Intermittent cold stimulation affects energy metabolism and improves stress resistance in broiler heart.

To investigate the effect of intermittent cold stimulation on cardiac energy metabolism and cold resistance of broilers, 288 broilers were divided into 3 groups: control group (CC) and 2 cold stimulation groups (CS3 and CS9). The CS3 and CS9 groups received cold stimulation at temperatures of 3°C and 9°C lower than CC group for 5 h from d 15 to 35. Three groups were subjected to acute cold stress (ACS) of 10°C for 12 and 24 h at 44 d. Performance, cardiac histopathological changes, heat shock proteins (HSPs), and lipid metabolism levels were measured. Results showed that the performance was not different among groups at 22 and 29 d (P > 0.05), but the mRNA levels of Acyl CoA synthase long-chain family member 1 (ACSL1) and acyl-coenzyme oxidase (ACO) in CS group were upregulated compared to CC group (P < 0.05). At 36 d, the performance of the CS3 group was better than the other 2 groups, myocardial structure was normal and other lipid metabolism indexes, except for peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) levels, were similar to those of CC group (P > 0.05). The myocardial fiber disorder, Triglyceride (TG), and leptin (LEP) contents were significantly lower in CS9 group than in CC and CS3 groups at 36 d (P < 0.05). The HSP protein levels were significantly higher in CS group than in CC group before ACS (P < 0.05). After 24 h of ACS, the mRNA of lipid metabolism genes, the protein levels of HSP40 and HSP60, and the contents of TG and LEP in the CS3 group were upregulated compared to other groups. The CC and CS9 groups showed myocardial structure was destroyed, with lower TG and LEP levels compared to before ACS (P < 0.05). Therefore, cold stimulation at 3°C lower than the normal feeding temperature for 5 h did not impair performance but can increase the resistance of broilers to ACS by promoting lipid metabolism.

Cold stimulation causes oxidative stress, inflammatory response and apoptosis in broiler heart via regulating Nrf2/HO-1 and NF-κB pathway.

To investigate the effect of cold stimulation on heart, 300 1-day-old female broilers were divided into control (CON) and two cold stimulation (CS3 and CS9) groups. Birds in CON group were reared in normal ambient temperature during day 1-43; while birds in CS3 and CS9 groups were reared at 3 °C and 9 °C below CON group for 5 h at 1-day intervals from day 15 to day 35, respectively. Heart tissues were collected at day 22, 29, 36, and 43 to determine the indexes related to oxidative stress, inflammation and apoptosis. The H&E staining displayed that inflammatory cell infiltration and myocardial fiber break were obviously observed in CS9 group, and cardiac pathological score in CS9 group was higher than CON and CS3 groups (P < 0.05) at day 22, 36, and 43. Overall, compared to CON group, the concentrations of MDA and H2O2 were elevated, the activities of SOD, CAT, GPx, and T-AOC were reduced, and mRNA expression of CAT, GPx, SOD, nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) was downregulated in CS9 group at each time-point (P < 0.05). Compared to CON group, mRNA expression of NF-κBp65, COX-2, iNOS, PTGEs, TNF-α, and IL-1ß, and mRNA and protein expression of Bax, Bak, Cyt-c, caspase-3, and caspase-9 were increased, while Bcl-2 and Bcl-2/Bax ratio were decreased in CS9 group (P < 0.05) at the most detected time-points. There were no significant differences in the levels of indexes associated with oxidative stress, Nrf2/HO-1 antioxidant system, inflammation, and apoptosis between CON and CS3 groups at the most detected time-points (P > 0.05). Therefore, this study suggests that severe cold stimulation at 9 °C below normal rearing temperature induces cardiomyocyte inflammation and apoptosis by regulating Nrf2/HO-1 pathway-related oxidative stress in broilers, and mild cold stimulation of CS3 group can improve the adaptability of hearts to cold environment.

Appropriate cold stimulation changes energy distribution to improve stress resistance in broilers.

