A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 pl from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 ,ug/,ul, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 ,ug/ml for the commercial product and 0.8 to 51.7 1g/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 ,Ig/ml), and RNase could not be inhibited at all (MIC, >7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 ILg of the standard humic acids per ml and 0.24 to 0.48 ,ug of the coextracted humic acids per ml. The addition ofT4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 ,ug/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 ,ug and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUNI, at concentrations of 100 ;ig/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids. Recovery rates of soil-seeded microorganisms, carrying a mammal-derived DNA sequence as a marker gene, were 72 to 79%o for Corynebacterium glutamicum ATCC 13032 pUNI, 81 to 85% for E. coli DH5-a pUN1, and 86 to 92% for Hansenula polymorpha LR9-Apr8, compared with that for DNA extracted from the respective pure cultures. Soil-extracted DNA was pure enough to detect 105 C. glutamicum pUN1 cells per g of soil by transformation ofE. coli HB101, 104 cells ofH. polymorpha per g of soil by slot blot DNA-DNA hybridizations, and 10 cells of H. polymorpha per g of soil by polymerase chain reaction amplification of the mammalian marker gene.

The gastrointestinal tract is a dynamic ecosystem containing a complex microbial community. In this paper, the indigenous intestinal bacteria and the microbial fermentation profile particularly short chain fatty acids (SCFA), lactate, and ammonia concentrations are reviewed. The intestinal bacterial composition changes with age. The bacterial density of the small intestine increases with age and comprises of lactobacilli, streptococci, enterobacteria, fusobacteria and eubacteria. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium spp., Clostridium spp., Lactobacillus spp., Fusobacterium spp. and Bacteroides) are predominating caecal bacteria in young broilers. Data from culture-based studies showed that bifidobacteria could not be isolated from young birds, but were recovered from four-week-old broilers. Caecal lactobacilli accounted for 1.5-24% of the caecal bacteria. Gene sequencing of caecal DNA extracts showed that the majority of bacteria belonged to Clostridiaceae. Intestinal bacterial community is influenced by the dietary ingredients, nutrient levels and physical structure of feed. SCFA and other metabolic products are affected by diet formulation and age. Additional studies are required to know the bacterial metabolic activities together with the community analysis of the intestinal bacteria. Feed composition and processing have great potential to influence the activities of intestinal bacteria towards a desired direction in order to support animal health, well-being and microbial safety of broiler meat.

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Medium-chain fatty acids (MCFAs) are found at higher levels in milk lipids of many animal species and in the oil fraction of several plants, including coconuts, palm kernels and certain Cuphea species. Medium-chain triglycerides (MCTs) and fatty acids are efficiently absorbed and metabolized and are therefore used for piglet nutrition. They may provide instant energy and also have physiological benefits beyond their energetic value contributing to several findings of improved performance in piglet-feeding trials. MCTs are effectively hydrolyzed by gastric and pancreatic lipases in the newborn and suckling young, allowing rapid provision of energy for both enterocytes and intermediary hepatic metabolism. MCFAs affect the composition of the intestinal microbiota and have inhibitory effects on bacterial concentrations in the digesta, mainly on Salmonella and coliforms. However, most studies have been performed in vitro up to now and in vivo data in pigs are still scarce. Effects on the gut-associated and general immune function have been described in several animal species, but they have been less studied in pigs. The addition of up to 8% of a non-esterified MCFA mixture in feed has been described, but due to the sensory properties this can have a negative impact on feed intake. This may be overcome by using MCTs, allowing dietary inclusion rates up to 15%. Feeding sows with diets containing 15% MCTs resulted in a lower mortality of newborns and better development, particularly of underweight piglets. In conclusion, MCFAs and MCTs offer advantages for the improvement of energy supply and performance of piglets and may stabilize the intestinal microbiota, expanding the spectrum of feed additives supporting piglet health in the post-weaning period.

Weaned piglets were fed diets containing 57 (low) or 2425 (high) mg kg(-1) analytical grade ZnO for a period of 5 weeks. Intestinal contents were sampled in weekly intervals and analyzed for bacterial cell numbers and main bacterial metabolites. The most severe effects of high dietary zinc were observed 1 week after weaning in the stomach and small intestine. Pronounced reductions were observed for Enterobacteriaceae and the Escherichia group as well as for Lactobacillus spp. and for three of five studied Lactobacillus species. The impact of high dietary zinc diminished for enterobacteria with increasing age, but was permanent for Lactobacillus species. Bifidobacteria, enterococci, streptococci, Weissella spp. and Leuconostoc spp. as well as the Bacteroides-Prevotella-Porphyromonas group were not influenced by high dietary zinc throughout the trial. High dietary zinc reduced bacterial metabolite concentrations and increased molar acetate ratios at the expense of propionate in the proximal intestine, but differences diminished in older animals. Lower lactate concentrations were observed in the high dietary zinc group throughout the feeding trial. This study has shown that the application of dietary zinc at high concentrations leads to transient and lasting effects during the development of the intestinal microbiota, affecting composition as well as metabolic activity.