| In poultry industry,increasing age leads to gradual decline in laying performance and egg quality during late laying period.Poor egg-laying rate and low eggshell quality are the main consequences in the late laying period,which could cause tremendous economic losses to poultry industry.It is of great significance to explore feed additives with therapeutic potential for declined laying performance and egg quality,which is helpful for extending the laying period of aged breeder hens.In addition,the increase of breeder age could affect the innate immunity of chicken offspring.Nowadays,the use of antibiotics has been prohibited in China,which brings huge challenges to poultry industry.Due to the fact that the intestinal immune function is not fully developed at the early growth stage,the intestine is vulnerable to the invasion of exogenous pathogenic microorganisms,which could lead to the intestinal mucosal damage.Recent studies have shown that maternal nutrition intervention could effectively enhance the intestinal innate immunity of offspring.The manipulation of breeder hens’nutrition might improve the intestinal immune function of chickens in the early growth stage.Stevioside has been proven to exert a number of biological activities in mammals,including antioxidant,anti-inflammatory and immunoregulatory properties.However,data are lacking in elucidating the application effects of stevioside in the production of breeder hens.Therefore,in this study,we mainly investigated the effects and potential molecular mechanisms of dietary supplementation with stevioside on the production performance of aged breeder hens and intestinal immune function of their chicken offspring.1.Effects of dietary stevioside supplementation on the lipopolysaccharide（LPS）-induced intestinal mucosal damage of broiler chickensThe objective of experiment 1 was to investigate whether stevioside could exert its biological activities using the LPS-induced intestinal mucosal damage model.In experiment1,a total of 192 1-day-old male Ross 308 broiler chicks with similar hatching weight were randomly divided into 4 treatments:broiler chickens fed with a basal diet（CON）,broiler chickens fed with a basal diet supplemented with 250 mg/kg stevioside（STE）,broiler chickens fed with a basal diet+LPS-challenge（LPS）and broiler chickens fed with a basal diet supplemented with 250 mg/kg stevioside+LPS-challenge（LPS+STE）.LPS-challenged groups received an intraperitoneal injection of LPS at 17,19 and 21 d,whereas the CON and STE groups received saline injection.The results showed that:（1）Compared with the CON group,the ADFI and ADG of the STE group significantly increased,whereas the ADFI and ADG of the LPS group significantly decreased（P<0.05）.Compared with the LPS group,the ADFI of the LPS+STE group significantly increased（P<0.05）.（2）In both jejunal and ileum mucosa of broiler chickens,compared with the CON group,LPS challenge significantly increased the protein expression level of p-NF-κB and p-IκBα.LPS challenge also increased the expression level of inflammation-related genes（NF-κB and IL-1β）,and reduced the expression level of tight-junction related genes（OCLN）and antioxidant-related genes（SOD1,CAT and HO-1）（P<0.05）.Compared with the LPS group,the LPS+STE group exhibited decreased protein expression level of NF-κB,p-NF-κB and p-IκBα.LPS+STE group also exhibited decreased expression level of inflammation-related genes（TLR4,MYD88 and TNF-α）,and increased expression level of tight-junction related genes（ZO-1）and antioxidant-related genes（SOD2 and NRF2）in the jejunal mucosa（P<0.05）.（3）LPS challenge could cause obvious intestinal hyperemia and neutrophil infiltration.Dietary stevioside supplementation could alleviate increased serum diamine oxidase,intestinal morphological damage,enhanced intestinal apoptotic index,and reduced intestinal cell proliferation induced by LPS challenge.