| Eggshell quality,generally evaluated by eggshell strength and eggshell thickness,is an important factor affecting the egg quality and commercial value.Improving the eggshell quality to improve the egg quality and reduce the rate of broken eggs is an important issue for the laying hen breeding industry.Many factors affect eggshell quality,including blood hormone levels.Progesterone(P4)which is closely related to eggshell quality is an important reproductive hormone.It is of great scientific significance to study the effect of P4 on eggshell quality and its possible mechanism to guide the regulation of eggshell quality in production.In this paper,the change of P4 levels in laying hens with different eggshell strengths during the eggshell calcification was investigated,and the relationship between eggshell strength and P4 level in laying hens was analyzed.The genes related to eggshell strength were screened as candidates in uterus and their functions were shown.Accordingly,the effect and mechanism of P4 on eggshell quality were studied by injecting P4.And then it was explored whether it was feasible to improve the eggshell quality by adding agnus castus extract and octacosanol.The main research contents and results are as follows1.Differences in blood progesterone levels and uterine transcriptome analysis in laying hens with different eggshell strengthsTwo hundred and ten 42-week-old Hy-Line brown laying hens were caged individually.The oviposition time of each laying hen was observed and recorded for 10 consecutive days.During this period,the eggshell strength of eggs from each hen was measured every day.Accordingly,the laying hens with a stable oviposition time and stable eggshell strength were selected.The thirty hens with eggshell strength more than 40 N were selected as the high strength group(HS)and the another thirty hens with eggshell strength less than 30 N were selected as the low strength group(LS).The ovulation time of each hens was predicted using oviposition(Ovulation occurs about 15 min after oviposition of previous egg).In initiation(6-9 h after ovulation),growth(14-16 h after ovulation)and termination(21-23 h after ovulation)of calcification periods respectively,10 laying hens were selected from each group for sampling and hormone levels and uterine transcriptome were studiedThe main results are as follows(1)The mammillary layer thickness,mammillary knob width and the ratio of mammillary layer in eggshells from HS group were significantly lower than those in LS group(P<0.05),and the effective layer thickness and effective layer ratio were significantly higher in the HS group compared to the LS group(P<0.05)(2)In the initiation of calcification,the blood P4 and 1,25-(OH)2D3 concentrations in the HS group were significantly higher compared to the LS group(P<0.05)(3)There were 1777 differentially expressed genes(DEGs)during the initiation period of calcification,16 DEGs during growth period of calcification and 8 DEGs during termination period of calcification in uterus,respectively.30 DEGs related to eggshell strength were screened in the initiation period,among them,H+ and HCO3-transporter genes and OPN have greater effects on eggshell strength2.The effects of progesterone on eggshell quality and its mechanismTrial ? was used to determine the effects of P4 on eggshell quality.According to the results of Trial ?,trial ? studied the mechanism of P4 affecting eggshell qualityTrial ? The effects of progesterone on eggshell qualitySixty 40-week-old Hy-Line brown laying hens with eggshell strengths ranging from 25 to 35 N were caged individually and randomly divided into 5 groups named group 1-5.The oviposition time of each laying hen was observed and recorded for 10 consecutive days.The ovulation time of each hens was predicted using oviposition(Ovulation occurs about 15 min after oviposition of previous egg).The laying hens in group 1 and 2 were injected with P4 5 h after ovulation and the doses were 1.0 and 0.15 mg/kg body weight(BW)respectively.The laying hens in group 3 were injected with P4 2 h after ovulation and the doses was 0.15 mg/kg BW.The laying hens in group 4 were injected with P4 1 h after ovulation and the doses was 0.15 mg/kg BW Group 5 was considered a control without P4 injection.The main results are as follows(1)The effects of P4 on eggshell quality were related to the injection time.P4 injected 5 h after ovulation reduced eggshell quality(P<0.05).P4 injected 2 h after ovulation increased eggshell quality(P<0.05),P4 injected 1 h after ovulation did not affect eggshell quality(P>0.05)(2)The P4 injected 2 h after ovulation significantly increased effective layer thickness(P<0.05)and decreased mammillary layer thickness(P<0.05),improving the eggshell ultrastructure.The P4 injected 5 h after ovulation significantly decreased effective layer thickness(P<0.05)and did not affect mammillary layer thickness(P>0.05),reducing the eggshell ultrastructure.It indicated that P4 changed the the eggshell quality by affecting the eggshell ultrastructureTrial ? The regulation mechanism of progesterone on eggshell qualityThree hundred and five 45-week-old Hy-Line brown laying hens were caged individually.The oviposition time of each laying hen was observed and recorded for 10 consecutive days to predict the ovulation time(Ovulation occurs about 15 min after oviposition of previous egg).Synchronously,the eggshell strength of eggs from each hen was measured every day.Accordingly,150 laying hens with a stable oviposition time and an eggshell strength of 25.0-30.0 N were selected.The selected hens were randomly divided into 5 groups each with 30 birds,namely control,oil-2 h,oil-5 h,P4-2 h,and P4-5 h groups.The control group received no injection.The oil-2 h and oil-5 h groups received an injection of 0.2 mL peanut oil without P4 2 and 5 h after ovulation,respectively.The