SERUM BIOCHEMISTRY, HAEMATOLOGY AND GUT ECOSYSTEM OF BROILER CHICKENS FED DIET TREATED WITH ORGANIC ACIDS

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1 SERUM BIOCHEMISTRY, HAEMATOLOGY AND GUT ECOSYSTEM OF BROILER CHICKENS FED DIET TREATED WITH ORGANIC ACIDS ABSTRACT Ndelekwute 1, E. K., Ebenso 1, I. E., Okereke 2, C. O. and Assam 1, E. D. 1 Department of Animal Science, University of Uyo, Uyo, Nigeria. 2 Livestock Unit, National Root Crops Research Institute, Umudike, Nigeria. *Correspondence author: ndelekwute.ek@gmail.com Tel: An experiment was conducted to determine the effect of organic-acids (OA)-treated-diets on serum biochemistry, heamatological indices; ph, viscosity and microbiology of gut digesta of broilers. The OAs were acetic acid (AA) butyric acid (BA), citric acid (CA) and formic acid (FA). One hundred and fifty (150) day old Abor-Acre chicks were used. There were five dietary treatments. Diet 1 (control) contained no OA, while diets 2, 3, 4 and 5 respectively contained 0.25% AA, BA, CA and FA. Each treatment was replicated three times having 10 birds per replicate, arranged in completely randomized design (CRD). Birds were fed and water given ad libitum for 8 weeks the experiment lasted. Results showed that there were no significant differences (P>0.05) in the number of white blood cells, haemoglobin, mean corpuscular haemoglobin concentration and mean corpuscular volume. The number of red blood cells was significantly higher in AA and BA groups. Diets did not significantly (P>0.05) alter blood biochemical indices except cholesterol which was significantly (P<0.05) lowered by CA and FA diets. AA diet showed more capacity to reduce the digesta ph followed by CA (P<0.05). The viscosity in the ileum and caecum was the same (P>0.05). However, AA and BA diets significantly (P<0.05) reduced viscosity in the duodenum. The gut bacteria population was significantly (P<0.05) reduced by OA diets. It was therefore concluded that the OAs reduced digesta viscosity; impacted no deleterious effect on the blood and could be added to broiler diets to reduce gut bacteria population. Keywords: broilers, gut ph, organic acids, microbiology, serum biochemistry, viscosity INTRODUTION For maximum productivity, adequate diet both in quality and quantity should be provided for broiler chickens. Hence, diet plays an important role for the well being of broiler chickens considering the significant part diet plays in development of the gut (Choct, 2009). The gut is where diet is processed; the nutrients are absorbed and distributed to the different parts of the body where they are further processed by the different organs. The gut must be in good condition to be able to digest feed. According to several reports the gut health determines the way nutrients are partitioned and utilized for organ development, tissue growth and immune system maturation ( Sun, 2004). According to Choct (2009) gut health status should be considered among other factors from the point of view of microbiology, otherwise the number and type of microflora or the bacteria community of the gut could undermine nutrient utilization (Choct, 2009). Not only could the bacteria population in the gut affect the gut health and its ability to process feed, Lee et al. (2004) reported that high digesta viscosity could negatively affect gut efficiency. According to several reports also, the ph of the stomach could regulate digestibility especially protein in young monogastric animals (Paul et al., 2007). It is pertinent to regulate the gut through dietary means so as to achieve the desired performance of broiler chickens in a short period of time. To this end, according to Choct (2009) formulating diets for its effect on gut health is fast becoming a reality in the monogastric animal industry. One of the oldest ways of modulating the gut is by addition of pharmaceutical antibiotics to the feed, but the practice is currently either restricted or banned in certain countries because of reported problem of antibiotic resistance (Dibner, 2004). Because of this, some dietary products are being introduced to replace antibiotics in poultry diets. The products include; probiotics prebiotic, yeast culture essential oils, spices and organic acids (Paul et al., 2007). Farmers have been expressing fears that addition of organic acids to broiler diets could induce