Effect of feeding Pediococcus acidilactici on performance of broiler chicken and microstructures of intestinal villus

Transcription

1 Indian Journal of Poultry Science (2012) 47(3): ; Research Article Effect of feeding Pediococcus acidilactici on performance of broiler chicken and microstructures of intestinal villus Y. SATHEESH, K. KONDAL REDDY*, P.S.P. GUPTA, P.V.R. MALLIKARJUNA, Y. RAMANA REDDY AND M. KISHAN KUMAR Department of Livestock Products Technology, College of Veterinary Science, S V Veterinary University, Rajendranagar, Hyderabad (A.P.) (Received on June 25, 2012; accepted for publication on October 13, 2012) ABSTRACT Satheesh, Y., Reddy, K.K., Gupta, P.S.P., Mallikarjuna, P.V.R., Reddy, Y.R. and Kumar, M.K Effect of feeding Pediococcus acidilactici on performance of broiler chicken and microstructures of intestinal villus. Indian Journal of Poultry Science, 47(3): The effect of feeding encapsulated and non-encapsulated Pediococcus acidilactici on body weight gain, feed conversion efficiency, meat quality and ultra structural changes of intestinal (ileum) villus in commercial male broiler chicks was evaluated. Group 1 (Control) was fed with feed without supplementation of any probiotic organism, Group 2 was fed with non-encapsulated P.acidilactici 106 cfu/ g supplemented diets. Groups 3 and 4 were fed with low and high level of encapsulated Pediococcus acidilactici diets (EPAL and 10 6 cfu/g and 10 9 cfu/g, respectively. The body weight gain during the (0-3) and (0-6) week in the control group was significantly (P<0.05) lower than NEPA, EPAL and EPAH groups. EPAL and EPAH groups showed significantly (P<0.05) better feed conversion ratio (FCR) compared to control group at the end of 3 rd and 6 th week. The sensory evaluation scores of meat revealed a significant (P<0.05) improvement in juiciness, tenderness and overall acceptance in probiotic fed broilers compared to control group. The Scanning Electron Microscopy of ileum revealed increased villus width and length (area) in EPAH group compared to all other groups (Control, NEPA and EPAL). However, NEPA and EPAL fed chicks have higher villus area compared to control group. An excellent adhesion of Pediococcus acidilactici on intestinal walls was seen in EPAL and EPAH birds compared to control and NEPA group. Encapsulated Pediococcus acidilactici enhanced the colonization and adhesion of probiotics on the surface of intestines besides increasing the density, length and width of intestinal villus resulting in better performance and meat quality in broilers. Key words: Pediococcus acidilactici, performance, broiler chicken and intestinal villus INTRODUCTION The progressive reduction in the use of nontherapeutic dosages of antibiotics in animal feed as growth promoters has raised renewed the interest in finding alternatives to antibiotics in poultry production (Chichlowski et al., 2007). Some possible alternatives to antibiotics for growth promotion and improvement of feed efficiency in domestic avian species are probiotics, prebiotics and organic acids (Biggs et al., 2007). To achieve health benefits, probiotic bacteria must be viable and available at a higher concentration, typically between CFU/g of feed (Shah et al., 2000). Encapsulation in hydrocolloid beads has been investigated as a means to protect and improve viability of probiotic microorganisms in food products and in the intestinal tract *Corresponding author : e.mail - kkkreddy5@rediffmail.com (Rao et al., 1989). Pediococcus acidilactici is reported to exert positive effects on the balance and function of intestinal flora (Jin et al., 2000) with further benefits on improvement of animal performance. It strengthened the microbial ecosystem in poultry, contributed to disease resistance and protected poultry from stress due to vaccination, environmental temperature etc. An improvement in feed intake, weight gain, and feed conversion rate was observed by Stella et al. (2005). Therefore, the present study was planned to evaluate the effect of feeding encapsulated and non-encapsulated Pediococcus acidilactici on broiler performance, sensory evaluation of meat and ultra structural changes in intestinal (ileum) villus, using commercial male broiler chicks. MATERIALS AND METHODS A feeding trial of 6 weeks duration was conducted on 120 day-old commercial male broiler chicks procured

