【作者】 刘松柏；

【导师】 罗绪刚；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2012， 博士

【摘要】 本论文共通过五个系列试验，研究肉仔鸡对饲料原料标准磷利用率、非植酸磷及标准可利用磷的需要量，并用原位结扎灌注肠段法研究无机磷在肉仔鸡小肠各段的吸收规律和机制。试验一包括3个试验，旨在建立测定肉鸡对常用饲料原料（豆粕）标准矿物元素（钙、磷、铜、锰和锌）利用率的方法。在试验1中，12只24日龄商品代AA肉仔公鸡用于测定肉仔鸡排空消化道食糜的适宜绝食时间。设1个处理组，包括4个重复，每个重复3只鸡。肉仔鸡自由采食玉米-豆粕型饲粮2小时后截料，然后收集截料后12、24、36或48小时的排泄物。结果表明，肉仔鸡至少绝食24小时才能保证其消化道食糜排空。在试验2中，48只22日龄商品代AA肉仔公鸡分2个处理组（无矿物质和豆粕饲粮组），每个处理组24只鸡（每个处理组6个重复笼，每个重复笼4只鸡）。经过3天的适应期后，24日龄肉仔鸡绝食24小时后饲喂无矿物质或豆粕饲粮4小时，从采食开始收集28小时（截料后24小时）或52小时（截料后48小时）的排泄物用于测定肉仔鸡对豆粕的标准矿物元素利用率。结果表明，测定肉仔鸡对豆粕的标准矿物元素利用率适宜的收集排泄物时间为从采食开始收集排泄物52小时。24日龄肉仔鸡对豆粕的标准钙、磷、铜、锰、锌利用率分别为51.1、50.2、36.3、30.8及49.7%。在试验3中，采用试验2相同的试验方法测定36日龄肉仔鸡对同一豆粕的标准矿物元素利用率。共48只34日龄商品代AA肉仔公鸡分2个处理组（无矿物质和豆粕饲粮组），每个处理组24只鸡（每个处理组6个重复笼，每个重复笼4只鸡），然后适应3天至36日龄进行试验。结果表明，36日龄肉仔鸡对豆粕的标准磷（51.6%）、铜（36.4%）、锰（31.9%）、锌（43.4%）利用率与试验2中的相应测定结果基本一致（P>0.12），但标准钙（41.5%）利用率低于试验2中的测定结果，说明在本试验期间内肉仔鸡日龄对豆粕的标准磷、铜、锰和锌利用率没有显著影响。总之，本试验建立的简单、快速的平衡试验技术新方法可以用于测定肉鸡对常用饲料原料（如豆粕）的标准矿物元素利用率。试验二包括2个试验，用于测定肉鸡对玉米、豆粕及玉米-豆粕型饲粮的标准磷利用率，同时验证标准磷利用率在玉米-豆粕型饲粮中的可加性。每个试验均用96只体重相近的22日龄商品代AA肉仔公鸡，分4个处理组（无磷、玉米、豆粕及玉米-豆粕饲粮组），每个处理组24只鸡（每个处理组6个重复笼，每个重复笼4只鸡）。经过3天的适应期后，绝食24小时，然后分别自由采食无磷、玉米、豆粕及玉米-豆粕型饲粮4小时（试验1）或72小时（试验2），收集排泄物28或52小时（试验1）与96或120小时（试验2）（截料后48小时）。试验1的结果表明，收集排泄物52小时（截料后48小时）是测定标准磷利用率适宜的收集排泄物时间。肉仔鸡通过采食无磷饲粮测定的基础内源磷排泄量为123mg/52小时·只。通过基础内源磷校正计算的肉仔鸡对玉米、豆粕的标准磷利用率分别为37.6和50.5%。同时，测定玉米-豆粕型饲粮的标准磷利用率为44.4%，与预测值43.5%相近（P>0.79）。试验2的结果表明，收集排泄物96小时（截料后24小时）是测定标准磷利用率适宜的收集排泄物时间。肉仔鸡通过采食无磷饲粮测定的基本内源磷排泄量为85.4mg/96小时·只。通过基础内源磷校正计算的肉仔鸡对玉米、豆粕的标准磷利用率分别为40.2和52.9%，与试验1的相应测定结果相近（P>0.48），但测定的玉米-豆粕型饲粮的标准磷利用率为39.7%，显著低于预测值46.0%（P <0.001），这可能与肉仔鸡的磷进食量较高有关。试验结果表明，无磷饲粮可用于测定肉仔鸡基础内源磷的排泄量进而用于估测肉鸡对植物性饲料原料的标准磷利用率。但是，关于标准磷利用率在混合饲粮中的可加性还有待进一步研究。试验三包括2个试验，用于测定肉仔鸡对无机矿物质磷源（磷酸氢钙、磷酸二氢钙及磷酸二氢钾）的标准磷利用率。每个试验均用96只体重相近的22日龄商品代AA肉仔公鸡随机分成4个处理组（无磷、磷酸氢钙、磷酸二氢钙及磷酸二氢钾饲粮组），每个处理组24只鸡（每个处理组6个重复笼，每个重复笼4只鸡）。经过3天的适应期后，绝食24小时，然后饲喂无磷、磷酸氢钙、磷酸二氢钙及磷酸二氢钾饲粮4小时（试验1）或72小时（试验2），收集排泄物52小时（试验1）或120小时（试验2）（截料后48小时）。试验1的结果表明，肉仔鸡通过无磷饲粮估测的基础内源磷排泄量为109mg/52小时·只。通过基础内源磷校正估测的肉仔鸡对磷酸氢钙、磷酸二氢钙及磷酸二氢钾的标准磷利用率分别为68.7、69.8及76.6%。试验2的结果表明，收集排泄物96小时（截料后24小时）即可用于测定无机矿物质磷源的标准磷利用率。肉仔鸡通过无磷饲粮测定的基础内源磷排泄量为49.2mg/96小时·只。通过基础内源磷校正估测的肉仔鸡对磷酸氢钙、磷酸二氢钙及磷酸二氢钾的标准磷利用率分别为71.8、70.6和78.3%。试验1和2中测定的结果很一致(P <0.001)，表明无磷饲粮可以用于测定肉仔鸡基础内源磷的排泄量，且快速平衡法可用于测定肉鸡对无机矿物质磷源的标准磷利用率。试验四采用单因子的完全随机试验设计，用540只1日龄商品代AA肉仔公鸡进行试验，研究饲粮不同非植酸磷水平对肉仔鸡生长性能、骨骼发育参数及组织中磷代谢关键功能基因mRNA表达水平的影响，从而估测1-21日龄肉仔鸡玉米-豆粕型饲粮中非植酸磷及标准可利用磷的适宜需要量。试鸡按体重随机分为9个处理组（每个处理组6个重复笼，每个重复笼10只鸡），分别饲喂在玉米-豆粕型基础饲粮（非植酸磷含量实测为0.08%）中以CaHPO4·2H2O形式分别添加磷水平为0.10、0.15、0.20、0.25、0.30、0.35、0.40、0.45及0.50%的处理饲粮。各处理饲粮钙水平保持一致（1.0%）。试验期21天。结果表明，日增重、胫骨抗压强度、胫骨灰分含量、胫骨矿物质含量、胫骨骨密度及中趾骨灰分含量是评价肉仔鸡非植酸磷需要量的敏感指标，其中胫骨矿物质含量及其骨密度为评价肉鸡非植酸磷需要量新的特异敏感指标，而血清及胫骨碱性磷酸酶活性、胫骨灰分磷含量、胫骨灰分钙磷比、中趾骨灰分磷含量、十二指肠钠磷协同转运蛋白（NaP-IIb）mRNA和胫骨非组织特异性碱性磷酸酶（TNAP）mRNA表达水平更适于评价肉鸡对饲粮磷的生物学利用率。同时十二指肠NaP IIb mRNA表达水平受饲粮非植酸磷水平的显著（P <0.03）影响。根据以上敏感反应指标估测1-21日龄肉仔鸡非植酸需要量分别为0.34、0.39、0.37、0.37、0.39及0.36%。综合各指标，1-21日龄肉仔鸡玉米-豆粕型饲粮中非植酸磷适宜需要量为0.39%，标准可利用磷的适宜需要量为0.37%。试验五包括2个试验，用原位结扎灌注肠段法研究无机磷在肉仔鸡十二指肠、空肠及回肠的动力学吸收特点及其与NaP-IIb基因表达的相关性。试验1通过研究不同灌注时间点时肉仔鸡小肠各段无机磷吸收率变化的规律，以确定研究肉仔鸡结扎小肠各段磷吸收动力学的适宜灌注时间。