饲料中黄麴毒素含量对1～5周龄台湾土鸡之影响

郑永祥⁽¹⁾    詹德芳⁽²⁾     吴继芳⁽³⁾

(1)国立宜兰农畜产科   

(2)国立中兴大学畜产科

(3)养猪科学研究所        

摘   要

        本试验目的在探讨饲料中黄麴毒素对1～5周龄台湾土鸡之影响。供试鸡只系采中兴大学育成之BxDL品系台湾土鸡，试验以完全逢机设计(CRD)，5处理，每处理2重覆(公母各半)，计240只。试验期间采笼饲，饲料、饮水自由摄取。试验饲粮以玉米－大豆粕为主之日粮，粗蛋白质为22.5％，代谢能为3100kcal／kg，再添加黄麴毒素Ｂ₁粗制品，使饲粮中黄麴毒素Ｂ₁含量为0、100、200、400及800ppb。

        藉由测定体增重、饲料利用效率、肝、脾、胰、肝重／体重及血中血球容积比。麸胺草醋酸转移鶤(GOT)、麸胺丙酮酸转移鶤(GPT)、总蛋白质(TP)、总胆红质(T-BIL)、血清球蛋白(GLO)、血清白蛋白(ALB)与肝脏组织切片。以评估台湾土鸡对黄麴毒素之敏感性。结果显示：1～5周龄台湾土鸡之各处理间的体增重、饲料采食量及饲料利用效率均无显着差异(P＞0.05)。台湾土鸡各处理组之脏器重量，以800ppb组之肝脏重显着较其他处理组为轻(P＜0.05)，但肝脏总脂质含量及肝重／体重比不受影响。血液生化分析方面，台湾土鸡各处理间GOT、PCV、T-BIL、ALB、GLO无显着差异，但800ppb处理组之GPT活性显着较其他处理组为高(P＜0.05)。肝脏组织病理切片方面，台湾土鸡各处理组肝脏均正常。综合本研究之成果，显示台湾土鸡对黄麴毒素之耐受性颇高，全期饲养鸡只均无死亡，且800ppb处理时生长性能之表现，仍未受影响。

(关键语：黄麴毒素，台湾土鸡，生长性能，血液生化值)

绪   言

        黄麴毒素为麴菌属中Aspergillus flavus及Aspergillus parasiticus所产生之二次代谢产物(Sargeant et al., 1961)具强裂之肝毒性与致癌性，对人畜健康威胁至钜。

        黄麴毒素对鸡只造成之毒害有：生长性能及饲料利用率变差(Smith and Hamilton, 1970)，脂肪肝(Hamilton and Carlich, 1971)，胰脏酵素活性降低(Osborne et al., 1981 a,b)，免疫功能减弱(Giamborne et al., 1978；Campbell et al., 1983；Virdi et al., 1989)，血凝延迟(Doerr and Hamilton, 1981)，白鸟症(Tyczkowski and Hamilton, 1987；Schaeffer et al., 1988 a,b)，低蛋白血症(Huff and Hamilton, 1975)，体内能量代谢扰乱(Raj and Venkitasubramanian, 1974；Rajion and Farrell, 1976)等诸症状，造成家畜业界潜在性之重大经济损失。

        国内有关黄麴毒素对家禽影响之研究仅见於沈等(1987,1988)及吴等(1990)，两者之试验分别使用白肉鸡与台湾土鸡。

        而台湾土鸡消费量逐年增加，其经济价值亦大为提高。故本试验之目的旨在探讨黄麴毒素含量对1～5周龄台湾土鸡之影响，藉由测定生长性能，血液生化及肝脏组织病变以了解台湾土鸡黄麴毒素之敏感性，并可供业界及未来相关研究之参考。

试验材料与方法

一、试验动物与饲养管理

        本试验系选用中兴大学畜牧系育成之BxDL品系台湾土鸡於出生日经性别鉴定与马立克疫苗接种，经平饲育雏一周後，鸡只个别秤重并挂上识别翼号，依体重逢机分配至各处理组中供试。鸡只并於三日龄时施以新城鸡瘟与传染性支气管炎(ND＋IB)疫苗接种，再於第二、四周龄时补强新城鸡瘟疫苗。

        本试验采完全逢机设计(CRD)，5种毒素含量处理，每处理二重覆，以每笼为一重覆之试验单位，含24只鸡，计10笼，共240只鸡。

        试验期间均饲以玉米－大豆粕为主之基础日粮，其粗蛋白质与代谢能分别为22.5％、3100kcal／kg。日粮中添加黄麴毒素Ｂ₁之粗制品，使饲粮中黄麴毒素含量分别为0，100，200，400及800ppb，日粮组成列於表1。

Table  1.   Composition    of     basal    diet.

