The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress Caused by Salmonella enterica

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1 Acta Scientiae Veterinariae, : 34. RESEARCH ARTICLE Pub. 34 ISSN The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress Caused by Salmonella enterica Ali Maddahian,2, Ali Asghar Sadeghi, Parvin Shawrang 3 & Mehdi Aminafshar ABSTRACT Background: Feed restriction is limiting feed in one period of breeding and has advantages such as decreasing ascite and sudden death syndrome. During feed restriction, colonization of pathogens. A great number of researches have shown the positive effects of feed restriction or prebiotic on performance in separate, but few have been done on their effects on blood parameters, especially in pathogen challenging condition. The aim of this study was to investigate the impacts of prebiotic and feed restriction, alone and together, on serum biochemical parameters of broilers under microbial stress caused by Salmonella enterica challenge. Materials, Methods & Results: A total of 320 male broilers (Ross 308) at a2 2 2 factorial arrangement in randomized complete block design were divided into eight treatment groups with four replicates per each. The impact of main effects of prebiotic, feed restriction and Salmonella challenge and their interactions on serum parameters were measured at days 2 and 42 of age. The addition of prebiotic to diet decreased serum cholesterol (P < 0.0) and low density lipoprotein (P < 0.0), but increased high density lipoprotein (P < 0.05), total protein (P < 0.05) and albumin (P < 0.05) at day 42 of age. The feed restriction increased triglyceride (P < 0.00) at day 2 of age. The Salmonella challenge decreased total protein (P < 0.0) and albumin (P < 0.0) at day 2 of age. Interaction between prebiotic and feed restriction on serum high density lipoprotein (P < 0.05) at day 42 of age, and the interaction between feed restriction and Salmonella challenge on serum high density lipoprotein (P < 0.05) and interaction among prebiotic, feed restriction and Salmonella challenge on serum glucose (P < 0.05) at day 2 of age were significant. Serum uric acid was not affected by the main effects and their interactions (P > 0.05). Discussion: It seems that prebiotics are beneficial to lowering blood cholesterol concentration by helping the rise in Lactic acid bacteria count. The stress caused by limitation in feed access can lead to increase in corticosterone hormone secretion. Corticosterone increases lipolysis in tissues therefore TG level rises in blood. Prebiotic caused an increase in serum high density lipoprotein level. Serum low density lipoprotein level in prebiotic supplemented groups significantly decreased at day 42 of age. Probably prebiotics can cause difference in lipid metabolites in blood. It seems that Salmonella and prebiotic affect amino acid absorption in intestine by decreasing and increasing nutrient digestibility, respectively. Salmonella challenge stress can lead to more secretion of corticosterone hormone and consequently higher level of blood glucose, whereas prebiotics cause decrease in blood glucose level by reducing the stresses and therefore the secretion of this hormone. The results of this study showed that prebiotics can be effective on decreasing blood lipid metabolites such as cholesterol. This can probably cause a decrease in fat deposition in carcass of broilers. Also, when pathogens like Salmonella attack the broilers, prebiotics can remove these pathogens from the gut and increase the nutrient digestibility, consequently increase blood metabolites such as glucose and protein. Feed restriction severity used in this experiment was not effective on decreasing blood lipid metabolites. Possibly, more severity or longer duration of feed restriction is needed for it to be effective on blood lipid metabolites. Keywords: prebiotic, feed restriction, Salmonella, serum biochemical parameters. Received: 22 February 205 Accepted: 0 September 205 Published: 6 October 205 Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Agriculture, Payame Noor University, Tehran, Iran. 3 Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, Karaj, Iran. CORRESPONDENCE: A.A. Sadeghi [a.sadeghi@srbiau.ac.ir - Fax: +98 (26) ]. Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.

