肉鸡中生长性能、养分利用、血清生化指标及骨矿化指标检测方案(定氮仪)

智能文字提取功能测试中

Taylor & FrancisTaylor & Francis GroupItalian journal of Animal ScienceISSN：(Print)(Online)journal homepage： https：//www.tandfonline.com/loi/tjas20 ITALIAN JOURNAL OF ANIMAL SCIENCE2021， VOL. 20， NO. 1， 1282-1291https：//doi.org/10.1080/1828051X.2021.1953409Taylor & FrancisTaylor & Francis Group Full Terms & Conditions of access and use can be found at Effect of azomite on growth performance，nutrientutilization， serum biochemical index and bonemineralization of broilers fed low protein diet Shoaib Ahmed Pirzado， Faiz ul Hassan， Muhammad Asif Arain， Wu Zhengke，Cai Huiyi， Tesfay Hagos Haile & Liu Guohua To cite this article： Shoaib Ahmed Pirzado， Faiz ul Hassan， Muhammad Asif Arain， Wu Zhengke，Cai Huiyi， Tesfay Hagos Haile & Liu Guohua (2021) Effect of azomite on growth performance，nutrient utilization， serum biochemical index and bone mineralization of broilers fed low protein diet，Italian Journal of Animal Science， 20：1，1282-1291， DOI： 10.1080/1828051X.2021.1953409 To link to this article： https：//doi.org/10.1080/1828051X.2021.1953409 8 ◎ 2021 The Author(s). Published by Informa UK Limited， trading as Taylor & Francis 曲 Published online： 28 Sep 2021. Group. Submit your article to this journal C Hil A Article views： 512 View related articles C CrossMark View Crossmark data C Citing articles： 2 View citing articles https：//www.tandfonline.com/action/journallnformation?journalCode=tjas20 The present study investigated the effect of Azomite (AZO) on the growth performance， nutrientutilisation， and bone mineralisation of broilers fed low protein diet. A total number of 180 one-day-old male chicks were randomly distributed to three treatment groups having six replicatesof 10 birds each. Experimental diets included； positive control (PC) with 21.5% CP (starter) and19.0% CP (finisher)， negative control (NC) with 2% lower CP than PC in each period and treat-ment group (AZO-0.25) in which NC was supplemented with 0.25%AZO. Azomite significantly(p<.05) improved live body weight， average daily gain and feed conversion ratio， but not thefeed intake， as compared to NC. Eviscerated， breast muscle and leg muscle percentages weresignificantly (p<.05) higher in the birds fed AZO-0.25 than NC， while abdominal fat (AF) per-centage was significantly (p>.05) higher in NC. Azomite improved the digestibility of dry matter(DM)， crude protein (CP)， apparent metabolisable energy (AME)， calcium (Ca) and phosphorus (P)(p<.05) compared to the NC. Trypsin activity was increased (p<.05) in birds fed diet with AZO-0.25 compared with NC. Blood creatinine， growth hormone (GH) and calcitonin (CT) was alsohigher (p<.05) in birds supplemented with AZO-0.25. Treatment also affected (p<.05) the tibiabreaking strength (TBS)， ash， Ca and P contents. Overall， AZO improved the growth perform-ance， nutrient utilisation and tibia mineralisation in broilers fed with low CP diet， which looks aneconomically promising approach. HIGHLIGHTS The dietary supplementation of Azomite improved the growth and nutrient utilisation inbroiler chicken. · The efficiency of digestive enzymes and digestibility was enhanced by dietary Azomite. ·Dietary Azomite improved the tibiabreakingg strengthand bone mineralisationinbroiler chicken. ARTICLE HISTORY . Received 24 March 2021Revised 21 June 2021Accepted 6 July 2021 KEYWORDS Azomite； growthperformance； nutrientutilisation； bonemineralisation； broilers Introduction ln poultry nutrition， much attention is paid to proteinsources， owing to importance of protein as a majorconstituent of biologically active compounds in thebody. It is also principally involved in the synthesis ofbodyytissues，rrepairr and growth . of ttheebodyFurthermore， all enzymes and hormones which playkey roles in the physiology of any living organisms areprotein in nature (Beski et al. 2015). The reduction ofprotein content in the poultry diet not only reducesthe feed price for economical poultry production butalso rminimisesthe potentialforreenvironmenta pollution from nitrogen waste. Numbers of researchstudies have been conducted to establish the min-imum level of crude protein (CP) that would supportthe optimumperformance. However， conflictingresults from these studies do