The aim of this study was to assess the effects of 2 different phytogenic products on performance, ileal nutrient digestibility, and composition of the intestinal microbiota. The 2 phytogenic products contained different essential oil mixtures (EOM) characterized by either menthol (Mentha arvensis; EOM-M) or cinnamon aldehyde (Cinnamomum aromaticum; EOM-C) as main constituents. Three treatments consisted of control diet without EOM addition and diets supplemented with EOM-M or EOM-C. Reproducibility of the effects was examined in 4 trials with a total of 300 male castrated and female piglets weaned at 25 d of age. The number of pens per treatment in trials I through III were 7, 9, and 9, respectively, for research station conditions, and 10 in trial IV for simulated farm conditions. In research station conditions, the experimental unit consisted of flat deck pens with 2 piglets per pen, whereas it consisted of floor pens with straw bedding with 5 piglets per pen in farm conditions. The feed additives had no effect on feed intake or BW gain. Improvements (P < 0.05) in G:F were observed for EOM-M supplemented diets in 2 of 4 trials as well as for the combined data of all trials. These improvements were associated with greater (P<0.05) apparent ileal digestibility of CP and of most AA. The effect of EOM-C on these response criteria was intermediate between control and EOM-M. Real-time PCR analysis of the gastrointestinal contents for 7 bacterial groups (Lactobacillus spp., Enterococcus spp., Clostridium coccoides and Clostridium leptum cluster, Escherichia spp., and Escherichia coli toxin estII) indicated no effect of treatments on the gastrointestinal microbiota. It was concluded that EOM-M consistently improved feed efficiency in weaned piglets, and it was associated with improved ileal protein and AA digestibility. In general, however, the effectiveness of EOM as feed additives differs considerably depending on the constituents.

An interdisciplinary research group granted by the German Research Foundation (FOR 438) tested various hypotheses and tired to develop a model for the mode of action of probiotics in pigs. The study included the fields of animal nutrition/digestion physiology, anatomy and histology of the intestinal mucosa, transport and secretory properties of the mucosa, microbiology of the intestinal tract, immune system (classes of intraepithelial lymphocytes, Immoral responses), gene expression of the mucosa and finally the in vitro and in vivo resistance against infection with Salmonella.Five trials with ten sows per treatment each and their piglets and two probiotic strains were included in this study. The studied bacterial strains were Enterococcus faecium NCIMB 10415 and Bacillus cereus var. toyoi NCIMB 40112.Concluding from our studies and the published data of others, the effects of probiotics on performance are rarely significant. However, with one exception the incidence of post-weaning diarrhoea under the effect of both probiotics was significantly reduced in the trials of the research group. Furthermore, the identification frequency of various E. coli sero-pathovars relevant in post weaning diarrhoea was reduced in these animals. On the other hand, no significant modifications were found for the morphology and histology of the intestinal mucosa and also not on transport properties of this tissue. A further important finding was that the mode of action for probiotics is not unique but species or even strain specific. Most probably the studied probiotics act directly and/or via modifications of the intestinal microbiota on the immune system (intraepithelial lymphocyte population).

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.

1. Three bacterial phytases derived from Bacillus, Escherichia coli or Klebsiella were compared with a phytase derived from Aspergillus niger in vitro and in vivo. 2. The in vitro results indicated that Aspergillus, E. coli and Klebsiella phytase displayed their activity optima in an acid pH range while Bacillus phytase did so in neutral pH. 3. The trials also revealed that only Bacillus phytase is more resistant to heat treatments, while E. coli and Klebsiella phytases are more stable against proteolytic inactivation. 4. In vivo phytases derived from Aspergillus, Bacillus, E. coli, Klebsiella or a combination of Bacillus and E. coli improved the utilisation of phosphorus (P balance) significantly to 0·54, 0·54, 0·55, 0·55 or 0·58, respectively, compared to 0·42 in the negative control. 5. The phytases used in this study seemed to be equally effective in improving P utilisation regardless of proposed intestinal site of activity. Combination of phytases acting in the gizzard with phytases acting in the intestine seems to be a promising way to further improving in vivo efficacy of phytases in poultry.