negative effect on the birds especially on the blood. But Ndelekwute et al. (2013) opined that because organic acids are part of body biochemical processes, such as glycolysis and Kreb cycle they could be safe for use in broiler diets. Therefore, the objective of this research was to determine the dietary effects of acetic, butyric, citric and formic acids on serum biochemistry, haematology, digesta ph, viscosity and gut bacteria population of broiler chickens under intensive management. MATERIALS AND METHODS The experiment was conducted at the Teaching and Research Farm of Michael Okpara University of Agriculture Umudike; Nigeria (latitude 5 o 28 1 N and longitude 7 o 32 1 E). Completely randomized design (CRD) was used. One hundred and fifty (150) day old chicks of Abor-Acre strain were used. They were divided into five treatments replicated three times having 10 birds per replicate. Each treated group received acetic acid, butyric acid, citric NJAFE VOL. 12 No. 1,

2 acid or formic acid at 0.25% level of their diets, while the control group received neither of the organic acids. The birds were fed organic acids for eight weeks. The diets contained similar crude protein (22: 20%); energy (2850 : 2800kcalME/kg); calcium (1.2 : 1.0%), phosphorus (1.0 : 0.8%); fat (3.25 : 4.05%); fibre and (3.5 : 4.5%) for the starter and finisher feeds respectively. Feed and water were given ad libitum and Newcastle and Gumboro diseases vaccines were given. Determination of digesta ph, viscosity and microbiological analysis At the end of the experiment, the ph and bacteria load of the digesta were determined Digesta was collected from different segments of the digestive system (crop, gizzard, duodenum, ileum, caecum, and large intestine). Three birds per treatment representing one per replicate were killed and used for that purpose. Each digesta was collected into a McCartney bottle and quickly stored in a refrigerator at 4 o C pending analysis.. A ph meter (PHep, Hanna Instruments, Italy) was used to determine the ph according to Nisbet et al. (1994) and the viscosity was measured using a Viscometer (Bohlin CS 50 Rheometer, manufactured by Bohlin Reologi, Muhlacker, Germany) as described by Lee et al. (2004). Serial dilution technique after incubation of the digesta according to Fawole and Oso (1988) was used to determine the bacteria load. Blood collection and analysis Blood was collected and both haematological and serum chemistry analyses were carried out according to Fawole and Oso (1998). Blood collection was through the jugular vein by the use of hypodermic syringe into a 10ml capacity clinical Mackartney bottles containing dipotassium salt of ethelene-diamine-tetra-acetic acid (EDTA) as anticoagulant. Containers containing blood samples for serum biochemistry had no EDTA. Data transformation and statistical analysis Bacteria count expressed in colony forming units (cfu) were transformed using Log 10 according to Alshawabkeh and Tabbaa (2002). All Data collected were subjected to analysis of variance (ANOVA). Significant means were separated using Duncan New Multiple Range Test. RESULTS Haematological Indices Dietary effect on haematological indices (Table 1) shows that there were no significant differences (P>0.05) in the number of white blood cells, haemoglobin, mean corpuscular haemoglobin concentration and mean corpuscular volume. The number of red blood cells was significantly (P<0.05) higher in acetic and butyric acid groups than the control and the other organic acids. Packed cell volume of acetic acid group was significantly higher than those of butyric and formic acids. Mean corpuscular haemoglobin was higher (P<0.05) in acetic and citric acid groups than the control. Serum chemistry The serum chemistry indices (Table 2) were not significantly (P>0.05) different among the broilers fed organic acid diets and control diet except cholesterol level. Cholesterol level was significantly (P<0.05) reduced in broilers fed citric and formic acid diets than in those fed control and other organic acid diets. Digesta ph The results of the effect of diets on the ph value of the content of different gastro intestinal tract segments (Table 3) indicated that diets with organic acids resulted in significant alteration of the ph in all the segments except the crop and the ileum. In the proventriculus, the ph was significantly