【作者】 张世栋；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 兽医， 2006， 博士

【摘要】 针对国内外SPF（Specific Pathogen Free）鸡产业的发展趋势及存在的问题，本研究从以下方面对SPF鸡进行研究和分析，以促进我国SPF鸡产业相关科研和生产水平的提高。1．SPF鸡的营养需要及饲料生产技术的建立本部分进行了⁶⁰Co辐照灭菌的最小有效剂量和高压蒸汽灭菌法最适灭菌条件的研究，分析比较了不同灭菌方式处理后SPF饲料的营养成分破坏程度及相应补偿办法和标准。在此基础上分别采用辐照灭菌饲料和高压蒸汽灭菌饲料饲喂SPF鸡，比较分析了不同处理灭菌饲料对SPF鸡生长和生产性能的影响。并根据SPF鸡特殊的生活环境条件及生物学特性，对SPF鸡的营养需要进行研究。结果表明：SPF鸡饲料采用10～15kGy的辐照剂量难以完全杀死全部细菌；而经20kGy以上的剂量辐照后，在30天之内未检到有细菌生长，可以认为20kGy的辐照剂量可以达到SPF鸡饲料灭菌的要求。采用高压蒸汽灭菌法时，灭菌物品的包装大小和形状对灭菌效果影响很大。饲料包装体积不宜过大，以扁平状为宜，厚度应小于15cm，饲料袋之间要有间隙，以加强蒸汽的通透性。在饲料包装符合要求的前提下，121℃下灭菌20min可以达到理想灭菌效果。采用辐照灭菌法处理SPF鸡饲料，除对维生素B₁、B₁₂、叶酸和生物素的破坏比较严重外，对其它营养成分的破坏比较轻；而采用高压蒸汽灭菌法时，饲料粗蛋白、大部分维生素成分及赖氨酸、精氨酸和胱氨酸的破坏都比较严重，特别是对赖氨酸及大部分维生素的破坏必须在生产实际中得到重视和补偿。采用两种灭菌方式处理过的饲料，通过进行相应的营养成分补充后，未见对SPF鸡生长性能产生显著的不同影响。而且辐照后饲料明显有异臭味，变得特别细碎，粉尘增多，适口性变差。相对而言，鸡反而喜欢采食高压蒸汽灭菌饲料。SPF鸡长期采食高压蒸汽灭菌饲料，在产蛋率以及种蛋合格率方面没有发现不良影响。2．SPF鸡群的遗传控制和生长及生产性能研究本部分采用RAPD方法进行了不同品系SPF鸡群的遗传结构分析，对不同品系SPF鸡的生长及生产性能进行测定分析并对同品系不同世代的SPF鸡生长性能进行比较，在此基础了对我国SPF鸡的育种方案进行探讨。不同品系SPF鸡群RAPD分析结果表明，RAPD随机引物较多，但具有多态性且重复性好的引物较少，本实验采用了60个随机引物进行筛选，结果12个引物没有产生条带或条带不清晰而无法判断，其它48种引物均获得清晰条带，其中13个引物产生了稳定、清晰、重复性好的RAPD标记，其余引物均表现单态。13种引物共产生条带91个，其中多态性片段数64条，占总片段数的70％，这说明RAPD多态性丰富，能迅速提供大量DNA标记。对照系的品系内平均相似系数为0.968，而三个SPF鸡品系的品系内平均相似系数在0.673～0.903之间，其中B系为0.903，面F系和M系分别为0.673和0.755，说明F系和M系的纯合程度均较低，而B系较高；聚类分析结果发现F系与M系、B系与对照H系分别聚为一类，说明F系与M系的遗传距离较近，而与B系之间距离较远。三者亲缘关系以F系与M系较近。这真实地反映了国内引进SPF鸡种源不一、纯度不同、遗传结构较为丰富的情况。SPF鸡的育种可以以现有SPF鸡群组成基础群，育种目标以生产性能和病原敏感性指标为主，育种方法应采用组选择法结合分子标记辅助选择法，近交程度不必要很高。性能测定结果表明，三个品系SPF鸡的前期生长性能基本一致，但育成后期体重有一定的差异性，在相同的饲养条件下，B系后期体重增加较快，而F系与M系基本相同，但开产前体重三个品系趋于一致。不同品系SPF鸡的产蛋性能有明显的差异，以M系产蛋性能最好，F系与B系差别不大；不同品系SPF鸡群的生长性能在各世代基本稳定，没有出现明显的近交衰退现象。3．SPF鸡胚的应用效果研究本研究采用SPF鸡胚和普通鸡胚分别接种新城疫、传染性支气管炎、禽脑脊髓炎和传染性法氏囊病毒，对病毒在不同鸡胚上的增殖规律进行了研究。结果表明，普通种蛋卵黄中ND抗体水平普遍较高(≥2^6.9)，且不均匀，IBD抗体阳性率为87.5％～100％。LaSota病毒在SPF鸡胚上的繁殖速度比普通鸡胚快，病毒含量高5～20倍。接种LaSota病毒后96h，SPF鸡胚的死亡率高于65％，而普通鸡胚死亡率一般低于5％；SPF鸡胚尿囊液中病毒HA效价较普通鸡胚尿囊液病毒效价平均高2～2.5个滴度。MA₅毒株接种普通鸡胚后死亡率较低，病毒液的EID₅₀相差较大；而SPF胚的鸡胚死亡率较高，病毒液EID₅₀为普通鸡胚的10倍以上，且稳定性好。接种AEV后，普通鸡胚的病变率分别为4.5％和5％，而SPF鸡胚病变率为85％以上，病毒滴度大于10⁶EID₅₀／ml。传染性法氏囊病毒在普通胚上繁殖困难，鸡胚死亡率非常低，不能用于疫苗生产；SPF鸡胚的病变率高（86.7％～97％），可利用率很高。总之，不同病毒在SPF鸡胚上的增殖都远远好于普通鸡胚，SPF鸡胚是生产禽用疫苗的优质原材料。更多还原

【Abstract】 More and more SPF chicken production industry are developed in recent years, but theproduction level is still comparatively low, and we have to do a lot of work to improve thesituation. In order to raise the research and production standard of SPF chicken, the studyhad been done on the following respects.1. The nutrition requirement of SPF chicken and the production of its feedThe SPF chicken’s feed was irradiated by different dose of ⁶⁰Co ray in order todetermine the minimum dose which is able to kill the bacteria in the feed completely. TheSPF chicken’s feed was sterilized by high pressure steam under different conditions, andthe optimum condition was determined. The nutrient component lose of sterilized feed bydifferent method was examined, then the compensation