Weaned piglets were fed a wheat based diet either non-supplemented or supplemented with a multi-enzyme preparation or a xylanase mono-enzyme preparation, respectively. Both enzyme preparations increased live weight gain nonsignificantly, but only animals of the xylanase group showed a trend (p = 0.076) for an improved feed conversion. Only precaecal digestibility of total amino acids was improved significantly when the mono-enzyme preparation was added. Improvements of digestibility of crude fat, crude protein and starch did not reach the significance level. Both enzyme preparations reduced jejunal viscosity, however viscosity in the colon was only reduced by the mono-enzyme preparation. Both enzymes significantly reduced Lactobacillus spp. cell numbers as well as bacterial metabolites in the stomach and showed similar nonsignificant modifications in jejunum contents except for acetate in the mono-enzyme group. Total jejunal bile acids were unchanged. Compared to the control, the ratio of the main conjugated to the main deconjugated bile acid was significantly higher in the mono-enzyme group. This study has shown that the mono- and multi-enzyme preparation can lead to improved performance in wheat based diets for piglets. Like in poultry, the main mode of action seems to be the reduction of small intestinal viscosity. However, the generation of fermentable carbohydrates by the multi-enzyme preparation may mask beneficial effects on performance due to the development of an active bile acid deconjugating microbiota in the small intestine.

Two probiotics of different ecological origin, Enterococcus faecium NCIMB 10415 and Bacillus cereus var. toyoi, were chosen as model organisms. Feed for sows during gestation/lactation and for piglets pre-/postweaning was supplemented with either of these probiotics. To evaluate the effect of different starting points of E. faecium NCIMB 10415 initiations the probiotic was administered to piglets of sows, which have not received the probiotic, from birth onwards or just postweaning. Here we report the impact of these variants on probiotic distribution in the gut, on the gut microbiota, on diarrhea and on performance. Both probiotic strains were detected immediately after the start of the supplementation in feces of sows and piglets. The vertical transfer of both probiotic strains with sow feces to piglets could be demonstrated already before suckling piglets had access to the supplemented diets. Both probiotics were recovered from all intestinal segments of piglets. The dominant autochthonous colonic microbiota of young piglets as revealed by denaturing gradient gel electrophoresis was more similar within than between treatment groups (control vs. probiotic). Both probiotics reduced the incidence of postweaning diarrhea (p<0.05). For the E. faecium probiotic the relative magnitude of this effect was largely independent of dietary probiotic concentration or starting time of supplementation. Significant overall influence on piglet performance was observed only with the B. cereus probiotic.

The effect of two microbial phytases of fungal origin (Peniophora and Consensus phytase) were evaluated for their effectiveness in improving performance and digestibility of P and Ca. A total of 44 multiparous sows were used during gestation (days 28 to 69) and lactation (days 1 to 19). Gestating and lactating sows were fed adequate P (9.9 and 27 g P per day) or low P diets (7.6 and 15.9 g per day) with or without supplementation of Peniophora (pregnancy) or Consensus phytase (lactation) providing 500, 750 and 1000 or 500 and 1000 units per kg diet, respectively. The obtained data generally indicated that graded levels of both phytases were effective in improving apparent phosphorus digestibility during gestation or lactation. The additional phosphorus release achieved when administering the recommended supply of Peniophora phytase (750 PPU/kg feed) was 0.1 g/kg feed in gestating sows, which is equivalent to 0.6 g of monocalcium-phosphate. Consensus phytase administered at the recommended dosage of 500 units per kg for lactating sows provided a release of 0.6 g P per kg feed from phytate, which corresponds to 2.5 g monocalcium-phosphate. The effectiveness of the studied phytases in pregnant or lactating sows is comparable to that in growing pigs.

Two 6-week feeding trials were conducted on a total of 112 newly weaned piglets to examine the recently reported growth promoting effects of dietary rare earth elements (REE) in European pig production. Rare earth element-diets were supplemented with a REE-citrate premix of lanthanum and the light lanthanoides cerium, praseodymium and neodymium at 200 mg/kg for 6 weeks after weaning. Overall for both trials, growth performance of REE-citrate and control fed piglets did not differ significantly (p > 0.05). An early enhancive tendency for REE-citrate in trial 1 (feed conversion ratio, FCR -3%, p = 0.15) proved irreproducible in trial 2. In the late period of trial 1, in-feed addition of REE-citrate significantly impaired piglet performance (FCR + 8%, p = 0.01). A cultivation-independent molecular approach, polymerase chain reaction-denaturing gradient gel electrophoresis was further applied to assess REE induced alterations in the predominant faecal microbiota from weaning pigs. Calculation of various ecological characteristics does not indicate (p > 0.05) an often discussed selective effect on local microbial composition of dietary REE.

