Departament de Bioquímica i Fisiologia, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona i Institut de Recerca en Nutrició i Segur tat Alimentària (INSA·UB). Departament de Nutrició, Ciències de l'Alimentació i Gastronomia, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona i INSA·UB. Departament Biologia, Sanitat i Medi Ambient, Facul tat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona. Industrial Técnica Pecuaria, Barcelona. Nutrition Animal Welfare, IRTA, Generalitat de Cata lunya. *Presenting author

A 26-day trial with 18 Pietrain×(Landrace×Duroc) pigs was conducted to investigate the effect of two dose levels of a specifically selected Bacillus spp. direct-fed microbial (DFM) product, on the emission of environmentally harmful gasses (methane, ammonia and hydrogen sulphide) from manure. Pigs were assigned to one of three treatments in a randomized complete block design according to their sex and initial BW. Each treatment contained three replications with two pigs per pen. The test treatments included a Bacillus spp. DFM containing 3×108 colony-forming unit/g, added at a low (250 mg/kg) and high (500 mg/kg) dose to an antibiotic free high fibre-based diet, and a non-supplemented control diet. Manure from pigs fed with the supplemented diets emitted lower amounts of atmospheric contaminants. The most significant reduction was observed with low DFM supplementation, in which methane and ammonia volatilization decreased (P<0.05) by >40% and 50%, respectively, on fresh weight basis in relation to the control. Microbiome analysis of manure by high through put sequencing techniques on eubacterial and archaeal 16S rRNA genes highlighted the complex interactions between indigenous gut microflora and inoculated Bacillus spp. The tested Bacillus DFM could be considered as a best available technique in reducing the environmental impacts of growing pigs fed with high fibre-based diets.

A trial was conducted to evaluate the efficacy of a carbohydrase enzyme complex under liquid feeding conditions in pigs fed a rye-based diet. One hundred and forty-four pigs (in 36 pens; 29 kg BW; 1/2 males and 1/2 females) were fed 2 diets consisting of a rye-based basal diet (Control; without enzyme) and the same diet supplemented with 100 mg/kg of an enzyme complex (Enzyme). The enzyme complex used (Ronozyme MultiGrain, DSM Nutritional Products, Basel, Switzerland) is derived from Trichoderma reesei and has endo-1,4-beta-glucanase (EC 3.2.1.4; 800 U/g), endo-1,3(4)-beta-glucanase (EC 3.2.1.6; 700 U/g), and endo-1,4-beta-xylanase (EC 3.2.1.8; 2,700 U/g) activities. Pigs' performance was evaluated for 98 d, and nutrient apparent total tract digestibility (ATTD) at 56 and 98 d of trial was evaluated using acid insoluble ash as an indigestible marker. At 56 d of trial, the basal diet contained 50% rye, and the enzyme supplementation improved (P < 0.01) ATTD for DM (79.8 vs. 83%), CP (72.8 vs. 78%), fat (61.1 vs. 68.6%), GE (79.5 vs. 83.2%), NDF (31.5 vs. 41.6%), and ADF (11.3 vs. 27.6%). At 98 d of trial, the basal diet contained 70% rye, and enzyme supplementation also improved ATTD for DM (80.4 vs. 82.1%; P < 0.01), CP (73.7 vs. 77.5%; P < 0.01), fat (51.1 vs. 57.0%; P < 0.01), GE (80.6 vs. 82.3%; P < 0.01), and ADF (17.4 vs. 24.6; P < 0.05). It is concluded that the tested enzyme complex improves nutrient ATTD of rye-based diets fed to pigs under liquid feeding.

