Bent Borg Jensen

Effects of Dietary Fat Source and Subtherapeutic Levels of Antibiotic on the Bacterial Community in the Ileum of Broiler Chickens at Various Ages

ABSTRACT The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed−1 and salinomycin at 40 mg kg of feed−1) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.

Microbial production of skatole in the hind gut of pigs given different diets and its relation to skatole deposition in backfat

The intestinal production of skatole and its deposition in backfat was investigated in 35 uncastrated crossbred male pigs. The pigs were fed five purified non-commercial diets containing either casein or brewers yeast slurry as protein source. The yeast slurry diet was used alone or supplemented with either wheat bran (200 g/kg), sugar-beet pulp (200 g/kg), or soya oil (100 g/kg). Skatole concentrations in backfat, and in digesta in different sections of the gastro-intestinal tract were measured at slaughter (mean weight 112 kg). There were large variations in skatole concentrations in the hind gut of different animals given the same diet. Although there was some correlation between skatole in intestinal contents and deposition in adipose tissue, there were also large variations between individual animals in their response to intestinal skatole. Nevertheless, there was a clear effect of diet on both intestinal skatole production and skatole deposition in backfat. The use of casein as a protein source decreased microbial skatole production, the total amount in the gut, and the concentration in the backfat. Addition of sugar-beet pulp to the yeast slurry diet increased microbial activity in the intestine (measured as ATP content, concentration of short-chain fatty acids, and lowering of digesta pH ). There was a decreased rate of skatole production during in vitro incubations of intestinal content, and less skatole in the hind gut and backfat. In vitro fermentations of freeze-dried Heal effluent inoculated with faecal bacteria, and addition of substrates to in vitro incubations of intestinal contents, demonstrated that tryptophan availability rather than microbial activity was the limiting factor for skatole production. The results show that skatole production depends on the amount of protein entering the hind gut and the proteolytic activity of the intestinal microbiota. Protein fermentation in the hind gut can be decreased either by using more readily digestible protein sources (for example casein rather than yeast slurry) which reduce the amount of protein passing through to the hind gut, or by adding an alternative energy source which is more readily metabolized by the hind gut microbiota (for example supplementation of the yeast slurry diet with sugar-beet pulp). This provides a basis for the rational design of diets which will decrease skatole concentrations in the carcass.

The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens

1. The influence of feed grinding (coarsely or finely ground feed) and feed form (mash or pellets) on the intestinal environment was investigated in a growth experiment with broiler chickens taking the intestinal microflora, intestinal viscosity, and the activities of pancreatic digestive enzymes into consideration. 2. As compared to mash the feeding of pellets was associated with a significantly higher body weight due to increased feed intake and improved feed utilisation. 3. Pellet-fed birds had significantly decreased gizzard weights, a higher gizzard pH and a lower intestinal pH than mash-fed birds. 4. Pellet-fed birds had significantly lower relative pancreas weights and lower activities of pancreatic digestive enzymes (amylase, lipase, chymotrypsin), which indicates the existence of a feedback mechanism, which may have been triggered by the intestinal concentration of enzymatically hydrolysed products or of the respective digestive enzymes. 5. Pellet-fed birds had larger numbers of coliform bacteria and enterococci in the ileum and a reduced number of Clostridium perfringens and lactobacilli in the distal end of the digestive tract (caeca and rectum). Microbial fermentation in terms of volatile fatty acid (VFA) concentration was found to be significantly higher in the caeca of pellet-fed birds than in mash-fed birds.

Digestion of polysaccharides and other major components in the small and large intestine of pigs fed on diets consisting of oat fractions rich in β-D-glucan

The digestibility of polysaccharides and other major components and the metabolic response of the microflora in the small and large intestines to oat diets varying in mixed linked beta(1-->3; 1-->4)-D-glucan (beta-glucan) were studied in experiments with ileum-cannulated pigs. The oat fractions for diets were prepared in a dry milling process in which oat groats were milled into two endosperm fractions (oat flour 1 and oat flour 2) and oat bran. The digestibility of polysaccharides and the metabolic response of the microflora were followed for the two contrasting diets, oat flour 1 and oat bran, from ingestion to excretion while the digestibility of oat groats and oat flour 2 were estimated only at the ileum and in faeces. There was no degradation of beta-glucan from either oat flour 1 or bran in the stomach and the first, middle and distal thirds of the small intestine (average digestibility approximately 0), while in the terminal ileum digestibility increased to 0.30 to 0.17 respectively. The digestion of starch in the first third of the small intestine was lower for the high-beta-glucan oat-bran diet (0.49) than for the low-beta-glucan flour diet (0.64). However, digestibility differences between the two diets levelled out as the digesta moved aborally in the small intestine and the digestibility at the terminal ileum was almost complete (0.970-0.995) for all diets. Oat non-starch polysaccharides (NSP) were an easily digestible energy source for the microflora in the large intestine less than 13% of dietary NSP being recovered in faeces. The bulk of beta-glucan which survived the small intestine was degraded in the caecum and proximal colon while arabinoxylan was more slowly degraded. The amount of residues passing the ileo-caecal junction has little impact on the density of micro-organisms in the large intestine, which on the flour and bran diets were in the range of 10(10)-10(11) viable counts/g digesta, but a high impact on the activity of the flora in colon. Oat bran resulted in a higher proportion of butyric acid in large intestinal content compared with the flour diet. The faecal bulking effect of oat bran was mainly caused by an increased excretion of protein and fat, presumably of bacterial origin. Of all the diets tested the oat-bran diets had the lowest digestibilities of protein and fat at the terminal ileum and in the faeces.

Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with Brachyspira hyodysenteriae

ABSTRACT Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.

Influence of dietary zinc oxide and copper sulfate on the gastrointestinal ecosystem in newly weaned piglets

ABSTRACT Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as Lactobacillus amylovorus (OTU171), Lactobacillus reuteri (OTU173), and Streptococcus alactolyticus (OTU180). The colonies on Rogosa plates were dominated by the two Lactobacillus phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO4-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO4 inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO4 was limited compared to that of ZnO.

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen

Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.

Effects of physical properties of feed on microbial ecology and survival of Salmonella enterica serovar Typhimurium in the pig gastrointestinal tract.

ABSTRACT A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of Salmonella enterica serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (P < 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of S. enterica serovar Typhimurium DT12 in content from the stomach (P < 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (P < 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of S. enterica serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (P < 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (P < 0.05) and colon (P < 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of Salmonella during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.

Enteral feeding induces diet-dependent mucosal dysfunction, bacterial proliferation, and necrotizing enterocolitis in preterm pigs on parenteral nutrition

Preterm neonates have an immature gut and metabolism and may benefit from total parenteral nutrition (TPN) before enteral food is introduced. Conversely, delayed enteral feeding may inhibit gut maturation and sensitize to necrotizing enterocolitis (NEC). Intestinal mass and NEC lesions were first recorded in preterm pigs fed enterally (porcine colostrum, bovine colostrum, or formula for 20-40 h), with or without a preceding 2- to 3-day TPN period (n = 435). Mucosal mass increased during TPN and further after enteral feeding to reach an intestinal mass similar to that in enterally fed pigs without TPN (+60-80% relative to birth). NEC developed only after enteral feeding but more often after a preceding TPN period for both sows colostrum (26 vs. 5%) and formula (62 vs. 39%, both P < 0.001, n = 43-170). Further studies in 3-day-old TPN pigs fed enterally showed that formula feeding decreased villus height and nutrient digestive capacity and increased luminal lactic acid and NEC lesions, compared with colostrum (bovine or porcine, P < 0.05). Mucosal microbial diversity increased with enteral feeding, and Clostridium perfringens density was related to NEC severity. Formula feeding decreased plasma arginine, citrulline, ornithine, and tissue antioxidants, whereas tissue nitric oxide synthetase and gut permeability increased, relative to colostrum (all P < 0.05). In conclusion, enteral feeding is associated with gut dysfunction, microbial imbalance, and NEC in preterm pigs, especially in pigs fed formula after TPN. Conversely, colostrum milk diets improve gut maturation and NEC resistance in preterm pigs subjected to a few days of TPN after birth.

Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs

Necrotizing enterocolitis (NEC) remains the most severe gastrointestinal disorder in preterm infants. It is associated with the initiation of enteral nutrition and may be related to immature carbohydrate digestive capacity. We tested the hypothesis that a formula containing maltodextrin vs. a formula containing lactose as the principal source of carbohydrate would predispose preterm pigs to a higher NEC incidence. Cesarean-derived preterm pigs were given total parenteral nutrition for 48 h followed by total enteral nutrition with a lactose-based (n = 11) or maltodextrin-based (n = 11) formula for 36 h. A higher incidence (91% vs. 27%) and severity (score of 3.3 vs. 1.8) of NEC were observed in the maltodextrin than in the lactose group. This higher incidence of NEC in the maltodextrin group was associated with significantly lower activities of lactase, maltase, and aminopeptidase; reduced villus height; transiently reduced in vivo aldohexose uptake; and reduced ex vivo aldohexose uptake capacity in the middle region of the small intestine. Bacterial diversity was low for both diets, but alterations in bacterial composition and luminal concentrations of short-chain fatty acids were observed in the maltodextrin group. In a second study, we quantified net portal absorption of aldohexoses (glucose and galactose) during acute jejunal infusion of a maltodextrin- or a lactose-based formula (n = 8) into preterm pigs. We found lower net portal aldohexose absorption (4% vs. 42%) and greater intestinal recovery of undigested carbohydrate (68% vs. 27%) in pigs acutely perfused with the maltodextrin-based formula than those perfused with the lactose-based formula. The higher digestibility of the lactose than the maltodextrin in the formulas can be attributed to a 5- to 20-fold higher hydrolytic activity of tissue-specific lactase than maltases. We conclude that carbohydrate maldigestion is sufficient to increase the incidence and severity of NEC in preterm pigs.