2 A. Maddahian, A.A. Sadeghi, P. Shawrang & M. Aminafshar The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress... Acta Scientiae Veterinariae. 43: 34. INTRODUCTION The recent concern in poultry industry is the harmful effects of antibiotic growth promoters (AGP), and the probability of remaining drugs in carcass, therefore antibiotic usage has been limited in many countries [2]. One of these alternatives is prebiotic which is an indigestible feed ingredient selectively used by useful gut bacteria [6,4,8,9]. In addition, feed restriction is necessary for decreasing metabolic disorders like ascite and sudden death syndrome, reducing feed costs and improving feed efficiency, and reduction of fat deposition in carcass and abdominal cavity [23]. Feed restriction and feeding diet without antibiotic could result in incidence of intestinal infections, because of longer remaining feed and digesta motility. In the literature review, there is no information regarding the effects of prebiotic and feed restriction, alone and in combination, on blood parameters such as lipid metabolites [2], especially under feed restriction and bacterial challenging. It seems that prebiotics are indirectly advantageous to lipid metabolites in blood such as cholesterol and triglyceride by changing intestine s micro-flora [26]. Therefore, this study was done to investigate the individual and combined effects of feed restriction and prebiotic supplementation on broiler chickens blood parameters under microbial stresses caused by oral gavage of Salmonella Enteritidis. MATERIALS AND METHODS The prebiotic was used at the amount of 0.% of the diet. This product consists of mannanoligosaccharide (MOS) and β-, 3-Glucan and has been obtained from Saccharomyces cerevisiae. Animals Three hundred twenty one-day-old male broilers (Ross 308) were obtained from a commercial hatchery. They were divided into eight treatment groups with four replicates and 0 broilers in each replicate at a factorial arrangement in randomized complete block design, and were then put in special cages. A diet based on corn and soybean meal (Table ) was used for feeding chickens. Methodology Based on a valid method [22], from day 7 to day 4, the daily feed intake of the groups getting ad libitum consumption was measured, and 80% of this feed was given to feed restricted treatment groups the next day. Salmonella enterica (PTCC 709) 2 was provided freeze-dried from the Persian Type Culture Collection 3 isolated from the liver of chickens. Freezedried inoculum was grown in nutrient broth media at 37 C for 8-h and passed to fresh nutrient broth for 3 incubation periods. Counting colony-forming units (cfu) was done with Neobar lam at day 0 of age. After counting, chickens in the challenged groups received cfu/chick of passaged medium through oral gavage with utilizing micropipette on the same day. Treatment groups The treatment groups included :) a control group with no supplementation and challenge (C); 2) a group under feed restriction (R); 3) a Salmonella challenged group (S); 4) a group under feed restriction and Salmonella challenge (R+S); 5) a prebiotic treated group (P); 6) a prebiotic treated and feed restricted group (P+R); 7) a prebiotic treated under Salmonella challenge (P+S), and 8) a prebiotic treated under feed restriction and Salmonella challenge (P+R+S). Blood samples At days 2 and 42 of age, 3 ml of blood sample was collected from wing vein of two birds in each replicate (8 birds per each treatment), centrifuged at 500 g for 0 min and the serum was separated, then stored at -20 C until assayed to measuring blood parameters including cholesterol, triglycerides (TG), high density lipoprotein (HDL) cholesterol, total protein (TP), albumin, glucose and uric acid using commercial kits (Pars Azmoon reagents kit) 4 according to the manufacturer s protocols. The low density lipoprotein (LDL) cholesterol was calculated using following formula [5]: LDL = cholesterol - HDL - (TG/5) Statistical analysis All data were subjected to GLM procedures of SAS software (SAS User s Guide: Statistics.2002.) 5 as a factorial arrangement of treatments in randomized complete block design that included prebiotic supplementation, feed restriction and Salmonella challenge as the main factors and their respective interactions. The treatment means were compared by Duncan s multiple range tests. Probability values of less than 0.05 (P < 0.05) were considered significant. 2

