Effects Of Vitamin D And PI3K Inhibitor On Calcium And Phosphorus Transporter Gene Expression In Small Intestine Of Broiler Chickens

Calcium(Ca)and phosphorus(P)are essential minerals of animals,which are absorbed in intestines,reabsorbed in kidney,and deposited in bone.Vitamin D3(VD3)and 25-hydroxyvitamin D3(25-OH-D3)are widely used as poultry feed additives to regulate Ca and P absorption in animal intestines.The effects of dietary vitamin D level on Ca and P transporter gene expression in the small intestine of broilers should be clarified.Therefore,this study was conducted to investigate the effects of dietary VD3 and 25-OH-D3 concentration on growth performance,bone mineralization,and gene expression of intestinal Ca and P transporters in broiler chickens.The results will provide theoretical basis for promoting Ca and P absorption and improving Ca and P utilization in poultry.Experiment 1: Gene expression of Ca and P transporters in the duodenum,jejunum,and ileum of broiler chickensThe expression of Ca and P transporter genes in duodenum,jejunum and ileum of broilers was studied.Ca transporters detected in this study included calcium-binding protein 28-k Da(Ca BP-D28k),plasma membrane calcium ATPase 1b(PMCA1b),and sodium-calcium exchanger 1(NCX1).P transporters included type IIb sodium phosphate cotransporter(Na Pi-IIb),inorganic phosphorus transporter 1(Pi T-1)and inorganic phosphorus transporter 2(Pi T-2).A total of 100 one-day-old male Arbor Acres broilers with similar body weight were randomly divided into 10 replicates with 10 chickens per replicate.Broilers were fed a normal nutrient level diet.At 21 days of age,ten chickens were randomly selected(one chicken per replicate)and killed.The mucosa samples of the duodenum,jejunum,and ileum were collected.The expression levels of Ca and P transporter genes were analyzed by quantitative q RT-PCR.Results showed that the m RNA expression levels of Ca transporters(Ca BP-D28 k,PMCA1b,and NCX1)were the highest in the duodenum,followed by the jejunum and ileum(P<0.05).No significant difference in the m RNA expression levels of Ca BP-D28 k and PMCA1 b was noted between jejunum and ileum(P>0.05).The m RNA expression level of Na Pi-IIb was higher in the duodenum than in the jejunum and ileum(P<0.05).By contrast,the m RNA expression levels of Pi T-1 and Pi T-2 in the duodenum were lower than those in the jejunum and ileum(P<0.05).These data indicated that the capacitry of Ca and P in the the three intestinal segments(duodenum,jejunum,and ileum)of broilers is different.Experiment 2: Effects of different levels of VD3 on the expression of Ca and P transporter genes in the small intestine of broilersThis experiment was conducted to investigate the effects of different levels of VD3 on the expression of Ca and P transporter genes in small intestine of broilers.Six diets were designed.The basal diet(without vitamin D)was supplemented with 0,3.125,6.25,12.5,25 and 50 μg/kg VD3,respectively.A total of 420 one-day-old male Arbor Acres broilers with similar body weight were randomly divided into 6 treatments with 5 replicates per treatment and 14 broilers per replicate.The chickens were slaughtered at 21 days of age.The mucosa samples of duodenum,jejunum and ileum were scraped and collected.The gene expression levels of Ca and P transporters were analyzed by quantitative q RT-PCR and western blotting.Results showed that increasing dietary VD3 levels elevated the weight gain and feed intake,enhanced the leg bone quality,and decreased the feed conversion ratio of 1-21-day-old broilers(P<0.05).VD3 supplementation significantly increased the m RNA expression levels of CABP-D28 k,PMCA1b and NCX1 in duodenum of broilers at 21 days of age(P<0.05).Dietary VD3 also enhanced the m RNA expression levels of P transporters(Na Pi-IIb,Pi T-1,and Pi T-2)in the duodenum(P<0.05).Similar results were detected in the jejunum and ileum.The m RNA expression level of Ca BP-D28 k increased by 536,1745,and 27 folds in the duodenum,jejunum,and ileum,respectively,after the addition of 25 μg/kg VD3(P<0.05).The m RNA expression levels of other Ca and P transporters increased by 1.00–4.00 folds after adding 25 μg/kg VD3(P<0.05).Adding 6.25 μg/kg VD3 increased the protein expression level of Ca BP-D28 k in duodenum of broilers(P<0.05),but did not affect the protein expression level of Na Pi-IIb and phospho-PI3K(P>0.05).These