lowered by acetic and citric acid diets than the control. In the gizzard, acetic, citric and formic acid treated diets produced lower ph than the control diet. All the organic acid treated diets produced lower ph than the control diet in the duodenum. In the caecum, only the acetic acid diet significantly reduced the ph more than the control diet. Digesta viscosity The effect of treatment on digesta viscosity in the gastrointestinal tract (Table 3) shows only significant effect (P<0.05) on duodenal digesta. Acetic and butyric acid treated diets significantly reduced the viscosity of the duodenal digesta than the control diet. Though the viscosity of digesta in duodenum of citric and formic acid groups was the same with the control, they show promising ability to reduce gut viscosity. Bacteria load of gastro intestinal tract (GIT) The total bacteria load (Salmonella, E. coli, and Staphylococcus) in the foregut (crop; gizzard, duodenum) are shown on Table 4. Organic acid diets significantly (P<0.05) reduced the total bacteria load in all the segments of the gastro-intestinal tract (GIT) as compared to the control. Among broilers fed organic acid diets, total bacteria load was significantly (P<0.05) reduced in the crop by citric acid diet, followed by formic, butyric and acetic acids in that order. Reduction of number of Salmonella and Staphylococcus followed the same pattern as the total load. The number of E. coli was higher in the GIT of broilers fed acetic acid than in the other organic acid diets. In the gizzard, butyric acid diet reduced total bacteria load more than the control, citric and formic acid diets. Salmonella, E. coli and Staphylococcus loads were significantly (P<0.05) higher in the gizzard of broilers fed control diet than organic acids (acetic, citric and formic acid) diets. Total load and number of staphylococcus were statistically the same in all the organic acid dietary groups in duodenum. The number of salmonella though lower in butyric acid dietary group was similar to citric acid dietary group. The number was the same in acetic, citric NJAFE VOL. 12 No. 1,

3 and formic acid dietary groups. E. coli occurred higher in citric than in acetic acid dietary group but the same as in butyric and formic acid groups. In acetic, butyric and formic acid dietary groups the number of E. coli was the same. Table 1: Effect of organic acid treated diets on haematological parameters of broilers (± SEM) Parameters (%) CON AA BA CA FA White blood cells (x10 3 /mm 3 ) ± ± ± ± ±1.50 Red blood cells (x10 6 /mm 3 ) 2.15 b ± a ± a ± b ± b ± 0.14 Packed cell volume (%) ab ± a ± b ± ab ± b ±2.88 Haemoglobin (g/100ml) ± ± ± ± ±0.26 MCH (%) b ± a ± ab ± a ± ab ±2.11 MCHC (%) ± ± ± ± ±0.77 MCV (mg / 100ml) ± ± ± ± ±3.23 abc. Means along the same row with different superscripts are significantly different (P<0.05). CON = control, AA = Acetic acid, BA = Butyric acid, CA = Citric acid, FA = Formic acid. MCH = Mean Corpuscular Haemoglobin, MCHC = Mean Corpuscular Haemoglobin Concentration, MCV = Mean Corpuscular Volume. SEM = Standard error of the means. n = 30 Table 2: Effect of diets treated with organic acids on serum chemistry of broilers. (± SEM) Parameters CON AA BA CA FA Total protein (mg per 100 ml) 3.23± ± ± ± ±0.30 Albumin (mg per 100 ml) 2.00 ± ± ± ± ±0.37 Globulin (mg per 100 ml) 1.23 ± ± ± ± ±0.19 Urea (mg per 100 ml) 1.80 ± ± ± ± ±0.18 Cholesterol (mg per 100 ml) 142 a ± ab ± ab ± c ± c ±4.88 Creatinine (mg per 100 ml) 0.45 ± ± ± ± ±0.04 Glucose (mg per 100 ml) ± ± ± ± ±5.22 ALP (mg per 100 ml) ± ± ± ± ±3.69 ALT (iu per l) ± ± ± ± ±1.78 AST (iu per l) ± ± ± ± ±4.23 a,b,c: Means along the same row with different superscripts are significantly (P<0.05) different. SEM= Standard error of mean. CON=control, AA= Acetic acid, BA= Butyric acid, CA= Citric acid, FA= Formic acid. ALP= Alkaline phosphatise, ALT = Alanine amino transferase, AST = Aspartate amino transferase. SEM = Standard error of the means. n = 30. The observation made in the hindgut (ileum, caecum and large intestine) in Table 5 indicates that in the ileum, total bacteria load was significantly reduced in broilers fed acetic acid diets followed by those fed butyric, citric and formic acid diets. The number of salmonella and staphylococcus was the same in all the broilers fed organic acid diets. E. coli population in the ileum of broilers was