EFSA Supporting PublicationsVolume 13, Issue 2 1001E External scientific reportOpen Access Preparatory work to support the re-evaluation of technological feed additives (OC/EFSA/FEED/2013/01) N. Tous, N. Tous IRTASearch for more papers by this authorJ. Brufau, J. Brufau IRTASearch for more papers by this authorA. Pérez-Vendrell, A. Pérez-Vendrell IRTASearch for more papers by this authorB. Vilà, B. Vilà IRTASearch for more papers by this authorM. Francesch, M. Francesch IRTASearch for more papers by this authorD. Torrallardona, D. Torrallardona IRTASearch for more papers by this authorM. Castellari, M. Castellari IRTASearch for more papers by this authorJ. Diogene, J. Diogene IRTASearch for more papers by this authorM. Viñas, M. Viñas IRTASearch for more papers by this authorV. Castell, V. Castell ACSASearch for more papers by this authorE. Vicente, E. Vicente ACSASearch for more papers by this authorI. Badiola, I. Badiola ACSASearch for more papers by this authorE. Esteve-Garcia, E. Esteve-Garcia ACSASearch for more papers by this author N. Tous, N. Tous IRTASearch for more papers by this authorJ. Brufau, J. Brufau IRTASearch for more papers by this authorA. Pérez-Vendrell, A. Pérez-Vendrell IRTASearch for more papers by this authorB. Vilà, B. Vilà IRTASearch for more papers by this authorM. Francesch, M. Francesch IRTASearch for more papers by this authorD. Torrallardona, D. Torrallardona IRTASearch for more papers by this authorM. Castellari, M. Castellari IRTASearch for more papers by this authorJ. Diogene, J. Diogene IRTASearch for more papers by this authorM. Viñas, M. Viñas IRTASearch for more papers by this authorV. Castell, V. Castell ACSASearch for more papers by this authorE. Vicente, E. Vicente ACSASearch for more papers by this authorI. Badiola, I. Badiola ACSASearch for more papers by this authorE. Esteve-Garcia, E. Esteve-Garcia ACSASearch for more papers by this author First published: 26 February 2016 https://doi.org/10.2903/sp.efsa.2016.EN-1001 The present document has been produced and adopted by the bodies identified above as authors. This task has been carried out exclusively by the authors in the context of a contract between the European Food Safety Authority and the authors, awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Published date: 26 February 2016 Question number: EFSA-Q-2016-00147 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, 1– 65. EFSA (European Food Safety Authority), 2008a. Guidance for the preparation of dossiers for the reevaluation of certain additives already authorised under Directive 70/524/EEC. The EFSA Journal 2008, 779, 1– 9. EFSA (European Food Safety Authority), 2008b. Technical guidance for assessing the safety of feed additives for the environment. The EFSA Journal 2008, 842, 1– 28. EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2011a. Guidance on the assessment of the additives intended to be used in pets and other non food-producing animals. EFSA Journal 2011; 9(2):2012, 3 pp. doi:10.2903/j.efsa.2011.2012 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2011b. Technical Guidance: Tolerance and efficacy studies in target animals. EFSA Journal 2011; 9(5):2175, 15 pp. doi:10.2903/j.efsa.2011.2175 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012a. Guidance for the preparation of dossiers for technological additives. EFSA Journal 2012; 10(1):2528, 23 pp. doi:10.2903/j.efsa.2012.2528 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012b. Guidance for the preparation of dossiers for additives already authorised for use in food. EFSA Journal 2012; 10(1):2538, 4 pp. doi:10.2903/j.efsa.2012.2538 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012c. Guidance on studies concerning the safety of use of the additive for users/workers. EFSA Journal 2012; 10(1):2539, 5 pp. doi:10.2903/j.efsa.2012.2539 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L268, 18.10.2003, 29– 43. Volume13, Issue2February 20161001E ReferencesRelatedInformation

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne salmonellosis in humans. Poultry is the single largest reservoir, and the consumption of incorrectly processed chicken meat and egg products is the major source of infection. Since 2006, the use of antibiotics as growth promoters has been banned in the European Union, and the dietary inclusion of β-galactomannans (βGM) has become a promising strategy to control and prevent intestinal infections. The aim of this study was to investigate the effect of various βGM-rich products on intestinal morphology in chickens challenged with Salmonella Enteritidis. To assess this effect, a total of 280 male Ross 308 chickens were studied (40 animals per treatment housed in 5 cages). There were 7 treatments, including controls: uninoculated birds fed the basal diet (negative control) and inoculated birds fed the basal diet (positive control) or the basal diet supplemented with Salmosan (1 g/kg), Duraió gum (1 g/kg), Cassia gum (1 g/kg), the cell walls of Saccharomyces cerevisiae (0.5 g/kg), or the antibiotic colistine (0.8 g/kg). The birds were fed these diets from the d 1 to 23, except the animals in the colistine group, which were fed the diet containing the antibiotic only from d 5 to 11. The inoculated animals were orally infected on d 7 with 10(8) cfu of Salmonella Enteritidis. Bird performance per replicate was determined for the whole study period (23 d), and the distal ileum and cecal tonsil of 5 animals per treatment (1 animal per replicate) were observed at different magnification levels (scanning electron, light, and laser confocal microscopy). In the images corresponding to the treatments containing βGM we observed more mucus, an effect that can be associated with the observation of more goblet cells. Moreover, the images also show fewer M cells, which are characteristic of infected animals. Regarding the morphometric parameters, the animals that received Duraió and Cassia gums show greater (P = 0.001 and P = 0.016, respectively) villus length compared with the animals in the positive control, thus indicating the capacity of these products to increase epithelial surface area. However, no effect (P > 0.05) on microvillus dimensions was detected. In conclusion, the results obtained indicating the beneficial effects of these βGM on intestinal morphology give more evidence of the positive effects of these supplements in poultry nutrition.

