Purification methods to reduce interference by dextran sodium sulfate with quantification of gene expression in intestinal tissue samples from a piglet model of colitis.

Dextran sodium sulfate (DSS) is commonly used to induce intestinal (i.e., colonic) inflammation in a variety of animal models. However, DSS is known to cause interference when using quantitative-real time polymerase chain reaction (qRT-PCR) methods, thereby invalidating accurate and precise measurement of tissue gene expression. Therefore, the goal of this study was to determine whether different mRNA purification methods would reduce DSS-interference. Colonic tissue samples were collected at postnatal days (PND) 27 or 28 from pigs that had not been administered DSS (Control), and two independent groups of pigs that received 1.25 g of DSS/kg of BW/d (DSS-1 and DSS-2) from PND 14 to 18. Tissue samples collected were subsequently stratified into three purification methods (i.e., 9 total treatment × method combinations), including: 1) no purification, 2) purification with lithium chloride (LiCl), or 3) purification using spin column filtration. All data were analyzed using a one-way ANOVA in the Mixed procedure of SAS. The average RNA concentrations across all treatments were between 1,300 and 1,800 µg/µL for all three in vivo groups. Although there were statistical differences among purification methods, the 260/280 and 260/230 ratios fell between acceptable limits of 2.0 to 2.1 and 2.0 to 2.2, respectively, for all treatment groups. This confirms the RNA quality was adequate and not influenced by purification method in addition to suggesting the absence of phenol, salts, and carbohydrate contamination. For pigs in the Control group that did not receive DSS, qRT-PCR Ct values of four cytokines were achieved, though these values were not altered by purification method. For pigs that had undergone DSS dosing, those tissues subjected to either no purification or purification using LiCl did not generate applicable Ct values. However, when tissues derive from DSS-treated pigs underwent spin column purification, half of the samples from DSS-1 and DSS-2 groups generated appropriate Ct estimates. Therefore, spin column purification appeared to be more effective than LiCl purification, but no method was 100% effective, so caution should be exercised when interpreting gene expression results from studies where animals are exposed to DSS-induced colitis.

Dextran sodium sulfate (DSS) is a chemical used to experimentally induce colonic inflammation in animal models. However, DSS causes chemical inhibition of processes involved with quantitative real-time polymerization chain reaction, thereby inhibiting the measurement of gene expression in tissues. In this study, differing methods of RNA purification were applied to remove DSS inhibition. Because no purification methods were 100% effective in alleviating this interference, caution should be exercised when interpreting gene expression results from studies where animals are exposed to DSS-induced colitis.

Oral gamma-cyclodextrin-encapsulated tributyrin supplementation in young pigs with experimentally induced colitis.

Disruption of intestinal integrity and barrier function due to tissue inflammation has negative implications on overall growth and well-being in young pigs. In this study, we investigated the effects of oral gamma-cyclodextrin-encapsulated tributyrin (TBCD) in young pigs experiencing dextran sodium sulfate (DSS)-induced colitis. Pigs (n = 32 boars) were weaned from the sow at postnatal day (PND) 2, allotted to treatment based on the litter of origin and body weight (BW), and reared artificially over a 26-d feeding period. Treatment groups included: 1) nutritionally adequate (control) milk replacer, no DSS (Control n = 8), 2) control milk replacer plus oral DSS (DSS, n = 7), and 3) control diet supplemented with 8.3 g of TBCD per kg of reconstituted milk replacer plus oral DSS (TBCD + DSS, n = 8). Colitis was induced by administering DSS at 1.25 g of DSS/kg BW daily in a reconstituted milk replacer from PND 14-18. Milk replacer and water were provided ad libitum throughout the 26-d study. All the data were analyzed using a one-way ANOVA using the MIXED procedure of SAS. Control and DSS pigs had similar BW throughout the study, while TBCD + DSS pigs exhibited decreased (P < 0.05) BW starting at approximately PND 15. Additionally, average daily gain (ADG) before and after initiation of DSS dosing, along with over the total study duration, was decreased (P < 0.05) in pigs receiving TBCD + DSS compared with the Control. Milk disappearance was decreased (P < 0.05) in TBCD + DSS pigs when compared with Control and DSS groups. Both the concentration and molar ratio of cecal butyrate concentrations were increased (P < 0.05) in TBCD + DSS pigs compared with the Control group. The DSS and TBCD + DSS treatments also increased (P < 0.05) butyrate concentrations in the luminal contents with the proximal colon compared with Control. TBCD + DSS and DSS pigs had increased (P < 0.05) mucosal width in the distal colon compared with Control, thereby indicating heightened intestinal inflammation. Overall, oral supplementation of encapsulated tributyrin increased the concentration of butyrate in the colon, but was unable to mitigate the negative effects of DSS-induced colitis.

There are negative implications in young pigs when the integrity and function of the intestine are disrupted due to colonic inflammation. Volatile compounds have been used as dietary supplements to alleviate intestinal inflammation, but little work has been completed on the use of encapsulated tributyrin in newly weaned pigs. In this study, pigs received 1 of 3 treatments: 1) a standard milk replacer without the induction of intestinal inflammation, 2) the same standard milk replacer with the induction of intestinal inflammation, or 3) milk replacer supplemented with encapsulated tributyrin with the induction of intestinal inflammation. Throughout the study period, growth performance was decreased in pigs receiving supplemental tributyrin compared with other treatments. Additionally, experimentally induced colitis increased butyrate concentrations in the cecum, while tributyrin supplementation increased butyrate concentrations in the proximal colon. Pigs undergoing intestinal inflammation had increased thickness of the mucosal layer in the distal colon compared with sham-challenged pigs. Overall, the supplementation of encapsulated tributyrin increased colonic butyrate concentrations, but did not mitigate the negative effects of inflammation in the large intestine.