Isfahan , IRAN; 2 Department of Animal Sciences, College of Agriculture, Shahrekord Branch, Islamic Azad University, Shahrekord, IRAN

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1 Effect of Dietary Butyric Acid Supplementation on Blood Metabolites, Ileal Microbial Population and Nutrient Digestibility in Laying Hens Fed Corn- or Wheat-Based Diets Rahman Jahanian 1* and Morteza Golshadi 2 1 Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan , IRAN; 2 Department of Animal Sciences, College of Agriculture, Shahrekord Branch, Islamic Azad University, Shahrekord, IRAN * Corresponding Author: r.jahanian@cc.iut.ac.ir ABSTRACT The present trial aimed to investigate the effect of different levels of butyric acid (BA) on blood biochemical metabolites, ileal microbial ecosystem and nutrient digestibility in Leghorn laying hens. A total of 150 Hy-Line W-36 laying hens (56 wk of age) were used in a completely randomized design. Dietary treatments consisted of the levels of 0, 0.25 and 0.50% of glyceride form of BA, which added into the two basal diets (diets based on cornsoybean meal or diets containing wheat-corn-soybean meal) and fed to the birds during a 77 d period including 7 d for adaptation and 70 d as main recording period. Both serum triglycerides and cholesterol contents were affected (P<0.01) by the type of basal diet so that, the least concentrations of these blood metabolites were observed in laying hens fed wheatbased diets. On the other hand, dietary BA supplementation increased (P<0.01) serum triglycerides and cholesterol contents. Dietary treatments affect ileal microbial ecosystem with greatest alterations was shown in Escherichia coli (E. coli) populations and dietary BA supplementation reduced (P<0.05) E. coli number in ileal digesta. Interestingly, feeding wheat-based diets caused significant (P<0.05) increase in ileal lactobacillus population. Although ileal digestibility of protein wasn't influenced by basal diet type, BA level or respective interaction, but dietary inclusion of BA improved ileal fat (P<0.01) and ash (P<0.05) digestibilities. The present findings indicated that dietary BA supplementation improves nutrient digestibility and this effect is more obvious in wheat-based diets. Keywords: Laying hens, Butyric acid, Wheat, Serum cholesterol and triglycerides, Ileal microbial ecosystem, Nutrient digestibility Introduction The use of organic acids as feed additives in poultry nutrition has been recently investigated by several authors (Gunal et al., 2006; Abdel-Fattah et al., 2008; Biggs and Parsons, 2008). It has been shown that use of organic acids in broiler rations increases body weight gain and improves feed efficiency (Abdel-Fattah et al., 2008). Among organic acids, butyric acid appears to play an important role in development of the intestinal epithelium. It has been 1

