9 l KEYWORDS Apparent phosphorus digestibility; chicks; low-phosphorus; phosphorus output. PAGES 9 – 17 REFERENCES Vol. 2 No. 1 (2015) ARTICLE HISTORY Submitted: January 29, 2015 Revised: March 29, 2015 Accepted: March 31 2015 Published: April 26 2015 CORRESPONDING AUTHOR Shu Geng Wu, Feed Research Institute, Chi nese Academy of Agricultural Sciences, Beijing, China Zhongguancun Nandajie 12, 100081, Beijing, China e-mail: wushugeng@caas.cn phone: +86 1082106097 fax: +86 1082106054 JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Effect of phytase supplementation on apparent phosphorus digestibility and phosphorus output in broiler chicks fed low- phosphorus diets. Xian R. Jiang1, 2, Fa H. Luo1, Shu G. Wu1*, Hai J. Zhang1, Valentino Bon tempo2, Ming R. Qu3, Hong Y. Yue1, Guang H. Qi1 1 Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, China. 2 Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Università di Milano, Italy. 3 Animal Science and Technology College, Jiangxi Agricultural University, China. ABSTRACT. This study was conducted to evaluate the effect of supplemental phy tase in broiler chicks fed different low levels of total phosphorus (P) on the apparent phosphorus digestibility (APD) and phosphorus output (PO) in the faeces and ileal digesta. After fed a standard broiler starter diet from day 0 to 14 post-hatch, a total of 144 male broiler chicks were allocated to 6 groups for a 7-d experiment with a 2 × 3 fac torial design comparing phy tase (supplemented without (CTR) or with 400 FTU/kg phytase (PHY)) and total P levels (2.0, 2.5 and 3.0 g/kg). The faecal samples were collected from day 17 to 2 1 post-hatch. At 22 days of age, all the chicks were slaughtered and collected the ileal digesta. Phytase supplementation signific antly (P < 0.01) increased APD and decreased PO in the faeces and ileal digesta in comparison with the CT R group. In addition, PO in the faeces expressed as g/kg DM diets and faeces (Diet × P level, P = 0.047 and < 0.01, respectively) as well as PO in the ile al digesta expressed as g/kg DM digesta (Diet × P level, P = 0.04) were affected by diet and P level, which were due to the significant reduction (P < 0.01) by PHY supplementation to the diets with 3.0 g/kg total P. The results evidenced that supplemental phytase improved the APD and PO when chicks was fed 3.0 g/kg total P diet, while lower total P levels may limit exogenous phytase efficacy. X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 10 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 1 Introduction Monogastric animals, such as poultry and pig, have virtually no phytase activity of th eir own. Thus, the availability of phosphorus (P) in feedstuffs of plant origin is generally very low , ranging from 30 to 40% (Nelson et al., 1968). To increase P bioavailability, the mos t common ly used method is supplemen ting high dosage of inorganic P in feed, which leads the excretion of large amounts of P in animal manure. Consequently, the cost of feed and the environmen tal adverse impact are increased. Moreover, ph ytate limits th e availability of s everal oth er essential nutrien ts, such as minerals, protein and amino acids (Bieh l and Baker, 1996). Man y studies showed that microbial phytase can be used to increase the availability of P and reduce its excretion (Waldroup et al., 2000; Paik, 2003). Previous studies have mainly focused on th e utilization of 3-phytase (EC 3.1.3.8) derived from the Aspergillus niger (Panda et al., 2007) as feed additiv es for broilers. Shieh and Ware (1968) reported that 3 -phytase can catalyze th e conversion of myo-inositol hexakisphosphate and water to 1L-myo-inositol 1, 2, 4, 5, 6- pentakisphosphate and orthoph osphate. The interes t in the supplem entation of ph ytas e in low P diets for monogastric animals has got great atten tion du e to environmen tal concerns and high cost of inorganic P. Man y previous studies demonstrated that phytase supplemen tation to low -P diets improved performance and P utilization of broiler chicks (Viveros et al., 2002; Rutherfurd et al., 2004; 2012; Jiang et al., 2013). However, it might be also interesting to investigate wheth er phytas e supplementation could consistently im prov e P use of broiler chicks fed th e P deficient diets even far from th e requiremen t for normal growth. Therefore, we conducted this study to evaluate the effect of supplemen tal ph ytase on th e apparen t P diges tibility and P output in th e faeces and ileal digesta of broiler chicks fed differen t low -P lev els of diets. 