用22日龄商品代肉仔公鸡96只，按体重随机分为6个处理组（每个处理组8个重复笼，每个重复笼2只鸡），灌注6mmol/L的无机磷（磷酸二氢钾），共设0、2.5、5、10、20和40分钟6个采样时间点。结果表明，无机磷在肉仔鸡结扎十二指肠、空肠及回肠中的吸收率均随灌注时间的延长而增加，且在0-40分钟内呈渐近线变化；十二指肠磷吸收率在灌注后20分钟时显著（P <0.04）高于空肠和回肠磷吸收率，提示十二指肠可能是肉仔鸡磷吸收的主要部位。由于在20分钟时各肠段磷吸收率均达到了最大吸收率的87.0%以上，故在试验2中采用的适宜灌注时间为20分钟。试验2通过结扎肉仔鸡小肠各段灌注不同磷浓度（0、1.5、3、6、12、24或48mmol/L，磷酸二氢钾），观测无机磷吸收速率与灌注磷浓度之间的关系，揭示无机磷在肉仔鸡小肠各段的吸收动力学模式，并通过小肠各肠段NaP-IIb mRNA表达水平来研究NaP-IIb在结扎肉仔鸡小肠各段磷吸收转运中的作用。用22日龄商品代肉仔公鸡56只，按体重随机分为7个处理组（每个处理组8个重复笼，每个重复笼1只鸡）。肉仔鸡小肠各段磷吸收动力学模型表明，十二指肠磷吸收以饱和载体转运为主，而空肠和回肠磷吸收以非饱和扩散为主。在不同的磷灌注浓度（0、6或48mmol/L）下，十二指肠NaP-IIb mRNA表达水平都显著（P <0.0001）高于空肠和回肠，说明十二指肠磷的饱和载体吸收与其NaP-IIb密切相关。综上所述，本研究建立了测定肉鸡对饲料原料和全价饲粮中标准矿物元素利用率的快速平衡试验技术新方法，并以此测得的肉仔鸡对玉米、豆粕、玉米-豆粕型饲粮、磷酸氢钙、磷酸二氢钙及磷酸二氢钾的标准磷利用率分别为37.6、50.5、44.4、68.7、69.8及76.6%；筛选到胫骨矿物质含量及其骨密度是评价肉仔鸡磷需要量的特异性敏感新指标，并用其评价获得的1-21日龄肉仔鸡玉米豆粕型饲粮中非植酸磷适宜需要量为0.39%，标准可利用磷需要量为0.37%；无机磷在肉仔鸡十二指肠中的吸收以饱和载体为主，而在空肠和回肠则以非饱和扩散为主，与其NaP-IIb表达密切相关。以上研究新成果对于我国肉鸡生产中合理、高效使用磷源，以精准满足肉鸡的可利用磷需要量，进而降低饲料成本，减少磷排放对环境的污染，都具有重要的理论和现实指导意义。更多还原

【Abstract】 Five studies were conducted in this dissertation to investigate the standardized phosphorus (P)availabilities in feedstuffs, non-phytate P and standardized available P requirement, as well as theabsorption mechanism of inorganic P in the ligated small intestine segments of broiler chicks.Firstly, three experiments were conducted to develop a procedure for estimating the standardizedmineral (calcium, Ca; phosphorus, P; copper, Cu; manganese, Mn; zinc, Zn) availabilities (SMA) in themost commonly used model ingredient (soybean meal) for broilers. In experiment1, twelve24-d-oldcommercial AA male chicks were used to determine the length needed to empty the total tract of feedresidues during the pre-experimental fasting period. The chicks were randomly allotted to four replicatecages of three chicks per cage. Birds were fed the corn–soybean meal basal diet for2h, then feeds wereremoved and excreta samples were collected for12,24,36or48h after feed withdrawal. The resultsindicated that the excreta collection time of24h after feed withdrawal was sufficient to empty the totaltract of feed residues. In experiment2, forty-eight22-d-old commercial AA male chicks were used todetermine SMA in soybean meal. The chicks were randomly allotted to one of the two treatments(mineral-free or soybean meal diet group) with six replicate cages of four chicks per cage. After3-dacclimation, chicks were fasted for24h and then fed mineral-free or soybean meal diet for4h, andexcreta samples were collected for28h (24h after feed withdrawal) or52h (48h after feed withdrawal).The results showed that the extended excreta collection time of52h was adequate for estimating SMA.The standardized availability values of Ca, P, Cu, Mn and Zn in soybean meal were51.1,50.2,36.3,30.8and49.7%, respectively. In experiment3, a similar bioassay was conducted with forty-eight36-d-oldcommercial AA male chicks to measure SMA in the same soybean meal. Forty-eight34-d-old chickswere randomly allotted to one of the two treatments (mineral-free or soybean meal diet group) with sixreplicate cages of four chicks