（4）Dietary stevioside supplementation also reduced the malondialdehyde content in both jejunal and ileum mucosa,enhanced the jejunal total antioxidant capacity and activities of jejunal catalase,jejunal glutathione peroxidase and ileal superoxide dismutase,resulting in the alleviation of LPS-induced oxidative stress injury in the intestinal mucosa.The above results showed that stevioside could exert its anti-inflammatory and antioxidant activities in the chicken body,dietary stevioside supplementation could alleviate LPS-induced intestinal mucosal damage in broiler chickens.The data of experiment 1 provided a theoretical basis for subsequent experiments on breeder hens.2.Effects of dietary stevioside supplementation on the laying performance and egg quality of aged breeder hensThe objective of experiment 2 was to investigate whether dietary stevioside supplementation could improve the lay performance and egg quality of aged breeder hens.In experiment 2,a total of 120 Jinmao breeder hens（54-week-old,average body weight 2402±57g）during the late laying period were randomly divided into 2 treatments:breeder hens fed with a basic corn-soybean diet（CON）and breeder hens fed with a basic corn-soybean diet supplemented with 0.25 g/kg stevioside（STE）.The experiment lasted for 5 weeks.The results showed that:（1）Dietary stevioside supplementation improved the daily egg production,daily egg mass,feed conversion rate,eggshell strength and eggshell thickness of aged breeder hens（P<0.05）.Dietary stevioside supplementation also reduced serum triglyceride and uric acid levels,and enhanced the antioxidant capacity of the ovary and shell gland（P<0.05）.（2）Dietary stevioside supplementation increased the serum estrogen level,and up-regulated the gene expression level of hypothalamic GNRH1,hypothalamic ERβand hypophyseal GNRHR（P<0.05）.Dietary stevioside supplementation could also enhanced the gene expression level of FSHR,CYP17A1,17β-HSD,CYP19A1 and ERβin the small yellow follicles（P<0.05）.（3）Dietary stevioside supplementation increased the calcium concentration in both serum and shell gland（P<0.05）.The activity of Ca2+-ATPase and gene expression level of calcium-binding-related genes（ERα,ERβ,CABP-28K,THBS2,CDH6and KCNA1）in the shell gland were increased after stevioside supplementation（P<0.05）.（4）Dietary stevioside supplementation significantly decreased the apoptotic index in the luminal epithelium and glandular epithelium of shell gland.It also increased the luminal epithelium height and glandular density,which lead to improved histomorphology of shell gland.The above results showed that dietary stevioside supplementation could improve the laying performance and eggshell quality through increasing estrogen synthesis,calcium level and antioxidant capacity of reproductive organs in aged breeder hens.3.Effects and potential mechanisms of maternal stevioside supplementation on the intestinal immune function of chicken offspringThe objective of experiment 3 was to investigate the effects of maternal stevioside supplementation on the intestinal immune function of chicken offspring,and to explore its potential molecular mechanisms.In experiment 3,a total of 200 breeding eggs of aged breeder hens from each group in experiment 2 were collected and incubated in the same incubator.The chicken offspring were randomly selected and divided into two treatments:the offspring of hens fed a basal diet（CON）and the offspring of hens fed a stevioside-supplemented diet（STE）.The chicken offspring during embryonic stage,one-day-old and28-day-old chicken offspring were involved in this study.The results showed that:（1）Maternal stevioside supplementation significantly increased the relative embryo weight,relative embryo length and length of tibia during the embryonic stage（P<0.05）.Maternal stevioside supplementation increased the villus height and villus height-to-crypt depth ratio of the jejunum,causing an improved intestinal integrity.Maternal stevioside supplementation could also increase the ratio of PCNA-positive cells in the jejunal crypt of newly-hatched chicken offspring（P<0.05）.（2）The data of RNA-sequencing showed that the transcriptional profiles in the jejunum of the STE group were obviously changed compared with the CON group.The data of q RT-PCR verified that maternal stevioside supplementation significantly increased the expression level of genes related to TLR4 signaling pathway（TLR4,TRIF and IRF7）,cytokine（IFN-α,IFN-γ,IL-4 and IL-18）and endotoxin tolerance（PECAM1,STAT1,SOCS1 and SOCS3）in the jejunum of chicken offspring（P<0.05）.At 28 days of age,the STE group exhibited higher jejunal s Ig A and serum interferons levels compared with the CON group（P<0.05）.