Effect of a mannan oligosaccharide used as a food additive for broilers

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1 Effect of a mannan oligosaccharide used as a food additive for broilers C. GARCÉS-NARRO 1 *, J.I. BARRAGÁN 1, M. SOLER 1, J.M. ROS, J. ORTEGA 2, M.L. MOCÉ 1, M. MATEOS 1, M.T. GÓMEZ-MUÑOZ 2, M.C. LÓPEZ-MENDOZA 1, and V. RODILLA 3. 1 Dpt. Producción Animal y Ciencia y Tecnología de los Alimentos. 2 Dpt. Atención Sanitaria, Salud Pública y Sanidad Animal. 3 Dpt. Fisiología, Farmacología y Toxicología. Universidad Cardenal Herrera-CEU. Ed. Seminario s/n Moncada. Spain. *Corresponding author: cgarces@uch.ceu.es This experiment was carried out to determine the effect of an additive based on a mannan oligosaccharide in the nutrition of broilers during the first 43 days of their life. The experiment was performed in a commercial farm in the region of Valencia (Spain). A total of 720 broilers divided into groups of 60 were each placed in 4 m 2 pens (2x2 m), thus maintaining the same animal density as in the rest of the farm. Half the animals (6 pens) were given standard feed, whereas the other half were fed the same food supplemented with the additive at a rate of 500 g/t. Throughout the study mortality and morbidity were assessed daily. On days 1, 22 and 43, the weight of the broilers was recorded and samples were obtained to investigate the presence of oocysts, the presence of bacterial pathogens and to assess the integrity of the intestine mucosa. During the first three weeks, we detected differences between the two groups of broilers, in terms of average daily gain (ADG) and feed conversion rate (FCR); ADG (41.95 vs 43.26; p<0.05) and FCR (1.82 vs 1.76; p<0.05) were better in broilers fed the standard feed than those receiving the additive. However these differences were not significant when the whole period of study was considered. We found no effects either in the number of Eimeria oocysts or infections by Salmonella or other pathogens. No differences were detected on mortality and morbidity between the two groups. Keywords: broilers; mannan oligosaccharide, nutrition; additive, gut integrity Introduction The European poultry industry has recently been affected by some important changes in legislation and has had to make costly adaptations in order to maintain its competitivity. Recent legislation aimed to protect consumers, public health and to minimize risk resistance has prohibited the use of antimicrobial growth promoters. The elimination of antimicrobial agents in the feed may reduce farm productivity and animal performance (Emborg et al., 2002), but also may increase the frequency of several pathologies caused by presence of Salmonella spp., Escherichia coli o Campylobacter spp. To maintain productivity but reducing the presence of food-borne pathogens without the use of antimicrobial growth promoters, several feed-related strategies have to be implemented. Such strategies may involve maintaining the gut s epithelial integrity, and/or to facilitate the development of a beneficial microbial flora to avoid, in as much as possible, pathogen proliferation or to reinforce the immune system (Canibe et al., 2002; Engberg, 2002). Prebiotics such as inuline, fructo-oligosaccharides (FOS), isomaltooligosaccharides (IMO) and mannan oligosaccharides (MOS) have been defined by Gibson and Roberfroid (1995) as microbial food supplements that beneficially affect the host by improving its intestinal microbial balance and have been used to change the composition of colonic microbiota. Prebiotics have been reported to produce a beneficial effect upon the animal that receives them. This is due to the proliferation of