per cage, then birds recovered and acclimated for3d. The standardizedavailability values of P (51.59%), Cu (36.37%), Mn (31.94%) and Zn (43.37%) were similar (P>0.12)to the results of experiment2except for Ca (41.49%), indicating that the age of birds had no effect onthe standardized availabilities of P, Cu, Mn or Zn in soybean meal. The results from this study suggestedthat this simple and rapid procedure could be used to determine SMA in feedstuffs (e.g. soybean meal)for broiler chicks.Secondly, two experiments were conducted to estimate standardized phosphorus (P) availabilities(SPA) of corn, soybean meal (SBM) and corn-soybean meal (C-SBM) diet in broilers chicks, and verifythe additivity of SPA for feed formulation of broilers. Ninety-six22-d-old commercial AA male broilerswith similar bodyweight were used in each experiment. The chicks were randomly allotted to one of thefour treatments (P-free, corn, SBM or C-SBM diets group) for six replicate cages of four chicks per cage.After3-d acclimation, chicks were fasted for24h and then fed P-free, corn, SBM or C-SBM diets,respectively, for4h in experiment1or72h in experiment2, and collected excreta for52h (48h afterfeed withdrawal) in experiment1or120h (48h after feed withdrawal) in experiment2. In experiment1, the results showed that the excreta collection time of52h (48h after feed withdrawal) was adequate forthe estimation of SPA. The basal endogenous P loss (EPL) of chicks fed the P-free diet was estimated tobe123mg/52h per bird. The values of SPA corrected by basal EPL were37.6and50.5%for corn andSBM, respectively. The determined value of SPA for the C-SBM diet was very close (P>0.79) to thepredicted summation of SPA from corn and SBM (44.4vs.43.5%). In experiment2, the results showedthat the excreta collection time of96h (24h after feed withdrawal) was sufficient for the estimation ofSPA. The basal EPL of chicks fed the P-free diet was estimated to be85.4mg/96h per bird. The valuesof SPA corrected by basal EPL were40.2and52.9%for corn and SBM, respectively. The determinedvalue of SPA of the C-SBM diet was lower (P <0.001) than the predicted summation of SPA from cornand SBM (39.7vs.46.0%), which might be due to the effect of higher total P intake. The results from thecurrent study demonstrated that the P-free diet could be used for measuring basal EPL in broiler chicks,and then estimating the SPA values of plant feedstuffs for broiler chicks. However, the additivity of SPAin the diet formulation needs to be further studied.Thirdly, two experiments were conducted to estimate standardized phosphorus (P) availabilities(SPA) of dicalcium phosphate (DCP), monocalcium phosphate (MCP) and monopotassium phosphate(KCP) in broilers chicks. Ninety-six22-d-old commercial AA male chicks with similar bodyweight wereused in each experiment. The chicks were randomly allotted to one of the four treatments (P-free, DCP,MCP or KCP diets group) with six replicate cages of four chicks per cage. After3-d acclimation, chickswere fasted for24h and then fed P-free, DCP, MCP or KCP