To investigate the potential protective effect of prior cold stimulation on broiler intestine induced by acute cold stress (ACS). A total of 384 one-day-old broilers were divided into control (CON), ACS, cold stimulation Ⅰ (CS3+ACS), and cold stimulation Ⅱ (CS9+ACS) groups. Broilers in CON and ACS groups were reared normally, and birds in CS3+ACS and CS9+ACS groups were reared at 3℃ and 9℃ below CON group for 5 h, respectively, on alternate days from d 15 to 35. Broilers in ACS, CS3+ACS, and CS9+ACS groups were subjected to 10℃ for 24 h on d 43. Eventually, small intestine tissues were collected for histopathological observation and indexes detection. The results showed that intestinal tissues in all ACS-broilers exhibited inflammatory cell infiltrates, microvilli disruption, reduced villus length in jejunum and increased crypt depth in jejunum and ileum. Whereas these phenomena were relatively light in CS3+ACS group. Compared to CON group, mRNA expression of the TLR4/MyD88/NF-κB pathway-related genes (TLR4, MyD88, NF-κBp65, COX-2, iNOS, PTGEs, TNF-α), Th1/Th17-derived cytokines (IL-1β, IL-2, IL-8, IL-12, IFN-γ, IL-17), and HSPs (HSP40, HSP60, HSP70, HSP90) was upregulated (P < 0.05), and that of Th2-deviated cytokines (IL-4, IL-6, IL-10, IL-13) and IκBα was downregulated (P < 0.05) in small intestine in almost all ACS-broilers. Compared to ACS group, mRNA expression of most of the TLR4/MyD88/NF-κB pathway-related genes, Th1/Th17-derived cytokines, and HSPs was downregulated and that of Th2-derived cytokines was upregulated in CS3+ACS group (P < 0.05). Protein expression levels of TLR4, MyD88, p-p65/p65, p-IκBα/IκBα, IKK, TNF-α, IL-1β, IL-10, and HSPs were similar to their mRNA expression. The concentration of sIgA and activities of CAT, SOD, and GSH-px were decreased and MDA and H₂O₂ were increased in ACS and CS9+ACS groups compared to CON group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to gut immune dysfunction; while mild cold stimulation at 3℃ below normal rearing temperature alleviated cold stress-induced intestinal injure and dysfunction by modulating the TLR4/MyD88/NF-κB pathway in broilers.

Keel bone damage negatively affects the welfare, production performance, egg quality, and mobility of laying hens. This study aimed to investigate whether abnormal bone metabolism causes keel bone damage in laying hens. Eighty Hy-line Brown laying hens were housed in eight furnished cages with 10 birds per cage and studied from 18 to 29 weeks of age (WOA). Accordingly, keel bone status was assessed at 18, 22, 25, and 29 WOA using the X-ray method, and the serum samples of laying hens with normal keel (NK), deviated keel (DK), and fractured keel (FK) that occurred at 29 WOA were collected across all the time-points. Subsequently, the serum samples were used to measure markers related to the metabolism of Ca and P and activities of osteoblast and osteoclast. The results showed that FK laying hens had lighter bodyweight than NK and DK birds throughout the trial (p < 0.05), while the keel bone length and weight were not different in NK, DK, and FK hens at 29 WOA (p > 0.05). Moreover, bone hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining indicated that damaged keel bone had evident pathological changes. In the FK hens, serum P level was reduced but serum 1,25-dihydroxy-vitamin D₃ (1,25-(OH)₂D₃) and 25-hydroxyvitamin D₃ (25-OHD₃) levels were elevated compared to NK hens (p < 0.05). Additionally, DK hens had higher levels of serum 1,25-(OH)₂D₃, parathyroid hormone (PTH) and calcitonin (CT), and lower level of serum 25-OHD₃ than the NK birds (p < 0.05). Furthermore, serum alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), TRAP, and corticosterone (CORT) levels were elevated in DK and FK hens compared to NK hens (p < 0.05). The levels of serum Ca, P, PTH, ALP, TRAP, OPG, OC, and CORT in laying hens fluctuated with the age of the birds. Generally, the results of this study indicate that keel bone damage, especially fractures, could be associated with abnormal bone metabolism in laying hens.

