J. Zentek

Effects of Dietary Inulin on the Intestinal Short Chain Fatty Acids and Microbial Ecology in Broiler Chickens as Revealed by Denaturing Gradient Gel Electrophoresis

Inulin can stimulate the growth of the intestinal bacteria as well as alter the ratio among various short chain fatty acids (SCFA) produced. In the present study, we analyzed the effect of dietary inulin on the intestinal bacterial community as determined by denaturing gradient gel electrophoresis analysis of universal 16S rDNA after amplication with PCR and SCFA profile. Broilers were fed a diet primarily composed of corn-soybean meal or same diet with 1% inulin for 42 d. The relative weight of digesta-filled ceca of the inulin-fed group was higher (P<0.01) than in the control group. Amongst SCFA, only acetate could be detected in the jejunal digesta, which tended to be higher (P=0.09) in inulin-fed group compared with the control group. Inulin did not affect the total concentration of SCFA in the cecal digesta. The relative proportion of n-butyrate was elevated (P=0.05) with a concomitant decrease in the concentration of n-valerate (P<0.05) in the inulin-fed group compared with the control group. Dietary inulin did not affect the number of PCR-denaturing gradient gel electrophoresis bands nor their diversity in the jejunal and cecal digesta. Intragroup similarities were not different between the groups, nor were any differences between intra-and intergroup similarities in the jejunal and cecal samples. In conclusion, inulin altered the cecal microbial metabolic activity without any major impact on the composition of intestinal bacterial communities as measured by the present techniques.

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography-mass spectrometry.

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI-) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.

The Impact of the Fusarium Mycotoxin Deoxynivalenol on the Health and Performance of Broiler Chickens

The aim of the present experiment was to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on morphometric indices of jejunum and to follow the passage of deoxynivalenol (DON) through subsequent segments of the digestive tract of broilers. A total of 45 1-d-old broiler chickens (Ross 308 males) were randomly allotted to three dietary treatments (15 birds/treatment): (1) control diet; (2) diet contaminated with 1 mg DON/kg feed; (3) diet contaminated with 5 mg DON/kg feed for five weeks. None of the zootechnical traits (body weight, body weight gain, feed intake, and feed conversion) responded to increased DON levels in the diet. However, DON at both dietary levels (1 mg and 5 mg DON/kg feed) significantly altered the small intestinal morphology. In the jejunum, the villi were significantly (P < 0.01) shorter in both DON treated groups compared with the controls. Furthermore, the dietary inclusion of DON decreased (P < 0.05) the villus surface area in both DON treated groups. The absolute or relative organ weights (liver, heart, proventriculus, gizzard, small intestine, spleen, pancreas, colon, cecum, bursa of Fabricius and thymus) were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls. DON and de-epoxy-DON (DOM-1) were analyzed in serum, bile, liver, feces and digesta from consecutive segments of the digestive tract (gizzard, cecum, and rectum). Concentrations of DON and its metabolite DOM-1 in serum, bile, and liver were lower than the detection limits of the applied liquid chromatography coupled with mass spectrometry (LC-MS/MS) method. Only about 10 to 12% and 6% of the ingested DON was recovered in gizzard and feces, irrespective of the dietary DON-concentration. However, the DON recovery in the cecum as percentage of DON-intake varied between 18 to 22% and was not influenced by dietary DON-concentration. Interestingly, in the present trial, DOM-1 did not appear in the large intestine and in feces. The results indicate that deepoxydation in the present study hardly occurred in the distal segments of the digestive tract, assuming that the complete de-epoxydation occurs in the proximal small intestine where the majority of the parent toxin is absorbed. In conclusion, diets with DON contamination below levels that induce a negative impact on performance could alter small intestinal morphology in broilers. Additionally, the results confirm that the majority of the ingested DON quickly disappears through the gastrointestinal tract.

Effects of B-trichothecenes on luminal glucose transport across the isolated jejunal epithelium of broiler chickens.