3 A. Maddahian, A.A. Sadeghi, P. Shawrang & M. Aminafshar The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress... Acta Scientiae Veterinariae. 43: 34. Table. Ingredient (%, as fed), composition and nutrient content of diets fed in this experiment. Ingredient Yellow corn Soybean meal (44%) Fish meal (62%) Soybean oil Di-calcium phosphate Limestone Common salt DL-Methionine Vitamin and Mineral premix Prebiotic 2 (or Sand) Nutrient Metabolizable energy (kcal/kg) Crude protein (%) Lys (%) Met (%) Met + Cys (%) Calcium (%) Available phosphrous (%) Sodium (%) Starter (days 0 to 2) Grower (days 2 to 42) Vitamin and Mineral premix provided per kilogram of diet: vitamin A, IU; vitamin D3, IU; vitamin E, 7200 IU; vitamin K3, 800 mg; vitamin B, 720 mg; vitamin B9, 400 mg; vitamin H2, 40 mg; vitamin B2, 2640 mg; vitamin B3, 4000 mg; vitamin B5, 2000 mg; vitamin B6, 200 mg; vitamin B2, 6 mg; choline chloraid, mg; manganeze, mg; iron, mg; zinc, mg; copper, 4000 mg; iodine, 400 mg; selenium, 80 mg; 2 The prebiotic (TechnoMOS) has the same amounts of mannan-oligosaccharide and β-,3-glucan. RESULTS Table 2 shows the effects of various treatments on serum cholesterol, triglyceride, HDL and LDL of broilers at days 2 and 42 of age. There was no significant difference of serum cholesterol among treatment groups at day 2 of age (P > 0.05), but at day 42 of age the main effect of prebiotic on reducing the serum cholesterol was significant (P < 0.0). There was a significant difference of serum TG among treatment groups at day 2 of age which was caused by the main effect of feed restriction (P < 0.00). TG concentration in the serum of broilers was higher in feed restricted groups, but the difference of serum TG was not significant among treatment groups at day 42 of age (P > 0.05). Serum HDL level was different among treatment groups at days 2 and 42 of age. The interaction between feed restriction and Salmonella challenge had a significant effect on serum HDL level at day 2 of age (P < 0.05), and at day 42 of age, the main effect of prebiotic (P < 0.05) and the interaction between prebiotic and feed restriction (P < 0.05) on serum HDL level was significant. At day 2 of age, there was no significant difference of serum LDL level among treatment groups (P > 0.05), but serum LDL level in prebiotic supplemented groups significantly decreased at day 42 of age (P < 0.0). The effects of various treatments on total protein, albumin, glucose and uric acid are shown in Table 3. At day 2 of age, the main effect of Salmonella challenge significantly decreased serum TP and albumin (P < 0.0), but the main effect of prebiotic significantly increased them at day 42 of age (P < 0.05). There was no significant difference of serum glucose level among treatment groups at day 2 of age (P > 0.05), but the effect of interaction among prebiotic supplementation, feed restriction and Salmonella challenge on serum glucose level was significant at day 42 of age (P < 0.05). None of the main effects and interactions had a significant effect on blood uric acid level at days 2 and 42 of age (P > 0.05). 3

4 A. Maddahian, A.A. Sadeghi, P. Shawrang & M. Aminafshar The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress... Acta Scientiae Veterinariae. 43: 34. Table 2. Serum cholesterol, triglyceride, HDL and LDL levels in different treatment groups at days 2 and 42 of age. Treatment Cholesterol day 2of age Triglyceride HDL LDL Cholesterol day 42 of age Triglyceride HDL LDL C ab(2) a a ab ab R a 5.75 ab ab b a S b 0.75 ab ab a bc R+S a 6.75 ab ab b abc P ab ab bc ab 9.80 bc P+R a b abc a 2.45 c P+S ab 4.25 ab ab ab abc P+R+S a 8.50 ab c a.90 c SEM Probability 3 Main effect P NS NS NS NS ** NS * ** R NS *** NS NS NS NS NS NS S Interaction NS NS NS NS NS NS NS NS P R NS NS NS NS NS NS * NS P S NS NS NS NS NS NS NS NS R S NS NS * NS NS NS NS NS P R S NS NS NS NS NS NS NS NS C: control group; R: restriction in feed; S: Salmonella challenge; P: prebiotic supplementation; 2 a,b,c Within the same column, means with different superscripts are significantly differ (P < 0.05); 3NS: not significant; *P < 0.05; **P < 0.0; ***P < Table 3. Serum TP, albumin, glucose and uric acid levels in different treatment groups at days 2 and 42 of age. day 2 of age day 42 of age Treatment Albumin Glucose Uric acid Albumin Glucose Uric acid TP (g/dl) TP (g/dl) (g/dl) (g/dl) C 2.93 ab(2).20 ab b.00 bc b 4.63 R 2.63 ab.3 ab ab.3 abc ab 3.88 S 2.8 ab 0.98 ab ab 0.98 c a 4.48 R+S 2.5 b 0.85 b ab.5 abc b 3.60 P 3.30 a.45 a a.30 a b 4.0 P+R 2.95 ab.43 a a.25 ab b 4.40 P+S 2.30 ab 0.93 ab ab.5 abc b 4.80 P+R+S 2.30 ab 0.93 ab ab.5 abc b 4.63 SEM Probability 3 Main effect P NS NS NS NS * * NS NS R NS NS NS NS NS NS NS NS S Interaction ** ** NS NS NS NS NS NS P R NS NS NS NS NS NS NS NS P S NS NS NS NS NS NS NS NS R S NS NS NS NS NS NS NS NS P R S NS NS NS NS NS NS * NS C: control group; R: restriction in feed; S: Salmonella challenge; P: prebiotic supplementation; 2 a,b,c Within the same column, means with different superscripts are significantly differ (P < 0.05); 3NS: not significant, *P < 0.05; **P <