Keel bone fractures affect welfare, health, and production performance in laying hens. A total of one hundred and twenty 35-wk-old Hy-line Brown laying hens with normal keel (NK) bone were housed in furnished cages and studied for ten weeks to investigate the underlying mechanism of keel bone fractures. At 45 wk of age, the keel bone state of birds was assessed by palpation and X-ray, and laying hens were recognized as NK and fractured keel (FK) birds according to the presence or absence of fractures in keel bone. The serum samples of 10 NK and 10 FK birds were collected to determine bone metabolism-related indexes and slaughtered to collect keel bones for RNA-sequencing (RNA-seq), Micro-CT, and histopathological staining analyses. The results showed that the concentrations of Ca, phosphorus, calcitonin, 25-hydroxyvitamin D₃, and osteocalcin and activities of alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP) in serum samples of FK birds were lower than those of NK birds (P < 0.05), but the concentrations of parathyroid hormone, osteoprotegerin, and corticosterone in serum samples of FK birds were higher than those of NK birds (P < 0.05). TRAP staining displayed that FK bone increased the number of osteoclasts (P < 0.05). Micro-CT analysis indicated that FK bone decreased bone mineral density (P < 0.05). Transcriptome sequencing analysis of NK and FK bones identified 214 differentially expressed genes (DEGs) (|log2FoldChange| > 1, P < 0.05), among which 88 were upregulated and 126 downregulated. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis indicated that 14 DEGs related to skeletal muscle movement and bone Ca transport (COL6A1, COL6A2, COL6A3, PDGFA, MYLK2, EGF, CAV3, ADRA1D, BDKRB1, CACNA1S, TNN, TNNC1, TNNC2, and RYR3) were enriched in focal adhesion and Ca signaling pathway, regulating bone quality. This study suggests that abnormal bone metabolism related to keel bone fractures is possibly responded to fracture healing in laying hens.

Environmental enrichment can improve animal welfare. As a method of environmental enrichment, the effect of different auditory stimulations on the behavior response and welfare of laying hen chicks has yet to be investigated. Therefore, this study was aimed at exploring the impact of various auditory exposures on the behavior, learning ability, and fear response of 4-week-old laying hen chicks. A total of 600 1-day-old chicks were randomly assigned to five different groups: C (control group), LM (Mozart’s String Quartets, 65 to 75 dB), LN (recorded ventilation fans and machinery, 65 to 75 dB), HN (recorded ventilation fans and machinery, 85 to 95 dB), and HM (Mozart’s String Quartets, 85 to 95 dB). The experiment was conducted from day 1 until the end of the experiment on day 28. Groups LM and LN were exposed to music and noise stimulation ranging from 65 to 75 dB. Groups HN and HM, meanwhile, received noise and music stimulation ranging from 85 to 95 dB. The control group (C) did not receive any additional auditory stimuli. During the experimental period, continuous behavioral recordings were made of each group of chicks from day 22 to day 28. On day 21, the PAL (one-trial passive avoidance learning) task was conducted. On days 23 and 24, OF (open field) and TI (tonic immobility) tests were performed, and the levels of serum CORT (corticosterone) and DA (dopamine) were measured. The results indicated that exposure to music and noise at intensities ranging from 85 to 95 dB could reduce comforting, preening, PAL avoidance rate, the total number of steps and grid crossings of OF, and the concentration of DA in 4 WOA chicks (p < 0.05), increase the freezing times of OF (p < 0.05); 65 to 75 dB of noise stimulation could reduce preening and total number steps of OF in 4 WOA chicks (p < 0.05), increase the freezing times of OF (p < 0.05); and 65 to 75 dB of music exposure could reduce the concentration of CORT in 4 WOA chicks (p < 0.05). Therefore, 65 to 75 dB of music exposure could produce positive effects on chicks and showed relatively low CORT level, whereas 85 to 95 dB of music and noise exposure could reduce comforting and preening behavior, impair learning ability, and increase the fear responses of chicks.

Keel fracture has negative effects on the health and welfare of laying hens. We investigated effects of keel fracture on stress, inflammation, and the orexin system in laying hens. Ninety 17-week-old Lohmann white laying hens were palpated and euthanatized at 42 weeks old, and marked as normal keel (NK)/fractured keel (FK) from absence/presence of keel fracture. Serum, brain, liver, and abdominal-muscle samples were collected from 10 NK and 10 FK hens to determine the stress and inflammatory responses and the activity of orexin systems by corticosterone content, expression of heat shock proteins (TNF-α 60, 70, 90), and inflammatory factors (tumor necrosis factor (TNF)-α, nuclear factor-kappa Bp65 (NF-κBp65), inducible nitric oxide synthase (iNOS), prostaglandin E synthases (PTGEs), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β)), orexin (ORX), and orexin-receptor 1/2 (ORXR1/ORXR2). The FK hens had higher serum corticosterone content, Hsps, and inflammatory factor mRNA expression levels than NK hens, although levels of iNOS in the liver and TNF-α in the muscle were similar. Protein levels of Hsp70 and Hsp90 in the brain and liver, iNOS and COX-2 in the liver, NF-κBp65, iNOS, and COX-2 in the brain of FK hens were increased compared with NK hens. Furthermore, FK hens had lower mRNA expression of ORX, ORXR1, and ORXR2 than NK hens. Therefore, keel fracture causes stress and inflammation, and inhibits the expression of the orexin system in laying hens.