2 certain beneficial bacteria such as Bifidobacterium spp. and Lactobacillus spp. or an increase in their metabolic activity (Gibson and Roberfroid, 1995). The activity of these prebiotics is to a certain degree unspecific and many papers have been published reporting their effects. Apajalahti et al. (2004) reported that the 90% of the bacteria in the chicken gastrointestinal tract are previously unknown species. Inulin, FOS and IMO are reported to be substrates for certain species of beneficial bacteria (Chung and Day, 2004) whereas MOS appear to act due to their capability to interlink the fimbriae of E coli and Salmonella spp, blocking the ability of these bacteria to connect via their fimbriae, with the epithelial cells and therefore facilitating their excretion (Spring et al., 2000; Duncan et al., 2005). Also, the MOS have a positive effect on the immunity preventing the infections (Shashidahara and Devegowda, 2003). The aim of this paper is to assess the effects of a MOS obtained from the cell wall of Saccharomyces cerevisiae on the body weight, average daily gain (ADG), feed conversion rate (FCR), mortality, presence of Salmonella spp. in the gut, count of Eimeria spp. oocysts and intestinal integrity of broilers. Material and methods EXPERIMENTAL DESIGN A total of 720 broilers divided into 12 groups of 60 were each allocated to a 4 m2 pen (2x2 m). The pens were placed inside a commercial broiler farm with controlled housing environment on which a total of birds were placed at a density of 15 animals per square metre. Animal density was initially the same inside the pens as in the rest of the broiler house. Pens were distributed uniformly throughout the broiler house. Half the animals (6 pens) were given standard feed (), whereas the other half were fed the same food supplemented with the additive (MOS) at a rate of 500 g/t (AF). Throughout the study mortality and morbidity were assessed daily. On days 1, 22 and 43, the weight of the broilers was recorded by means of an electronic weighing scale and a caged platform with capacity for all the birds. The amount of feed used for each pen at those intervals, was also calculated. On days 22 and 43 a bird from each pen was killed by an intracephalic injection of sodium pentobarbital (20 mg/kg), and samples of the gut were obtained to investigate the presence of Eimeria oocysts, the presence of bacterial pathogens and to assess the integrity of the intestine mucosa. Additionally three samples of the litter in each of the pens as well as simples from outside the pens were taken to investigate the presence of Salmonella, Eimeria oocysts and for determination of moisture. The system described here aims to expose the experimental broilers to the same environment found at a commercial farm including similar levels of general contamination, temperature, ventilation, etc as well as similar stress in the animals caused by bird density or human presence. FEEDSTUFFS All the broilers in the experiment were given the same type of foodstuff according to the classical feeding program (starter feed from 0 to 21 days and a growing feed from 22 to 43 days) The composition of the foodstuffs used (starter and growing feeds) is listed in Table 1. Each type of food was prepared as a Standard feed () and additive-supplemented feed (AF) as earlier described.

3 Table 1. Composition of the basal diets (g/kg) Ingredient Starter diet Grower diet Wheat Soymeal 44% Maize Full fat Soy 50 0 Fat mix CaHPO CaCO Na Cl 3 3 L-Lysine Alimet Vitamin-mineral premix 4 4 Phytases 1 1 Enzymes 1 1 Antococcidial Chemical composition (g/kg) Metabolizable energy (kcal/kg) Crude protein Lysine Methionine Fat SALMONELLA ANALYSIS Gallbladder and rectal swabs were obtained from each animal, adequately stored in culture media and transported to the laboratory, where they were cultured in a Salmonella specific medium within three hours. Litter samples were also collected form each pen and processed identically. After incubation suspected colonies were isolated and subjected to specie confirmation using API strips OOCYSTS COUNT Rectal samples from sacrificed broilers and from the litter were used to determine presence of Eimeria oocysts. The samples were homogenized, diluted in saline salt solution and spun down at 400 g. The pellet obtained from centrifugation was reconstituted in a saturated salt solution. A volume of 0.3 ml from this solution was used to determine the number of oocysts, according with a modification of the McMaster technique (Ministry of Agriculture, Fisheries and Food, 1986). If the sample was from the rectal content the results were expressed as number of oocysts per gram of fresh matter but if the sample was from litter they were expressed as number of oocysts per gram of dry matter. MOISTURE CONTENT IN THE LITTER Samples (100g g each) of the litter were obtained from each pen and the rest of the farm. Each sample was homogenized, and 5 g of each were incubated at 103 ºC to dryness (constant weight). Moisture (water content in the sample) was calculated from the difference between initial and final weight of each litter sample and expressed as a percentage. HISTOPATHOLOGICAL EXAMINATION OF GUT SAMPLES. Samples of jejunum (approximately 4 cm proximal to Meckel s diverticulum) were obtained from the sacrificed broilers to determine the height of villi and the depth of the crypts of Lieberkühn and to calculate the relation of each other. The samples obtained were washed in saline solution and fixed in 10% neutral buffered formaldehyde. The samples were then dehydrated through graded alcohols before being embedded in paraffin wax. Several sections 3 µm thick were cut from each sample and stained with haematoxylin and eosin (Segura Gil et al., 2004). A histopathological assessment of the sections was carried out and random views were photographed and the images processed with image analysis software (EclipseNet, v1.20.0, Laboratory Imaging) to determine the measurements of the crypts of Lieberkühn and the villi.

