (GnRH) in letrozole treatment group (LET) were significantly increased than those in control group (CN) (P<0.05); estradiol (E₂) and ROS in LET were significantly lower than those in CN (P<0.05). Through transcriptomic and proteomic analysis, we identified 189 differently expressed genes (DEGs) and 64 differentially expressed proteins (DEPs) in the comparison of LET and CN. DEGs and DEPs Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) items are mainly enriched in steroid biosynthetic process, cell differentiation and proliferation, lipid metabolic process, oxidation-reduction process and electron transfer activity. Furthermore, 8 genes including STAR, CYP17A1, NSDHL, SULT1E1, EHF, NRNPA1, PLIN2 and SDHA were identified as key genes for letrozole to regulate semen quality in aged roosters. These results indicate that letrozole can up-regulate the expression of genes related to steroid hormone synthesis, cell differentiation and proliferation, electron transfer activity, and enhance mitochondrial activity, increase testicular weight, and ultimately improve the semen quality of aged roosters.

The egg-laying performance is closely related to ovulation of chicken. Ovulation is very complex and highly coordinated physiological process. However, little is known about the ovulation-related synergistic actions of molecules and their signaling. Here, we performed a comparative transcriptome analysis based on preovulatory, ovulatory, and post-ovulatory follicle data of thirty-week-old Lohmann Pink laying hens. Gene set enrichment analysis (GSEA) indicated that the ovulating follicles exhibited the significant changes in plasma membrane structure and cell morphology, and the immune response mediators and T-cells were activated in response to ovulation, and meanwhile multiple biological processes (such as oxidative phosphorylation and steroidogenesis) and biological functions of ribosomes and lysosomes were inhibited. The analyses of our ovulation RNA-seq data and previously reported forkhead box L2 (FOXL2) ChIP-seq and FOXL2 RNA-seq data revealed that FOXL2 gene might directly regulate extracellular matrix (ECM) structure-related genes, thus modulating the expression levels of genes related to DNA replication and cell cycle, ultimately affecting ovulation. FOXL2 was involved in the regulation of DNA replication and cell cycle at 22 h before ovulation, oocyte meiosis and actin cytoskeleton regulation at 3 h before ovulation, and ECM structure change and cell senescence after ovulation. Overall, this study reveals regulatory mechanism of FOXL2 on chicken ovulation at transcriptomic level. Our findings lay a foundation for further identifying key nodes regulating ovulation.

Heat stress is a major constraint to productivity and physiological homeostasis in laying hens. This study investigated integrated responses to acute heat stress using a multi-omics approach, including performance traits, serum biochemical parameters, histology, hepatic transcriptomics, cecal metagenomics, and metabolomics. Acute heat stress impaired productive performance, as reflected by changes in egg production and reduced eggshell strength, and induced systemic physiological disturbances, including increased stress- and injury-related blood indicators and disrupted metabolic and electrolyte balance. Histological analysis confirmed liver and intestinal tissue damage. Hepatic transcriptomics revealed inflammatory activation and suppression of metabolic pathways, particularly those involved in lipid metabolism, energy production, and redox homeostasis. Cecal metagenomic and metabolomic analyses showed altered microbial composition and functional potential, along with disruptions in amino acid, lipid, and energy metabolism. Collectively, these findings suggest that acute heat stress is associated with coordinated inflammatory responses and metabolic reprogramming, together with liver and intestinal injury and gut microbiota–metabolite alterations. The study provides a framework for understanding early heat stress responses and highlights potential targets for nutritional and microbiota-based interventions in poultry production. Importantly, serum biochemical indicators such as D-lactic acid and aspartate aminotransferase may serve as potential early biomarkers for monitoring heat-stress-induced physiological disturbances.

Heat stress (HS) represents a significant challenge in poultry production, impairing thermoregulation, intestinal function, and productive performance. This study utilized acute (6 h) and chronic (14 d) HS models at 36°C in laying hens to characterize stage-dependent responses and evaluate the protective effects of a ten-ingredient traditional Chinese medicine (TCM) formulation. Both acute and chronic HS significantly increased rectal temperature and respiratory rate. Egg production declined by approximately 18% following acute HS and was further compromised under chronic exposure, along with reduced eggshell strength and weight. Dietary TCM supplementation (0.5%) alleviated physiological stress and partially restored laying performance, with more pronounced recovery observed under chronic HS. Serum analysis and histopathology indicated that TCM attenuated HS-induced impairment of ileal barrier function. Metabolomic profiling revealed stage-dependent responses: acute HS primarily disturbed redox balance, whereas chronic HS induced broader remodeling related to energy and nutrient utilization. TCM supplementation modulated metabolic functions to support immediate stress buffering under acute HS while stabilizing long-term energy support and intestinal capacity under chronic HS. Metagenomic analysis indicated that TCM selectively promoted microbial groups related to intestinal metabolism and nutrient utilization, aligning with metabolomic findings. Correlation analyses linked these TCM-associated microbial and metabolic signatures with improved thermoregulatory responses, oxidative status, and intestinal barrier indicators. Collectively, these results demonstrate that TCM supplementation enhances heat resilience in laying hens through stage-dependent modulation of the gut microbiota-metabolome axis, supporting its application as a nutritional strategy to maintain productivity under thermal challenge.

Heat stress is a major systemic challenge in poultry, but the role of circulating extracellular vesicles (EVs) in liver-directed adaptation remains unclear. This study investigated whether plasma-derived EVs from heat-stressed chickens (HS_EV) mediate hepatoprotective responses under thermal stress. EVs were isolated from the plasma of control and heat-stressed chickens and characterized by morphology, size distribution, and marker expression. Their biodistribution in vivo and uptake by primary hepatocytes in vitro were also evaluated. Hepatocyte injury was induced by heat exposure, and the effects of HS_EV on proliferation, apoptosis, inflammatory cytokine production, transcriptomic reprogramming, and MYD88/NF-κB/MAPK signaling were assessed. Heat stress induced systemic injury in chickens, increased the release of plasma-derived EVs, and promoted their preferential accumulation in the liver. Whole-transcriptome analysis further showed that HS_EV enhanced glutathione metabolism and related metabolic pathways while suppressing apoptosis- and inflammation-related signaling. In primary hepatocytes, HS_EV, but not control EVs, restored proliferative capacity, reduced apoptosis, suppressed the expression and secretion of IL-1β, IL-6, and TNF-α under heat stress, and was associated with attenuation of the MYD88/NF-κB/MAPK axis. These findings suggest that circulating EVs participate in adaptive intercellular communication during heat stress and identify HS_EV as a potential endogenous mediator of hepatoprotection in chickens.