Regulation Of Ala-Gln On Intestinal Mucosal Barrier Function And Absorption Capacity Of Weaned Piglets

The study was conducted to explore the Ala-Gln regulation of piglet intestinal mucosabarrier function and intestinal absorption mechanism of action with modern techniques. Theresults would provide a theoretical basis for the applications of Ala-Gln in the piglet feed.Experiment I: The effect of Ala-Gln on the growth performance, intestinalmorphology and function of the weaned pigletsOne hundred weaned piglets(21days old) with similar genetic background and bodyweight were selected and completely randomly divided into4groups: pigs in control groupwere fed with basic diet and diets of treatment groups were basic diet supplemented with0.15%,0.30%and0.45%Ala-Gln. Each group had5repeats which included5piglets inevery repeat. The experiment lasted for21days and the diarrhea incidence was recordedevery day for computing diarrhea rate. Piglets in every repeat were weighed and feedconsumption was also recorded for the computing of average daily feed intake, averagedaily gain and feed gain ratio every week. At the7th day,14th day and21st day of theexperiment, one pig was selected from every repeat randomly and the pigs were slaughteredto collect samples as follows: serum, the mucosa, mucus and segments of intestine.Results indicated that:(1)Ala-Gln could significantly improve average daily weightgain, feed gain ratio and decrease diarrhea rate of weaned piglets. Compared with thecontrol group, the piglets in experimental group daily feed intake and daily gain weresignificantly increased (P<0.05) at21~28,28~35days of age, and0.30%Ala-Gln highlysignificantly increased daily gain (P<0.01) and lower the piglet feed conversion ratio (P<0.05) of the piglet at21~28days of age. With0.45%Ala-Gln in the diet, piglets diarrhearate and diarrhea index of piglets were significantly lower than the control group (P<0.05)at the whole time of the experitment.(2) Ala-Gln diets could effectively improve pigletduodenum, jejunum, ileum villus height and V/C, reduce the crypt depth. Among thetreatment groups, effects of0.15%,0.30%Ala-Gln were the most obvious (P<0.05).Transmission electron microscope found that jejunal microvilli of the control group wasdisorganized, ectopic and accompanied by shedding. Jejunum villi of treatment group wastrim, especially for the group with0.30%Ala-Gln.(3)0.30%Ala-Gln significantly reducedDAO activity (P<0.05) of35-day-old piglets serum, and0.15%,0.30%Ala-Glnsignificantly reduced DAO activity (P<0.05) of42-day-old piglets serum. With theincreasing of Ala-Gln in the diet, the content of the42-day-old piglets cPLA2linearlyincreased (P<0.05), group with0.45%Ala-Gln was significantly higher (P<0.01) than control group. Piglet intestinal mucus lysozyme activity was significantly higher (P<0.05)in three stages compared with control group. In summary, Ala-Gln can reduce piglet serumDAO activity, increase the content of intestinal mucosa the cPLA2and mucus lysozymeactivity, improve the morphology of the villi of the small intestine, thus contributing tosmall intestinal mucus barrier function, and improve the growth performance of piglets.Experiment II: Effect of Ala-Gln on the localization and expression of tightjunction (TJ) protein occludin.The objective of the present study was to determine the effect of dietary Ala-Glnsupplementation on intestinal barrier permeability and expression of tight junction proteinOccludin by the model of porcine intestinal epithelial cells.The cells were placed onsix-well plates and coverslip, grown in a culture medium composed of high glucose DMEMwith indicated concentration of Ala-Gln (0,0.25,0.5,1,2,4mM). When the cells grew andintegrated for more than70%, started to immunofluorescence localization and western blot.Results showed that (1) Occludin protein was localized in cytoplasm with0mMAla-Gln, the positive staining of cell junction was not obvious. With increasing of Ala-Gln,positive Occludin appeared as hexagon-like structures encircling the cells at the cellmembrane junction, while fluorescence signal in cytoplasm was reduced.(2) Occludinexpression in each treatment group were significantly higher than those in the control group(P<0.01). Occludin expression showed a trend of decreasing after increasing with theincreasing of Ala-Gln content. Occludin expression levels reached the peak with theaddition level of2mM, and were significantly higher than the other treatments (P<0.01).Thus, Ala-Gln can increase IPEC-1cell tight junction protein Occludin expression, improveintestinal epithelial cell tight connection structure.Experiment III: Effect of Ala-Gln on intestinal mucosa enzyme activity andexpression of Sodium Glucose Cotransporter-1and glucose transporter-2The experiment was conducted to study Effect of Ala-Gln dipeptide on intestinalmucosa enzyme activity and expression of Sodium Glucose Cotransporter-1and glucosetransporter-2. Na+/K+-ATPase, lactase, maltase, sucrose enzyme activity were measuredafter feeding trial. To investigate SGLT-1and GLUT-2gene expression, an optimizedRelative Quantification-RT PCR was constructed to measure the level.The results show that:(1) At the age of28days, the enzyme activity of Na+/K+-ATP(P<0.05) and maltase (P<0.01) can be significantly increased in group B. At the age of35days, the test group increased both the Na+/K+-ATP enzyme activity and the disaccharidesactivity (P<0.05).At the42days of age, group D can be significantly increased the sucrase and maltase (P<0.01) activities.(2) There was no significant difference on SGLT1andGLUT2mRNA expression of weaned piglets intestine mucosa except jejunal. Resultsindicate that Ala-Gln shows the active effects on the activity of disaccharides, which leadsto the enhacement of carbohydrate digestion. However there is no significant difference onthe absorptive function of glucose at transcriptional level of SGLT1and GLUT2.