Effects Of Dietary Different Iron Levels On Iron Deposition,gut Microbiota,and Transcriptome In Piglets

Iron plays an important role in maintaining the growth and metabolism of pigs,while piglets have low iron storage capacity and are more susceptible to iron deficiency-caused growth retardation.In addition,due to the low absorption rate of iron in the intestinal tract of piglets,excess supplementation of iron is widely occurred in feed,which also leads to the growth obstruction and body damage of piglets caused by iron overload.Now,the effect of supplementation with Fe SO₄·H₂O on piglets is still less.Therefore,the purpose of this study was to investigate the effects of dietary supplementation with different levels of Fe SO₄·H₂O on growth performance,diarrhea,intestinal morphology and iron deposition of weaned piglets,and to reveal the potential mechanisms of iron-mediated growth and metabolism of piglets basing on gut microbiota and transcriptome.It provides theoretical basis for the reasonable addition of iron in piglets’diet.50 21-day-old weaned“Duroc×Landrace×Yorkshire”ternary hybrid piglets with body weight 6.60±0.13 kg were selected and randomly divided into 5 groups,feeding 50,100,200,500 and 800 mg/kg Fe SO₄·H₂O,respectively.The prefeeding period was 7 days and the experimental period was 21 days.The main results are as follows:（1）Effects of dietary iron levels on growth performance of weaned pigletsThe addition of different levels of Fe SO₄·H₂O to the basic diet had no significant effect on feed intake,daily gain,and feed conversion rate of weaned piglets（P>0.05）;The results of fecal score showed that weaned piglets from 800 mg/kg Fe SO₄·H₂O group had marked diarrheal ratio（P<0.05）;Compared with 100 and 500 mg/kg Fe SO₄·H₂O groups,the diarrhea rate in 50 and 800 mg/kg Fe SO₄·H₂O groups was significantly increased（P<0.05）,suggesting that both low and high fe diets can induce diarrhea in piglets.（2）Effects of dietary iron levels on iron metabolism and deposition in weaned pigletsCompared with the 50 mg/kg Fe SO₄·H₂O group,the content of serum iron in 100 and800 mg/kg groups was significantly increased（P<0.01）;The content of unsaturated iron binding capacity was decreased in 100,200 and 800 mg/kg groups（P<0.05）.Meanwhile,the total iron binding capacity was decreased in 500 and 800 mg/kg groups（P<0.05）;In addition,the content of serum immunoglobulin A in 100,200,500 and 800 mg/kg Fe SO₄·H₂O fed piglets was significantly lower than that in 50 mg/kg group（P<0.01）.Dietary 100,200,500 and 800 mg/kg Fe SO₄·H₂O promoted the emission of iron in ileal contents,colon contents,cecal contents and feces,and the content of 800 mg/kg fed piglets was the increased.In the jejunum,the iron deposition in 500 and 800 mg/kg Fe SO₄·H₂O groups was significantly higher than that in 50 mg/kg groups（P<0.01）;In the colon,iron deposition was the highest in 800 mg/kg Fe SO₄·H₂O fed piglets（P<0.01）;In the liver,iron deposition in 200,500 and 800 mg/kg Fe SO₄·H₂O groups was significantly higher than that in 50 mg/kg groups（P<0.01）.（3）Effects of dietary iron levels on intestinal morphology of weaned pigletsDietary different levels of Fe SO₄·H₂O had no significant effect on the villus height of duodenum,jejunum and ileum;However,with the increase of dietary iron level,the depth of duodenal recess was significantly increased（P<0.01）.Meanwhile,the dietary addition of500 mg/kg Fe SO₄·H₂O significantly reduced the duodenal villus to crypt ratio compared with 100 mg/kg group（P<0.01）.There were no significant differences between jejunum and ileum in crypt depth and villus to crypt ratio（P>0.05）.（4）Effects of dietary iron levels on gut microbial in weaned piglets16S r DNA microbial sequencing analysis of colonic contents showed that the addition of 800 mg/kg Fe SO₄·H₂O significantly reduced Ace and Shannon indexes（P<0.05）compared with the 50 mg/kg group,suggesting that excessive iron supplementation can reduce gut microbialα-diversity;In addition,there was no significant difference in the effects of dietary iron levels on gut microbiota compositions at the phylum level（P>0.05）;At the genus level,compared with 50 mg/kg Fe SO₄·H₂O group,dietary supplementation with 800 mg/kg Fe SO₄·H₂O significantly increased the Lactobacillus and reduced the relative abundance of Ruminococcaceae＿UCG-005 and[Eubacterium]＿copro stanoligenes＿group（P<0.05）;At species level,supplementation of 200、500 and 800 mg/kg Fe SO₄·H₂O significantly increased the relative abundance of Lactobacillus＿amylovorus（P<0.05）;800 mg/kg Fe SO₄·H₂O significantly increased the Lactobacillus＿salivarius abundance（P<0.05）;500 and 800 mg/kg Fe SO₄·H₂O groups significantly reduced the abundance of Dorea＿longicatena compared with 100 mg/kg Fe SO₄·H₂O groups（P<0.05）.Correlation analysis showed that colon contents iron content was significant negatively correlated with Ruminococcaceae＿UCG-005 and Christensenellaceae＿R.7＿group（P<0.05）.The serum iron content was significant negatively correlated with abundance of Lachnospiraceae＿XPB1014＿group、Ruminococ-caceae＿UCG-005、Lachnospiraceae＿AC2044＿group、Ruminococcaceae＿UCG.002、Rikenellaceae＿RC9＿gut＿group、Christensenellace AE＿R.7＿group and Treponema＿2（P<0.05）.The serum iron content was significant positively correlated with abundance of Holdemanella（P<0.05）,suggesting that these microorganisms are involved in iron response and metabolism.（5）Effects of dietary iron levels on intestinal transcriptional map of weaned pigletsThe colon tissues from 50 and 800 mg/kg Fe SO₄·H₂O groups were selected for transcriptome sequencing analysis.424 differential genes were screened,of which 254 were up-regulated and 170 were down regulated.Go functional enrichment analysis showed that G protein coupled receptor activity and chemokine activity were significantly enriched;KEGG enrichment analysis showed that the cytokine receptor interaction pathway was significantly enriched;PPI network analysis revealed a highly relationship between these genes,such as interferon-γand signal transducer and activator of transcription 2.In conclusion,dietary different levels of Fe SO₄·H₂O had no significant effect on the growth performance of weaned piglets.The minimum iron supplemental level of 50 mg/kg Fe SO₄·H₂O can guarantee normal growth performance,but high dose could induce diarrhea and intestinal injury.Meanwhile,the increase of dietary iron level can promote its deposition in the body and affect gut microbial diversity,compositions and transcriptional map.