2 reported that butyrate derived from the fermentation of non-starch polysaccharides developed epithelial cells in humans (Brons et al., 2002). Also, Jahanian (2011) showed that butyric acid increased villi height in jejunal epithelial cells. In the study by Chowdhury et al. (2009), supplementation of citric acid at the level of 0.5% had positive effects on growth, feed intake, feed efficiency, carcass yield, bone ash, and immune status of broilers. Therefore, citric acid might be a useful additive maintaining growth performance and health status of broilers (Chowdhury et al., 2009). To our knowledge, there is no research study to investigate the interactive effects of organic acids and dietary crop source on gut bacterial populations and ileal digestibility. The present study was designed to investigate this possible interaction in laying hens. Materials and Methods The present study was performed at the Poultry Research Station of Isfahan University of Technology (Isfahan, Iran) and all procedures used were approved by the Isfahan University of Technology Animal Care and Use Committee. A total of 150 Hy-Line W-36 laying hens of 56 weeks of age were randomly allocated into the 6 dietary treatments with 5 replicates of 5 birds each. Dietary treatments were consisted a 3 2 factorial arrangement of treatments with 2 basal diets (corn-sbm based or wheat-corn-sbm based diets) and 3 different butyric acid levels (0, 0.25, or 0.5% of diet). The experimental diets were formulated to be isocaloric and isonitrogenous, and all of the diets had the similar nutrient composition. The experimental period lasted for a total of 10 wk started immediately after one week adaptation period. At d 50 of main trial period, blood samples were collected from two birds per replicate, thereafter the sera samples were analyzed for serum triglycerides, cholesterol and high-density lipoproteins (HDL) concentrations. Chromic oxide-containing diets (supplemented with 0.5% chromic oxide) were fed for five day during 66 th to 70 th d of trial to investigate ileal nutrient digestibility. At the final day, two birds per each replicate were killed by cervical dislocation and ileal contents were collected into the sampling vials to count populations of Escherichia coli, Salmonella spp., and Lactobacillus spp. Also, the intestinal tract was removed and the contents of the tract from Merckel s diverticulum to the ileal-cecal-colon junction were collected directly into 250 ml specimen cups (Scott and Boldaji, 1997). Samples were held on ice, frozen ( 20 C), freezedried, and stored for analysis. Feed samples and freeze-dried excreta were ground (0.5 mm screen) prior to chemical analysis. The samples were analyzed for crude protein, ether extract, and total ash according to standard procedures of AOAC (2002). Data obtained were subjected to analysis of variance using General Linear Model procedures of SAS statistical software (SAS Institute, 1999) and treatment means were compared by Duncan s multiple range tests at P<0.05 statistical level. Results and Discussion Data on serum biochemical parameters were presented in Table 1. As shown, both serum triglycerides and cholesterol contents were affected (P<0.01) by the type of basal diet so that, the least concentrations of these blood metabolites were observed in laying hens fed wheatbased diets. On the other hand, dietary BA supplementation increased (P<0.01) serum triglycerides and cholesterol contents. The repots show that organic acids improve nutrient digestibility within the gastrointestinal tract (Boling et al., 2000; Ricke, 2003) If organic acid could increase digestibility of energy, then the excess energy could increase serum triglycerides. 2

3 Table 1. Effect of dietary supplementation of glyceride form of butyric acid on serum metabolites (mg/dl) in laying hens fed on corn- or wheat-based diets Butyric acid level (% of diet) Triglyceride Cholesterol High-density lipoproteins Corn-based Wheat-based Probability Type of basal diet *** ** NS Butyric acid level ** ** NS Acid level NS NS NS NS: not significant; ** P<0.01; *** P< Dietary treatments had significant effect (P<0.05) on ileal microbial populations with greatest alterations was shown in Escherichia coli populations and dietary BA supplementation reduced (P<0.05) Escherichia coli number in ileal digesta (Table 2). Interestingly, feeding wheat-based diets caused significant (P<0.05) increase in ileal lactobacillus population. Organic acids have a long history of being utilized as food additives and preservatives for preventing food deterioration and extending the shelf life of perishable food ingredients. Specific organic acids have also been used to control microbial contamination and dissemination of foodborne pathogens in preharvest and postharvest food production and processing (Ricke, 2003). The antibacterial mechanism(s) for organic acids are not fully understood, and activity may vary depending on physiological status of the organism and the physicochemical characteristics of the external environment. Given the weak acid nature of most of these compounds, ph is considered a primary determinant of effectiveness because it affects the concentration of undissociated acid formed (Davidson, 2001). Table 2. Effect of dietary supplementation of glyceride form of butyric acid on ileal bacterial populations (Log 10 cfu/g fresh digesta) in laying hens fed on corn- or wheat-based diets Butyric acid level (% of diet) Total microbial count Escherichia coli Salmonella spp. Lactobacillus spp. Corn-based Wheat-based Probability Type of basal diet * ** NS * Butyric acid level ** * NS NS Acid level * ** NS NS NS: not significant; * P<0.05; ** P<