the same with acetic, citric and formic acid diets but significantly higher with butyric acid diet. In the caecum total bacteria load among the organic acids was significantly reduced by acetic and citric acid diets compared to butyric and formic acid diets. Organic acid diets did not have significant effect on the number of salmonella. The number of E. coli and staphylococcus were significantly reduced by acetic and citric acid diets compared to butyric and formic acid diets which were statistically similar. In the large intestine, effect of organic acid diets on the various bacteria was not significant. Table 3: Effect of organic acid treated diets on digesta ph and viscosity (± SEM) ph (-log H + ) Crop 5.40 ± ± ± ± ±0.20 Proventriculus 4.80 a ± b ± b ± b ± b ±0.21 Gizzard 3.73 a ± c ± ab ± bc ± bc ±0.16 Duodenum 6.63 a ± b ± b ± b ± b ±0.19 Ileum 6.40 ± ± ± ± ±0.27 Caecum 5.50 a ± b ± b ± ab ± a ±0.41 Viscosity Duodenum 1.38 a ± b ± b ± ab ± ab ±0.03 Ileum 1.84± ± ± ± ±0.13 Caecum 1.91± ± ± ± ±0.31 abc Means along the same row with different superscripts are significantly different (P<0.05). SEM = Standard error mean. CON = control, AA = Acetic acid, BA = Butyric acid, CA = Citric acid, FA = Formic acid, GIT = gastro intestinal tract. SEM = Standard error of the means. n = 30 NJAFE VOL. 12 No. 1,

4 DISCUSSION Haematological results indicated that the diets did not produce any deleterious effect on the haemalogolical parameters. This is because all the values fell within the normal range for mature broiler chickens reported by Banerjee (2007) to be white blood cells 9 31 x 10 3 per mm 3, red blood cells 2 4 x 10 6 per mm 3, haemoglobin 7 13 g per 100ml, packed cell volume 25 45%, mean corpuscular haemoglobin 33 57%, mean corpuscular haemoglobin concentration 26 35% and mean corpuscular volume mg per 100ml. Reduction of serum cholesterol by citric and formic acids is significant in that they could be used to reduce cholesterol in broilers. This result was in line with Abdel-Fattah et al. (2008) who reported that feeding of organic acids reduced serum cholesterol in broiler chickens. It was also observed that all the blood biochemical indices were within the normal range reported by Banerjee (2007). Lower ph at different segments of the gut could be as a result of organic acids in the diets according to Dibner (2004). This is significant in the sense that low ph in the stomach has been linked to better protein digestibility in young monogastrics (Canibe et al., 2008). This work is in line with numerous reports that organic acids in broiler diets could reduce the ph of the gastro intestinal tract (Dibner, 2004). The non significant difference in the large intestine showed that the concentration of the acids probably had reduced as the acids moved down the tract and cannot effectively work against the bacteria. Table 4: Effect of organic acid treated diets on bacteria load in the foregut of broilers. (± SEM) Crop Total load(x10 4 ) a ± b ± c ± e ± d ±0.10 Salmonella (x10 4 ) 8.25 a ± b ± c ± e ± d ±0.12 E. coli (x10 4 ) 4.66 a ± b ± c ± d ± cd ±0.02 Staphylococcus (x10 3 ) 1.63 a ± b ± c ± e ± d ±0.02 Gizzard Total load (x10 3 ) a ± b c ± c ± b ± b ±0.09 Salmonella (x10 3 ) 9.42 a ± b ± c ± b ± b ±0.03 E. coli (x10 3 ) 4.33 a ± b ± b ± b ± b ±0.02 Staphylococcus (x10 3 ) 1.90 a ± b ± c ± b ± b ±0.02 Duodenum Total load (x10 3 ) a ± b ± b ± b ± b ±0.13 Salmonella (x10 3 ) a ± b ± c ± b c ± b ±0.09 E. coli (x10 3 ) 8.17 a ± c ± bc ± b ± bc ±0.04 Staphylococcus (x10 3 ) 0.41 a ± b ± b ± b ± b ±0.01 abc Means along the same row with different superscripts are significantly different (P<0.05). SEM Standard error mean. CON=Control. AA = Acetic acid, BA = Butyric = acid, CA = Citric acid, FA = Formic acid, GIT = gastro intestinal tract. SEM = Standard error of the means. n = 30. Table 5: Effect of organic acid treated diets on bacteria load in the hindgut of broilers. (± SEM) Ileum Total load (x10 4 ) 3.67 a ± d ± c ± b ± b ±0.05 Salmonella (x10 3 ) 1.74 a ± b ± b ± ab ± b ±0.08 E. coli (x10 3 ) 1.68 a ± c ± b ± c ± c ±0.03 Staphylococcus (x10 3 ) 0.86 a ± b ± b ± b ± b ±0.02 Caecum Total load (x10 4 ) 5.44 a ± c ± b ± c ± b ±0.08 Salmonella (x10 4 ) 2.70 a ± b ± b ± b ± b ±0.04 E. coli (x10 4 ) 1.13 a ± c ± b ± c ± b ±0.03 Staphylococcus (x10 3 ) 0.92 a ± c ± b ± c ± b ±0.03 Large intestine Total load (x10 4 ) 2.27 