The restrictions on the use of antibiotics as animal growth-promoters have encouraged the use of essential oils as alternatives; among them, clove oil, which has eugenol as the major constituent [1]. The metabolism of eugenol has been described in rat [2], but not in chicken, for which clove oil is used as feed additive. Thus, the objective of the present study was to investigate the in vivo metabolism, following oral administration, of eugenol in chickens for fattening. A single dose of eugenol dissolved in refined sunflower oil, was administered by oral gavage, at a dose of 1.5 mmol/kg (246 mg/kg) to the treated group (n = 3) of chicken. Two control groups (n = 3) were included in the experiment, one without any treatment and the other administered with refined sunflower oil. Faeces were collected at 4, 24, 29 and 48h. Plasma, liver, kidney, muscle and fat (skin and fat) were collected at 48h. Samples were analysed by GC-MS for identification of the metabolites, with and without hydrolysis with ß-glucosidase and sulphatase, in order to know the content of the free and conjugated fractions of the compounds. Quantitative analyses were performed by GC-MS, using SIM acquisition mode and azulene as internal standard. LOD and LOQ, which were determined for each sample matrix, ranged from 0.02 to 0.14 ppm and 0.12 to 0.81 ppm, respectively. Relative extraction recoveries for eugenol from different types of samples ranged from 70% to 116%. After oral administration of a single dose of 246 mg/kg of eugenol to broiler chickens, the compound was excreted mainly as conjugated eugenol (99.6%). No metabolites were detected in faeces or chicken tissues of the treated animals. The excretion of eugenol is very fast, 98% of the excreted eugenol appearing in faeces in the first 24h after oral administration. After 48h of the administration, no free or conjugated eugenol was detected in plasma, kidney, muscle and fat of treated chickens.

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1. An experiment was conducted to study the effect of yeast (Saccharomyces cerevisiae) cell wall (YCW) supplemented in diets of broiler chickens challenged with Escherichia coli lipopolysaccharide (LPS). 2. One-day-old broiler chicks were randomly distributed into 24 cages (6 replicate cages; 8 birds/cage) and were inoculated with 0 or 1 mg/kg body weight E. coli-LPS (d 4 and 9) and 0 or 500 mg YCW/kg feed, resulting in a 2 × 2 factorial arrangement of treatments. Experimental diets did not include coccidiostats, in-feed antibiotics or enzymes. 3. On d 21, the inoculation of E. coli-LPS reduced weight gain and feed intake and increased feed conversion ratio (FCR) of birds, an effect maintained until 28 d. In contrast, chickens given diets with YCW improved the FCR at both 21 and 28 d of age. 4. E. coli-LPS challenge reduced the relative weight of bursa of Fabricius, except when chickens were given YCW, which resulted in an interaction. Supplementation of broiler diets with YCW exacerbated the cellular immune response as measured by the delayed cutaneous hypersensitivity response test. 5. The results of this study suggested a benefit on feed efficiency when YCW was added to diets fed to broiler chickens challenged with E. coli-LPS. Part of the mode of action of YCW might be related to better maintenance of immune status in response to microbial challenge.

Gums and yeast cells are natural mannose-rich products that can be used as subtracts for adhesion of gram-negative bacteria. The aim of the study was to investigate the role of dietary locust bean gum (LBG; Salmosan, ITPSA, Spain) or live yeast (Saccharomyces cerevisiae Sc47; Actisaf, Lesaffre, France) as immunological enhancers of the intestinal function of piglets. Treatments included a noninfected group fed with a control diet and 4 other groups orally challenged with 1x10(8) cfu of Escherichia coli K88. The challenged groups were fed the control or the control diet supplemented with colistin, LBG, or yeast. Twenty-five Landrace × Duroc piglets weaned at 4 wk were used. The animals were group housed, challenged on day 14, and euthanized 2 d later. Blood, bile, ileum, and mesenteric lymph node (MLN) samples were obtained for analysis of C-reactive protein (CRP), secretory immunoglobulin A (sIgA), and Toll-like receptors 2 (TLR2) and 4 (TLR4). Challenge increased the level of CRP of piglets fed the control diet, but this did not happen in the piglets fed the supplemented diets (P < 0.001). Challenge upregulated TLR2 in MLN and TLR4 in ileum and MLN (P < 0.05). This was prevented by yeast supplementation for the TLR2 in MLN and TLR4 in ileum. Pigs fed colistin and LBG diets showed intermediate but nonsignificant responses. In conclusion, the results suggest that colistin, live yeast, and LBG reduce the levels of CRP in blood of piglets challenged with E. coli, and yeast reduces the expression of Toll-like receptors in the intestine.