Trichothecenes are closely-related sesquiterpenoids (ring structure) with a 12, 13 epoxy ring and a variable number of hydroxyl, acetyl or other substituents. In chickens, D-glucose and amino acid absorption occurs via carrier-mediated transport. Recently, it has been observed that deoxynivalenol (DON) alters the gut function and impairs glucose and amino acid transport in chickens. The purpose of this work was to determine the effects of different B-trichothecenes [DON, Nivalenol (NIV), 15-Ac-DON and Fusarenon X (FUS X)] on intestinal carrier-mediated sodium co-transport of D-glucose in the small intestine of broiler chickens. Intestinal transport was determined by changes in the short-circuit current (Isc), proportional to ion transmembrane flux, in the middle segment of the jejunum of broilers with the Ussing chamber technique. D-glucose produced an increase of the Isc, and this effect was reverted by different B-trichothecene mycotoxins, indicating that the glucose induced Isc was altered by B-trichothecenes. The addition of glucose after pre-incubation of the tissues with B-trichothecenes had no effect (p > 0.05) on the Isc, suggesting that B-trichothecenes afflicted the Na(+)-D-glucose co-transport. However, FUX had no obvious effect on the measured parameters. It could be concluded from the present study that the glucose co-transporter activity appears to be more sensitive to DON, NIV and 15-Ac-DON suppression than by FUS X in the jejunum of broilers.

Effects of a probiotic Lactobacillus acidophilus strain on feed tolerance in dogs with non-specific dietary sensitivity.

This study investigated the effects of the probiotic Lactobacillus acidophilus strain DSM 13241 in dogs with non-specific dietary sensitivity (NSS). Six adult German Shorthair Pointers with NSS consecutively received a control dry diet and the same diet supplemented with the probiotic (6 · 106 cfu/g) for 12 weeks each, followed by another control period of four weeks. Frequency of defecations, faecal quality and nutrient digestibility were determined. Faeces were cultured for Clostridium perfringens, Escherichia spp., lactobacilli and bifidobacteria and quantitative fluorescence in situ hybridisation (FISH) was performed. Feeding the probiotic improved faecal consistency, faecal dry matter and defecation frequency (p < 0.05). Faecal concentrations of culturable lactobacilli and bifidobacteria increased numerically, but not significantly, also the decrease of the numbers of C. perfringens and Escherichia spp. did not reach the level of significance. Results from FISH showed more constant bacterial populations. It can be concluded that L. acidophilus DSM 13241 can stabilise the digestive processes in dogs with NSS.

Cytotoxicity and metabolic stress induced by deoxynivalenol in the porcine intestinal IPEC-J2 cell line

The digestive tract is a target for the Fusarium toxin deoxynivalenol (DON), a major cereal grain contaminant of animal and public health concern. Toxic effects of DON range from diarrhoea, vomiting and gastrointestinal inflammation to necrosis of several tissues. Following ingestion of contaminated food or feed, intestinal epithelial cells are exposed to a high concentration of ingested DON, potentially affecting intestinal functions. Pigs are considered to be the species most sensitive to DON toxicity. However, only few studies directly evaluated DON effects on porcine intestinal epithelial cells. Therefore, we used the porcine intestinal cell line (IPEC-J2) to assess short-term effects of DON on functional characteristics of the intestinal epithelial cells. The cytotoxic effect of DON on IPEC-J2 cells was evaluated by measuring the count of living cells and the activity of lactate dehydrogenase (LDH) released in the culture media at a DON concentration range from 0, 0.5, 2.5 and 10 μm. We demonstrated that DON at concentrations of 2.5 and 10 μm decreased significantly (p < 0.001) the cell count in a dose-dependent manner. At a concentration of 10 μm, DON caused cell damage, including rounding of cells, autolysis and cell loss from the monolayer. The mycotoxin, DON, increased LDH release into the culture medium compared with the control value. The alterations of LDH showed a good agreement with the decrease in cell count. Deoxynivalenol decreased the l-lactate concentration in the fluid supernatant of IPEC-J2 cells at 2.5 μm (p < 0.05) with a maximal effect at 10 μm of DON. To determine whether the altered lactate production may be linked to alterations of energy balance, we measured cellular ATP levels in IPEC-J2 cells. A significant decrease in ATP levels was seen at 48 h in a dose-dependent manner. It could be demonstrated that DON has a distinct cytotoxic effect on IPEC-J2 cells.