diets, respectively for4h in experiment1or72h in experiment2, and collected excreta for52h (48h after feed withdrawal) in experiment1or120h (48h after feed withdrawal) in experiment2. In experiment1, the basal endogenous P loss (EPL) ofchicks fed the P-free diet was estimated to be109mg/52h per bird. The values of SPA corrected bybasal EPL were68.7,69.8or76.6%for DCP, MCP and KCP, respectively. In experiment2, the resultsshowed that the excreta collection time of96h (24h after feed withdrawal) was sufficient for theestimation of SPA. The basal EPL of chicks fed the P-free diet was estimated to be49.2mg/96h perbird. The values of SPA corrected by basal EPL were71.8,70.6and78.3%for DCP, MCP and KCP,respectively. The results from the current study demonstrated that the P-free diet could be used formeasuring basal EPL in broilers, and then estimating the SPA values of inorganic P sources for broilerchicks.Fourthly, one experiment was conducted to investigate the effects of dietary non-phytatephosphorus (NPP) levels on performance, bone characteristics and tissue gene expression of Pmetabolism-related function gene, so as to determine the non-phytate P and standardized available Prequirement of broiler chicks fed a corn-soybean meal basal diet from1to21days of age. A total of540one-day-old commercial AA male chicks were assigned to one of nine treatments (eight replicate cagesof ten chicks per cage) in a completely randomized design. The diets included a basal corn-soybean mealdiet (0.08%of NPP) supplemented with0.10,0.15,0.25,0.30,0.35,0.40,0.45, or0.50%of inorganic Pin the form of CaHPO4·2H2O, respectively. All diets contained the same Ca content of1.0%. The resultsindicated that average daily gain, tibia bone strength, tibia ash percentage, tibia bone mineral content (BMC), bone mineral density (BMD) and middle toe ash percentage were sensitive indices forestimating P requirement of broilers, and tibia BMC and BMD were new specific and sensitive criteriafor estimating the optimal dietary NPP requirement for broiler chicks. Serum and tibia ALP activities,tibia ash P concentration, tibia Ca and P ratio, middle toe ash P concentration, duodenum NaP-IIb andtibia TNAP mRNA levels were suitable for determining dietary P bioavailabilities for broiler chicks. Inaddition, the duodenum NaP-IIb mRNA level was significantly (P <0.03) affected by dietary NPP level.Based on above sensitive response indices, the estimated NPP requirements were0.34,0.39,0.37,0.37,0.39and0.36%, respectively. According to the above results, the NPP requirement was0.39%, and thestandardized available P requirement was0.37%for broiler chicks from1to21days old fed acorn-soybean meal basal diet.Finally, two experiments were conducted to study the kinetics of inorganic P absorption in theligated duodenum, jejunum, and ileum intestinal segments of broiler chicks and whether NaP-IIbtransporter was involved. Experiment1was conducted to investigate the P absorption with time, so as todetermined appropriate post-perfusion time in the subsequent