a ± b ± b ± b ± b ±0.10 Salmonella (x10 3 ) 1.20 a ± b ± b ± b ± b ±0.04 E. coli (x10 3 ) 0.60 a ± b ± b ± b ± b ±0.02 Staphylococcus (x10 3 ) 0.36 a ± b ± b ± b ± b ±0.02 abcd. Means along the same row with different superscripts are significantly different (P<0.05). CON = Control. AA = Acetic acid, BA = Butyric = acid, CA = Citric acid, FA = Formic acid, GIT = gastro intestinal tract. SEM = Standard error of the means. n = 30 Apart from the antibacteria action of the organic acids to reduce the gut bacteria load the low viscosity in the duodenum of acid treated groups could have played a significant role in reducing the bacteria population. Longer stay (caused by higher viscosity) of digesta in the duodenum could lead to higher multiplication of bacteria (Lee et NJAFE VOL. 12 No. 1,

5 al., 2004). This result strongly agrees Lee et al. (2004) who stated that organic acids could improve digesta flow in the small intestine. The present results indicate that organic acids had varying capacities to reduce bacteria load in different segments of the gastro intestinal tract. Nevertheless, it could be established that organic acids have antibacteria effect. Low number of bacteria count recorded in different segments of the gastro intestinal tract of birds that consumed acid treated diets could be traced to low the ph. Low ph in the gut has been noted to reduce microbial population by penetrating the cell of bacteria in undissociated form, in that process releasing a proton (H + ) which is injurious to the bacteria cell (Dibner, 2004). This result is supported by earlier reports (Mchan, 1992) that organic acids in broiler diets reduced microbial load. CONCLUSION The results of this research indicated that acetic acid, butyric, citric and formic acids had antibacterial effect, reduced ph of proventriculus, gizzard and duodenum. The digesta viscosity was also reduced. Therefore, the organic acids could be recommended to be incorporated into the diets of broilers for improved gut health digesta flow and low level of cholesterol. REFERENCE Alshawabkeh, K. and Tabbaa, J Using dietary propionic acid to limit Salmonella gallinarum colonization in broiler chicks. Asian-Australian Journal Animal Science. 15(2): Abdel-Fattah, A., Faten, A. A., Nematallah, I. and Ali, G. M Growth performance and some blood parameters of growing Japanese quail as influenced by dietary different protein levels and microbial probiotic supplementation. Egyptian Poultry Science. 21: Banerjee, G. C A Text Book of Animal Husbandry. 8 th ed. Oxford and IBH Pub. Co. Pvt Ltd., New Delhi, India: P Canibe, N., Emberg, R. M. and Jensen, B. B An overview of the effect of organic acids on gut flora and gut health. Danish Institute of Agricultural Sciences Bulletin. 23: 4-7 Choct, M Managing gut health through nutrition. Brit. Poult. Sci., 50(1): Dibner, J Organic acids: Can they replace antibiotic growth promoters. Feed International. 25(12): Fawole, M.O. and Oso, B.A., Laboratory Manual of Microbiology. Spectrum Books Ltd., Ibadan, Nigeria: Lee, K. W., Everts, H., Kappert, H. J., Van Der Klien, J., Lemmens, A. G., Frehner, M. and Beynen, A. G., Growth performance, intestinal viscosity, fat digestibility and plasma cholesterol in broiler chickens fed a rye-containing diet without or with essential oil components. International Journal Poultry Science. 3(9): Mchan, F Effects of butyric and lactic acids on continued colonization of Salmonella typhimurium in chick ceca when added to chick diets. Poultry Science. 71 (sup. 1): 115. Ndelekwute, E. K., Amaefule, K. U., Uzegbu, H. O. and Okereke, C. O Effect of finisher diets treated with organic acids on carcass and internal organs of broiler chickens. Nigerian Journal of Animal Production. 40(1); Nisbet, D. J., Corrier, D. E. and Deloach, J. R Effect of mixed cecal microflora maintained in continuous culture and of dietary lactose on Salmonella typhinurium colonization in broiler chicks. Avian Diseases. 37: Paul, S. K., Samanta, A. S., Halder, G. G. and Biswas, P Effect of combination of organic acid salts as antibiotic replacer on the performance and gut health of broiler chickens. Livestock Research Rural Devision. 19 (11): Sun, X Broiler performance and intestinal alterations when fed drug free diets. M.Sc. Thesis, Virginia Polytechnic Institute and State University, Virginia, USA: 96. NJAFE VOL. 12 No. 1,