大豆粉

鱼粉

大豆油

磷酸氢钙

石灰石粉

食盐

DL-甲硫胺酸

氯化胆硷

维生素预伴料^a

矿物质预伴料^b

球 粉-60

合        计

Soybean meal

Fish meal, 65%

Soybean oil

Dicalcium phosphate

Limestone, pulverized

Salt

DL-methionine

Choline chloride, 50%

Vitamin premix

Mineral premix

Coxistac-60

Total

30.00

7.00

2.00

1.90

0.60

0.30

0.12

0.10

0.10

0.10

0.05

100.00

粗蛋白质

代谢能

含硫胺基酸

钙

有效磷

Crude protein, %

ME, kcal/kg

Sulfur amino acid, %

Calcium, %

Available phosphorus

22.5

3100

0.89

1.07

0.50

b.每公斤饲粮添加(Provided the following contents per kg of diet):FeSO 200mg;ZnO 60mg;CuSO₄ 16mg;KI 0.4mg;Na₂ SeO₃ 0.08mg;ScSO₄ 1.2mg.

        试验於鸡只5周龄时，自各处理组中逢机取32只鸡，经采血後备用，并以毛细吸管(9μL，32mm)吸取血液，经血球容积比测定仪(PCV meter，Minizentrifuge，Compar M1100)测定血球容积比。鸡只牺牲後，取肝脏、胰脏、脾脏秤重并记录之。

        血液经远心分离(3000RPM，1610xg，15分钟)所得血清，贮存於－20℃冷冻柜中；待作分析之用。并取肝脏右叶尖端约5gm，两块，一供肝脏总脂质(total lipids)浓度测定，另一个定於10％之福马林溶液中供组织病理切片之用。

        试验采笼饲，试验期为1～5周龄，试验期间之饲料与饮水自由摄取，并於试验结束时，鸡只予以个别评量，同时记录饲料消耗量。

二、黄麴毒素之制备及定性定量分析

        黄麴毒素粗制品之产制系综合Shotwell et al. (1966)；Smith and Hamilton (1969)；West et al. (1973)之方法。

        粗制品经萃取与净化处理後，再经silica gel管柱进行清净化，以己烷；冰醋酸(9：1)流洗管柱，流洗液废弃。以40ml二氯甲烷：丙酮(4：1)冲提管柱并用100ml茶色浓缩瓶收集之，40℃水浴加热；减压旋转浓缩至乾，以丙酮洗至7ml茶色瓶，50℃加热下，通氮气吹乾。

        三氟乙酸(trifluoroacetic acid, TFA)衍生化处理：於茶色瓶加入200μl正己烷及50μl TFA，混合摇动30秒，於40℃烘箱加热10分钟，冷却後加1ml水；乙氰(9：1)，混合摇动30秒静置10分钟後以高性能液相层析法(HPLC)分析(Tarter et al., 1984)。

四、生化分析

        血清中麸胺草醋酸转移鶤(glutamate oxaloacetate transaminase, GOT)、麸胺丙酮酸转移鶤(glutamate pyruvate transaminase, GPT)酵素活性及总胆红质(total bilirubin, T-BIL)、总蛋白质(total protein, TP)、血清白蛋白(serum albumin, ABL)及球蛋白(globulin, GLO)浓度乃以全自动血液生化分析仪(Hitachi 7050 chemistry analyzer)辅以Wako公司生产之套组测定之。

五、统计分析

        试验所得数据均先经变方分析，後以邓肯氏新多变域测定法(Duncan's New Multiple Range Test)(Steel and Torrie, 1980)测定各处理间之差异显着性。

