Abstract: This experiment was conducted to evaluate the combined effects of Citric Acid (CA) and Microbial Phytase (MP) on the serum concentration and digestibility of some minerals in broiler chicks. This experiment was conducted using 360 Ross-308 male broiler chicks in a completely randomized design with a 3x2 factorial arrangement (0, 2.5 and 5% CA and 0 and 500 FTU MP). Four replicate of 15 chicks per each were fed dietary treatments. The concentration of zinc, copper and manganese of serum and their digestibility and also digestibility of apparent metabolizable energy (AMEn) was evaluated. The results showed that interaction effect of CA and MP on concentration of copper, zinc and manganese in serum of broilers fed with low available phosphorus diets was significant (p<0.05). Adding 2.5% CA into low available phosphorus diets increased digestibility of zinc in comparison to diets without CA and with 5% CA in broiler. Adding CA into low available phosphorus diets increased manganese digestibility on based corn-soybean meal diets (p<0.01).
INTRODUCTION
The environment contamination with phosphorus which is caused by animals, recently, has been an important issue. Mono gastric animals consume diets based on oil seed meals and crops. These diets contain high amounts of phosphorus in phytase or phytic acid forms. Commonly, phytase, which has known activity in the intestine of poultry, is not available (Nelson, 1976). Various feed additives are used in order to increase the use of phosphorus and decrease the excretion of phosphorus in poultry and swine. Akyurek et al. (2005) demonstrated that phytase supplementation did not affect broiler performance whereas it significantly increased calcium and phosphorus retention in broiler chicks. It is known that the phytase (Edwards, 1993; Biehl et al., 1995; Biehl and Baker, 1996; Gordon and Roland, 1997; Bingol et al., 2009; Ziaei et al., 2009; El-Sherbiny et al., 2010) vitamin D and its products (Edwards, 1993; Biehl et al., 1995; Angel et al., 2001; Edwards, 2002; Snow et al., 2004) and citric acid (Boling et al., 2000; Boling-Frankenbach et al., 2001; Rafacz et al., 2003; Snow et al., 2004) can affectively be use to develop the availabilities of phytate in non-ruminant animals. Ebrahim Nezhad et al. (2008a) concluded that, Adding 300 FTU kg-1 microbial phytase to the corn-soybean meal based diets with 0.1% phosphorus, increased the concentration of serum copper and manganese and phosphorus of tibia ash in laying hens at 53-64 week age.
There is little information to say that if organic acids (except of citric acid) can improve the availability of phytate phosphorus in poultry. Ebrahim Nezhad et al. (2008b) reported that, Supplementing low available phosphorus diets with different levels of citric acid, increased the concentration of serum copper and the percentage of tibia ash in laying hens at 53-64 week age. Results obtained in our previous study suggest that contrary to the effects of citric acid in broiler chicks, this organic acid couldn’t enhance phytase effectiveness in laying hens, probably due to high levels of Ca in laying hens diets (Ebrahim Nezhad et al., 2007). Ebrahim Nezhad et al. (2008c) reported that, the Addition 2% citric acid to low available phosphorus diets improves quality characteristics such as specific gravity, eggshell thickness, eggshell weight, eggshell ash and Hough unit in laying hens. The EDTA is an organic acid which has similar potential with citric acid, and it increases availably of same minerals. Ethylenediaminetetraacetic acid (EDTA) is a strong chelate and it improves the absorption rate of minerals of diets in poultry.
Previous studies indicated that, supplementing diets which contain plant protein with EDTA, improved absorption of (Zn++) in turkey chicks (Kratzer et al., 1959) and chicks (O’Dell et al., 1964). Maenz et al. (1999) showed that EDTA increased the hydrolyzation of phytate phosphorus from canola meal when associated with microbial phytase in vitro experiments. It seems that EDTA comparatively links to the calcium and decreases its ligand to the phytate. Consequently it bounds the formation of insoluble calcium-phytate complexes and makes phytate of the diet sensitive to the endogenous and exogenous phytase.
The aim of this research was to study the combination effect of citric acid and microbial phytase on the serum concentration and digestibility of some minerals in broilers and its effect on efficacy of microbial phytase in corn-soybean diets with low available phosphorus level.