2 Materials and Methods 2.1 Bird husbandry and dietary treatments All the experim ental procedu res w ere approved by the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academ y of Ag ricultural Sciences. The feeding trial of this study was carried out in Nan Kou pilot base of the Chinese Academ y of Agricultural Sciences. Two hundred Arbor Acres male broiler chicks were obtained from a local hatch ery, wing-banded, and reared in electrically heated battery brooders maintained at temperatures of 33°C from day 1 to day 7 pos t-hatch and 30° C from day 8 to day 14 post-hatch. During this time, chicks w ere provided free access to water and a standard maize soybean meal starter diet containing 230 g of CP/kg, 13.39 MJ of MEn/kg, 10.0 g of Ca/kg, and 6.8 g of total P/kg. At day 15 post-hatch, after overnigh t feed withdrawal, a total of 144 chicks were w eighed and divided in to 6 homog eneous g roups. Six replicate cages (60×50×50 cm, length×width×h eight) were used per treatm ent with 4 chicks per cage. Chicks had free access to feed and water from d 15 to 21 post-hatch, and battery temperatu re was maintained at 27°C du ring this period. Lights w ere con tinuously on the first day post-hatch, after which a X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 11 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 23L:1D lighting schedule was maintained all through the duration of the feeding trial. Feed and water were provided ad libitum throughout the trial. The experimen t was a 2×3 factorial arrangem ent of th e treatm ents with diet (unsupplemen ted control [CTR] or supplemented with 400 FTU/kg phytase [PHY]) and 3 total phosphorus levels (2.0 g/kg [2.0P], 2.5 g/kg [2.5P] and 3.0 g/kg [3.0P] ). 1 FTU is the amoun t of enzym e which liberates 1 μmol of inorganic phosphate per minute from sodium phytate at pH 5.5 and 37°C. The phytase, whose type and source of extraction were 3 -phytase (EC 3.1.3.8) derived from Aspergillus Neiger, was purchased from BASF Vitamins Co. , Ltd., China with activity of 5,000 FTU/g. Soybean meal was th e only ph osphorus source. Diets (Table 1) were semipurified, consisting primarily of cornstarch, dextrose, and soybean meal, and were formulated deceed the NRC (1994) recommendations to ensure maximum responses with phytase. Chromium oxide (Cr2O3) was incorporated into diets (3.5 g/kg, as-fed) to calculate nutrien t utilization via the index m ethod. Diets in pellet form and water were provided a d libitum. Mortalities were recorded daily. 2.2 Sample Collection and Chemical Analyses After two adaptation days at 15 and 16 days pos t-hatch, faecal samples w ere collected twice per day at 6 AM and 6 PM from pans beneath each cage from day 17 to day 21 pos t -hatch for 5 days. At end of each collection period, cage samples were pooled. All the faecal samples were frozen and stored at -20°C before sending to laboratory for digestibility assays. At 22 day post-hatch, chicks were euthanized via carbon dioxide asph yxiation, and ileal dig esta samples were collected by dissecting a segm ent of the ileum defin ed as extending from Meck el’s diverticulum to the ileocecal junction. Contents of this segment were squeezed into a plastic container, pooled per cag e of 4 chicks, and subsequently lyophilized. Freeze-dried digesta an d faecal samples and, along with air-dried diet samples, were g round through a 0.75-mm sieve using a grinding mill to facilitate chemical analyses. Determination of dry matter (DM) was performed using the Association of Analytical Communities (AOAC, 20 05) official method AOAC 930.15. The contents of phosphorus and chromium were analyzed as described by Dilg er and Adeola (2006). 