4 It has been traditionally assumed that undissociated forms of organic acids can easily penetrate the lipid membrane of the bacterial cell and once internalized into the neutral ph of the cell cytoplasm dissociate into anions and protons (Eklund, 1983; Cherrington et al., 1990; Davidson, 2001). Generation of both of these species potentially presents problems for bacteria that must maintain a near neutral ph cytoplasm to sustain functional macromolecules. Export of excess protons requires consumption of cellular adenosine triphosphate (ATP) and may result in depletion of cellular energy (Davidson, 2001). As noted in Table 3, although ileal digestibility of protein wasn't influenced by basal diet type, BA level or respective interaction, but dietary inclusion of BA improved ileal fat (P<0.01) and ash (P<0.05) digestibilities. Table 3. Effect of dietary supplementation of glyceride form of butyric acid on ileal nutrient digestibility (%) in laying hens fed on corn- or wheat-based diets Butyric acid level (% of diet) Crude protein Ether extract Total ash Corn-based Wheat-based Probability Type of basal diet NS NS * Butyric acid level NS ** * Acid level ** NS NS NS: not significant; * P<0.05; ** P<0.01. In general, the present findings suggest that supplementation of layer diets with butyric acid could decrease adverse microbial populations of gastrointestinal tract and improve nutrient digestibility. These effects were more obvious in wheat-based diets. References ABDEL-FATTAH, S.A., M.H. El-SANHOURY, N.M. El-MEDNAY, F. ABDEL-AZEEM, 2008: Thyroid activity, some blood constituents, organs morphology and performance of broiler chicks fed supplemental organic acids. Int. J. Poult. Sci. 7, AOAC, 2002: Official Methods of Analysis of AOAC International. 17th ed., Washington, DC., USA. BIGGS, P., C.M. PARSONS, 2008: The effects of several organic acids on growth performance, nutrient digestibilities, and cecal microbial populations in young chicks. Poult. Sci. 87, BOLING, S.D., D.M. WEBEL, I. MAVROMICHALIS, C.M. PARSONS, D.H. BAKER, 2000: The effects of citric acid on phytate phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78,

5 BRONS, F., B. KETTLITZ, E. ARRIGONI, 2002: Resistant starch and the butyrate revolution. Trends Food Sci. Technol. 13, CHERRINGTON, C.A., M. HINTON, I. CHOPRA, 1990: Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli. J. Bacteriol. 68, CHOWDHURY, R., K.M.S. ISLAM, M.J. KHAN, M.R. KARIM, M.N. HAQUE, M. KHATUN, G.M. PESTI, 2009: Effect of citric acid, avilamycin, and their combination on the performance, tibia ash, and immune status of broilers. Poult. Sci. 88, DAVIDSON, P.M., 2001: Chap. 29. Chemical preservatives and natural antimicrobial compounds. In: DOYLE, M.P., L.R. BEUCHAT, T.J. MONTVILLE: Food Microbiology Fundamentals and Frontiers. 2nd ed. ed. American Society for Microbiology, Washington, DC, EKLUND, T., 1983: The antimicrobial effect of dissociated and undissociated sorbic acid at different ph levels. J. Appl. Bacteriol. 54, GUNAL, M., G. YAYLI, O. KAYA, N. KARAHAN, O. SULAK, 2006: The effects of antibiotic growth promoter, probiotic or organic acid supplementation on performance, intestinal microflora and tissue of broilers. Int. J. Poult. Sci. 5, JAHANIAN, R., 2011: Effect of varying levels of butyric acid glycerides on performance, immune responses and jejuna epithelium morphology of broiler chicks. 18th European Symposium on Poultry Nutrition. Izmir, Turkey, RICKE, S.C., 2003: Perspectives on the Use of Organic Acids and Short Chain Fatty Acids as Antimicrobials. Poult. Sci. 82, SAS INSTITUTE, 1999: SAS Statistics User s Guide. Statistical Analysis System. 5th rev. edition. Cary, NC., SAS Institute Inc. SCOTT, T.A., F. BOLDAJI, 1997: Comparison of inert markers [chromic oxide or insoluble ash (CeliteTM)] for determining apparent metabolizable energy of wheat- or barley-based broiler diets with or without enzymes. Poult. Sci. 76,