not allow a clear conclu-sion on the effects of low protein diets in practicalbroiler production (Moran and Stilborn 1996)， (Kamranet al. 2008； Hernandez et al. 2013； Yang et al. 2015).Reduction of 2 to 3% CP in the broiler diet resulted indecreased rate and efficiency of growth and poorquality of carcase traits even when diets fulfilled withall other nutrient requirements6(Bregendahl et al ( CONTACT Dr. Li u Guohua an d D r . Shoaib Ahm e d Pirzado ) liu g uoh u a@caa s .cn， sa pi rzad u @ s a u .edu.pk Key Lab o ratory of Feed Biotechnology of Agricultural Ministry， Feed Research I nstitute， Chinese Academy of Agricultural Sciences， Beijing 1 0 0081， China ) ( C 2021 T h e Author(s). Published by I n forma UK Limited， trading as T a ylor & Francis Group. This i s an O pen Access ar t icle d i stributed under t h e te r ms of the Cre a tive Com m ons Attrib u tion Licen s e (http： / /creat ive co mmo n s.or g / l ice nse s/ by /4.0 /)， whi c h perm i ts unrestricted u se， d istribution， and r eproduction in any me d ium， prov i ded the o r iginal work i s pro p erly cited. ) 2002； Sterling et al. 2002； Waldroup et al. 2005).Furthermore， low protein diet has a negative impacton the digestibility (Temim et al. 1999)， morphology ofintestinal villi (Laudadio et al.2012)， protein metabol-ism. (Awadet al.2017)and nitrogenretention(Nahm 2002). The large portion of dietary nitrogen intake is notretained by the animal body but excreted in the envir-onment which causes the environmental pollution.Reducing the protein content of diet could thereforebe utilised as a tool to minimise the excretion of nitro-gen and ammonia from poultry houses (Fergusonet al. 1998； Khajali and Moghaddam 2006； Namroudet al. 2008). Therefore， scientists are paying moreattention to reduce the environmental pollution with-out compromising the growth performance of broiler.ln recent years， poultry industry has focussed on theuse of synthetic amino acids (Nukreaw and Bunchasak2015) and several other feed additives like exogenousenzymes (Nabizadeh et al. 2017) phytogenic substan-ces (Paraskeuas et al. 2016； Arain et al. 2018； Saeedet al. 2018； Arif et al. 2019； Nabi， Arain， et al. 2020)，and mineral supplementation (Nabi， lqbal， et al. 2020)to improve the production performance of poultrybirds. Although， fast growth rate may cause metabolicdisorders such as high incidence of skeletal disordersand increased fat deposition (Yagoub and Babiker2008； Nabi et al. 2018) which raises major concern forboth producers and consumers. Excessive body fatdeposition in broiler results in poor energy metabol-ism and overall feed utilisation (Pasternak and Shalev1983； Saeed et al. 2017) which results in the economiclosses for producers. Therefore， low protein diet canbe supplemented with suitable natural feed additivesto reduce the economic losses without compromisingthe performance of broilers. Recent research developments have expanded thephysiological and economic benefits of dietary inclu-sions of aluminosilicates， particularly with respect tohydrated sodium calcium aluminosilicates (l(HSCAS)AzomiteQ (AZO) is a product marketed as a hydratedsodium calcium aluminosilicate that is comprised oftrace minerals and rare earth elements. Azomiteis anacronym which stands for 'A to Zof minerals includingtrace elements'. Although， AZO has been used in agri-culture for over 70 years， there are limited scientificstudies on the use of this product in animal nutrition.Recently， AZO has got attention as a mineral boosterand natural feed additive in poultry and aquacultureto improve the growth performance and digestibilityof nutrients. It is a mixture of residues from animalsand plants along with minerals， containing more than 70 trace and other minerals (Fodge and Fodge 2014).Some previous studies reported that dietary supple-mentation of AZO in tilapia (Oreochromis niloticus XOreochromis aureus) increased the growth perform-ance， feed efficiency， nutrient digestibility， and digest-ive enzyme activity (Liu et al. 2009； Batool et al. 2018).In addition， efficiency of nutrient utilisation， digestiveenzymes activity and immunity was improved in whiteshrimp (Tan et al.1.2014) and grass carp(Ctenopharyngodon idellus) supplemented with Azo(Liu et