5 A. Maddahian, A.A. Sadeghi, P. Shawrang & M. Aminafshar The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress... Acta Scientiae Veterinariae. 43: 34. DISCUSSION In our experiment, prebiotic supplementation decreased serum cholesterol at day 42 of age. The most important way of cholesterol excretion is synthesis of bile acids from cholesterol [25]. A study reported that increase in lactic acid bacteria amount and activity decreases intestine s ph which contributes to the reduction of serum cholesterol concentration, because solution of bile acids is less in low ph and this leads to their higher excretion in feces [2]. Eventually, the liver sends less cholesterol to the tissues to re-establish the hepatic cycle of bile acids, and the blood cholesterol concentration decreases [6]. It seems that prebiotics are beneficial to lowering blood cholesterol concentration by helping the rise in Lactic acid bacteria count [26]. Our findings are in agreement with results of one study, which expressed that the prebiotic extracted from yeast and copra meal reduces the serum cholesterol concentration [9]. Also other researchers reported serum cholesterol reduction by using mannan-oligosaccharide in diet [2,0,27]. On the contrary, according to another work, there was no significant decrease in serum cholesterol concentration using inulin-type prebiotic [24]. Feed restriction caused a rise in serum TG level at day 2 of age. Generally, TG is affected by two factors: diet and hormones. More feed intake causes less lipolysis which leads to decrease of blood TG, therefore feed restriction can increase blood TG. Also, the stress caused by limitation in feed access can lead to increase in corticosterone hormone secretion. Corticosterone increases lipolysis in tissues therefore TG level rises in blood [3]. One research reported that feed restriction in broilers increases TG level at day 42 of age [5]. However another results found no significant difference in serum TG level among feed restricted groups and the control group at day 28 in broilers [4]. Similar results were found in another study [23]. It seems that these contradictive results are because of the effect of other factors in the experiments, such as Salmonella and prebiotic in ours. Researchers [0,27] observed no significant difference in serum TG level using MOS which is in agreement with our findings, but a study [9] reported that MOS decreased serum TG in broilers. Serum HDL level was different among treatment groups at days 2 and 42 of age. It seems that prebiotic caused an increase in serum HDL level. Serum LDL level in prebiotic supplemented groups significantly decreased at day 42 of age. Probably prebiotics can cause difference in lipid metabolites in blood. Literature registered that prebiotics may cause simultaneous reduction of hepatic expression and activity of lipogenic enzymes and consequently reduction of fatty acids synthesis [24]. Also probiotics can reduce the activity of acetyl-coa carboxylase which is the main regulatory enzyme of fatty acids synthesis [20]. Prebiotics and probiotics have similar effects in this case. But using MOS in the diet, it was observed no significant effect on serum HDL and LDL levels [2], or only on HDL level [0]. On the other hand, another research reported increase in serum HDL and decrease in serum LDL levels using Lactobacillus probiotic [8]. Salmonella challenge causes a decrease in serum TP and albumin levels at day 2 of age, but prebiotic causes an increase in them at day 42 of age. It seems that Salmonella and prebiotic affect amino acid absorbtion in intestine by decreasing and increasing nutrient digestibility, respectively. Similar results were obtained using ß-glucan prebiotic and Salmonella challenge together [9], but a research did not observe any significant difference in serum TP and albumin between prebiotic supplemented and control groups []. There was a report that showed serum TP and albumin levels were not significantly affected by feed restriction which is in agreement with our finding [3]. The Salmonella challenge group had higher level of serum glucose at days 2 and 42 of age compared to other groups, and at day 42 of age prebiotic supplemented groups had less serum glucose level than Salmonella challenge and feed restriction groups. Salmonella challenge stress can lead to more secretion of corticosterone hormone and consequently higher level of blood glucose, whereas prebiotics cause decrease in blood glucose level by reducing the stresses and therefore the secretion of this hormone. Literature reported decrease in blood glucose level using ß-glucan prebiotic [9]. Serum glucose level increases in broilers under feed restriction which can be explained by higher feed efficiency and better nutrient absorption in intestine [3]. Also chickens keep the blood glucose level constant during fasting and hypoglycemia is prevented by gluconeogenesis [], and blood glucose level increases in feed restricted broilers [5]. However, another study concluded that feed restriction had no significant effect on blood glucose [23]. 5