Media with 1,3-1,4-beta -glucans as selective markers were used for isolation of non-starch-polysaccharide (NSP) degrading bacteria from the intestinal tract of broiler chicken. Formerly unknown 1,3-1,4-beta endoglucanase activities in various bacterial species were identified in this study. E. faecium , Streptococcus , Bacteroides and Clostridium strains seem to be responsible for degradation of mixed linked beta -glucans in the small intestine and in the hind gut of chickens. Strict anaerobic bacteria (Bacteroides ovatus , B. uniformis , presumably B. capillosus and Clostridium perfringens ) as well as an unidentified bacterium with 98% 16S rDNA homology to an uncultered chicken cecum bacterium were isolated. Additionally, Streptococcus bovis with 1,3-1,4-beta -endoglucanase activity was also detected. Different 1,3-1,4-beta -endoglucanase activity profiles were observed in SDS/PAGE zymograms.

rRNA extracts from the jejunum of rearing piglets fed a wheat/rye based diet with or without a xylanase feed additive were hybridized against eight Lactobacillus spp. specific digoxigenin labelled oligonucleotide probes. Known concentrations of sample- or culture RNA extracts were used to calibrate and quantify hybridization signals from different sample extracts and hybridizations. The xylanase feed additive tended to increase total eubacterial rRNA in the terminal jejunum, probably a result of increased Lactobacillus spp. rRNA in the central and terminal jejunum. However, individual animal variations were high. L. reuteri was the major Lactobacillus species followed by L. acidophilus and L. mucosae. L. amylophilus and L. crispatus were not detected in all RNA extracts. The results of this study show that the xylanase feed additive modifies Lactobacillus spp. populations on the species level in piglets in addition to their well known effects on digesta viscosity and nutrient digestibility. The implications of these effects for the intestinal microbiota require further analysis.

As part of an interdisciplinary research project, the performance response of sows and their litters to the probiotic strain Enterococcus faecium NCIMB 10415, as well as some health characteristics of the piglets, were studied. Gestating sows (n = 26) were randomly allotted into 2 groups. The probiotic was administered by dietary supplementation to 1 group of sows and their respective litters (probiotic group), whereas the second group (control group) received no probiotic supplementation. The duration of the treatment was nearly 17 wk for sows (d 90 ante partum until d 28 postpartum) and 6 wk for piglets (d 15 to 56). Body weight and feed consumption were recorded weekly. The frequency of 4 toxin and 5 adhesion genes of putative pathogenic Escherichia coli was monitored weekly (d 7 to 35) by multiplex PCR assays, and fecal consistency of weaned piglets was studied daily. Probiotic treatment of lactating sows led to an overall pre-weaning mortality of 16.2% compared with 22.3% in the control group (P = 0.44). Animal losses during the first 3 d of the suckling period were decreased in the probiotic group (P = 0.09). For piglets (n = 153), which were weaned at 28 d, there were no overall treatment differences in BW gain, feed intake, or feed efficiency. Probiotic supplementation, however, led to nearly a 40% reduction (P = 0.012). The actual percentage of piglets with postweaning diarrhea in the probiotic group was 21% compared with 38% in the control group (P = 0.05). The study on virulence factors of dominant fecal E. coli isolates revealed a high diversity with varying frequency and distribution of each single pathogenicity gene. The 440 isolates carried 29 different pathogenicity gene combinations as well as each of the 9 pathogenicity genes alone. Altogether, isolates with more than 2 pathogenicity genes were quite rare (< or = 10%), and up until d 28 isolates without any pathogenicity gene occurred most frequently. Depending on the time of sampling, one-third or more of all isolates contained est2 or est1b as single gene or in combination with other pathogenicity genes.