结果与讨论

        试验鸡只生长性能之测定包括体增重、饲料采食量及饲料利用效率(表2)。日粮中黄麴毒素含量对1～5周龄台湾土鸡各处理组之体增重、饲料采食量及饲料利用效率之影响，各处理间均无显着差异(P＜0.05)。黄麴毒素对生长性能之影响，包括生长及发育受阻，饲料利用效率变差(Smith and Hamilton, 1970；Huff et al., 1986；Doerr et al., 1983)，此乃由於高量或纯化AFB之添加所导致之毒害作用；Giambrone et al. (1985)指出800ppb会降低白肉鸡之生长及饲料利用效率。Bryden et al. (1980)指出鸡只品系与黄麴毒素间具交感作用；且黄麴毒素显着降低饲料转换率，但采食量无影响。吴等(1990)指出黄麴毒素400ppb对台湾土鸡，亦无生长抑制现象，认为台湾土鸡对黄麴毒素受性颇高。本研究中由台湾土鸡之结果显示对800ppb处理时，仍未有体重显着降低之现象。Donaldson et al. (1972)指出黄麴毒素抑制鸡只肝脏脂肪酸合成酵素及链加长鶤系统之作用。黄麴毒素症之典型病徵为脂肪肝，因肝中脂肪的蓄积乃为肠道吸收经门脉而来之脂肪酸蓄积，又因脂蛋白合成受黄麴毒素抑制，使脂肪自肝中移出少，而进入不断增加所致。(Tung et al., 1972)。

Table 2. Effect of aflatoxin in diet on the growth performance of Taiwan country chickens during 1~5 weeks of age.

Items

SE

Avg. weight gain, g

饲料消耗量

Feed intake, g

增重／饲料

gain／feed

840

0.491

841

0.498

838

0.492

826

0.498

837

0.486

7.00

0.02

Based on aflatoxin B₁。

Table 3. Effect of aflatoxin in diet on organ weight and hematocrit of Taiwan country chickens during 1~5 weeks of age.

Items

SE

Liver wt., g

肝重／体重

Liver wt.／Body

肝脏总脂质，g

Liver total lipid, g

脾脏重，g

Spleen wt., g

胰脏重，g

Pancrease wt., g

血球容积比，％

Hematocrit, %

2.20

1.48

0.78

1.11

26.67

2.19

1.53

0.69

1.13

25.96

2.25

2.05

0.82

1.01

26.68

2.27

1.47

0.84

0.95

26.43

2.10

1.97

0.72

1.09

24.87

3.67

12.37

3.58

3.60

4.12

Means of the same row with different superscript letters are significantly different (P<0.05)。

(表3)显示肝脏总脂质含量各处理组均无显着差异，故无脂肪肝情形。800ppb造成肝重显着降低应为初期黄麴毒素症，使鸡只生长受抑制，肝重亦呈同步减少，故肝重／体重比值时，各处理组呈无显着差异。此与Merkley et al. (1987)及(Huff et al. (1986)所指称初期黄麴毒素症，肝重降低之结果一致。

        一般用GOT与GPT活性以测知肝脏疾患之指标，日粮中黄麴毒素对台湾土鸡血液生化值之影响(表4)、GOT活性并不受黄麴毒素之影响，但GPT於800ppb处理组时活性则显着地较其他处理组为高，由於GPT活性具有肝脏专一性，此与肝脏重量(表3)可相印证。Avidar et al. (1986)指出肝脏受黄麴毒素影响使功能减弱，肝细胞之渗透性增加致GPT活性提高。且以血液生化值为指标，评估鸡只对黄麴毒素之敏感性较生长性能高(表2)为敏感。

Table 4. Effect of aflatoxin in diet on blood biochemistry of Taiwan country chickens during 1~5 weeks of age.

Items

SE

Glutamate oxaloacetate transaminase, U／I

麸胺丙酮酸转移，U／l

Glutamate pyruvate transaminase, U／l

总胆红质，mg／dl

Total bilirubin, mg／dl

总蛋白质，g／dl

Total protein, g／dl

血清白蛋白，g／dl

Serum albumin, g／dl

血清球蛋白，g／dl

Serum Globulin, g／dl

172.28^b

0.50

3.83

1.43

2.40

178.89^b

0.54

4.00

1.41

2.59

169.84^b

0.46

3.95

1.41

2.52

169.94^b

0.55

3.81

1.42

2.39

197.28^a

0.48

3.72

1.43

2.29

3.94

1.84

1.88

1.74

1.88

Means of the same row with different superscript letters are significantly different (P<0.05)。

