| Intestinal inflammation induced by heat stress is an important problem that restricts the health and production of broilers.Chicken embryo thermal manipulation is to increase the conventional incubation temperature in the critical period of development of chicken embryo to obtain postnatal heat resistance.In view of the importance of intestinal health to poultry production,this paper investigated the effect of chicken embryo thermal manipulation（16-18days of embryonic age,39.5℃,65%RH,3 h/d）on intestinal inflammation of postnatal heat-stressed broilers and explored its potential mechanism,to provide the technical support for green and healthy breeding of broilers.The results can provide theoretical basis for epigenetic mechanism of chicken embryo thermal manipulation.Experiment 1:Effects of chicken embryo thermal manipulation on heat tolerance and intestinal inflammation of postnatal heat-stressed broilers.A total of 234 Arbor Acres broiler embryos were randomly divided into 3 groups with 6 replicates per group and 13 embryos per replicate.The treatment groups were as follows:control group（Con）:no thermal manipulation during the embryonic stage（37.8℃,56%RH）,and no heat stress after birth（23±1℃,60%RH）;Heat stress group（HS）:no thermal manipulation in embryonic stage（37.8℃,56%RH）,and heat stress during postnatal d 22-42（35±1℃,60%RH,8 h/d）;Thermal manipulation group（TM）:thermal manipulation（39.5℃,65%RH,3 h/d）during embryonic d 16-18,and heat stress during postnatal d 22-42（35±1℃,60%RH,8 h/d）.Six healthy broilers were randomly selected from each group and sampled after 10 h of fasting,at day 1（d 1）and the beginning（d 22）,middle（d 31）and end（d 42）of heat stress.In terms of production performance and heat resistance,the results showed that embryo thermal manipulation did not affect the hatchability（P>0.05）;the brooding time was advanced by 8 h earlier;the performance of postnatal heat-stressed broilers was improved（P<0.05）;the increase of rectal temperature in heat-stressed broilers was delayed（P<0.05）.In terms of intestinal mechanical barrier,embryo thermal manipulation improved intestinal morphological parameters of heat-stressed broilers（P<0.05）,and alleviated the decreased m RNAexpression of tight junction protein-related genes induced by heat stress,only in jejunum（P<0.05）.In terms of intestinal microbial barrier,at the phylum level,embryo thermal manipulation alleviated the increase in the abundance of Bacteroidetes induced by heat stress and the decrease in the ratio of Firmicutes/Bacteroidetes（P<0.05）.At the genus level,embryo thermal manipulation mitigated the increase of Bacteroides abundance and the decrease of Oscillospira abundance（P<0.05）.Beta diversity analysis showed that the structure of microflora was changed by thermal manipulation.LEf Se analysis showed that the characteristic microorganisms of TM group were mainly beneficial bacteria such as Akkermansia,while those of HS group were mostly conditioned pathogenic bacteria.In terms of the intestinal inflammation,embryo thermal manipulation decreased serum LPS content of heat-stressed broilers（P<0.05）,alleviated the increased m RNA expression of proinflammatory cytokines（IL-1β,IL-6,TNF-α）in duodenum and jejunum of broilers under heat stress（P<0.05）.In the jejunum with the most obvious anti-inflammatory effect,embryo thermal manipulation alleviated the activation of NF-κB signaling pathway in heat-stressed broilers（P<0.05）.In terms of regulatory mechanism,m RNA expression of six TRP ion channels（TRPV1-4,TRPA1,TRPM8）in intestine of heat-stressed broilers was detected,but only three TRP ion channels（TRPV1,TRPV4,TRPA1）m RNA could be detected.The results showed that embryo thermal manipulation could alleviate the increase of TRPV4 m RNA and protein expression in jejunum induced by heat stress（P<0.05）,and also increased DNA methyltransferase m RNA expression（DNMT1,DNMT3A）in jejunum of heat-stressed broilers（P<0.05）.DNA methylation levels in the TRPV4 promoter region of jejunum were further detected.Among the16 Cp G units detected,the methylation level of one Cp G unit in HS group was significantly lower than that in Con group（P<0.05）,4 Cp G units in the TM group had significantly higher methylation levels than those in the HS group（P<0.05）,and 3 Cp G units were significantly higher than those in Con group and HS group（P<0.05）.These results indicate that chicken embryo thermal manipulation can improve heat resistance,improve intestinal barrier function and relieve intestinal inflammation of heat-stressed broilers,especially in jejunum.DNA methylation-mediated TRPV4 may be a key target for thermal manipulation relieving intestinal inflammation in postnatal heat-stressed broilers.Experiment 2:Validation of TRPV4 in an inflammatory model of intestinal epithelial cells.The LPS-induced inflammation model was constructed by primary cultured chicken intestinal epithelial cells,and the effect of TRPV4 was further verified by GSK2193874,a specific inhibitor of TRPV4.Cells were divided into 4 treatment groups with 6 replicates per group:control group（Con）;100 ng/m L LPS treatment（LPS）;100 n M GSK2193874 treatment（GSK）;15 min of 100 n M GSK2193874 pretreatment followed by adding 100 ng/m L LPS treatment（LPS+GSK）.Cells were harvested 3 h after treatment.The results showed that after TRPV4inhibition,the LPS-induced Ca2+influx was significantly decreased（P<0.05）,the activation degree of NF-κB was significantly decreased（P<0.05）,and the m RNA expression of proinflammatory cytokines（IL-1β,IL-6）was significantly decreased（P<0.05）.The results show that TRPV4 is a switch to control intestinal inflammatory response,and inhibition of TRPV4 can alleviate intestinal inflammatory response through NF-κB signaling pathway.In conclusion,chicken embryo thermal manipulation can relieve intestinal inflammation induced by postnatal heat stress,especially in jejunum.This may be due to the increased abundance of beneficial anti-inflammatory microorganisms and increased DNA methylation in the TRPV4 promoter region,which reduces TRPV4 expression and thereby reduces Ca2+influx,ultimately reducing inflammation by affecting the NF-κB signaling pathway. |