4 STATISTICAL ANALYSIS Statistical analysis was carried out using a statistical package (SAS, 1999). Comparisons between the two groups ( and AF) were performed using General Linear Models. Differences in mortality, Salmonella presence and number of oocysts were assessed using the test of the chi-square. Data from histological assessment were analyzed using the MIXED procedure with food type as factor and animal as random effect. Results The broiler chicks were weighed upon arrival to the farm and randomly allocated to each pen. As would be expected no significant differences were found amongst the weight of chicks in the different pens (Table 2). No differences were found in animal growth performance except at day 22: broilers feed Standard feed had a higher body weight, ADG and FCR. These differences were not maintained during the rest of the experiment (Table 2). Table 2. Growth performance parameters and mortality in the broilers throughout the breeding period. Feed Control day Body weight g ADG g/d FCR Mortality % AF ± ± 0.33 AF ± 9.33* ± 0.64* 1.82 ± 0.02* ± 6.22* ± 0.64* 1.76 ± 0.03* 1.6 AF ± ± ± ± ± ± AF 59,55 ± ,10 ± ,8 Overall 60,23 ± ,09 ± ,3 The * indicates statistically significant differences (p<0,05) between the two treatments. The weight of chickens at 22 days was introduced as a covariable on the weight at 43 days and on ADG, to correct for the possible bias caused by earlier weights. Data (except mortality) are expressed as the average ± SEM. Mortality was calculated as the percentage of dead animals in each pen for each period of study (1 to 22 days, 23 to 43 days and 1 to 43 days). AF: Additive-supplemented Feed; : Standard Feed Salmonella growth was in all cases negative, although there was growth of Proteus mirabilis in some samples (Table 3). All litter samples both from inside the pens and the rest of the farm were positive for.p mirabilis as were 80 % of rectal swabs and gallbladder from chickens fed, or 60% of rectal swabs and 20% gallbladder swabs from chickens fed AF on day 43. No bacterial growth was recorded in samples obtained at 22 days in the Salmonella medium (Table 3).. Feed Table 3. Number of oocysts, Salmonella and litter moisture in samples. Control day Number of rectal oocystsa Number of Oocysts in litterb Salmonella Litter moisture % AF AF No significant differences were found in any of the parameters evaluated between the two groups of broilers in the study a Number of rectal oocysts per gram of fresh matter. a Number of oocysts per gram of dry weight of litter sample. AF: Additive-supplemented Feed; : Standard Feed No significant differences were obtained in the number of Eimeria spp. oocysts either from samples from the litter or from rectal swabs (Table 3). During the starter phase, no oocysts were

5 detected either in the broilers or the litter, but they could be detected in the growth phase both in the rectum of broilers and in the litter (Table 3). The height of small intestine villi and the depth of the Lieberkühn crypts from broilers receiving the two types of food were compared but no statistically significant differences (p>0.1) between the two groups were detected. The ratio depth crypt/villus height between the two groups of broilers were also compared and no differences were found (Table 4). Tabla 4. Villi height, depth of Lieberkühn crypts (both in µm) and ratio crypt depth to villus height in jejunum from 22 day old chicks. Feed Villi height depth of Lieberkühn crypts ratio depth crypt/villus height AF 847 ± ± ± ± ± ± The absence of * indicates no significant differences (p>0,05) between the two treatments. Data are expressed as the average ± SEM. Discussion The results obtained show that the additive at the doses used, does not have a positive effect on growth performance in chicks under the conditions described. In fact, during the first stages of growth it has a slight negative effect which is compensated at later stages. In contrast to what Sims et al. (2004) and Stanley et al. (2004) have shown FCR and ADG in our broilers did not improve after an additive was included in the food. Although during the initial stages growth performance seems to be reduced in chickens consuming the food with the additive, growth performance improved with time and overall there were no significant differences between chickens receiving both types of food. However the variability of field studies is so great that although our results show partial similarity to those reported by Stanley et al. (2004) this group is able to prove differences in growth performance between additive supplemented and standard feed. We cannot comment on the effect of the additive used in our experiments on Salmonella spp. since we were not able to detect these bacteria in any of the samples analysed throughout the experiment. The effect of the additive used in our experiments on the number of Eimeria spp. oocysts was not statistically significant, although in most of the samples analysed there were no oocysts detected. The low incidence of Eimeria spp. could have been due to farm disinfection and the use of clean litter with every batch of chicks in the farm as well as the use of anticoccidial agents during the starter and growth period. Although the differences are statistically not significant when oocysts were detected, samples from chickens fed the additive always seem to have lower frecuencies of oocysts. Similar results have also been reported by other groups (Stanley et al. 2004). It would be interesting to demonstrate the effect of the additive on the number of oocysts using animals experimentally infected. Deeper crypts are indicative of faster tissue turnover to permit renewal of the villi as needed in response to normal sloughing or inflammation from pathogens and their toxins (Yason et al., 1987; Miles et al., 2006). In our case significant differences were not found in the villi height and the depth of the crypts between the animals fed with AF and those fed with, which would indicate that probably nutrient absorption was similar when standard feed or additive-supplemented feed were used. This argument is also supported by the few differences found in the growth performance parameters evaluated between the two groups. Acknowledgements The authors wish to thank R. Conejos and SADA p.a. for their assistance. This research was supported by the Generalitat Valenciana (GV185/05).

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