Digestive microflora is partly responsible for physiological gut conditions. Measurements of redox potential (Eh) and pH of digesta may give a basis for understanding microbial activity and dynamics of fermentation. However, few studies have assessed the Eh of the gastrointestinal tract of pigs. Twenty-four pigs of 30 kg BW were slaughtered to measure Eh and pH of cecum content in situ and to obtain samples of ileum, cecum, and colon contents for VFA determinations. Pigs had previously been fed with a nonmedicated starter feed for 5 wk. Measurements of Eh and pH were recorded first at 2 min and then at 5-min intervals for 35 min to estimate kinetics and the delay to reach stabilization of the Eh value. Cecum Eh rapidly decreased (P < 0.001) from -115 to -180 mV after 15 min of insertion of the electrodes and then slowly decreased until -185 mV at 35 min. Cecal pH started at 5.74 and decreased (P < 0.01) slowly afterwards until 5.53 after 35 min. The Eh value after stabilization was negatively correlated (r = -0.64; P < 0.001) with final pH. Acetic, propionic, and butyric acids accounted for 58.7, 24.0, and 12.8%, respectively, of total VFA production of cecum content. The VFA production of ileal content was lower (P < 0.001) when compared to cecum or colon (50.8, 142.1, and 130.8 μmol/g, respectively) and a higher proportion of formic and lactic acids was detected (32.3 and 27.0%, respectively). Proportions of acetic and propionic acids were negatively (r = -0.53; P < 0.01) and positively (r = 0.66; P < 0.001) correlated with Eh, respectively. In conclusion, Eh measured after stabilization seemed to be a meaningful predictor of hindgut fermentative activity.

Salmonella is presently one of the microorganisms of higher concern for food safety in poultry products. The present study examined the effect of feeding galactomannans from carob bean gum on nutrient digestibility and performance in chickens, and on the prevalence of Salmonella enterica var. Enteritidis in challenged animals. Four experiments were performed with either broiler or leghorn chickens, challenged with 106CFU (colony-forming units) of S. Enteritidis at 1day of life, and feeding carob bean gum at different concentrations (25, 50 or 100g/kg, depending on the experiment), alone or in combination with β-mannanase, cellulase or α-galactosidase at 8.3U/g; or feeding D-mannose at 25g/kg, or depolymerized carob bean gum or guar gum at 100mg/kg. Trials lasted 3or 4weeks. Body weight and feed intake were determined and feed conversion ratio calculated (feed:gain). Faeces were collected during the last week on trial for evaluation of nutrient balance (energy, lipids and protein), using chromium oxide as inert marker. Viscosity of the ileal content was also determined at the end of the second experiment. Salmonella presence in caeca was determined two and 3weeks after challenge. Performance and nutritive value of diets were impaired in birds fed carob bean gum, with higher effect at higher inclusion rates. D-mannose impaired performance variables only whereas depolymerized gums did not affect bird performance or nutritive value of the diets. Of the enzymes tested, only β-mannanase significantly decreased the viscosity of the intestinal contents of birds fed carob bean gum and partly counteracted the impairment in bird performance and the reduction in the nutritive value of the diets. The number of Salmonella-positive birds varied among experiments and was lower in the third week post-challenge compared to the second week post-challenge. However, the reduction in the number of Salmonella-positive birds was more constant and marked when carob bean gum was present in the diet. The inclusion of carob bean gum in the diet of chickens at the high concentrations used in the present experiment reduced the presence of Salmonella in challenged birds, but it also impaired performance and nutrient digestibility. These impairments were partially counteracted by the addition of β-mannanase to the diet. Carob bean gum might be used to reduce the incidence of Salmonella in chickens, while its negative effects on performance and nutrient digestibility could be counteracted by β-mannanase.

We review current trends in the analysis of antimicrobial agents in animal feeds. After a brief introduction to feed-industry figures and the unavoidable problem of cross-contamination, we provide an overview of the European Union legislative framework for feedingstuffs. We devote the core of the article to analytical methodology developed over the past 10 years for monitoring residues of antibiotics and coccidiostats in feedingstuffs in order to ensure that feeds comply with current legislation and are of high quality and safety for both livestock and consumers. We consider the potential and the limitations of analytical methods and devote special attention to their validation and performance characteristics.