al. 2011). According to a recent study (Pirzadoet al. 2020)， AZO in conjunction with a low-energydiet exhibited positive effects on the growth perform-ance， bone parameters， and nutrient digestibility inbroilers. Furthermore， supplementation of AZO at 0.25and 0.50% in the diet of broiler improved the growthperformance， immune functions and tibia breakingstrength (Pirzado et al. 2021). Azomite possesses a number of potential benefitslike improving the growth performance， feed utilisa-tion whilee reducing the environmentall ppollutionAlthough， the dietary inclusion of AZO may providepotential benefits as observed previously with theindividual addition of aluminosilicate clays or REE’s inpoultry production， there is limited research on explor-ing the utilisation of AZO in poultry. Keeping in viewof potential advantages of AZO， this study was aimedto evaluate the effect of AZO on the growth perform-ance， nutrient utilisation and bone mineralisation inbroilers fed with a low protein diet. Materials and methodsBirds， treatments and management conditions A total of 180 one-day-old broiler chicks (Ross 308)were purchased from a commercial hatchery (BeijingHuatu Broiler Company Limited) and randomly allo-cated to three treatment groups having six replicatesof 10 birds each. The birds were kept in solid floorpens covered with wood shavings andn. managedunder recommended temperature (32°C during 1stweek and the gradual decrease by2℃ each weekuntil it reached the 22°C) and relative humidity (55 to65%). Up to day 7， the broilers were subjected to lightschedule of 23h light and one hour darkness. Fromday 7， the birds were subjected to gradually decline inlight schedule (one hour/week) and fixed at 18h lightand six hours darkness following the European Unionstandards on the protection of animals used for scien-tific purposes. Fresh drinking water and feed wereprovided ad libitum to all birds during the entireexperimental period. The experiment was conducted Table 1. Ingredient composition of experimental basal diet. Ingredients% Positive control Negative control AZO-0.25 Starter Finisher Starter Finisher Starter Finisher Corn 57.47 59.47 63.94 66.24 63.94 66.24 Soy bean Oil 1.50 4.32 0.52 3.45 0.52 3.45 Soy Bean 30.96 25.05 25.51 19.1 25.51 19.1 CSM 5.00 7.00 5.00 7.00 5.00 7.00 Salt 0.35 0.35 0.35 0.35 0.30 0.30 CaHPO 1.53 1.39 1.50 1.40 1.50 1.40 Lime stone 1.54 1.40 1.61 1.44 1.51 1.34 Lys 0.24 0.22 0.21 0.26 0.21 0.26 Met 0.14 0.15 0.11 0.13 0.11 0.13 Cys 0.07 0.04 0.05 0.02 0.05 0.02 Chol 0.20 0.01 0.20 0.01 0.20 0.01 Premix° 0.50 0.10 0.50 0.10 0.50 0.10 Zeolite 0.50 0.50 0.50 0.50 0.40 0.40 Azomite 0.25 0.25 Total 100 100 100 100 100 100 Nutritional value of diet ME (kJ/kg) 12342.8 12761.2 12342.8 12761.2 12342.8 12761.2 Protein (%) 21.50 19.00 19.50 17.00 19.50 17.00 Lys (%) 1.200 1.050 1.050 0.950 1.050 0.950 Met (%) 0.450 0.440 0.400 0.400 0.400 0.400 Met+Cys (%) 0.900 0.800 0.800 0.710 0.800 0.710 Thr (%) 0.866 0.724 0.778 0.640 0.778 0.640 Trp (%) 0.311 0.248 0.279 0.212 0.279 0.212 Ca (%) 0.990 0.904 0.990 0.906 0.987 0.898 Total P (%) 0.679 0.669 0.661 0.61 0.661 0.61 Avail P (%) 0.456 0.552 0.451 0.549 0.451 0.549 The premix provided (for 1 kg of diets) VA 10000IU， VB1 1.8 mg， VB2 40 mg， VB12 0.71 mg， VD3 2000 IU， VE 10IU， VK3 2.5 mg，biotin 0.12 mg， folic acid 0.5 mg， D-pantothenic acid 11 mg， Cu (as copper sulfate) 8 mg， Fe (as ferrous sulfate) 80 mg， Mn (asmanganese sulfate) 60 mg， Zn (as zinc sulfate) 40 mg， I (as potassium iodide) 0.0.35 mg and Se (as sodium selenite) 0.15mg. Intwo phases： starter ((1-21))and finisher phase(22-42). Three basal diets were prepared for the study，positive control comprising 21.5% CP (starter) and19.0% CP (finisher)， negative control with 2%age unitCP lower in each phase and in AZO-0.25 than PC，(Table 1). Azomite was purchased from a commercialcompany (Taiwan Lytton Company， New Taipei CityTaiwan) and was mixed in feed mill at a concentrationof 0.25%.The detailed composition of AZO is pre-sented in Table 2. The basal diet was in mash formand formulated to meet the nutritional requirementsof 1 to 6 week-old broiler chicks. Recording of data On days 21 and 42 of the experiment， live bodyweight (LBW) and feed consumption were recordedfor