2 (Vencobb, Hyderabad) and allotted randomly to four groups. Each group was divided into six replicates of five chicks each and raised in battery brooders. The freeze dried culture of Pediococcus acidilactici 252 strain was obtained from National Dairy Research Institute, Karnal, Haryana. The birds were offered commercial feed formulated according to NRC (1994) recommendations for both starter and finisher phases was purchased from Vimala Animal Feed Agencies, Hyderabad. The body weight, weight gain, feed consumption and feed conversion ratio were recorded in all the groups at weekly intervals. Histological observations were made on six birds from each group that were sacrificed on day 42, to study the histo-morphological changes of intestines (ileum) by using scanning electron microscopy (SEM). Encapsulation of Pediococcus acidilactici (extrusion technique) Utilizing the extrusion technique of encapsulation, microorganisms were added into a hydrocolloid solution (alginate) and then the cell suspension was extruded through a syringe needle to form droplets, which free-fall into a hardening solution or setting bath. Alginate is used commonly as supporting material in this technique. The concentration of alginate used was 1% to form a gel with 0.5M CaCl 2 (Krasaekoopt et al., 2003). Sensory evaluation of meat The organoleptic attributes of cooked breast meat samples were assessed by subjecting them to sensory analysis for colour, flavour, juiciness and tenderness by semi-trained 5 member taste panel drawn from the staff of the College of Veterinary Science, Hyderabad. Cooked cubes of approximately 1.5 cm cut from the breast muscle were served to the taste panel members as coded samples, and the panelists were provided with a nine point score card to assess the colour, flavour, juiciness and tenderness of the samples served. Scanning electron microscopy (SEM) of ileum For microscopic studies, samples of duodenum were transferred to vials and fixed in 2.5% gluteraldehyde in 0.05 M phosphate buffer (ph 7.2) for 24 h at 4 o C and post-fixed in 2% aqueous osmiumtetroxide in the same buffer for 1 h. After the fixation, the samples were dehydrated in a series of graded alcohol, and processed for critical point drying with Electron Microscopy Science, CPD unit. The dried samples were mounted over the stubs. Finally, a thin layer of gold coating was applied over the sample using an automated sputter coater (JEOL JFC- 1600) for about 3 minutes (Bozzola and Russel, 1999). The samples were subsequently, scanned in scanning electron microscope (Model:JOEL-JSM 5600) at various magnifications at RUSKA Lab, College of Veterinary Science, Hyderabad, India. Statistical analysis The data were subjected to statistical analysis by applying one-way ANOVA using Statistical Package for Social Sciences (SPSS), the 15 th version. Differences between means were tested using Duncan s (1951) multiple comparison test and significance was set at P<0.05. RESULTS AND DISCUSSION Body weight gain The body weight gain during the (0-3) and (0-6) week in the control group was significantly (P<0.05) lower than non-encapsulated Pediococcus acidilactici (NEPA), encapsulated Pediococcus acidilactici low level (EPAL) and encapsulated Pediococcus acidilactici high level (EPAH) groups (Table 1). There was significant (P<0.05) gain in the body weight of EPAH, EPAL when compared with nonencapsulated Pediococcus acidilactici, during the six week trial. At the end of 6th week, there was a significant (P<0.05) gain in the body weights in EPAL and EPAH group compared to both NEPA and Control group. The body weight gain in EPAL and EPAH groups was significantly (P<0.05) higher than the other groups during the entire feeding period. This was in agreement with the findings of Sahin et al. (2009). The supplementation of P. acidilactici in the diet of chickens might have created a favorable environment by producing lactic acid (lowering ph) and allowing only P. acidilactici growth, resulting in unfavourable conditions for the growth of pathogens and probably also suppressing their growth by competitive exclusion (Ahmed, 2006). However, Karaoglu and Durdag (2005) used Saccharomyces cerevisiae as a dietary probiotic to assess performance and found no overall weight gain difference in broilers. It is probably due to the nature of probiotics, since the lactic acid bacteria modify the intestinal ecosystem which may not be the same for yeasts. Therefore the activity of probiotics may differ widely from one experiment to another depending on type of microorganisms (strains), concentrations used in the feed, the interaction of these additives with some added components 358 / Indian Journal of Poultry Science (2012) 47(3):

3 Table 1. Effect of feeding Pediococcus acidilactici supplemented diet to broilers on body weight gain (g), feed conversion ratio (FCR) and sensory properties of meat BWG FCR Sensory scores of meat Dietary (Weeks) (end of 6 th week) group (0-3) (4-6) (0-6) (end of (end of Colour Flavour Juciness Tenderness Overall 3 rd week) 6 th week) acceptance Control 652 d 760 d 1808 d 1.92 a 2.60 a 5.2 a a 5.8 a 5.5 a NEPA 678 c 794 c 1887 c 1.83 ab 2.47 a 6.3 b ab 6.1 b 6.0 ab EPAL 691 b 928 a 2059 b 1.82 b 2.48 b 6.4 b b 6.3 b 6.3 b EPAH 709 a 907 b 2121 a 1.80 b 2.34 c 6.5 b b 6.8 b 6.4 b SEM Note: NEPA-Non encapsulated Pediococcus acidilactici (10 6 cfu/g); EPAL Encapsulated Pediococcus acidilactici low level (10 6 cfu/g); EPAH - Encapsulated Pediococcus acidilactici high level (10 9 cfu/g) in the feed, the age of birds and their nutritional status and health (Ahmed, 2006). Feed conversion ratio The FCR recorded at the end of the 3rd week in the control group was 1.9, while the EPAL and EPAH groups showed an improvement in FCR (1.8) (Table 1). FCR was better in all the groups as compared to the control diet (1.9). The FCR recorded for control group at the end of the 6 th week was 2.6, while the NEPA, EPAL and EPAH groups showed better FCR (2.5, 2.5 and 2.3, respectively). In general the FCR of control group was higher than probiotics fed groups from 2 to 6 weeks of age. The results indicated that the addition of P.acidilactici induced a significant (P<0.05) improvement in the FCR of chickens similar to other studies (Vittorio et al., 2005) or other strains of lactic acid bacteria (Mountzouris et al., 2007). The positive effect of probiotics on the feed conversion ratio reflected a greater efficiency of nutrient utilization, which was consistent with the significant increase in the size and density of intestinal villi observed in this study (Awad et al., 2009). Sensory evaluation of broiler meat The sensory evaluation scores of meat revealed that there was a significant (P<0.05) improvement in juiciness, tenderness and overall acceptance in probiotic fed broilers compared to control group of broilers (Table 1). There were no significant differences between the sensory evaluation scores of EPAL and EPAH groups. The results of the present study are in agreement with Vargas et al. (2002). The positive effects of probiotics on meat may be due to intramuscular fat, which plays a major role in broiler meat quality including flavour and juiciness (Chizzolini et al., 1999), and with a negative correlation between muscle ph and tenderness. Histology of ileum The Scanning Electron Microscopy sections of the intestines (ileum) in non-encapsulated and encapsulated P. acidilactici supplemented diets (NEPA and EPAL) revealed increased villi width (Figs. 2 and 3), while EPAH (encapsulated P cfu/g) revealed an increased length and width of villi (Fig. 4) on the 42 nd day compared to control group (Fig. 1). The length and width of villus was 788 μm and 766 μm in EPAH group. The length of villus in NEPA and EPAL groups was 470 μm and 504 μm, while the width was 335 μm and 428 μm, respectively. Whereas, villus length and width was 784 μm and 184 μm in the control group (Fig. 1). An excellent adhesion of Pediococcus acidilactici on intestinal walls can be seen in EPAL and EPAH groups indicating the probiotics have the important property for colonization in intestines (Figs. 5 and 6) compared to control and NEPA