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Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.

The synthetic disaccharide lactulose is known to improve the intestinal microflora by stimulating the growth of selected probiotic bacteria in the gut. In our experiment the effects of lactulose in combination with the probiotic bacteria Enterococcus faecium on growth performance and morphology of the bovine intestine were examined. Calves aged 39^2 days were randomised to three feeding groups (no. ¼ 14 each group): control (L0), fed milk replacer (MR) containing E. faecium; a lactulose group (L1) containing additional 1% lactulose and a second lactulose group (L3) containing 3% lactulose dry matter. The calves were weighed weekly. After 19 weeks the calves were slaughtered and tissues were collected for histological studies. The average daily live weight gain tended to be higher (P , 0.1) for L3 (1350 g/day) than L0 (1288 g/day). Compared with L0, a reduction (P , 0.001) of ileal villus height due to lactulose treatment of approximately 14% in group L1 and 20% in L3 was determined. A significant decrease in the depth of the crypts about 12% in L1 and 8% in L3 was detected in the caecum. The surface area of lymph follicles from Peyer's patches was decreased by lactulose treatment. Results show that lactulose has an effect on the morphology of intestine. A significant effect on growth performance can not be confirmed. However, results permit the conclusion that lactulose feeding has the tendency to increase growth performance.

Prebiotics and probiotics could represent an effective alternative to the use of synthetic antibiotics in nutrition. The mechanisms by which prebiotics affect the immune system have not yet been investigated in detail. Most effects have been attributed to increases in the innate and acquired immune responses. This study was conducted to elucidate the long-term effects of orally administered lactulose on the immune response in the intestinal tract of probiotic-fed calves. Preruminant calves were randomized to 3 feeding groups: milk replacer containing 1) no lactulose, 2) 1% lactulose, or 3) 3% lactulose. All 3 milk replacers contained 10(9) cfu Enterococcus faecium/kg. Messenger RNA expression of different cell activation markers, pro- and antiinflammatory cytokines, and IgA Fc receptor was investigated in the ileum, mesenterial lymph node, spleen, and white blood cells. A significantly greater number of blood lymphocytes were detected in the 3% lactulose group (P = 0.02) than in the control group. The expression results in male calves indicated that the transcription of IgA Fc receptor in the ileal mucosa of the 1% lactulose treatment group increased significantly (P = 0.04) and also tended to increase in the 3% lactulose group (P = 0.07). Furthermore, decreases in IL-10 and interferon-gamma mRNA expression were observed in the ileum (P = 0.04). The CD4-presenting lymphocytes were decreased significantly in the ileum (P = 0.04) and mesenteric lymph node (P = 0.01), whereas CD8-presenting lymphocytes were increased in the blood (P = 0.03) of females. Other proinflammatory cytokines (IL-1beta, IL-8, and tumor necrosis factor-alpha) and antiinflammatory cytokines (transforming growth factor-beta1) did not show significant differences in mRNA expression among treatment groups. The results indicate that additional lactulose feeding had an immunomodulatory effect on the composition of T-cell subsets in different immune compartments and had minor effects on pro- and antiinflammatory cytokine mRNA expression.

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ABSTRACT:The study was conducted to elucidate the effects of orally administered lactulose in combination with Enterococcus faecium on immune response of the intestinal tract in pre-ruminant calves. The mRNA expression of pro-and anti-inflammatory cytokines and proliferation and apoptosis markers were investigated in jejunum, ileum, colon and caecum. Simmental calves were fed diets containing 1% (L1) or 3% (L3) lactulose and the probiotic strain of the genus E. faecium, and compared with a non treated control group. Primarily the high dose feeding with lactulose showed an effect on several mRNA gene expression parameters. In the jejunum a down-regulation of the anti-apoptotic marker Bcl-xl was determined and IL-10 mRNA gene expression was 2.6-fold up-regulated (P < 0.05). In the colon a 1.9-fold (P < 0.05) up-regulation of IL-10 and only in caecum an about 2-fold increase of TGF-β 1 (P < 0.05) was found for both lactulose feedings. Caspase 3 was up-regulated in caecum only in the 3% lactulose treated group (P < 0.05). The enhanced apoptotic rate of caspase 3 seems to be associated with a decrease in crypth depth due to lactulose supplementation. The results indicated that mainly the high 3% lactulose dose in probiotic-fed calves has an affect on the intestinal immune function and on diverse apoptotic markers.