The objective was to evaluate the effect of ZnO-Functionalised-Sepiolite (ZnO-Sepiolite) to fulfil Zn requirements and health status of weaning piglets. Pre-starter Basal Diet (BD, corn–soybean based, from weaning till 14days on trial) was calculated to provide 27mg Zn/kg feed from raw materials and had no added ZnO and no antibiotics or organic acids. Treatments during pre-starter period were: 1) BD+90% of NRC Zn requirements completed with ZnO (ZnO90); 2) BD+90% of NRC Zn requirements completed with ZnO-Sepiolite (ZnOS90); 3) BD+3000mg ZnO/kg of diet (ZnO3000); 4) BD+150mg added Zn/kg diet from ZnO-Sepiolite (ZnOS150). The starter feed (corn–soybean based, from 14 till 31days on trial) was common for all piglets, and met 90% NRC Zn requirements by adding ZnO. Diarrhea affected more than 50% of the animals of ZnO90, ZnOS90 and ZnOS150, and 33% of the ZnO3000 animals. Animals from ZnOS90 tended (P<0.10) to improve Gain to Feed ratio (G:F) compared to animals from ZnO90 (0.830kg/kg vs. 0.811kg/kg for G:F). Performance of animals from ZnO3000 was not significantly different from the other treatments, and was numerically similar to animals from ZnOS90. The inclusion of ZnO at 3000mg/kg of feed in the pre-starter period numerically decreased P in serum at the end of this period, with no effect on Ca level; normal levels were restored after 2weeks of feeding the same levels of Zn than other animals. Animals fed ZnO-Sepiolite diets had numerically higher serum Ca than ZnO90 and ZnO3000 at 12days and higher than ZnO90 at 28days. Serum Zn levels were significantly higher for ZnO3000 than the other treatments.

Benefits from probiotic micro-organisms have been recognised for over 100 years, and as being useful in poultry for 50 years. Fuller (1989) redefined probiotics as 'a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance'. Benefits derived from this improved intestinal microbial balance could be reflected in performance or prevention of pathogen colonisation. Probiotic micro-organisms use in poultry production has been widely accepted and new opportunities arose from the 2006 EU ban on antimicrobial growth promoters. The majority of microbial products for compound feeds are made up from a relatively small number of micro-organisms that are normally present in the GI tract. They include non-sporulated bacteria, sporulated bacteria, fungi or yeasts; and presented from single to multi-strain products. A review on the proposed modes of action is presented including recent approaches to quorum sensing interference.

1. Three experiments were carried out to study the effects of two experimental yeast cell wall (YCW) supplements, one from the yeast extract industry and the other from the brewery industry, added to maize or wheat based-diets, on performance and intestinal parameters of broiler chickens (Ross 308). 2. In the first and second experiments, a completely randomised block design with 4 experimental treatments was used: T-1) Negative control, no additives T-2) Positive control, avilamycin group (10 mg/kg feed), T-3) Yeast extract-YCW (500 mg/kg), and T-4) Brewery-YCW (500 mg/kg feed). There were 6 replicates of 20 (experiment 1) and 22 (experiment 2) chicks per treatment. 3. In experiment 1 (wheat based diets), yeast extract-YCW increased BW and daily feed intake (42 d). The effects were comparable to those of avilamycin. In experiment 2 (maize based diet), avilamycin, yeast extract-YCW and brewery-YCW treatments improved the feed conversion ratio with respect to the negative control group (0 to 14 d). 4. At 24 d, in both experiments, the ileal nutrient digestibility and ileal bacterial counts were not affected by any experimental treatment. In maize diets, lower intestinal viscosity was obtained with avilamycin, yeast extract-YCW and brewery-YCW than with the negative control. In wheat diets, yeast extract-YCW and brewery-YCW reduced intestinal viscosity. 5. A third experiment was conducted to study the effect of yeast extract-YCW on animal performance, intestinal mucosa morphology and intestinal viscosity. A 2 x 2 factorial arrangement of treatments was used; one factor was the dietary yeast extract-YCW supplementation (0 or 500 mg/kg feed) and the other the cereal in the diet (maize or wheat). 6. At 43 d, the heaviest BW was in chickens fed on yeast extract-YCW compared to those given the negative control. At 22 d, yeast extract-YCW increased villus height, mucus thickness and number of goblet cells with respect to negative control. 7. Results of these experiments suggest that supplementation of yeast extract-YCW to broiler chicken diets increased animal performance by favouring intestinal mucosal development.