（3）The data of 16S r RNA sequencing showed that maternal stevioside supplementation significantly increased the relative abundance of Lactobacillales,Staphylococcales,Peptostreptococcales-Tissierellales,Lachnospirales,Oscillospirales,Burkholderiales and Rhizobiales,whereas it reduced the abundance of Clostridiales in the meconium of newly-hatched chicken offspring（P<0.05）.At 28 days of age,the STE group had higher relative abundance of cecal Lactobacillales（P<0.05）.The relative abundance of Lactobacillales was positively correlated with intestinal immune-related factors.（4）The in ovo injection with high-dose steviol（the metabolite of stevioside in the body）resulted in severe embryonic mortality,whereas the in ovo injection with steviol had no effect on the embryonic development,hatching performance and immune-related gene expression level（P>0.05）.（5）The data of methylation-specific PCR and bisulfite sequencing PCR revealed that maternal stevioside supplementation could significantly down-regulate the overall methylation level of SOCS1 promoter in the jejunum of chicken offspring（P<0.05）.The above results showed that maternal stevioside supplementation could increase the intestinal immune function of chicken offspring.This intergenerational effect is highly related with the modulation of gut microbiota and down-regulation of SOCS1 promoter Cp G island in the jejunum of chicken offspring.4.Effects of maternal stevioside supplementation on the intestinal mucosal damage and gut microbiota of chicken offspring challenged with LPSThe objective of experiment 4 was to investigate whether maternal stevioside supplementation could alleviate intestinal mucosal damage and microbiota perturbation in the chick offspring challenged with LPS.In experiment 4,the chicken offspring in the experiment 3 were involved.After hatching,the male chicken offspring were randomly selected and divided into four treatments:the offspring of hens fed a basal diet（CON）,the offspring of hens fed a stevioside-supplemented diet（STE）,the CON group challenged with LPS（LPS）and the STE group challenged with LPS（LSTE）.LPS-challenged groups received an intraperitoneal injection of LPS at 24,26 and 28 d,whereas the CON and STE groups received saline injection.The results showed that:（1）LPS challenge significantly decreased the terminal body weight and the concentrations of serum triglyceride and glucose of chicken offspring（P<0.05）.（2）Maternal stevioside supplementation alleviated LPS-induced intestinal morphological damage,decreased number goblet cell,increased intestinal apoptotic index,and reduced intestinal cell proliferation induced by LPS challenge.（3）In both jejunal and ileal mucosa,LPS challenge significantly increased the expression level of inflammation-related genes（TLR4,IL-6,IFN-γand IL-1β）,and reduced the expression level of tight-junction related genes（OCLN and ZO-1）（P<0.05）.Compared with the LPS group,the LSTE group exhibited decreased TLR4 and increased MUC2 expression level in the jejunal mucosa（P<0.05）.（4）The data of 16S r RNA sequencing showed that maternal stevioside supplementation could prevent the impairment of bacterial diversity in LPS-challenged chicken offspring.The results of PCo A and UPGMA both showed that the cecal microbiota of the LPS group exhibited clear difference with the CON and LSTE groups.Compared with the LPS group,maternal stevioside supplementation markedly reduced the relative abundance of cecal Bacteroides（P<0.05）.Compared with the CON and LPS groups,the LSTE group exhibited a significantly higher abundance of Lactobacillus（P<0.05）.The above results showed that maternal stevioside supplementation could ameliorate intestinal mucosal damage and microbiota perturbation in the chick offspring challenged with LPS.In summary,stevioside could exert its biological activities in chickens.Dietary stevioside supplementation could improve the laying performance and eggshell quality through increasing estrogen synthesis,calcium level and antioxidant capacity of reproductive organs in aged breeder hens.Maternal stevioside supplementation could induce intergenerational effect,it could improve the intestinal immune function of chicken offspring potentially via modulating the gut microbiota and down-regulating the promoter methylation level of SOCS1.Maternal stevioside supplementation may be helpful for the prevention of intestinal mucosal damage in chickens at the early and middle growth stage. |
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