Effects of thyme as a feed additive in broiler chickens on thymol in gut contents, blood plasma, liver and muscle

BACKGROUNDnAromatic herbs as feed additives in animal production are encountering growing interest, but data on the fate of the aromatic compounds from the plant in the animal body are very scarce. In the present study, thyme (Thymus vulgaris) herb consisting of leaves and flowers without stems was used as an ingredient in the diet for broilers. The herb was fed for 35 days to five groups of broilers (0, 0.1, 0.2, 0.3, and 1% w/w in the diet). Animal performance and the concentrations of the main essential oil component from thyme, thymol, were measured in gut contents, plasma and liver and muscle tissues using solid phase microextraction and gas chromatography/mass spectrometry.nnnRESULTSnThere were no differences between the groups in feed intake, daily weight gain, feed conversion and slaughter weight. Thymol was detected in gut contents, plasma and liver and muscle tissues. Increased intestinal thymol concentrations were found in the group with 1% thyme compared with the other groups (Pu2009<u20090.05). In liver and muscle tissues the thymol levels were close to the limit of quantification.nnnCONCLUSIONnThe data do not indicate a positive effect of thyme on animal performance. With high dietary levels of thyme herb, thymol concentrations increased in gut contents and plasma but were very low in edible tissues such as liver and flesh.

Occurrence of deoxynivalenol (DON) and ochratoxin A (OTA) in dog foods

The commercially available dog food samples (29 dry foods and 11 wet foods) were analysed for deoxynivalenol (DON) and ochratoxin A (OTA) using ELISA. All (100%) dry foods were contaminated with DON with various amount of the toxin (22-1837 μg/kg). In wet food 3 samples were found to be positive for DON in the range of 95-170 μg/kg. There were a few samples contaminated with OTA: 3 samples in dry foods (7-40 μg/kg) and 2 samples in wet foods (45 and 115 μg/kg).

Effect of maternally supplied n-3 and n-6 oils on the fatty acid composition and mononuclear immune cell distribution of lymphatic tissue from the gastrointestinal tract of suckling piglets

Fatty acids are essential for immune cell function. Maternal dietary fatty acid supply influences body fat composition of their offspring. As a first step to study immunonutritional interactions at an early age of pigs, four sows were fed a diet containing sunflower oil or oil from seal blubber during pregnancy and lactation. Corresponding piglets were sacrificed at three consecutive time points in the suckling period and their mesenteric lymph nodes and spleen were analysed by gas chromatography for levels of fatty acid. At the same time mononuclear cells of these organs and of the intestinal lymphoid tissue from the jejunum were isolated and subpopulations characterised by flow cytometry. Levels of fatty acids from the lymphatic organs of the piglets were significantly influenced by the maternal diet. The concentration of the fatty acids 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the spleen and mesenteric lymph node of piglets suckled to sows of the test diet. Additionally, suckling time affected the levels of some long chain polyunsaturated fatty acids. Dietary effects were seen on some subpopulations including CD4−CD8α+ lymphocytes of the mesenteric lymph nodes and CD4+CD8α+ lymphocytes of the lamina propria, which were higher in the group fed seal blubber oil. The levels of CD21+ B-cells were higher in the group fed sunflower oil. The results indicate that the maternal diet and suckling time affect the fatty acid status of the investigated lymphatic tissues of piglets, but may have minor effects on the investigated lymphocyte subpopulations.

Phenotypic and functional aspects of the neonatal immune system as related to the maternal dietary fatty acid supply of sows

The maternal-fetal transfer and subsequent uptake of sow milk enriched with n-6- or n-3-polyunsaturated fatty acids may not only influence neonatal body fat but may also have an impact on the immune function of newborn piglets. Sows were fed a diet containing sunflower oil as n-6-source or oil from seal blubber with long chain polyunsaturated n-3-fatty acids during pregnancy and lactation. Sow serum was investigated during pregnancy and serum and milk during lactation; piglet serum and liver were investigated in the suckling period until day 19. Piglet leukocyte subpopulations were characterised by flow cytometry and leukocyte proliferation was tested after stimulation with mitogens. No differences were noted in performance. The serum and milk fatty acid status of the sows was markedly influenced by the diet. Eicosapentaenoic acid (20:5n-3), 22:5n-3 and 22:6n-3 were higher (p < 0.001) in serum and liver of piglets delivered from sows fed the seal blubber oil. Piglets at birth had lower lymphocyte counts (p < 0.01) than piglets 19 days after birth. However, no influence of feeding the different oil sources was noted on lymphocyte phenotyping and leukocyte proliferation test. The results of the present study show that the maternal diet affected the fatty acid status of neonates, but much more in the sucking period. Immunological traits were not affected, probably as the mononuclear cell lineage is too immature around birth. Effects of PUFA n-3 might only be seen at a later time point or in the polymorphonuclear cell lineage as they were dominating right after birth.