MATERIALS AND METHODS
A total of 360 feather sexed male day old Ross 308 broiler chicks were used in this experiment. Chicks were weighed individually and randomly assigned to battery pens so that pens had equal initial weight and weight distribution. The experiment was carried out using a completely randomized design with a 3x2 factorial arrangement (0, 2.5 and 5% CA and 0 and 500 FTU MP). Four replicates of 15 chicks per each were fed dietary treatments including (1) P-deficient basal diet [0.2% available phosphorus (aP)] (NC); (2) NC + 500 FTU MP per kilogram of diet; (3) NC + 2.5% CA per kilogram of diet; (4) NC + 2.5% CA + 500 FTU MP per kilogram of diet; (5) NC + 5% CA per kilogram and (6) NC + 5% CA + 500 FTU MP per kilogram of diet. All diets meet or exceed (NRC, 1994) recommendation except for aP (Table 1). The same ingredients were used for formulation of diets during 0-21 and 21-49 days of age (diet composition for period of 21-49 days of age are not presented). The supplied MP (Natuphos-500; BASF, Mt. Live, Nj) had 1000 FTU active phytase per gram. The citric acid used in this experiment was monohydrate 92% which was added to the diets after calculating purity percentage.
At day 44, chromium oxide (Cr2O3) was added to all diets at 0.1% level as a detectable marker for specifying of zinc (Zn++), copper (Cu++), manganese (Mn++) and also apparent metabolizable energy. To determine digestibility of minerals, special sacs fastened to back of two chickens that their weights were close to mean weight of cage and their feces were collected for three days. Samples of digested materials were freeze immediately after collection and then were dried in oven at 60 degree of centigrade.
| Table 1: | Composition and nutrient content of the diet during starter (0-21 days) period |
| aSupplied kg-1 of die; Vitamin A: 9000 IU, Cholecalciferol: 3000 IU, Vitamin E: 18 IU, Vitamin K3: 2 mg, Vitamin B12: 0.015 mg, Thiamin: 1.8 mg, Riboflavin: 6.6 mg, Folic acid: 1 mg, Biotin: 0.10, Niacin: 35 mg, Pyridoxine: 4 mg, Choline chloride: 250 mg, Ethoxyquine: 0.125, Supplied kg-1 of diet; Manganese sulphate: 100 mg, Copper sulphate: 10 mg, Selenium (sodium selenate): 0.2 mg, Iodine (EEI): 1 mg, Zinc sulfate: 100 mg, Fe: 50 mg, bNatuphos® (BASF Crop., Mt. Olive, NJ) was used to supply 500 FTU microbial phytase kg-1 of diet, ME: Metabolisable energy, CP: Crude protein, Ava. P: Available Phosphorous, TP: Total phosphorous | |
After drying of samples of digested materials, they grinded and use 1 mm pore filter to homogenize them. Also samples of foods were grinded and filtered in this leach. Gross energy of feed samples and feces samples were measured by automatic colorimeter (Automatic Colorimeter, AC-300, and Model 789-400) in nutrition laboratory of Tabriz University and then apparent metabolizable energy were calculated. Minerals concentration for feeds and feces samples was measured by atomic absorption in nutrition of Islamic Azad University-Shabestar branch and was expressed at percent. Chromium (Cr) concentration in feeds and feces samples were measured by method explained by Fenton and Fenton (1979) and by using spectrophotometer. Digestibility of minerals was calculated by following formula (Ravindran et al., 2000):
At the end of experiment, two birds were selected from each replication and 5 mL blood was taken from wing puncture. Blood samples centrifuged for 15 min (3000 rpm/min) and serum was separated. The concentration of zinc, copper and manganese measured by using ICP (Inductively Coupled Plasma Emission Spectrometer, Model JY-24, Jobin Yvon, Longjumeau, Cedex, France).
Statistical analysis: Data were statistically evaluated by the analysis of variance procedure of SAS software (SAS Institute, 1990), involving a factorial arrangement of main factor (citric acid and microbial phytase levels) in a completely randomized design. Significant differences between mean values were separated by the GLM procedure of SAS software (SAS Institute, 1990). Statistical significance was considered (p<0.05).
