| Calcium and phosphorus are the most important mineral elements for maintaining the health of laying hens,and they play a significant role in skeletal development and eggshell formation.This study focused on the calcium and phosphorus transporters?CaBP-D28k,PMCA1b,NPt2a,and NPt2b?in laying hens,and studied its spatial and temporal expression rules,influencing factors,and relationship with eggshell quality.For a clearer understanding of layer calcium and phosphorus transport.1.The spatial and temporal expression rules of calcium and phosphorus transporters in laying hensThe study choosed 1,4,6,12,18,20,28,and 80-week-old Hy-line brown laying hens and collected samples.The results showed that serum calcium levels increased significantly with growth?P<0.0001?,and serum ALP levels decreased significantly with growth?P<0.0001?.The protein expression of CaBP-D28k was almost undetected before 18-week-old in small intestine,and it was significant increased in laying period?P<0.0001?.The protein expression of PMCA1b was the highest in brooding and laying period,and the lowest in growth period?P<0.0001?.The protein expression of CaBP-D28k and PMCA1b were not change as much in kidney,and the expression level in brooding and laying period were higher than that in the growth period?P<0.05?.The protein expression of NPt2b was significantly higher in brooding and laying period of the small intestine than in growth period?P<0.05?.The mRNA expression of NPt2a increased with growth in kidney,and was highest in laying period?P<0.0001?,and declined at the end of the laying period.2.Effect of dietary P levels on the expression of calcium and phosphorus transporters30 Hy-line brown laying hens aged at 23 weeks were divided into three treatments randomly.The low P group was fed with 0.15%AP,the control group was fed with 0.41%AP,and the high P group was fed with 0.82%AP.The results showed that dietary P levels had no effect on egg quality?P>0.05?.Compared to 0.41%AP group,dietary high P significantly increased serum P,ALP levels?P<0.05?,and dietary low P significantly decreased serum P levels?P<0.05?,dietary P levels had no effect on serum Ca levels?P>0.05?.In control group,the duodual protein expression of CaBP-D28k and PMCA1b was significantly higher than jejunal and ileal?P<0.05?,and the jejunal protein expression of CaBP-D28k was significantly higher than ileal?P<0.05?,the duodenal mRNA expression of NPt2b was significantly higher than jejunal and ileal?P<0.05?,and the duodenal protein expression of NPt2b was significantly higher than jejunal?P<0.05?.Compared to 0.41%AP group,low P and high P diets significantly increased duodenal mRNA expression of PMCA1b?P<0.05?.Low P diets significantly increased ileal the mRNA expression of CaBP-D28k and NPt2b,and the low P diets significantly decreased the mRNA expression of CaBP-D28k in shell gland and the protein expression of NPt2b in duodenum?P<0.05?.3.Effect of dietary phytase on the expression of calcium and phosphate transporters in small intestine50 male ISA brown laying hens aged at 15 weeks were randomly divided into five treatments.The control group was fed with 0.4%AP diet,and the experimental groups were fed with low phosphorus+4 phytase diets respectively?0.2%AP?.On the day 10 of the experiment,Cr2O3 was added as an external indicator to each group in a 0.5%ratio.The results showed that there was no difference in serum Ca,ALP levels compared with the control group,and serum P levels in control group were significantly higher than the phytase A,C,and D groups?P<0.05?.In control group,the disappearance rate of calcium and phosphorus were increased significantly from duodenum to ileum?P<0.05?,while there were no difference in the disappearance rate of calcium and phosphorus between jejunum and ileum in the phytase treatment group?P>0.05?.There was no difference in the disappearance rate of phytate phosphorus from duodenum to ileum?P>0.05?.The protein expression of CaBP-D28k of the control group was not different significantly in small intestine?P>0.05?,and the duodenal protein expression of PMCA1b of the control group was significantly higher than jejunal and ileal?P<0.05?,and the duodenal protein expression of NPt2b was significantly higher than ileal?P<0.05?.The dietary phytase significantly inhibited the protein expression of CaBP-D28k in duodenum?P<0.05?,and promoted the protein expression of PMCA1b in ileum?P<0.05?,there was no effect on the protein expression of NPt2b in small intestine.4.Effect of ambient temperature on the expression of calcium and phosphorus transporters360 ISA brown laying hens aged at 13 weeks were randomly divided into five treatments?22,24,26,28,and 30oC?.They were treated at 10:00-18:00 every day for 8 hours,and then the ambient temperature returned to 22°C.Sample collection at 18-weeks-old.We found that temperature treatment had no significant effect on serum Ca,P,ALP?P>0.05?.The mRNA and protein expressions of CaBP-D28k and PMCA1b in small intestinal at 2426oC were significantly higher than those at other temperatures?P<0.05?.Furthermore,The mRNA expressions of CaBP-D28k,PMCA1b and the protein expressions of PMCA1b in small intestine were also promoted at 30oC?P<0.05?.24oC and 30oC promoted the mRNA expression of CaBP-D28k in the kidney?P<0.05?,and 22oC,30oC promoted the protein expression of PMCA1b in the kidney.26oC30oC was most suitable for the mRNA expression of NPt2b in the small intestine?P<0.05?,and 22oC28oC was most suitable for the protein expression of NPt2b in the small intestine?P<0.05?.The mRNA expression of NPt2a was higher at 24oC26oC than other temperatures in kidney?P<0.05?.5.Relationship between eggshell quality and the expression of calcium and phosphorus transporterThe experiment selected 20 poor eggshell quality laying hens and 20 normal eggshell hens for sampling and analysis.We found that egg weight,shell thickness,and eggshell hardness in the experimental group were significantly lower than the normal group?P<0.05?.There were no difference in serum Ca,P,ALP levels?P>0.05?.In the experimental group,the ileal protein expression of PMCA1b was lower than the normal group significantly?P<0.05?,and there were no difference in the expression of other calcium and phosphorus transporters in the small intestine,kidney,and eggshell glands.In summary,calcium and phosphorus transporters in small intestine and kidney of laying hens were mainly expressed during brooding and laying period.The calcium and phosphorus transporters in the duodenum were higher than those in the jejunum and ileum,but calcium and phosphorus absorption capacity was similar in each intestine segment.Phytase decomposes phytate phosphorus in duodenum,and inhibit the expression of CaBP-D28k in duodenum;dietary P levels affect the mRNA expression of calcium and phosphorus transporter rather than protein expression;2426oC is the most beneficial to the expression of intestinal CaBP-D28k,PMCA1b and renal NPt2a,2228oC is most helpful for the protein expression of NPt2b in the small intestine,and 22oC,30oC are most beneficial to the expression of calcium transporter in the kidney.The decrease of eggshell quality may be related to the decrease expression of PMCA1b in ileum,and the mechanism needs to be studied. |
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