Appropriate cold stimulation can improve stress resistance in broilers and alleviate the adverse impacts of a cold environment. To investigate the effects of intermittent mild cold stimulation (IMCS) on energy distribution in the livers of broilers, 96 healthy 1-d-old Ross-308 male broilers were randomly divided into the control group (CC) and the cold stimulation group (H5). The CC group was raised at a normal thermal temperature, i.e., 35 °C until 3 d, after which the temperature was dropped gradually by 0.5 °C/d until 20 °C at 33 d. This temperature was maintained until 49 d. The H5 group was raised at the same temperature as the CC group until 14 d (35 to 29.5 °C) and at 3 °C below the temperature of the CC group starting at 0930 hours for 5 h every other day from 15 to 35 d (26 to 17°C). The temperature was returned to 20 °C at 36 d and maintained until 49 d. At 50 d, all broilers were subjected to acute cold stress (ACS) at 10 °C for 6 and 12 h. We found that IMCS had positive effects on production performance. Using transcriptome sequencing of the broiler livers, 327 differentially expressed genes (DEG) were identified, and highly enriched in fatty acid biosynthesis, fatty acid degradation, and the pyruvate metabolism pathway. When compared to the CC group, the mRNA levels of ACAA1, ACAT2, ACSL1, CPT1A, LDHB, and PCK1 in the H5 group were increased at 22 d (P < 0.05). The LDHB mRNA level was upregulated in the H5 group at 29 d compared to the CC group (P < 0.05). After 21 d of IMCS (at 36 d), the mRNA expression levels of ACAT2 and PCK1 were found to be significantly increased in the H5 group compared to the CC group (P < 0.05). Seven days after the IMCS had ended (at 43 d), the mRNA levels of ACAA1, ACAT2, and LDHB in the H5 group were higher than in the CC group (P < 0.05). The mRNA levels of heat shock protein (HSP) 70, HSP90, and HSP110 in the H5 group were higher than in the CC group after 6 h of ACS (P < 0.05). The protein levels of HSP70 and HSP90 in the H5 group were downregulated after 12 h of ACS, compared to the CC group (P < 0.05). These results indicated that IMCS at 3 °C lower than the normal temperature could improve energy metabolism and stress resistance in the livers of broilers, alleviate the damage of short-term ACS on broilers, help broilers adapt to the low temperature, and maintain stable of energy metabolism in the body.

Cold stimulation has a great impact on broilers. Excessive cold stimulation can lead to damage, while intermittent mild cold stimulation (IMCS) can make broilers adapt to the cold environment. Low temperature will make the body produce lots of heat to maintain metabolic stability. The liver controls the energy metabolism, and the avian livers are the main organ regulating lipid metabolism. In this study, the broilers of different ages were subjected to cold stimulation training and then acute cold stress (ACS). We found that IMCS had positive effects on production performance. Through transcriptome sequencing, we found that the differentially expressed genes were highly enriched in the energy metabolism pathway, and the expression levels of the most key genes and heat shock proteins were upregulated. The stress resistance was also enhanced, which could alleviate the damage of short-term ACS to the body. The broilers gradually adapted to the low-temperature environment and finally established cold adaptation. The findings of this work will be helpful to the development of animal husbandry in cold regions and improve animal welfare.

Effect of intermittent mild cold stimulation on intestinal immune function and the anti-stress ability of broilers.

A total of 240 healthy 1-day-old Ross 308 male broilers were randomly divided into 3 groups (CS0 group, CS3 group, and CS6 group), with 5 replicates in each group and 16 broilers in each replicate, in order to evaluate the effects of intermittent mild cold stimulation (IMCS) on the intestinal immune function and anti-cold stress ability of broilers after acute cold stress. The mRNA expression levels of cytokines and Toll-like receptors (TLRs) in the duodenum and jejunum were detected at the end of cold stimulation (36 d), 2 wk after recovery (50 d), and after acute cold stress (Y6). In addition, the mRNA and protein expression levels of heat shock proteins (HSPs) were measured before and after acute cold stress. The experimental data were statistically processed using 1-way ANOVA and Duncan's multiple comparisons. The results showed that the mRNA expression levels of IL2, IL8, IFN γ, TLR7, and TLR21 in the duodenum and IL2 and IFN Î³ in jejunum were significantly higher in the CS6 group than in the CS0 and CS3 groups at 36 d (P < 0.05). All TLR levels in the jejunum were significantly lower in the CS3 group than in the CS0 and CS6 groups at 36 d (P < 0.05). After 6 h of acute cold stress, in the duodenum, the mRNA expression levels of IL6 and IL8 were significantly decreased in the CS0 and CS6 groups compared to levels at 50 d (P < 0.05), while levels in the CS3 group remained stable (P > 0.05). Compared with 50 d, the expression level of HSP mRNA in the jejunum in the CS3 group was relatively stable compared to that in the CS0 and CS6 groups after acute cold stress (P > 0.05). At the protein level, the HSP60 expression level in the duodenum and HSP40, HSP60, and HSP70 expression levels in the jejunum were significantly higher in the CS3 group than in the CS0 and CS6 groups after acute cold stress (P < 0.05). In conclusion, cold stimulation training at 3℃/3 h lower than the conventional feeding temperature can improve the intestinal immune function and anti-stress ability of broilers.