2.3 Calculations and Statistical Analyses Apparent P digestibility (i.e. faecal and ileal digestibility) was calculated using the index method according to the following equation: APD (%) = 100 − [(CrI/CrO) × (PO/PI) × 100] [1] where APD is apparent P diges tibility (calculated for ileal digesta or faecal samples ) expressed as a percen tage, CrI is the chromium concentration of dietary in take, CrO is th e chromium concentration of outpu t (as analyzed in ileal digesta or faeces), P O is the P X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 12 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 Table 1. Composition and nutrient content of th e experim ental diets (as fed basis) 1 CTR PHY 2.0P 2.5P 3.0P 2.0P 2.5P 3.0P Ingredient, g/kg Corn starch 589.4 490.8 389.3 589.4 490.8 389.3 Dextrose 90.0 90.0 90.0 90.0 90.0 90.0 Soybean meal 294.2 379.8 465.5 294.2 379.8 465.5 Soybean oil 12.0 25.0 40.0 12.0 25.0 40.0 Limestone 4.8 6.0 7.0 4.8 6.0 7.0 Salt 2.5 2.5 2.5 2.5 2.5 2.5 Lysine 3.0 2.0 2.0 3.0 2.0 2.0 Methionine 1.0 0.8 0.6 1.0 0.8 0.6 Choline chloride 0.6 0.6 0.6 0.6 0.6 0.6 Mineral premix 2 1.0 1.0 1.0 1.0 1.0 1.0 Vitamin premix 3 1.5 1.5 1.5 1.5 1.5 1.5 Phytase, FTU/kg -- -- -- 400 400 400 Calculated and analyzed composition Metabolizable energy, MJ/kg 12.27 12.27 12.31 12.27 12.27 12.31 CP, g/kg 132.1 169.6 207.0 132.1 169.6 207.0 Analyzed DM, g/kg 892.1 873.7 875.4 872.6 885.0 870.9 Analyzed Ca, g/kg 2.7 3.4 4.1 2.7 3.4 4.1 Analyzed total P, g/kg 2.1 2.7 3.0 2.1 2.7 3.0 1. CTR = basal diet without phytase supplementation; PHY = CTR + 400 FTU/kg phy tase; 2.0 P = 2.0 g/kg total P level; 2.5P = 2 .5 g/kg total P level; 3.0 P = 3.0 g/kg total P level. 2. Provided the following per kg of diet: Cu 8mg, Zn 75 mg, Fe 80 mg, Mn 100 mg, Se 0.15 mg, I 0.35 mg. 3. Provided the following per kg of diet: re tinyl acetate, 4.3 mg; cholecalcipherol, 0. 0625 mg; DL-alpha-tocopherol, 18.75 mg; menadione, 2.65 mg; cyanocobalamin, 0.02 5 mg; biotin, 0.0325 mg; folic acid, 1.25mg; niacin, 50 mg; D -pantothenic acid, 12 mg; riboflavin, 6 mg; thiamin, 2 mg concen tration of output (as analyzed in ileal dig esta or faeces ), and P I is the P concentration of dietary in take. All analyzed values were expressed as grams per kilo g ram of D M. X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 13 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 Calculation of P output, expressed on a dry matter in take (D MI) basis, utilized th e ratio of chromium intak e to chromium ou tput: PO-DMI (g/kg) = PO-DMO (g/kg) × (CrI/CrO) [2] where PO-DMI and P O-DMO repres ent P ou tput concentrations (as analyzed in ileal digesta or faeces) on DMI and DM output bases, respectively, and CrI and CrO represen t chromiu m concen trations of intak e and output (of either ileal dig esta or faeces), res pectively. Data were analyzed as a com pletely randomized block design by ANOVA, as implemen ted in the MIXED procedu re of SAS v. 9.2 (SAS Institu te Inc., Cary, NC). A model for a 2×3 factorial design was applied. The model statemen t included th e effects of diet (CTR and PHY), total P levels (2.0, 2.5 and 3.0 g/kg) and interactions among those factors. Cage served as th e experimen tal unit for all statistical analys es. Treatmen t differences were assessed by using th e least squares means with a Tukey adjustmen t. Treatment effects were considered significan t at P ≤ 0.05. 3 Results Graded lev els of dietary P in take and th e supplem entation of ph ytase, did not affect normal diges tive functions as was reflected by changes in apparen t DM diges tibility values (Table 2). Phytase supplem entation significantly ( P < 0.01) increased APD and decreased PO in the faeces and ileal digesta in comparison with the CTR group, when these were expressed as g/kg DM diges ta, faeces or diets. Compared with th e CTR chicks fed 3.0P, diet ary PH Y increased APD in faeces (lin ear contrast, P = 0.02) and ileal digesta (lin ear con trast, P = 0.02) of 3.0P chicks. PHY supplem entation decreased PO in the ileal diges ta of birds fed 2.5 g/kg (lin ear contrast, P < 0.01) and 3.0 g/kg (linear contrast, P = 0.010) total P diets compared to the CTR chicks fed 2.5P and3.0P, respectively, when th ese were expressed as g/kg DM digesta. Birds fed 2.0P had less PO in the faeces than fed 3.0P (linear con tras t, P < 0.01), when these were expressed as g/kg DM faeces. However, compared to th e birds fed 2.0P, chicks fed 2.5P had an increased APD in th e ileal diges ta (lin ear contrast, P = 0.04), and chicks fed 3.0P had an increased APD in the faeces (linear contrast, P = 0.02). Moreover, APD in th e faeces of 2.5P chicks and in the ileal digesta of 3.0P chicks had tendencies