groups. Adhesion or close association of Probiotic LAB to the epithelial cells of host may contribute to competitive exclusion of pathogenic and undesirable microbes. Besides adhesion of Lactobacilli to intestinal epithelial cells (IEC), the colonization of intestinal mucous seems to be very important for individual stability of lactic flora (Mikelsaar et al., 1998). The attachment of microorganisms to epithelial cells of the mucous is the result of a specific binding process involving surface adhesions on the bacteria and mucosa receptors on the cell membrane (Wadstrom and Aleljung 1989). Indian Journal of Poultry Science (2012) 47(3): / 359

4 Fig. 1. SEM of ileal villi showing length and width with marking on the 42 nd day in control group Fig. 2. SEM of ileal villi showing length and width with marking on the 42 nd day in NEPA group Fig. 3. SEM of ileal villi showing length and width with marking on the 42 nd day in EPAL group Fig. 4. SEM of ileal villi showing length and width with marking on the 42 nd day in EPAH group Fig. 5. SEM of ileum showing adherence of P.acdidilactici on the surface of the intestinal wall in EPAL group Fig. 6. SEM of ileum showing adherence of P.acdidilactici on the surface of the intestinal wall in EPAH group 360 / Indian Journal of Poultry Science (2012) 47(3):

5 These results were in agreement with Awad et al. (2009) who reported similar changes with Lactobacillus spp. supplemented diets in broilers. Chichlowski et al. (2007) found that the dietary supplementation with Bifidobacterium thermophilum and Enterococcus faecium increases jejunal villus height in broilers compared to control diets. The increased size of villi suggested that the surface area of intestine increased and thus absorption was higher in chickens supplemented with probiotic feeds (Caspary, 1992). The increased surface area of intestines is linked with induction of proliferation of intestinal epithelial cells after ingestion of probiotic diets (Ichikawa et al., 1999). The passive absorption of glucose and proline were increased in chickens receiving probiotics containing Lactobacilli, B. thermophilum and E. faecium (Chichlowski et al., 2007). Among the encapsulated P.acidilactici supplemented feeds, higher villus length and width (area) was observed in group fed higher levels ( cfu/g) compared to the lower levels ( cfu/g) of P.acidilactici. This may be due to the fact that probiotic inclusion level had a significant effect on nutrient apparent digestibility coefficients and cecal microflora composition (Mountzouris et al., 2010). For effective expression of probiotic characteristics in animals an effective level of 108 to 109 cfu/g/d was ideal (Ewing and Cole, 1994). The present study inferred that encapsulated P. acidilactici at different levels resulted in enhancing the broiler performance, meat quality expansion and also the ultrastructure of intestine villus. The efficacy of the encapsulated P. acidilactici was primarily due to their survivability in the gastrointestinal tract of broilers resisting gastric acidity and bile salts. Therefore, it can be suggested that encapsulated P. acidilactici can effectively be used in the poultry broiler diets. Encapsulation also enhanced the colonization and adhesion of probiotics on the surface of intestines besides increasing the density, length and width of intestinal villus. ACKNOWLEDGEMENTS The authors express thanks to the Agri-Biotech Foundation (APNL Project), ARI campus, Hyderabad for supporting this research work by award of a grant. REFERENCES Ahmed, I Effect of probiotics on broiler performance. International Journal of Poultry Science 5: Awad, W.A., Ghareeb, K., Abdel-Raheem, S. and Bohm, J Effects of dietary inclusion of probiotics and synbiotic on growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poultry Science, 88: Biggs, P., Parsons, C.M. and Fahey, G.C The effects of several oligosaccharides on growth performance, nutrient digestibilities and cecal microbial populations in young chicks. Poultry Science, 86: Bozzola, J.J., Russell, L.D Electron Microscopy Principles and Techniques for Biologists. Jones and Barlett Publishers, Boston. Caspary, W.F Physiology