experiment. Ninety-six commercial AAmale chicks with similar bodyweight were randomly allotted to one of the six treatments (sixpost-perfusion time points) with eight replicate cages of two chicks per cage. The intestinal loops wereperfused with solutions containing6mmol P/L as KH2PO4, and samples were collected at0,2.5,5,10,20or40min post-perfusion. The results showed that P absorption increased in an asymptotic response topost-perfusion time within40min in the all ligated segments, and P absorption in the duodenum washigher (P <0.04) than that in the other2segments at20min post-perfusion. The results indicated thatthe duodenum might be the main site of P absorption for chicks. In addition, the absorption at20minwas greater than87%of the maximum absorption in each segment. For this reason, the subsequentexperiment was carried out at20min post-perfusion. Experiment2was carried out to study the kineticof P absorption and the mRNA levels of NaP-IIb in the different ligated segments, in order to explain themechanisms of P absorption. Fifty-six commercial AA male chicks with similar bodyweight wererandomly allotted to one of the seven treatments (seven perfusion solutions) with eight replicate cages ofone chick per cage. Intestinal loops were perfused with solutions containing0,1.5,3,6,12,24, or48mmol P/L as KH2PO4. The mRNA levels of NaP-IIb in three intestinal loops in the0,6or48mmol P/Lgroups were analyzed. The kinetic curves showed that P absorption in duodenum depended on a saturatecarrier mediated process, whereas P absorption in the jejunum and ileum occurred with a non-saturatediffusion process. Moreover, the NaP-IIb mRNA levels in the duodenum were higher (P <0.02) thanthat in the ileum or jejunum in the0,6or48mmol P/L groups, further indicating that P absorption in theduodenum occurred mainly by a saturate carrier mediated process.In conclusion, series of present studies developed a procedure to determine the SPA in feedstuffs forbroilers chicks, and according to this procedure the estimated SPA values of corn, soybean meal,corn-soybean meal diet, DCP, MCP or KCP were determined as37.6,50.5,44.4,68.7,69.8and76.6%,respectively. The results of current studies also indicated that tibia BMC and BMD were the specificsensitive criteria for estimating NPP requirement of broiler chicks. Based on the new criteria, the optimal NPP requirement of broiler chicks was0.39%, and the standardized available P requirement was0.37%for broiler chicks from1to21days old fed a corn-soybean basal diet. In addition, P absorption in theduodenum depended on a saturate carrier mediated process, in which NaP-IIb transporter might beinvolved, whereas P absorption in the jejunum and ileum occurred with a non-saturate diffusion process.The above new findings in the current studies had important theoretical and practical significances forreasonable and efficient use of P in feed formulation to accurately meet available P requirement inbroiler production, so as to decrease feed cost and reduce environmental pollution from P excretion ofbroilers.更多还原