P4-2 h and P4-5 h groups received an injection of 0.2 mL peanut oil containing P4 2 and 5 h after ovulation,respectively.the dose of P4 injected was 0.15 mg/kg BW.The main results are as follows(1)During the initiation of calcification,no significant differences were observed in the concentrations of blood E2,1,25-(OH)2D3,Ca and P after P4 injection(P>0.05),the concentrations of blood P4,LH and PTH were lower than control significantly(P<0.05).During the growth of calcification,no significant changes in the concentrations of blood E2,P4,LH and 1,25-(OH)2D3(P>0.05),and the blood PTH level in P4-2 h group was increased obviously(P<0.05)(2)Compared to control,the eggshell strength,eggshell thickness and effective layer thickness in P4-2 h group were increased(P<0.05,P<0.01),and the mammillary layer thickness and mammillary knobs width were decreased obviously(P<0.05,P<0.01).In P4-5 h group,the eggshell strength,eggshell thickness and effective layer thickness were lower than control(P<0.01),and the mammillary layer thickness and mammillary knobs width have no changes compared to control(P>0.05)(3)The concentrations of Thr,Cys,Leu,Lys and His in shell membrane from P4-2 h group were higher than control(P<0.05).The concentrations of Val and Lys in shell membrane from P4-5 h group were reduced compared to control(P<0.05)(4)During the initiation of calcification,no significant changes in the expression levels of duodenal CaBP-D28k,NCX1 and PMCA 1b were observed between the groups(P>0.05).During the growth of calcification,the expression levels of duodenal CaBP-D28k and PMCA 1b in the P4-2 h group was significantly higher than that in the P4-5 h group(P<0.05)(5)During the initiation of calcification,the Ca content in uterine tissues was not significantly different between the groups(P>0.05).During the growth of calcification,the Ca content in the uterine tissue of the P4-2 h group was significantly higher than that of the P4-5 h group(P<0.05)(6)During the initiation of calcification,the relative expression levels of OC-116,OPN,CA2,ATP2B2 and EDIL3 in P4-2 h group were decreased obviously(P<0.05,P<0.01)and the relative expression level of OVA was higher compared to control(P<0.01).In in P4-5 h group,the relative expression levels of OC-116,OPN,CA2,PR and EDIL3 were inhibited obviously(P<0.05).During the growth of calcification,the relative expression levels of OC-17,CA2,OCX-36,OVA and CaBP-D28k were increased(P<0.05,P<0.01)and was inhibited obviously(P<0.05)in in P4-2 h group.In in P4-5 h group,the relative expression levels of OCX-36,OPN and CaBP-Da8k were decreased significantly(P<0.05)(7)During the initiation and growth of calcification,P4 did not affect the expression levels of uterine HCO3-transporter genes and H+transporter genes(P>0.05)3.Effects of octacosanol and agnus castus extract on performance and eggshell quality of laying hensThis study investigated the effects of octacosanol(O)and agnus castus ectract(AC)on the performance,eggshell quality and plasma P4 level of laying hens,and explored the feasibility of using external additives to change the plasma P4 level of laying hens to improve eggshell qualityOne hundred twenty 60-week-old Hy-Line brown laying hens were randomly divided into 5 groups each with 24 birds.These groups were fed basal diets(BD)(Control),BD+15 mg/kg octacosanol(O-15),BD+30 mg/kg octacosanol(O-30),BD+100 mg/kg agnus castus(AC-100)and BD+200 mg/kg agnus castus(AC-200),respectivelyThe main results are as follows(1)Compared with the control group,the feed/egg ratio in the O-15 and O-30 groups decreased after 3 weeks,but did not significant(P>0.05).No significant changes in the blood P4 and Ca content,eggshell strength and eggshell thickness(P>0.05)(2)No significant changes in performance and eggshell quality of AC-100 group(P>0.05).While the egg production rate of laying hens in the AC-200 group was significantly reduced(P<0.05),the feed/egg ratio was increased,and the plasma P4 level was significantly reduced(P<0.05),and the eggshell strength and eggshell thickness showed a decreasing trendIn summary,this study come to the following conclusions1.During the periods of eggshell calcification,the initiation period is the key calcification period that affects the eggshell strength2.The eggshell quality was significantly increased by P4 injected 2 h after ovulation and decreased by P4 injected 5 h after ovulation,indicating P4 could improve or reduce eggshell quality,which related to the injection time of P43.P4 affects eggshell quality by affecting the ultrastructure.P4 injected 2 h after ovulation promoted the fusion of mammillary knobs to reduce the mammillary layer thickness by inhibiting the expression level of OPN,promoted the deposition of CaCO3 to increase the effective layer thickness by increasing the relative expression levels of OVA,OC-17,CaBP-D28k and CA2 and increased the content of Lys in shell membrane to improve the compactness of the shell membrane.While P4 injected 2 h after ovulation decreased the Ca2+ supply in uterus to reduced the effective layer thickness by inhibiting the relative expression level of CaBP-D28k and decreased the content of Lys in the shell membrane to cause a poor compactness in the shell membrane4.The relative expression levels of H+and HCO3-transporter genes were not affected by P4 injected,indicating P4 does not affect the eggshell quality by affecting uterine H+ and HCO3-transport5.The addition of agnus castus extract lowered the plasma P4 levels and the addition of octacosanol in the diet had no significant effect on the plasma P4 levels of laying hens,while they did not improve eggshell quality.It indicated that measures to regulate the plasma P4 levels of laying hens by external additives in dietary to improve eggshell quality ware temporarily not feasible. |
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