RESULTS AND DISCUSSION
The effect of CA and MP on concentration of zinc, copper and manganese in serum and also digestibility of these minerals and apparent metabolizable energy are presented in Table 2. The results showed that interaction effect between CAxMP on the percentage of zinc, copper and manganese at the end of breeding period in broilers that fed with low available phosphorous diets were significant p<0.01, p<0.0001 and p<0.05, respectively, Also adding of CA into low available phosphorus diets that supplemented MP, decreased the concentration of zinc in serum p<0.05. However, supplementing these diets with 5% CA without adding MP, increased zinc concentration in serum in comparison to diets without MP and 2.5% CA (p<0.05). The concentration on copper in serum increased when low available phosphorus diets were supplemented by CA in comparison to the control group (p<0.05), although adding 2.5% CA into low available phosphorous diets supplemented with MP, decreased copper in serum (p<0.05). Also, adding CA into low available phosphorous diets, increased manganese in serum in comparison to the control group (p<0.05). Likewise, manganese concentration in serum increased when 5% CA added to low available phosphorous diets supplemented with MP (p<0.05). These results showed that CA did not have effect on the efficiency of MP in low available phosphorous diets. In addition, it can be said that adding 2.5% CA into low available phosphorous diets supplemented with MP, caused reduced activity of MP. The exact reason of this reaction in not clear, but it might be because of the lack of response of MP activity to CA supplementation in low available phosphorous diets, maybe reflective of the fact that reduced pH in Gastrointestinal tract (GIT) caused by adding CA leads to lower MP efficiency, although CA is an organic acid that metabolized in body rapidly and it does not have much effect on the pH of intestine. These results are in agreement with the findings of Radcliffe et al. (1998) who observed that adding 1.5-3% CA to the piglet's diets had significantly decreased the MP activity in the gastric digesta of piglets after slaughtered. Brenes et al. (2003) reported, adding 2% CA into low available phosphorous diets, had no effect on the efficacy of MP that results of recent experiment was in agreement with findings of these researchers whereas Deepa et al., (2011) indicated that, the addition of phytase improved the growth performance and increased Ca, P and N retention in chicks. Also, inclusion of CA along with phytase caused an increase in mineral utilization. These researchers demonstrated that, proper combination of CA and phytase may represent a practical solution to improve growth performance, phytate P and other mineral utilization. Adding CA into low available phosphorous diets increased the digestibility of zinc and manganese in comparison to the control group. Also there was a significant difference between different levels of CA added to these diets. For instance, adding 5% CA, decreased digestibility of some minerals in comparison to 2.5% CA (p<0.05). In this experiment, adding different levels of CA into low available phosphorous diets, increased manganese digestibility (40%) in comparison to the control group (p<0.05). It seems that, reducing the pH of gastric due to CA added to the diet, would reduced the passage rate time from intestine and it provides enough time for absorbing of nutrient from intestine. Han et al. (1998) suggested that, may be organic acids like CA, increases solubility of phosphorous in digesta and also transitions time of digesta from intestine, consequently increases total absorbance of phosphorous.
Digestibility of copper and apparent metabolizable energy was not affected by treatments (Table 2).
| Table 2: | The Effects of CA and MP on the serum concentration and digestibility of some minerals in broiler chicks |
| Means in columns with no common superscript differ significantly (p<0.05), 1Natuphos® (BASF Crop., Mt. Olive, NJ) was used to supply 300 FTU microbial phytase per kilogram of diet, AME: Apparent metabolisable energy, dig.: digestibility | |
CONCLUSION
From the present study it can be concluded that supplementing low available phosphorus diets with different levels of CA, increases the digestibility of zinc and manganese in broiler chicks. Adding different levels of CA to the low available phosphorus diets which supplemented with MP, did not improved the efficacy of MP on the bioavailability of zinc, copper, manganese and Apparent metabolizable energy (AMEn) in broiler chicks. Adding 500 FTU kg-1 MP to the corn-soybean meal based diets with low available phosphorus, do not affect the bioavailability of zinc, copper and manganese in broiler chicks.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the excellent financial and technical assistance of Shabestar Islamic Azad University, and Shahid Sardari’s Educational and Research Center of Agricultural Jahad Ministry of East Azerbaijan.