Several non-starch polysaccharide (NSP)-degrading enzymes were studied for their suitability as feed enzymes in combination with soya bean meal (SBM) in broiler nutrition. In vitro experiments with SBM and several enzymes isolated from an Aspergillus aculeatus multi-enzyme preparation or respective enzyme specificities showed that only enzymes of the 1,4-beta-arabinogalactanase type led to effective solubilization of dry matter from SBM, while other enzymes needed very high concentrations to invoke this effect. 1,4-beta-galactomannanase activity also showed a high rate of sugar release from SBM, but the release of sugars from treated SBM (simulated stomach digestion with pepsin) was reduced, while an 1,4-beta-arabinogalactanase from Hunficola insolens still led to a high release of sugars on the same substrate. Synergistic effects on the release of sugars were observed when both galactanase and galactomannanase enzymes were used. Synergistic effects of galactanase and mannanase were also noted for two different endopolygalacturonases, partly purified from the multi-enzyme preparation. In feeding trials with broilers, increasing concentrations of the multi-enzyme preparation with numerous NSP-degrading enzymes showed beneficial effects on feed intake, but high in feed concentrations were needed. Further feeding trials with the galactanase preparation from H. insolens alone and in combination with a mannanase preparation (Gammanase) were conducted. However, due to weak in vivo stability of the galactanase, only a small effect on feed conversion was noticed. Additional supplementation of the mannanase reduced live weight gain and feed intake. It is concluded that enzymes of the 1,4-beta-arabinogalactanase type are the most likely candidates for a SBM-specific feed enzyme in broiler nutrition, possibly in combination with endopolygalacturonases. However, while a partial degradation of soya NSP may enhance digestibility, a high degree of hydrolysis may produce oligo- and monomers with anti-nutritive effects such as osmotic pressure or microbial fermentation in the small intestine. (c) 2005 Elsevier B.V. All rights reserved.

As part of an interdisciplinary research project, we studied the performance response of sows and their litters to the probiotic strain Bacillus cereus var. toyoi as well as feces consistency of piglets. Gestating sows ( n = 26) were randomly allotted into two groups. The probiotic B. cereus var. toyoi was administered by dietary supplementation to one group of sows and their respective litters (probiotic group) whereas the second group (control group) received no probiotic supplementation. The duration of the application was nearly 17 weeks for sows (day 90 ante partum until day 28 post partum) and six weeks for piglets (day 15-56). Piglets were weaned after 28 days. Body weight and feed consumption were recorded weekly and fecal consistency of weaned piglets was studied daily. B. cereus var. toyoi was recovered from feces of sows and piglets as well as from digesta of piglets in the probiotic group, while being absent from all samples of control animals. In addition, the probiotic was detected in piglet feces and digesta before pre-starter feed was offered, indicating a second route of uptake besides diet. Sows of the probiotic group nursed numerically more piglets and supported a higher sum of total nursing days of all piglets within each litter than control sows ( p = 0.04). In turn, body weight (BW) up to day 35 was greater for control piglets ( p <0.01), while average daily gain and gain to feed ratio ( G: F) in weeks six and eight postweaning was higher in the probiotic group ( p<0.05). The overall G: F of the total postweaning period was 680 g/kg and 628 g/kg in the probiotic group and control group, respectively (p = 0.009). During the trial a high prevalence of liquid feces with its maximum in the second week after weaning was observed. Probiotic supplementation led to a reduction in the incidence of liquid feces and postweaning diarrhea by 38% and 59%, respectively (p<0.001).

Bacterial growth inhibiting effects of dipicolinic acid (DPA), which occurs in spores of probiotic Bacillus spp. strains, was studied with a range of Lactobacillus spp. (13), Enterococcus spp. (14), and E. coli strains (8) in vitro. In vivo effects were studied in a broiler chicken nutrition trial and DPA supplemented feed (10 g/kg).In vitro studies showed that DPA inhibited growth of most Lactobacillus spp. strains to a larger extent than E. coli strains. Enterococci were generally influenced less by DPA. However, in each group of bacteria some strains were less inhibited by DPA than others. Intestinal lactic acid bacteria in the jejunum and ileum showed a trend (P[les ]0·1) for lower counts in birds fed the DPA-supplemented feed on day 14 and 21. Counts of enterobacteria were not significantly influenced by DPA. No differences in colony counts were observed in 28-day-old animals. Digesta pH was not statistically reduced, but a lower crop pH was recorded throughout the nutrition trial.The study showed that dipicolinic acid, present in probiotic Bacillus spp. spores, is inhibitory for in vitro growth of most lactobacilli and enterobacteria, while growth of enterococci was less inhibited. Intestinal colony counts in young broiler chickens fed a DPA-supplemented diet in part mirrored in vitro results.

In this contribution compositional shifts of intestinal bacterial communities in response to different nutritional factors (enzymes, probiotics, rare earth elements) are reported, as determined with nucleic acid based methods, i.e. 16S RNA probes, denaturing gradient gel electrophoresis, Real-time PCR, Multiplex PCR. These techniques enabled the detection of a promotion of jejunal G. reuteri activity by xylanase as well as a reduction of the E. faecalis/E. faecium ratio and frequency of potentially pathogenic E. coli in piglets by probiotic supplementation. While the probotic altered the faecal microbiota of sows, an influence of REE on species composition in faeces of weaned piglets was not detectable by DGGE.