| The aging of the intestinal tract of laying hens in the late laying period will reduce their ability to absorb calcium and lead to the deterioration of eggshell quality,an essential factor affecting egg quality.Poor egg quality will lead to the increase of egg breakage rate in the process of egg transportation and preservation,which will have a negative impact on economic benefits.Therefore,improving the quality of eggs in the late laying period is of positive significance to improve the absorption and utilization of calcium in the intestine from the perspective of nutritional regulation.Casein Phosphopeptides(CPP),as a bioactive peptide,play a good role in promoting calcium(Ca),iron(Fe)and other mineral elements absorption in the body.Therefore,in this study,different concentrations of CPP were added to the basal diet of laying hens to explore the effects of CPP on laying performance,egg quality,eggshell ultrastructure,antioxidant properties,and eggshell gland and intestinal-related functions of laying hens in late laying period,providing a reference for the application of CPP in laying production and the development of feed additives for laying hens in late laying period.A total of 800 healthy 58-week-old Hyline brown laying hens were randomly divided into five groups with eight replicates per group and 20 hens per replicate.Piglets in T1(control group)were fed a basal diet,and those in experimental groups T2,T3,T4,and T5 were fed the basal diet supplemented with 0.5,1.0,1.5,and 2.0 g/kg CPP,respectively.At the end of the experiment,five eggs were randomly selected from each replicate for egg quality determination.The ultrastructure of the eggshell was observed by scanning electron microscope,and the contents of Ca,P,and Fe in the eggshell were determined by kit.On the day of sampling,blood samples were collected under the wings,and samples of eggshell glands,and intestinal mucosa were retained.Serum biochemical indices,eggshell glands and intestinal enzyme activities were determined with relevant kits.mRNA expression levels related to eggshell glands and intestinal enzyme activities were first detected by ND-1000 ultraviolet spectrophotometer under OD260 and OD280.cDNA was synthesized by RNA reverse transcription,and then fluorescence quantitative PCR was performed.The chicken’s tibia was subjected to Instron3367 two-column electronic tester to detect its strength.The results show that:(1)After CPP supplementation,the breakage rate of the experimental groups was lower than that of a control group(P<0.05).With the increase of CPP supplemental level,the breakage rate showed a linear decrease(P=0.046)and a quadratic change(P<0.05).Dietary CPP supplementation had no significant effects on the laying rate,egg weight,feed intake,and feed-to-egg ratio of laying hens.(2)The yolk color in groups T2,T3,and T4 was higher than that in group T1(P<0.05).With the increase of CPP supplemental level,the yolk color showed a secondary change of first increasing and then decreasing(P=0.002),which had no significant effect on the yolk percentage(P>0.05).The eggshell thickness in the T4 group was higher than that in T1 and T2 groups(P<0.05),and the eggshell thickness increased linearly with the increase of CPP supplemental level(P=0.006).The eggshell color of the experimental groups was higher than that of a control group,and the eggshell color showed a linear increase and quadratic relationship with the rise of CPP supplemental level(P<0.05)but had no significant effect on eggshell rate(P>0.05).There were no significant differences in Haugh unit,egg shape index,and protein height among all groups(P>0.05).(3)Dietary CPP supplementation had linear increases in palisade layer thickness and crystalline layer thickness of eggshell(P<0.05).The effective layer thickness in T3,T4,and T5 groups was significantly higher than that in the T1 group(P<0.05).With the increase of CPP supplemental level,The effective layer thickness showed a linear increase(P=0.001)and a quadratic change(P=0.027).The number of papillary nodules in the T2 and T3 groups was significantly higher than that in the T1 group(P<0.05).With the increase of CPP supplemental level,the number of papillary nodules in T2 and T3 groups presented a quadratic change in first increasing and then decreasing(P=0.006).Dietary supplementation of CPP had no significant effects on the mastoid layer’s thickness and the mastoid space’s width among different groups(P>0.05).(4)The eggshell Ca content in T2 and T3 groups was higher than that in the T1 group(P<0.05),and with the increase of CPP supplemental level,the Ca content presented a quadratic curve change(P=0.021).CPP dietary supplementation had no significant effects on eggshell P content(P>0.05).Fe content of eggshells in the T2 and T3 groups was higher than that in the T1 group(P<0.05).(5)The serum Ca content of 0.5 g/kg CPP group was higher than that of the control group(P<0.05),and with the increase of CPP supplemental level,Ca content was firstly increased and then decreased in a quadratic curve(P=0.022);The serum ALP activity of 0.5 and 1.0 g/kg CPP groups was lower than that of the control group(P<0.05);Dietary CPP supplementation had no significant effects on serum P content and the activity of tartrate-resistant acid phosphatase(TRAP)(P>0.05).(6)The Ca2+=ATP activity in the duodenum was quadratically changed with CPP supplementation(P=0.007);The CA activity of the CPP group supplemented with 0.5 g/kg was higher than that of a control group(P<0.05).(7)Dietary CPP supplementation linearly increased the CA activity of the eggshell gland(P=0.01);The CaBP-D28K activity of 0.5 and 1.0 g/kg CPP groups was higher than that of the control group(P<0.05),and with the increase of CPP supplemental level,the CaBP-D28K activity showed a quadratic change(P=0.015).Dietary CPP had no significant effect on ALP activity in the eggshell gland(P>0.05).(8)Compared with the control group,the relative expression level of CaBP-D28k in the duodenum was up-regulated by adding 1.5 g/kg CPP(P<0.05),and the relative expression level of CaBP-D28k was linearly increased with the increase of CPP supplemental level(P<0.05).The relative expression level of NCX in diets supplemented with CPP was linearly increased(P<0.05),and the relative expression level of NCX in 1.0 and 2.0 g/kg CPP groups was higher than that in a control group(P<0.05).Compared with the control group,the relative expression level of PMC A in the diet supplemented with 1.5 g/kg CPP group was up-regulated(P<0.05),and the relative expression level of PMCA was linearly increased with the increase of the supplemental level(P<0.05).The relative expression level of CA in the dietary 0.5 g/kg CPP group was higher than that in the control group(P<0.05).With the increase of CPP supplemental level,the relative expression level of CA was linearly decreased(P<0.05).(9)Compared with the control group,the relative expression level of CA in the eggshell gland was down-regulated in different dietary CPP groups(P<0.05);The relative expression level of ALP in the diet supplemented with 2.0 g/kg CPP group was higher than that in a control group(P<0.05).With the increase of CPP supplemental level,the relative expression level of ALP presented linear and quadratic relationships(P<0.05).Dietary CPP supplementation had no significant effects on the relative expression levels of OPN and CaBP-D28K(P>0.05).(10)Dietary CPP supplementation had no significant effects on follicle number,liver antioxidant function,and intestinal tissue morphology of laying hens in the late laying period(P>0.05).Dietary CPP supplementation had no significant effects on tibia weight,ash content,Ca,and P contents(P>0.05).The tibial strength of the CPP group supplemented with 1.0 g/kg was higher than that of a control group(P<0.05).Conclusions:Under the conditions of this experiment,dietary 0.5-1.0 g/kg CPP can increase the related enzyme activities in the intestinal tract and eggshell gland,promote serum calcium content and tibia strength,and improve egg quality of Hyline Brown laying hens. |