each replicate of the treatment groups. The aver-age daily feed intake (ADFl)， average daily gain (ADG)and feed conversion ratio (FCR) was calculated. At theend of experimental trial (on day 42) two birds fromeach replicate were slaughtered to measure the evis-cerated， breast muscle， leg muscle， abdominal fat andimmune organ weights. Nutrient digestibility Titanium Oxide was used as an indigestible marker fordetermining the digestibility of nutrients. Faeces were Table 2. Mineral analysis of Azomite (provided by the TaiwanLytton Company China). Mineral g/100g/mg/kg Calcium oxide 5.17 g/100g Potassium oxide 5.18 g/100g Sodium oxide 2 g/100g Magnesium oxide 0.78g/100g Sulfur trioxide 0.21 g/100g Ferric oxide / iron 1.37 g/100g Manganese oxide 0.02 g/100g Zinc 64.3 mg/kg Cobalt 22.3 mg/kg Copper 13.5 mg/kg Molybdenum 12.6 mg/kg Fluorine 320 mg/kg Lithium 859 mg/kg Boron 29mg/kg Chromium 7.85 mg/kg Lanthanum 257 mg/kg Cerium 360 mg/kg Praseodymium 26.75 mg/kg Tungsten 31.00 mg/kg Vanadium 9.35 mg/kg Nickel 2.27 mg/kg Tin 2.90 mg/kg collected from days 39-41 and composited to get arepresentative sample. The faeces were dried at 65C°for 72 hrs， ground and then passed through 0.40 mmscreen. Nutrient analysis of faeces and basal diet wasconducted. Dry matter and ash1\were determinedaccording to the methods described by AOAC (2000).Crude protein (Dumatherm， Gerhardt， Germany)， GrossEnergy (GE)) tthrough calorimeter(OxygenBomb Calorimeter， C 2000 Version， IKA， Germany) while Caand P were analysed by using atomic absorption spec-trometer (novAA 400 P， analytikjena， Germany) andUV-VIS Spectrophotometer (Model： 1780， Shimadzu，Japan)，respectively. The contents of TiO2 in diets andfaeces were determined according to the methoddescribed previously (Short et al. 1996). The apparentdigestibility of nutrients was calculated using followingformula： Apparent digestibility of nutrients Intestinal enzymes activity The digesta was collected from jejunum and stored inliquid nitrogen. The enzymatic activities of lipase，amylase and trypsin were analysed using the commer-cial kits according to the manufacturer's instructions(Nanjing Jiancheng Bioengineering Institute，Nanjing， China). Blood biochemical indices At the end of the experimental trial blood samples(5 ml each) from subcutaneous veins were collected inanticoagulant labelled vacutainers from two birds perreplicate for serum biochemical analysis. The serumwas harvested and centrifuged at 3000 rpm for 15 minat 4C and stored at -20C until analysis. The con-tents of total cholesterol (TC)， creatinine (CREAT)， uricacid (UA)， glucose (GLU) and total protein (TP)， wereanalysed by using an automated IDEEX Vet TestChemistry Analyser (IDEEX Laboratories， Inc). The con-centrations of immunoglobulins， IgA， lgG and IgM inplasma were analysed using specific ELISA kits instruc-tions (Shang Hai Lengton Biosciences Co.， LTD， ShangHai， China). The serum concentration of growth hor-mone (GH)， Parathyroid Hormone (PTH) and calcitonin(CT) were determined by immunoradiometric assaymethod， and gamma counter (Bio SourceInternational， Camarillo， CA)， by using commercial kitsas per manufacturer instructions (Immutopics， Inc.， SanClemente，CA). Tibia sampling On day 42， tibial bones from two birds per replicatewere isolated from the skin， muscle， and soft tissues of slaughtered birds， dried and then stored at 4°C. Thetibiallkbiomorphometeric measurementincludingweight， length and diameter were done using weigh-ing balance， ruler， and Vernier calliper， respectivelyThe diameter was measured at the narrowest and wid-est points and then averaged. The bone breakingstrengthh was determined using TAxT2iTextureAnalyser (Stable Micro Systems， London， UK). After themeasurement of bone strength， the broken tibia wasplaced in plastic bags for the determination of ash，Caand P. Tibia bone samples were defated with ethanoland diethyl ether for 48 hrs. The samples were dried inthe oven at 100C for 24 hr then weighed and burntin muffle furnace at 600°C for 16hrs. Ash was dis-solved in 10 ml of hydrochloric acid (HCl) and 5 ml ofnitric acid (HNO3). Digested samples were filtered anddiluted with deionised water to the required volumeand analysed for Ca and P， using Atomic AbsorptionSpectrometer (novAAA4400 P， analytikjena， Germany)and UV-VIS Spectrophotometer (Model： 1780，Shimadzu， Japan) respectively. Statistical analysis Data for different parameters were tested for homo-geneity of the variances using Levene's test (Levene1960) before statistical analysis. After that data werestatistically analysed using two-way analysis of vari-ance (ANOVA) using SPSS software (SPSS， 19.0). Thesignificant differences among means of treatmentswere compared by Tukey’s test. The results were con-sidered significant at p<.05. Results Growth performance Dietary supplementation with AZO exhibited a positiveeffect on growth performance in broilers (Table 3).Birds fed diet supplemented with AZO had higher， (p<.05) LBW and ADG compared with NC diet inboth starter and finisher phases， whereas， no signifi-cant difference was observed between PC and AZO.The ADFI was not affected by the treatment in thestarter phase； however， it was lowest (p<.05) in PCtreatmentit infinisher phaseand overalll periods.Moreover， FCR was higher (p<.05) in birds on NC andthe lowest on AZO diet. Carcase traits Table 3. Effect of Azomite on growth performance in broilerchickens fed low protein diet. Parameters PC NC AZO-0.25 SEM P Value Starter 1-21 day One-day-old Weight 41.45° 41.44° 41.40° 0.02 0.989 LBW(g) 938° 906° 957° 1.29 0.043 ADG(g) 44.69ab 43.16° 45.57° 0.62 0.046 ADFI(g) 58.62 58.75 59.36 0.58 0.463 FCR 1.31 1.36° 1.30° 0.09 0.006 Finisher 1-42 day LBW 2735° 2565° 2790° 12.9 0.001 ADG 65.11° 61.07” 66.44° 0.87 0.008 ADFI 97.77 101.61° 101.86° 0.84 0.006 FCR 1.54° 1.66° 1.53 0.01 0.002 Means in same row with no common superscript differ significantly(p<.05). PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%； LBW：live body weight； ADG： average daily gain， ADFl： average daily feedintake； FCR： feed conversion ratio. Table 4. Effect of Azomite on carcase performance in broilerchickens fed low protein diet. Parameter PC NC AZO -0.25 SEM P Value EV% 70.74° 69.31° 71.68° 0.30 0.040 BM% 15.75° 14.45° 16.00° 0.10 0.015 LM% 10.98° 10.45° 11.14° 0.11 0.027 AF% 1.85° 2.15 1.68° 0.06 0.491 Thymus% 1.34 1.22 1.42 0.10 0.744 Bursa% 0.32 0.30 0.32 0.01 0.921 Spleen% 0.84 0.79 0.80 0.05 0.920 a， Means in same row with no common superscript differ significantly(p<.05).PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%； EV：eviscerated； BM： breast muscle； LM： leg muscle； AF： abdominal fat. percentage in comparison to those on NC treatment，while， no significant difference was observed betweenPC and AZO (Table 4). The broilers fed diet supple-mented with AZO exhibited lower abdominal fat per-centage as compared to the NC treatment. But noeffect of treatment was observed on thymus， bursaand spleen percentages (p>.05). Nutrient digestibility The dietary supplementation of AZO in low CP dietsignificantly increased the digestibility of DM， Ca(p<.05)， CP， AME and P (p<.01) compared to NCtreatment， but no difference was observed in compari-son with PC diet (Table 5). However， no effect of treat-ment was observed on the digestibility of ash. Enzymatic activity No effect of AZO supplementation was observed onthe activities of lipase and amylase enzymes (Table 6).However， AZO enhanced (p<.05) the activity of tryp-D-sin enzyme as compared to NC but no difference wasobserved when compared with PC diet. Table 5. Effect of Azomite on digestibility percentage ofnutrients in broiler chickens fed low protein diet. Parameter PC NC AZO-0.25 SEM P Value DM% 73.91ab 70.74° 75.67° 0.07 0.016 CP% 66.16° 64.86° 67.18° 0.07 0.037 ME% 76.98° 73.71° 77.85° 0.05 0.002 Ash% 62.40° 57.47"，b 63.26° 0.10 0.051 P% 45.55ab 42.72° 48.83° 0.01 0.037 Ca% 51.49° 46.72° 54.17° 0.01 0.002 ab， Means in same row with no common superscript differ significantly(p<.05). PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%； DM：dry matter； CP： crude protein； ME： metabolisable energy； Ca： calcium；P： phosphorus. Table 6. Effect of Azomite on enzyme activity in broiler chick-ens fed low protein diet. Parameter PC NC AZO-0.25 SEM P Value Lipase (U/mg of protein) 288.82 266 279 8.69 0.645 Amylase (U/mg of protein) 3.91 3.55 3.90 0.13 0.496 Trypsin (U/mg of protein) 178.38°118.88” 198.66° 12.51 0.022 Means in same row with no common superscript differ significantly(p<.05). PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%. Table 7. Effect of Azomite on serum biochemical parametersof broiler fed low protein diet. Parameter PC NC AZO -0.25 SEM P Value TC mmol/L 2.78 2.85 2.74 0.05 0.191 CREAT mmol/L 14.71° 13.63° 14.90° 0.22 0.007 UA umol/L 344.80 310.80 362.60 30.6 0.716 GLU mmol/L 9.64 9.34 10.20 0.27 0.474 IgA g/L 1.19 1.01 1.22 0.05 0.264 lgM g/L 0.83 0.64 0.95 0.06 0.175 lgG g/L 7.58 6.88 7.95 0.27 0.303 GH (ng/ml) 3.91° 3.17± 4.39° 0.18 0.012 CT pg./ml 88.1° 68.12° 103.9° 2.31 0.045 PTH pg./ml 24.43 26.34 22.5 1.43 0.056 a.b，Means in same row with no common superscript differ significantly(p<.05). PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%. TC：total cholesterol； CREAT： creatinine； UA： uric acid； GLU： glucose； Ig：immunoglobulin； GH： growth hormone； CT： Calcitonin； PTH： parathy-roid hormone. Blood biochemical indices Azomite supplementation had no effect (p>.05) ontotal cholesterol (TC)， uric acid (UA)， glucose (Glu)，immunoglobulin G， A， and M (IgG IgA， IgM) however，AZO supplementation enhanced (p<.05) the serumcreatinine (CREAT)， growth hormone (GH) and calci-tonin (CT) contents compared to NC (Table 7). Tibia parameters Azomite supplementation had no effect (p>.05) ontibia weight (TWT)， tibia length (TL) and tibia diameter(TD). Dietary supplementation of AZO resulted in anincrease in tibia breaking strength (TBS) and Ca con-tent (p<.05) compared to NC， but no difference wasnoted in comparison with PC. However， ash and P Table 8. Effect of Azomite on tibia breaking strength， Ash， Pand Ca contents% in broiler chickens fed low protein diet. Parameter PC NC AZO -0.25 SEM P Value TWT (g) 7.51 6.73 7.76 0.20 0.082 TL (cm) 8.77 7.68 9.02 0.26 0.091 TD (cm) 0.86 0.79 0.87 0.01 0.106 TBS (kg) 22.45° 19.57° 24.65° 1.17 0.033 Ash% 47.88° 46.65 50.18° 0.04 0.003 P% 7.72 7.41 8.56° 0.17 0.007 Ca% 17.22° 13.75° 18.23° 0.78 0.031 a.b. Means in same row with no common superscript differ significantly (p<.05). PC： Positive Control； NC： Negative Control； AZO-025： Azomite 0.25%； TWT： tibia weight； TL： tibia length； TD： tibia diameter； TBS： tibia breaking strength； Ca： calcium； P： phosphorus. content were higher (p<.05) in the AZO than othertwo groups (Table 8). Discussion Azomite has been recognised as a mineral boosterand feed additive for promoting the growth perform-ance and nutrient utilisation by improving the digest-ive functions in aquaculture farming (Liu et al. 2011；Batool et al. 2018). Azomite may be acting via multiplemechanisms including toxin binding， nutrient allow-ance， or the provision of some other benefit due to itsdistinct composition of hydrated sodium calcium alu-minosilicate， rare earth minerals， and other trace ele-ments. The current study provides new insights intothe use of AZO with low protein diet in broilerchicken.To the best of our knowledge， there is limitedinformation available regarding the potential use ofAZO in broiler diet and its effects on the growth per-formance and nutrient utilisation. In the present study，reduction of dietary CP in broiler diet showed nega-tive effects on LBW， ADG and FCR， however， Azomitesupplementation ameliorated these negative effectsand exhibited similar or even better growth perform-ance compared to standard diets (PC). It is well estab-lished that reduction of dietary CP levels in broilerdiet decreases the growth performance (Awad et al.2015； Rehman et al. 2018). Our findings are in agree-ment with earlier studies as NC in our study (low CPdiet) resulted in poor growth performance as com-pared to PC (diet with recommended CP levels).Another study demonstrated that the broilers fed withlow protein diets (containing 1% low CP than the rec-ommended level)， at constant ME， with the sameamino acids levels did not adversely affect growth per-formance， carcase parameters and liliver functionswhile dietary crude protein and crude fibre utilisationwere improved significantly (Salah 2016). Moreover，our findings revealed that AZO supplementation canincrease the growth performance. Earlier study (Batool et al. 2018) has reported that supplementation of AZOimproved theweightt gain andFCR in catfish(Pangasius hypophthalmus). Previous studies also indi-cated improved growth performance and FCR thatwasattributed to the enhanced digestibility andmetabolism of nutrients in response to AZO supple-mentation in the diet (Fodge et al.