6 A. Maddahian, A.A. Sadeghi, P. Shawrang & M. Aminafshar The Impacts of Prebiotic and Feed Restriction on Serum Biochemical Parameters of Broilers under Microbial Stress... Acta Scientiae Veterinariae. 43: 34. None of the main effects and interactions has a significant effect on blood uric acid level. It was reported no significant effect on serum uric acid level using different feed restriction methods [3]. Also ß- glucan prebiotic did not affect the blood uric acid level in comparison with the control group [9]. CONCLUSION The results of this study showed that prebiotics can be effective on decreasing blood lipid metabolites such as cholesterol. This can probably cause a decrease in fat deposition in carcass of broilers. Also, when pathogens like Salmonella attack the broilers, prebiotics can remove these pathogens from the gut and increase the nutrient digestibility, consequently increase blood metabolites such as glucose and protein. Feed restriction severity used in this experiment was not effective on decreasing blood lipid metabolites. Possibly, more severity or longer duration of feed restriction is needed for it to be effective on blood lipid metabolites. MANUFACTURERS TechnoMOS Company Biochem. Lohne, Germany. 2 Salmonella enterica (Persian Type Culture Collection, no 709). 3 Iranian Research Organization for Science and Technology. Tehran, Iran. 4 Pars Azmoon Co. Tehran, Iran. 5 SAS Institute. Cary, NC, USA. Ethical approval. Use of birds in this study was approved by the local institutional animal ethical committee (Protocol ; 90--5). Declaration of interest. The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper. REFERENCES Al-Kassie G.A.M., Al-Jumaa Y.M.F. & Jameel Y.J Effect of probiotic (Aspergillus niger) and prebiotic (Taraxacum officinale) on blood picture and biochemical properties of broiler chicks. International Journal of Poultry Science. 7: Ashayerizadeh A., Dabiri N., Mirzadeh K.H. & Ghorbani M.R. 20. Effect of dietary supplementation of probiotic and prebiotic on growth indices and serum biochemical parameters of broiler chickens. Journal of Cell and Animal Biology. 5(8): Boostani A., Ashayerizadeh A., Mahmoodian-Fard H.R. & Kamalzadeh A Comparison of the effects of several feed restriction periods to control ascites on performance, carcass characteristics and hematological indices of broiler chickens. Brazilian Journal of Poultry Science. 2(3): Cornejo S., Gadelha A.C., Pokniak J. & Villouta G Qualitative feed restriction on productive performance and lipid metabolism in broiler chickens. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 59(6): Friedewald W.T., Levy R.I. & Fredrickson D.S Estimation of the Concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry. 8(6): Gibson G.R. & Roberfroid M.B Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Journal of Nutrition. 25(6): Hooge D.M Meta-analysis of broiler chicken pen trials evaluating dietary mannanoligosaccharide, International Journal of Poultry Science. 3(3): Kalavathy R., Abdullah N., Jalaludin S. & Ho Y.W Effects of lactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. British Journal of Poultry Science. 44(): Kannan M., Karunakaran R., Balakrishnan V. & Prabhakar T.G Influence of prebiotics supplementation on lipid profile of broilers. International Journal of Poultry Science. 4(2): Khalaji S., Zaghari M. & Nezafati S. 20. The effects of mannan-oligosaccharides on cecal microbial populations, blood parameters, immune response and performance of broiler chicks under controlled condition. African Journal of Biochemistry Research. 5(5): Klasing K.C Comparative avian nutrition. New York (USA): Cab International Press. 2 Klaver F.A.M. & Van Der Meer R The assumed assimilation of cholesterol by lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity. Applied and Environmental Microbiology. 59(4): Kubikova L., Vyboh P. & Kostal A Behavioural, endocrine and metabolic effects of food restriction in broiler breeder hens. Acta Veterinaria Brno. 70():

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