Changes In Uterus,Intestine And Gut Microbiota Are Involved In Production Of Sandpaper-shelled Eggs(SE) And Probiotics-Induced Reduction Of The SE Rate At The Late Phase Of Laying Cycle In The Chicken

In laying hens,eggshell is formed in uterus,and calcium ion is its main component.At the late phase of a laying cycle,eggshell quality is often decreased markedly.For example,the rate of the sandpaper-shelled eggs(SE),a category of eggs with low eggshell quality,is significantly increased.SE is characterized by the presence of many small sandlike particles on the surface,and is usually thin and easy to break,thus causes a great loss to poultry industry.However,the mechanism by which SE is formed remains unclear.Moreover,although dietary probiotics can improve egg production and eggshell quality in chickens,it is unknown that if gut bacteria contribute to SE production and if dietary probiotics can reduce the SE rate.To address these questions,this study carried out the following experiments and analyses:1.Hy-line hens were choosing as an experimental animal because this strain is one of main strains for egg production in China.Twenty hens including normal eggs(NE)-and SEproducing hens were selected from a Hy-line population of 450-d-old hens based on 2-wklong observation,and uterus samples was then collected from these hens for histomorphological and transcriptome analyses after their euthanasia.The differentially expressed genes(DEGs)were subsequently identified.The expression of some of the DEGs was validated by real-time quantitative PCR.These DEGs were also used for gene ontology(GO)and KEGG pathway analysis.Data indicated that,compared with NE group,the uteruses of laying hens in SE group appeared to be in the process of atrophy.Moreover,a total of 211 DEGs were identified.These DEGs were clustered into 145 GO terms(FDR<0.05)and enriched in 12 KEGG pathways(P<0.10),which are mainly related to organ morphogenesis and development,cell growth,differentiation and death,signal transduction,ion transport,endocrine and cell communication,immune response,metabolism,and corticotropin-releasing hormone.It is noteworthy that corticotropin may be important to SE formation as the hormones are related to some of these biological processes.In addition,the downregulation of genes related to ion transport and matrix proteins also contribute to the SE production through their role in macromolecular assembly of eggshell.Together,these findings revealed that there were the structural and functional differences in the uterus between the NE and SE groups.2.To test if dietary probiotics can reduce the SE rate,1200 450-d-old Hyline hens were assigned into a control group and 3 treatment groups(n=300)with the control group fed basal diet and the treatment groups fed basal diet plus 500,1000,1500 mg/kg of probiotics(Clostridium butyricm and Bacillus subtilis),respectively.The treatment with 1000 mg/Kg of probiotics had the best performance.After 4 wk,supplementing probiotics significantly decreased the SE rate from 42.51%to 28.02%,increased the total laying rate from 82.9%to 84.4%,average egg mass from 52.3 to 53.6 g/hen/d and feed conversion ratio from 2.17 to 2.23.To test if probiotics reduced the rate of SE by improving intestinal structure and function,the hens only producing NE or SE based on two weeks of observation were selected from the test population.The SE-producing hens were then divided into 2 groups with one group called SE group(without probiotics)and another called SEP group(with probiotics).The hens in the NE and SE groups were fed basal diet,while those in the SEP group were fed basal diet supplemented with Clostridium butyricm and Bacillus subtilis(1000 mg/Kg).Each group had 10 hens.After 4 wk,8 birds from each group was selected for histomorphological analysis of intestinal tissues,and for quantitative PCR to determine the expression of relevant genes.Histomorphological analysis indicated that probiotics supplementation significantly increased the villus length and the ratio of villus length to crypt depth.Quantitative PCR analysis indicated that there were no significant differences in the expression of genes related to tight junction,nutrient transport or calcium absorption among the groups(except TRPV6 P<0.001).Together,these findings suggest that dietary supplementation reduces SE production and increases laying rate of aged hens potentially through modulating intestinal structure and function,however,the molecular mechanism underlying modulation of intestinal structure and function by probiotics need further research.3.To test if gut bacteria contribute to SE production,the ileal contents of 5 birds each group were collected,and the microbial composition in the contents was then determined by 16S rDNA sequencing analysis.Data showed significant differences in the gut microbiota between the NE,SE,and SEP groups.In the SEP group,the alpha diversity was the highest with the enriched genera of bacteria(Lactobacillus,Streptococcus,Gallicola,Facklamia,and Bacillus).Moreover,Firmicutes phylum was dominant in the NE and SEP groups,while Proteobacteria phylum was dominant in the SE group.However,it is noteworthy that the abundance of Lactobacillus in the SE group was lower than that in the NE and SEP group.In conclusion,deterioration of uterine structure and function as well as the change of gut microbiota may contribute to the SE production,and probiotics supplementation can significantly influence the composition of intestinal microbiota,which may lead to the reduction of SE rate in the late laying period of hens.