Effects Of Probiotics Supplementation In The Diet On Uterine Development And Eggshell Quality Of Tianfu Green-Shelled Laying Hens At Different Stages Of Egg Production

Antibiotics play an extremely important role in poultry farming,but their resistance is one of the most serious threats to human health,so research into alternatives to antibiotics has emerged.Probiotics are living microorganisms that not only do not result in drug resistance but also regulate micro-ecological balance,promote intestinal health,and boost immunity.In poultry farming,probiotics are favored by the farming industry as feed additives to improve the intestinal flora of hens and enhance egg production performance and egg quality.At the present,studies on the effect of probiotics on uterine development and eggshell quality in different laying stages of Tianfu green shell hens have not been reported.In this trial,360 4-week-old Tianfu green-shelled hens were randomly divided into a control group(fed the basal diet only)and a test group(the basal diet was supplemented with one-thousandth of the probiotic complex,the major components of the probiotic complex are Bacillus subtilis≥1×10~9 CFU/g,Lactobacillus≥2×10~7 CFU/g and Yeast≥1×10~7 CFU/g),with 6 replicates of 30 hens in each group.Probiotics were fed from 4 week to 66 week and then sampled at 20,30,and 66 weeks,respectively.The effects of exogenous probiotic addition on the uterine development and eggshell quality of Tianfu green-shelled hens were investigated b by measuring the mineral content of serum,egg quality,eggshell mechanical properties,mineral content and ultrastructure of the eggshell,uterine tissue morphology,intrauterine biomineralization-related gene expression and intrauterine microflora distribution in both groups.The main results are as follows:(1)The addition of probiotic to the diet improved shell strength,reduced egg weight and shell area,and had a significant difference at 30 week(P<0.05);Compared to the control group,the probiotics group increased albumen height,Haugh units,and eggshell fracture modules at all three periods,and a decreasing trend in eggshell thickness,which showed particularly significant at 30 and 66 weeks(P<0.05).However,there were no significant differences in egg shape index,yolk color,yolk weight,eggshell weight,eggshell ratio,and eggshell index.(2)The addition of probiotics to the diet reduced blood potassium levels,with significant differences at 66 week;Calcium and sodium levels were significantly lower at 20 and 30 weeks(P<0.05);However,the change of chloride level was not obvious difference(P>0.05).(3)The addition of probiotic to the diet significantly reduced the levels of potassium and calcium in the eggshell at 20 and 30 weeks(P<0.05);Sodium and magnesium levels significantly increased at 20 week(P<0.05);However phosphorus level showed an irregular fluctuations(P<0.05).(4)The addition of the probiotics significantly increased the length and width on the mucosal villi of the uterus and had highly significant differences at 20 and 30 weeks(P<0.01),but there was an inconspicuous change in the morphology of the uterine tissue.(5)The ultrastructural results of the eggshell showed that the addition of the probiotic promoted the structure of the cuticle and the pore density of the palisades layer,as well as increased the effective layer thickness and the total thickness of the fenestra layer,with highly significant differences at 30 week(P<0.01).(6)The addition of probiotics to the diet altered the expression of biomineralization-related genes in the uterus.Among them,the expression of CA9,ATP2B2,SLC26A9,TF,OC-17,OCX-32,OC-116,and OVAL were differently increased at 20 and 30 weeks(P<0.05),while the expression of CA2 and OC-116 were significantly decreased at 20 weeks(P<0.05).(7)The results of 16S rDNA high-throughput sequencing revealed that the diet with probiotics altered the microbial abundance and community structure of the uterine flora of laying hens.Alpha diversity showed that the addition of probiotics increased the relative abundance of Proteobacteria and Actinobacteriota,while decreased the relative abundance of Firmicute in the uterus with significant differences at 20 and 30 weeks(P<0.05).In summary,the supplementation of probiotics to diet promotes egg quality and shell ultrastructure by slowing down uterine tissue damage,regulating the expression of biomineralization-related genes,and improving the microbiome in the uterus.