method and compensation standardwere analyzed. SPF chickens were feed with different sterilized feed and the capability ofgrowth and egg production of the SPF chicken were compared. Then SPF chicken’snutrition requirement was determined according to its special environmental condition andbiological characteristics.The result showed that SPF chicken’s feed irradiated by ⁶⁰Co ray of 10～15 kGy wasnot sterilized completely. But feed irradiated by ⁶⁰Co ray of 20 kGy was considered to besterilized completely, for there are no bacteria observed after bacteria culture for 30 days.So feed irradiated by ⁶⁰Co ray of 20 kGy was safety to feed SPF chicken.When feed are sterilized by high pressure steam, the size and shape of the feedpackage have a potent effect on sterilization effect. The feed shouldn’t be packed too big insize, it should be packed in thin shape less then 15cm. and there should be staked out someplace through feed packages for high pressure steam to be unobstructed. Packed in perfected size and shape, SPF chicken feed should be considered to be sterilized completelyby steam of 121℃for 20 min.When feed are sterilized by ⁶⁰Co ray, its vitamin b₁, vitamin b₁₂, biotin and folacinloss obviously, but the other nutrition components loss little. However when feed aresterilized by high pressure steam, the crude protein, most of the vitamins, lysine, arginine,and cystine of the feed loss obviously. And the loss of lysine and vitamins must be dulynoted and added in addition.When SPF chicken were fed with steam sterilized feed and ⁶⁰C0 ray sterilized feedrespectively, in which the loss nutrition components were make up, there were no decideddifference found in their capability of growth and egg production. But SPF chicken didnot like to take the feed sterilized by ⁶⁰Co ray, because it became too dusty and haddisagreeable odor obviously. There are no obvious harmful effects found on the capabilityof growth and egg production of the SPF chicken which take feed sterilized by highpressure steam.2. Research on SPF chicken’s genetic control and capability of growth andegg productionThe genetic variation of different lines of SPF chickens were analyzed by RAPDmethod, the capability of growth and egg production of different lines of SPF chicken wereexamined and the capability of growth were compared between different generations. Thenthe breeding methods of SPF chicken were discussed.The genetic variation of nuclear DNA among 3 lines of SPF chickens（F,M,B）and thecontract pure line （H） were analyzed by RAPD method with 30 individuals, 60 randomprimers were used and 13 primers were selected, 91 amplified fragments were observedand 64 of them were polymorphic fragments.The genetic similarity of different SPF chicken lines were calculated according to RAPD fingerprinting maps. The genetic similarity of line F, line M and line B were 0.673,0.755 and 0.903 respectively, however the genetic similarity of