        台湾土鸡之T-BIL，TP，ABL，GLO之浓度均未受黄麴毒素处理之影响，显见肝脏功能丧失仅属早期，故对上述之功能均能维持正常；但若攻毒时间再加长，则上述浓度均可能因肝脏脂肪蓄积之影响而使浓度降低。Lanza et al. (1980)及Tung et al. (1972)试验显示TP及ALB乃侦测白肉鸡对黄麴毒素最敏感之指标。但此一论点显然并不适用於台湾土鸡。

        黄麴毒素喂饲台湾土鸡1～5周龄後，经切片检查发现0～800ppb处理组之台湾土鸡均未见有关肝脏病变之发生(故此不刊示)。显示台湾土鸡之肝脏组织形态上未受破坏。林(1984)指出致癌物之致癌作用取决於代谢活化及代谢去毒两势力之消长。由於黄麴毒素为强力之致癌物质，属非极性的脂溶性物，为利於排出体外；遂行代谢活化(metabolic activation)，产物主要为黄麴毒素环氧化物(aflatoxin epoxide)，续由粒线体混合功能氧化鶤(mixed funcation oxidase)进行代谢去毒(metabolic detoxification)将环氧化物之致癌性消除。试验结果发现台湾土鸡将毒素行代谢去毒之速率大於代谢活化，致黄麴毒素对肝脏组织不致有重大的破坏，但因GPT活性的提高(表4)显然肝细胞表面己受毒素之损伤。

        由上述之结果归纳得知，血液生化及肝脏重量在做为黄麴毒素中毒指标上为最敏感。故若现场疑有鸡只黄麴毒素中毒时，测定血液中GOT、GPT活性可做一客观之诊断，配合饲料中黄麴毒素含量之检测，可做为预防黄麴毒素症发生之重要措施。

志   谢

        本试验之完成，蒙台湾养猪科学研究所陈美云及吴敏娟小姐之协助黄麴毒素分析与中兴大学兽医系陈三多老师之指导肝脏组织切片，和畜产系李渊白老师慨赠试验鸡只谨此志谢。

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The Effect of Dietary Aflatoxin Content on Taiwan Country

Chickens During 1-5 Weeks of Age

Yeong-Shiang Cheng¹, Der-Fang Jang², Jih-Fang Wu³

¹Department of Animal Science, National I-Lan Institute of Agriculture and Technology.

²Deparment of Animal Science, National Chung-Hsing University.

³Pig Research Institute of Taiwan.

Abstract

        The purpose of this experiment was to investigate the effect of aflatoxin on growth performance, blood biochemistry. organ weight, and liver histopathology of Taiwan country chickens (TCC) during 1-5 weeks of age.

        The chickens come from B×DL strain bred by the Department of Animal Science, National Chung-Hsing University. According to their body weight the chickens were alloted to 5 treatment groups randomly with two replicates Chickens were fed with cornsoybean meal basal diet containing 22.5％ crude protein, 3100 Kcal／kg metabolizable energy and supplemented with aflatoxin B₁ 0, 100, 200, 400, or 800 ppb.

        The parameters determined were weight gain、feed efficiency、liver weight、GOT、GPT、total bilirubin、total protein、albumin、globulin of serum、hematocrit、viceral weight、 and liver histopathology examination.

        The results showed that the body weight gain, feed consumption, and feed efficiency during 1-5 week of age were not significantly different among treatment groups. The liver weight of the 800 ppb treatment group decrease significantly (P＜0.05) compared to the other groups, but the liver total lipids content and liver weight／body weight ration were not affected by aflatoxin supplement.

        Serum GOT, hematocrict, total bilirubin, albumin, and globulin were not significantly different among dietary treatments, but only GPT activity of the 800 ppb treatment group was significantly increased (P＜0.05). Liver histopathology revealed no aflatoxicosis histopathology.

        In conclusion, TCC has high tolerance for aflatoxin B₁. The growth performance of TCC is not influenced by feeding diet containing aflatoxin B1 up to 800 ppb.

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(Key Words:Aflatoxins, Taiwan Country Chickens, Growth Performance, Blood Biochemistry)