， 2011； Azamet al.，2016). Broiler fed low protein diet had lower evisceratedyield， breast muscle and leg muscle percentage， whileabdominal fat was higher in birds fed with low CPdiet in the present study. Similar findings have beenreported earlier in broilers fed with low CP diet hadhigher fat percentage (Namroud et al.2008；Abudabos2012). This may be due to the higher energy ratiowith low protein diet that results in insufficient energymetabolism and overall . nutrient utilisationInterestingly， our study indicated that Azomite supple-mentation significantly increased the percentage ofeviscerated yield， breast muscle and leg muscle. Thesefindings verified that AZO supplementation can signifi-cantly increase the breast muscle percentage in broilerchicken (Emerson and Hooge 2008). Moreover， AZOhas also shown to stimulate the muscle mass of cat-fish (Pangasius hypophthalmus) previously (Batool et al.2018). The improvementitinincarcasiee performanceobserved with AZO supplementation is mainly attrib-uted to the increased digestibility， absorption and util-isation of nutrients as revealed by higher digestibilityof DM， CP and ME in supplemented birds as comparedto birds fed low protein diet. Moreover， supplementa-tion of AZO in a low CP diet also enhanced the digest-ibility of Ca and P in the present study. These findingsagree with previous study which reported that dietaryAZO increased the digestibility of DM and CP in tilapiafish (Fodge and Fodge 2011). ltis suggested that improvements in nutrientdigestibility may be attributed to the increased activityof digestive enzymes.Very few studies have evaluatedthe effect of AZO on the activity of digestive enzymes.In the present study， AZO supplementation in a lowCP diet improved the trypsin activity in broiler. Ourfindings are in agreement with earlier studies whichreported an improvement in the trypsin and amylaseactivity in shrimp and Tilapia fish in response to AZOsupplementation (Fodge and Fodge 2014； Tan et al.2014). These findings collectively suggest that the useof AZO in diet can enhance the activity of digestiveenzymes and nutrient uptake in the small intestine.The increased secretion of digestive enzymes (mainlytrypsin) seemed to be one of the primary mechanisms by which AZO enhances the nutrient digestion andabsorption in the present study. Serum indices are critical indicators to monitor thehealth， diagnosis and treatment of disease and alsodetermine the nutritional status of birdsIs (Schmidtet al. 2007). Serum biochemical indicators of birds inthe present study were within the normal physio-D-logical range for chicken. However， CREAT concentra-tion was decreased in birds fed low CP diet. Theseresults are in agreement with previous study reportingsignificant decrease in the CREAT level in broilers fedwith a low CP diet (Arczewska-Wtosek et al. 2018)However， CREAT level was increased in response toAZO supplementation. It isIswell established thatCREATisa product of CREAT phosphate innthemuscles tissues and its production is associated withmuscle mass (Wyss and Kaddurah-Daouk 2000； Rajmanet al. 2006). Hence， increased muscle mass observed inthe present study may be attributed to the enhancedlevel of CREAT in broiler. Moreover， increase observed in the serum TP inresponse to the AZO supplementation is also in agree-ment with earlier study reported higher serum TP inAZO supplemented koi carp fingerlings (Jaleel et al.，2015). These findings may be attributed to enhancedldigestion， absorption， and utilisation of CP in thegastrointestinal tract (GIT) of broiler chicken. In add-ition， serum GH and CT levels were increased by AZOsupplementation in the present study. The enhancedsecretion of GH in the blood is directly associatedwith the accelerated protein synthesis and higher fatbreakdown to release the dietary energy necessary forthe tissue growth. Growth hormone is considered asan important regulator of the bone size， muscle massand body growth (Ohlsson et al. 1998). The improve-ment in CT level is generally indicated as an availableuptake and storage of dietary