line H was 0.968. Geneticsimilarity of B is the highest but F and M were much lower.The cluster analysis showed the genetic distance between line F and line M was theshortest, they were classified into one group first, then line B and line H were classified,The result was in accord with the importing history of the breeds. The SPF chicken shouldbe selected by the method of group selection and mark-assistant-selection, with theselection target of laying capability and pathogen sensitivity, the underlying group may becomposed of existing SPF chicken flocks, and the inbreeding degree shouldn’t be high.The result of capability examination showed that three lines of SPF chicken havesimilar capability of early growth, but they have different capability of late growth,chicken of line B grow much quickly but line M and line F grow slowly. However theyhave similar body weight before lay eggs.Different lines of SPF hens had markedly different laying capability, Hens of line Mhad the best laying capability, Hens of line B and line F had similar laying capability whichis lower than line M. Different generations of the same line had similar growth capability,there was no obviously inbreeding decline observed.3. Research on multiplication of different virus in SPF chicken embryoSPF chicken embryo and common chicken embryo were inoculated with NDV（Lasota）,IBV（MA₅）, AEV（Van Roeke） and IBDV(B₈₇) respectively in order to study themultiplication rule of different virus. The results showed that titer of NDV antibody indifferent batch common hatching eggs were high and uneven, The AGP antibody positiveratio of IBDV in common hatching eggs was from 87.5%～100%. The NDV multiplicationamount inoculated in SPF chicken embryo was 5～20 times of its amount in commonchicken embryo. The lethality rate of SPF chicken embryo was up 65% after inoculated inNDV for 96h, however the lethality rate of common chicken embryo was under 5%. TheHA titer of NDV in SPF chicken embryo allantoic fluid was 2～2.5 higher than common chicken. When inoculated with IBV（MA₅）, the lethality rate of SPF chicken embryo washigher than common chicken embryo, and the EID₅₀ of SPF chicken embryo was over 10times of common chicken embryo. When inoculated with AEV（Van Roeke）, themorbidity of SPF chicken embryo was over 85%, however the morbidity of commonchicken embryo was only 4.5%～5%, and the EID₅₀ of SPF chicken embryo was over 10⁶EID₅₀/ml. IBDV can hardly grow in common chicken embryo, its lethality rate was verylow, so common chicken embryo was not good for IBD vaccine production. But whenSPF chicken embryo were applied, the lethality rate was so high that it is able to reach86.7%～97%, SPF chicken embryo had a high use factor to produce IBD vaccine.In a word, the multiplication of above virus in SPF chicken embryo are more betterthan its multiplication in common chicken embryo, SPF chicken embryo is the excellentmaterial for vaccine production.更多还原