and pathophysiology of intestinal absorption. American Journal of Clinical Nutrition, 55: Chichlowski, M., Croom, W.J., Edens, F.W., Mc Bride, B.W., Qiu, R., Chiang, C.C., Daniel, L.R., Havenstein, G.B. and Koci, M.D Microarchitecture and spatial relationship between bacteria and ileal, cecal and colonic epithelium in chicks fed a direct-fed microbial, primalac, and salinomycin. Poultry Science, 86: Chizzolini, R.E., Zanardi, V., Dorigoni. and Ghidini, S Calorific value and cholesterol content of normal and low fat meat and meat products. Trends of Food Science and Technology, 10: Duncan, D.B A significance test for differences between ranked treatments in an analysis of variance. Variance Journal of Science, 2: Ewing, W.N. and Cole, D.J.A The Living Gut: An Introduction to Micro Organisms in Nutrition. Biotechnology Advances, 16: Ichikawa, H., Kuroiwa, T. and Inagaki, A Probiotic bacteria stimulate epithelial cell proliferation in rat. Digestive Diseases Sciences, 44: Jin, I.Z., Ho, Y., Abdullah, N. and Jaludin, S Digestive and bacterial enzyme activities in broilers fed diets supplemented with lactobacillus cultures. Poultry Science, 79: Karaoglu, M. and Durdag, H The influence of dietary probiotic (Saccharomyes cerevisiae) supplementation and different slaughter age on the performance, slaughter and carcass properties of broilers. International Journal of Poultry Science, 4: Krasaekoopt, W., Bhandari, B. and Deeth, H Evaluation of encapsulation techniques of probiotics for yoghurt. International Dairy Journal, 13: Mikelsaar, M., Mandar, R. and Sepp, E Lactic acid microflora in the human microbial ecosystem and its development in lactic acid bacteria, microbiology and functional aspects. In: Food Science and Technology. (Eds.) Salminen and von Wright. Marcel Dekker, Inc. pp Mountzouris, K.C., Tsirtsikos, P., Kalamara, E., Nitsch, S., Schatzmayr, G. and Fegeros, K Evaluation of the efficacy of probiotic containing Lactobacillus, Bifidobacterium, Enterococcus, and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities. Poultry Science, 86: Mountzouris, K.C., Tsitrsikos, P., Palamidi, I., Arvaniti, A., Mohnl, M., Schatzmayr, G. and Fegeros, K Effects of plasma immunoglobulins and cecal microflora composition. Poultry Science, 89: NRC Nutrient Requirement of Poultry. 9 th rev. edn, National Research Council, National Academy Press, Washington, D.C. Indian Journal of Poultry Science (2012) 47(3): / 361

6 Rao, A.V., Shiwnarain, N. and Maharaj, J Survival of microencapsulated Bifidobacterium pseudolongum in simulated gastric and intestinal juices. Canadian Institute of Food Science and Technology Journal, 22: Sahin, E.H. and Yardimci, M Effects of kefir as a probiotic on growth performance and carcass characteristics in geese. Journal of Animal and Veterinary Advances, 8: Shah, N.P., Ali, J.F. and Ravula, R.K Populations of L.acidophilus, Bifidobacterium spp., and Lactobacillus casei in commercial fermented milk products. Bioscience and Microflora, 19: Stella Alberto, V., Fava, M., Bersani, C., Del Degan, G., Savoini, G. and Chevaux, E Effets de l addition de pediococcus acidilactici dans la ration de poulet de chair sur les performances zootechniques et la microflore intestinale. 6eme JRA-St-Malo, pp Vargas, Jr. J.G, Toledo, R.S., Albino, L.F.T., Rostango, H.S., Oliveira, J.E. and Carvalho, D.C.O Caracteristicas de carcaca de frango de corte submetidos a racoes contend probioticos prebioticos e antibioticos In: XXXIX Reunião Anual da SBZ, Recife. Vittorio, S.A., Mauro, F., Carla, B., Giovanna, S.D. and Giovanni, S.E Effets de Effects l addition de Pediococcus acidilactici dans la ration de poulets de chair sur les performancesthe addition of Pediococcus acidilactici in the diet of broiler performance Livestock and intestinal microflora In Poultry Research. In: Proceedings of the 6th DaysRecherche Avicole, St Malo (FRA), pp Poultry Research, St Malo (FRA), pp Wadstrom, T. and Aleljung, P Molecular aspects of bowel colonization. In: The Regulatory and Protective Role of the Normal Microlflora. (Eds.) Gurbb, Midvedt and Norin. Stockton Press, Stockholm, Sweden. pp / Indian Journal of Poultry Science (2012) 47(3):