data indicated that VD3 improves growth performance and bone mineralization by stimulating Ca and P transporter gene expression in broiler chicken intestines.The optimum addition of VD3 in the basal diet for broilers is 25 μg/kg.Experiment 3: Effects of different levels of 25-OH-D3 on the expression of Ca transporter gene in the small intestine of broilersThis experiment was conducted to investigate the effects of different levels of 25-OH-D3 on the expression of Ca transporter gene in small intestine of broilers.Six diets were designed.The basal diet(without vitamin D)was supplemented with 0,3.125,6.25,12.5,25 and 50 μg/kg 25-OH-D3,respectively.A total of 420 one-day-old female Arbor Acres broilers were randomly divided into 6 treatments with 5 replicates per treatment and 14 broilers per replicate.The chickens were slaughtered at 21 days of age and weighed before slaughter to calculate the feed intake,body weight gain,and feed-to-gain ratio.The mucosa samples of duodenum,jejunum and ileum were scraped and the tibia and femur were removed.The Ca transporter gene expression was analyzed by quantitative q RT-PCR.Results showed that supplementation of 3.125 μg/kg 25-OH-D3 increased body weight gain,feed intake,and decreased feed-to-gain ratio of 1-21-day-old broilers compared with the basal diet(P<0.05).No difference in growth performance was observed in broilers fed with 3.125~50 μg/kg 25-OH-D3(P>0.05).The addition of 3.125~50 μg/kg 25-OH-D3 enhanced the weight,length,ash weight,and the contents of ash,Ca,and P of the tibia and femur(P<0.05).Dietary 25-OH-D3 upregulated the m RNA expression levels of intestinal Ca BP-D28 k,PMCA1b,and NCX1.The m RNA expression level of Ca BP-D28 k in the duodenum,jejunum,and ileum increased by 935-,2912-,and 50-fold after adding 50 μg/kg 25-OH-D3,respectively,compared with the basal diet.In contrast,the m RNA expression levels of PMCA1 b and NCX1 in the three intestinal segments were enhanced by 1.00~4.00 folds after adding 3.125~50 μg/kg 25-OH-D3.These data indicated that 25-OH-D3 improved growth performance and leg bone mineralization by upregulating the Ca transporter gene expression levels in the small intestine of broiler chickens from 1 to 21 days of age.The optimal supplemental level of 25-OH-D3 in the basal diet for broilers aged from 1 to 21 days is 12.5~25 μg/kg.Experiment 4: Effects of intraperitoneal injection of PI3 K inhibitor LY294002 on Ca and P transporter gene expression and MAPK signaling pathway in the small intestine of broilersThis experiment was conducted to investigate the effects of PI3 K inhibitor LY294002 on Ca and P transporter gene expression and MAPK signaling pathway in small intestine of broilers from 11 to 15 days of age.A total of 60 male 11-day-old broilers fed with nutrient-adequare-diets were randomly divided into 2 treatments with 5 replicates per treatment and 6 broilers per replicate.The two treatment broilers were intraperitoneally injected with saline and MAPK inhibitor LY294002,respectively.The injection dosages of saline and LY294002 were 0.3 mg/kg body weight.Saline and LY294002 were injected from 11 to 14 days of age and 1 time per day at 15:00 PM.The broilers were killed at 15 days of age.Results showed that intraperitoneal injection of LY294002 did not affect growth performance,bone mineralization,and m RNA expression levels of intestinal Ca and P transporters of broilers(P>0.05).Intraperitoneal injection of LY294002 significantly decreased the protein expression level of Na Pi-IIb and increased the protein expression level of phospho-ERK1/2 compared with intraperitoneal injection of saline(P<0.05).Intraperitoneal injection of LY294002 did not affect the protein expression levels of Ca BP-D28 k and phospho-p38MAPK(P>0.05).These data indicated that PI3 K signaling pathway may be the upstream of MAPK signaling pathway and that PI3 K participates in the regulation of intestinal P absorption in broilers.In conclusion,the m RNA expression levels of Ca and P transporter in different intestinal segments of broilers were different.Dietary VD3 and 25-OH-D3 improved the growth performance and bone quality of broilers,and up-regulated the m RNA expression levels of Ca and P transporters in the small intestine of broilers.PI3 K signaling pathway may be the upstream of MAPK signaling pathway.PI3 K may participate in the regulation of intestinal P absorption in broilers.