Upregulating Calcium Absorption Genes Expression In Small Intestine By Reducing Dietary Cation-Anion Difference To Prevent Hypocalcemia

To clarify the mechanism of reducing dietary cation-anion difference（DCAD）to prevent hypocalcemia has been receiving great attention over years.In this experiment,mammalian research model animals（SD rats）were used as experimental animals to investigate the effects of low DCAD on acid-base balance,blood calcium levels,expression levels of calcium absorption genes in the small intestine and transcriptome association of calcium pathway-related genes.After that,in view of the central role of rumen for ruminants,we investigated the effects of low DCAD diets on rumen fermentation and rumen microbial diversity using ewes as model animals,with the aim of elucidating the mechanism of low DCAD to prevent hypocalcemia and evaluating the application feasibility of low DCAD in the practice.The paper is divided into 3 trials.Trial 1.Reducing dietary cation-anion difference regulate the expression and localization of genes-related with calcium absorption in small intestine of ratsTwenty perinatal SD female rats were randomly divided into 2 groups of 10 rats in each group with 2 rats.Rats were fed in single cages and were consumed diets with DCAD levels at 117（CON）and-51（LD）for 25 days to determine urinary p H,blood calcium,small intestine（duodenum,jejunum,ileum）calcium absorption-related genes（Ca BP-D9k,TRPV5,TRPV6,PMCA1b,NCX1,Na-K ATP,Cav1.3）.The expression differences of Cav1.3 and PMCA1b proteins localization were determined.Compared with CON,urine p H was significantly reduced（7.63 vs.5.85,P<0.05）and blood calcium concentration was significantly increased（1.52 vs.2.35 mmol/L,P<0.05）in the LD.The genes expression abundance of Ca BP-D9k,TRPV5,TRPV6,PMCA1b,NCX1,and Na-K ATP m RNA in duodenum,jejunum,and ileum was significantly upregulated in LD,which were 1.8-,3.5-,and 5.8-times;2.0-,2.9-,and 3.5-times;3.4-,16.4-,and 27.8-times;8.3-,3.1-,3.5-times;39.5-,3.3-,8.3-times;and 3.9-,2.3-,2.8-times,respectively,over CON（P<0.05）.As verified by immunofluorescence,PMCA1b and Cav1.3 proteins were expressed in small intestinal epithelial cells,and the expression of PMCA1b protein in duodenum,jejunum,and ileum in LD was 2.5-,2.3-,and 2.4-times higher than that in CON,respectively（P<0.05）.Accordingly,Cav1.3 protein was 25.0-,2.8-,and 2.5-times higher,respectively,with significant differences（P<0.05）.It is suggested that feeding low DCAD can acidify the body,enhance the expression of calcium absorption-related genes,promote calcium absorption in small intestine,improve blood calcium levels,and prevent hypocalcemia.Trial 2.Transcriptome association analysis of genes related to the small intestinal calcium pathway in rats fed lower dietary cation-anion differenceOn the basis of trial 1,duodenum,jejunum,and ileum were selected for transcript sequencing.Compared with CON,21 genes were up-regulated and 8 genes were down-regulated in the duodenum,322 genes were up-regulated and 1513 genes were down-regulated in the jejunum,and 27 genes were up-regulated and 46 genes were down-regulated in the ileum in the LD,respectively.KEGG signaling pathway analysis on the upregulated genes in the LD compared to the CON was enriched to 2 major channels related to calcium ions（MAPK signaling pathway,Calcium signaling pathway）,and the predicted genes of Calcium signaling pathway were found to be PTPRR,CD38,MYLK2,IIGHM genes,and the predicted genes of MAPK signaling pathway were PTPRR,PRKCB,Rac2,NR4A1,PLA2G4B,MEF2C,NTRK2,TGFB1,ATP7A genes.The above genes are likely to be key genes involved in the enhancement of calcium ion transport in small intestine cells by low DCAD.Low DCAD may enhance the regulation of calcium absorption channel-related genes in small intestine.Trial 3:Effect of lower dietary cation-anion difference on blood calcium,rumen fermentation parameters and microbiota diversity of ewesForty ewes were randomly divided into 2 groups:control group（CON）,treatment group（low dietary cation-anion difference,LD）,20 replicates per group,2 ewes per replicate.Animals were fed diets with DCAD levels of 200 and-100,respectively.Reducing DCAD had no significant effect on feed intake（1212.88 vs.954.84,P>0.05）.Urine p H was significantly reduced in the LD compared to CON（8.15 vs.6.19,P<0.05）.Plasma biochemical parameters（ALP,TP,ALB,UA,α-AMY,Urea,ALT,AST,LDL-C,HDL,TG,TC,Glu,and Crea-S）were unaffected in the two groups（P>0.05）.Plasma calcium（2.62 vs.3.43 mmol/L）and blood magnesium（1.38 vs.1.80 mmol/L）were significant higher for LD than CON（P<0.05）.Feeding of LD diet did not change ruminal fermentation parameters（p H,buffering capacity,acetic acid,propionic acid,butyric acid,TVFA,A/P,and NH₃-N）between the two groups（P>0.05）.Ruminal bacterial flora diversity indices（Observed species,Chao 1,Shannon,Simpson,PD whole tree,ruminal bacterial phylum relative content analysis,and Beta diversity,principal component analysis,and UPGMA cluster analysis）were not significantly different for LD and CON（P>0.05）.Overall,reducing DCAD can prevent hypocalcemia by inducing compensatory mild metabolic acidosis,enhancing the expression of calcium absorption-related genes,and enhancing the regulation of small intestinal calcium absorption channel-related genes;and had no adverse effect on plasma metabolites,rumen fermentation parameters,and rumen microbial diversity of ewes.Our result provides a new insight for the mechanism understanding on reducing DCAD to prevent hypocalcemia.