Effects of Acute Cold Stress after Intermittent Cold Stimulation on Immune-Related Molecules, Intestinal Barrier Genes, and Heat Shock Proteins in Broiler Ileum.

Cold stress will have a negative impact on animal welfare and health. In order to explore the effect of intermittent cold stimulation training on the cold resistance of broilers. Immune-related and intestinal barrier genes were detected before and after acute cold stress (ACS), aiming to find an optimal cold stimulation training method. A total of 240 1-day-old Ross broilers (Gallus) were divided into three groups (G1, G2, and G3), each with 5 replicates (16 chickens each replicate). The broilers of G1 were raised at normal temperature, while the broilers of G2 and G3 were treated with cold stimulation at 3 °C lower than the G1 for 3 h and 6 h from 15 to 35 d, respectively, at one-day intervals. At 50 d, the ambient temperature for all groups was reduced to 10 °C for six hours. The results demonstrated that before ACS, IL6, IL17, TLR21, and HSP40 mRNA levels in G3 were apparently down-regulated (p < 0.05), while IL8 and Claudin-1 mRNA levels were significantly up-regulated compared with G1 (p < 0.05). After ACS, IL2, IL6, and IL8 expression levels in G3 were lower than those in G2 (p < 0.05). Compared to G2, Claudin-1, HSP90 mRNA levels, HSP40, and HSP70 protein levels were increased in G3 (p < 0.05). The mRNA levels of TLR5, Mucin2, and Claudin-1 in G2 and IL6, IL8, and TLR4 in G3 were down-regulated after ACS, while IL2, IL6, and IL17 mRNA levels in G2 and HSP40 protein levels in G3 were up-regulated after ACS (p < 0.05). Comprehensive investigation shows that cold stimulation at 3 °C lower than the normal feeding temperature for six hours at one day intervals can enhanced immune function and maintain the stability of intestinal barrier function to lessen the adverse effects on ACS in broilers.

Ameliorative effects of dietary selenium against cadmium toxicity on production performance and egg quality in laying hens.

In order to reveal the influences of supplemented dietary selenium (Se) on the suppressive effect of cadmium (Cd) toxicity on performance and egg properties of laying hens, the effects of co-treatment Se and Cd on the performance, egg quality, levels of amino acids and the antioxidant capacity of egg and serum were investigated. A total of 128 31-week-old laying hens were randomly distributed in four treatments, which were fed with the basic diet (0.2 mg/kg Se and 0.08 mg/kg Cd), and the basic diet with Se (1.1 mg/kg Se and 0.08 mg/kg Cd), Cd (0.2 mg/kg Se and 92.1 mg/kg Cd) and Se+Cd for 13 weeks, respectively. Hens supplemented with Cd led to an impairment on production performance and egg quality with decreased egg production (EP), egg mass (EM), feed intake (FI), eggshell color, eggshell thickness, yolk color, albumen height and haugh unit and increased the feed conversion ratio (FCR) (p < 0.05). Cd treatment decreased the contents of cysteine (Cys), histidine (His), lithium (Li), aluminum (Al), chromium (Cr), manganese (Mn), nickel (Ni), zinc (Zn), arsenic (As), Se, strontium (Sr), stannum (Sn), mercury (Hg) and thallium (Tl) and increased the contents of isoleucine (Ile) and Cd (p < 0.05). Cd destroyed the egg yolk and serum redox states with the increased concentration of malondialdehyde (MDA) and the decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) (p < 0.05). The expression levels of ovarian apoptotic genes (protein 53, Caspase9, Cytochrome c and Bcl-2 associated X protein) increased, and B-cell lymphoma 2 (Bcl-2) expression decreased in the Cd group (p < 0.05). Feeding Se significantly alleviated Cd-induced toxicity on performance and egg quality. Se+Cd treatment restored the balance between oxidation and antioxidant systems and modulated the elements' homeostasis and alleviated the changes in apoptotic-related genes expression levels. Se could alleviate the Cd toxicity to laying hens and their eggs but could not counteract all negative effects of Cd.