calcium， which posi-tively influences the metabolic pathway of the boneformation (Talmage and Grubb 1977). The potentiaeffect of AZO on serum CT concentration observed inthe present study might enhance the Ca metabolismsubsequently leading to inhibit the osteoclast (boneresorption) while activating the bone forming osteo-blasts that ultimately improves the Ca deposition inbones. As CT improves the Ca and P deposition in thebone but stops bone resorption， the net effect of CTis the enhanced level of Ca and P in the bone with aconcomitant increase of these minerals by absorptionin the blood (Frandson and Spurgeon 1992). Effect of AZO onbone mineralisationn I.is notreported yet. In the present study， AZO increased thetibia breaking strength， ash， P and Ca percentage in the tibia of birds as compared to NC. The positiveeffect of AZO might probably be attributed to anincreased digestion and absorption of Ca and P in thebone， whichled to the increasedd tibia breakingstrength andmineralisation. These findings are inagreement earlier studies reporting that breakingstrength and ash content of bones are associated withbone mineralisation in chicken (Onyango et al. 2003).Moreover，r，tthe bone mineralisation makesSbonesharder which enables the skeleton to withstand thegravity，additionalloading aandpreventittthee legdeformities in broilers (Shim et al.， 2012). Anotherstudy demonstrated that inclusion of organic mineralmixture (Mn， Zn， and Cu) enhanced the reproductiveperformance， egg shell quality， plasma profile， yolkmineralconcentration， yolk lipid oxidation， andimmune response in laying hens under high ambienttemperature (Saleh et al. 2020). In the present studyAZO seems to possess sufficient potential for dietarysupplementation in low CP diets in broiler chicken toaddress problems associated with low CP rations.Overall， our findings indicated an improvement in thegrowth performance， nutrient utilisation， and bonemineralisation in response to AZO supplementation ofa low protein diet， which is similar or even better incomparison with standard diet. Conclusion Azomite supplementation in a low protein diet signifi-cantly enhanced the growth performance， nutrientutilisation and trypsin activity in broilers. Azomite alsoimproved the tibia breaking strength， Ca and P con-tents of the tibia bone. Therefore， it is suggested thatAZO supplementation at 0.25% of the diet is enoughto entirely alleviate the negative impact of low proteindiet in broilers. Reducing the CP contents of the dietwith AZO supplementation seems aa promisingapproach to lower feed cost while improving thegrowth performance and nutrient utilisation in broilersdiet to harvest more economic returns. Acknowledgement Authors are grateful to the staff of the laboratory of FeedBiotechnologyyCoffAgriculturalMinistry，V，FeedResearchInstitute， Chinese Academy of Agricultural Sciences， Beijing，for the technical assistance. Ethical approval This animal study (short title： Azomite in poultry nutrition)was carried out in strict accordance with the recommendation of the ethical committee of GraduateSchool of Chinese Academy of Agricultural Sciences， BeijingP.R. China， with the approval number (IACUC#AEC-CAAS-FRI-：CAAS20191029). All procedures and experiments complywith the guideline and were approved by the local ethiccommittee of the Feed Research Institute， Chinese Academyof Agricultural Sciences， Beijing China with respect to animalexperimentation and care of animals under study， and allefforts were made to minimise suffering. Disclosure statement The authors of this manuscript declare that there is no con-flict of interests that could possibly arise. Funding This research work was funded by National Key Researchand Development Program of China [2018YFG0501401]. ORCID Muhammad Asif Arainn( http：//orcid.org/0000-0001-5858-8175 References ( Abudabos AM. 2012. Effect of enzyme supplementation to normal and low density broiler diets based on corn-soy- bean meal. Asian J Anim Vet Adv.7(2)：139-148. ) ( AOAC.2000 . C Official methods 5 of l analysis. 17th ed. Washington (DC)： ： Associationof Official Analytica Chemists. ) ( Arain MA， Mei Z， Hassan F， Saeed M， Alagawany M， Shar A. Rajput l. 2018. 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