to be higher than in th e corresponding parts of 2.0P birds (lin ear contrast, P = 0.051 and 0.056, respectively). In addition, PO in the faeces expressed as g/kg DM diets and faeces (Diet × P level, P = 0.047 and < 0.01, respectively) as well as PO in the ileal diges ta expressed as g/kg D M diets and digesta (Diet × P lev el, P = 0.04) were affected by diet and P level, and PO in th e ileal digesta expressed as g/kg DM diets had a tendency to be affected by diet and P lev el (Diet × P level, P = 0.059). The interactions can be attributed to the significan t reduction (lin ear contrast, P < 0.01) by PH Y supplementation to the diets with 3.0 g/kg total P, bu t no differences were found in others total P levels diets. X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 14 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 Table 2. Effect of ph ytase on total P ou tput, and apparent P digestibility in the faeces and ileal diges ta of chicks fed different levels of P CTR PHY P-value 2.0P 2.5P 3.0P 2.0P 2.5P 3.0P SEM Phytase P level Interaction No of pen 6 6 6 6 6 6 P intake, g/kg of DMI 2.3 3.1 3.4 2.4 3.1 3.5 - - - - Faeces ADD, % 81.32 80.23 81.21 80.78 81.54 80.83 1.00 0.87 0.98 0.60 APD, % 57.33 63.52 60.22 60.89 69.17 74.77 2.94 <0.01 0.02 0.16 PO g/kg of DMI 0.99b 1.15ab 1.34a 0.94b 0.96b 0.88b 0.08 <0.01 0.19 0.047 PO g/kg of DMF 5.24bc 5.80b 7.12a 4.88c 5.16bc 4.60c 0.19 <0.01 <0.01 <0.01 Ileal digesta ADD, % 80.07 78.84 77.95 78.89 82.55 78.66 1.20 0.28 0.15 0.14 APD, % 42.62 49.16 44.86 46.70 65.97 68.39 4.91 <0.01 0.03 0.16 PO, g/kg of DMI 1.33 1.60 1.85 1.28 1.06 1.11 0.14 <0.01 0.41 0.059 PO g/kg of DMD 6.79 ab 7.53ab 8.45a 6.05bc 6.03bc 5.15c 0.50 <0.01 0.69 0.04 a-c . Data in the same row with different superscripts differ significantly (P<0.05). CTR = basal diet without phytase supplementation; PHY = CTR + 400 FTU/kg phytase ; 2.0 P = 2.0 g/kg total P level; 2.5P = 2.5 g/kg total P level; 3. 0 P = 3.0 g/kg total P level; PO = P output; ADD = apparent dry matter digestibility; APD = apparent P diges tibility; DMD = dry matter digesta; DMF = dry matter faeces; DMI = dry matter intake. 4 Discussion The objectives of the s tudy w ere to determine wh ether dietary phytase added to th e low total phosphorus (P) diets of broiler chicks would consisten tly improve apparent P diges tibility (APD) and P ou tput (PO) in faeces and ileal digesta, and whether th ere would be a significan t interaction between ph ytas e supplem entation and levels of low total P in th e diets. Ou r previous work confirmed that dietary supplem entation of phytase can improve growth performance with a higher ash content in bon e of chicks fed with low-P diet (4.8 g/kg total P diets) during 8-21 days post-hatch period (Jiang et al., 2013), which may be du e to th e improvem ent the availability and absorption of nutrients through increasing the dig estibility of the ingested diets (A budabos, 2012; Attia et al., 2012). Dilger and Adeola (2006) also observed X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 15 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 that low-phytate SBM decreased P ou tput and increased P reten tion as compared to th e conventional SEM. In the present study, positive results were obtained using 3 -phytas e supplemented to diets with low P (2.0, 2.5 or 3.0 g/kg total P) on apparen t P diges tibility and P output in both faeces and ileal digesta from day 14 to day 21 post-hatch. Numerous studies have been conducted that confirm the finding of enhan ced P uptak e in and pou ltry after treatmen t with ph ytase (Nelson et al., 1968; Waldroup et al., 2000; Paik, 2003; Rutherfurd et al., 2004). The improvement in P utilization by supplemen ting phytas e may be related to th e increased dephosphorylation of ph ytate in th e small in testi n e. Rutherfurd et al. (2004 ) reported that microbial phytase supplemen ted to the diets of chicks improved total P digestibility and increased ph ytate P disappearance in the ileum. In this study, higher PO was observed in th e faeces of birds fed with 3.0 g /kg total P diets compared to 2.0 g/kg total P diets, which may be du e to the increased P intak e. However, th e interaction between diet and P level evidenced that the high PO in the maximum total P level group was attributed by the unsupplemen ted g roup. Mo reov er, the increased ADP in th e faeces and ileal digesta of chicks fed with the diets containing 3.0 g/kg total P were due to th e 3-phytase supplem entation. In the present study, APD in the faeces were 57 -63% and in the ileal digesta were 42-49% in the unsupplemen ted chicks fed with low-P diets, which were similar as the finding by Sebastian et al. (1996) and Rutherfurd et al. (2004). Whereas th e correspondin g value in the low-P diet supplem ented with 400 FTU/kg of 3-phytase were 61-75% and 47-68%, respectively. In the present study, phytase improved the ADP and PO in the faeces of broilers from day 14 to day 21 of ag e mainly reflected in th e birds fed with 3.0 g/kg total P diets, interestingly, the observations in the ileal digesta were in accordance with the findings in faeces. Our observation is in agreemen t with the finding by Dilger and Adeola (2006) that low - phytate SBM improved P ou tput (1.122 vs. 1.808 g/kg of DMI) and retention (65.5 vs. 54.1 %) compared to th e conven tional SEM mainly in th e groups contained 528 g/kg SEM (total P; 3.01 vs. 3.58 g/kg). The limited improvem ents of ADP and PO in the diets with 2.0 and 2.5 g/kg total P migh t be due to th e levels of th e dietary P concen trations. It is possible that chicks rapidly acclimated to these low P intak es in an attempt to main tain P homeostasis or even “buffer” body P homeostasis for long-term P deficiency. Moreov er, th e low total P intak e may provide limited phytate P for dietary phytase to “recycle”, and consequen tly, rare improvemen ts in P digestibility and excrete w ere observed in the 2.0 and 2.5 g/kg total P diets even supplemen ted with 400 FTU/kg 3-phytase. However, it seems likely that the diets con taining 3.0 g/kg total P may possess enough phytate P for dietary phytase to hydrolyze and even tually result in th e significant improvem ents in th e P utilization. Although Dilger and Adeola (2006) concluded that the P use and endogenous P loss were influenced by dietary ph ytate content when broiler chicks were fed P-deficient diets and the levels of total P used were in the ranges of 0.83-3.58 g/kg, it is well known that severe P reductions in starter chicks might have jeopardized th eir skeletal in teg rity, general health status and survival (Scott et al., 1982). The levels of low total P in diets of th e studies which eviden ced the improvemen t in grow th performance and P utilization of broiler chicks by that phytase supplem entation were in the rang es of 4.8-6.4 g/kg (Viveros et al., 2002; Rutherfurd et al., 2004; 2012; Jiang et al., 2013), which are much high er than the levels (2.0, 2.5 and 3.0 g/kg) used in this study. Thus, phytase supplementation may not consisten tly improve P utilization during the early life stag e of a commercial chicken strain when they are fed severe P-deficien t diets. X.R. Jia ng et al. - Int. J. of Health, Animal science a nd Food sa fety 1 (2015) 9 - 17 16 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 5 Conclusions The observations in this study demons trated that supplemen tation of phytase increased apparen t P diges tibility and deceased P output in both faeces and ileal diges ta when broiler chicks were fed the diets with 3.0 g/kg total P. However, the lower total P levels (2.0 and 2. 5 g/kg) may limit th e effect of exogenous phytas e on P utilization and could not be recomm ended to use. 6 Conflicts of interest The authors declare that there are no con flicts of interest. 7 Acknowledgements This work was granted by China Agriculture Research System Poultry-related Science and Technology Innovation Team of Peking, and the National Key Technology Research an d Developmen t Program (2011BAD26B04). References Abudabos M.A. (2012). Phytate phosphorus utilization and intestinal phytase activity in laying hens. Ital. J. Anim. Sci., 11: 41-46. AOAC (2005). Official Methods of Analysis, 18th ed., Association Official Analytical Chemis ts, Arlington, VA, USA. Attia Y.A., El-Tahawy W.S., Abd El-Hamid A.E., Hassan S.S., Nizza A., El-Kelaway M.I. (2012). Effect of phytase with or without multienzym e supplemen tation on performan ce an d nutrien t diges tibility of young broiler chicks fed mash or crumble diets. Ital. J. Anim. 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