飼料中黃麴毒素含量對1～5週齡台灣土雞之影響

鄭永祥⁽¹⁾    詹德芳⁽²⁾     吳繼芳⁽³⁾

(1)國立宜蘭農畜產科   

(2)國立中興大學畜產科

(3)養豬科學研究所        

摘   要

        本試驗目的在探討飼料中黃麴毒素對1～5週齡臺灣土雞之影響。供試雞隻係採中興大學育成之BxDL品系臺灣土雞，試驗以完全逢機設計(CRD)，5處理，每處理2重覆(公母各半)，計240隻。試驗期間採籠飼，飼料、飲水自由攝取。試驗飼糧以玉米－大豆粕為主之日糧，粗蛋白質為22.5％，代謝能為3100kcal／kg，再添加黃麴毒素Ｂ₁粗製品，使飼糧中黃麴毒素Ｂ₁含量為0、100、200、400及800ppb。

        藉由測定體增重、飼料利用效率、肝、脾、胰、肝重／體重及血中血球容積比。麩胺草醋酸轉移鋛(GOT)、麩胺丙酮酸轉移鋛(GPT)、總蛋白質(TP)、總膽紅質(T-BIL)、血清球蛋白(GLO)、血清白蛋白(ALB)與肝臟組織切片。以評估台灣土雞對黃麴毒素之敏感性。結果顯示：1～5週齡臺灣土雞之各處理間的體增重、飼料採食量及飼料利用效率均無顯著差異(P＞0.05)。臺灣土雞各處理組之臟器重量，以800ppb組之肝臟重顯著較其他處理組為輕(P＜0.05)，但肝臟總脂質含量及肝重／體重比不受影響。血液生化分析方面，臺灣土雞各處理間GOT、PCV、T-BIL、ALB、GLO無顯著差異，但800ppb處理組之GPT活性顯著較其他處理組為高(P＜0.05)。肝臟組織病理切片方面，臺灣土雞各處理組肝臟均正常。綜合本研究之成果，顯示臺灣土雞對黃麴毒素之耐受性頗高，全期飼養雞隻均無死亡，且800ppb處理時生長性能之表現，仍未受影響。

(關鍵語：黃麴毒素，臺灣土雞，生長性能，血液生化值)

緒   言

        黃麴毒素為麴菌屬中Aspergillus flavus及Aspergillus parasiticus所產生之二次代謝產物(Sargeant et al., 1961)具強裂之肝毒性與致癌性，對人畜健康威脅至鉅。

        黃麴毒素對雞隻造成之毒害有：生長性能及飼料利用率變差(Smith and Hamilton, 1970)，脂肪肝(Hamilton and Carlich, 1971)，胰臟酵素活性降低(Osborne et al., 1981 a,b)，免疫功能減弱(Giamborne et al., 1978；Campbell et al., 1983；Virdi et al., 1989)，血凝延遲(Doerr and Hamilton, 1981)，白鳥症(Tyczkowski and Hamilton, 1987；Schaeffer et al., 1988 a,b)，低蛋白血症(Huff and Hamilton, 1975)，體內能量代謝擾亂(Raj and Venkitasubramanian, 1974；Rajion and Farrell, 1976)等諸症狀，造成家畜業界潛在性之重大經濟損失。

        國內有關黃麴毒素對家禽影響之研究僅見於沈等(1987,1988)及吳等(1990)，兩者之試驗分別使用白肉雞與台灣土雞。

        而台灣土雞消費量逐年增加，其經濟價值亦大為提高。故本試驗之目的旨在探討黃麴毒素含量對1～5週齡台灣土雞之影響，藉由測定生長性能，血液生化及肝臟組織病變以瞭解台灣土雞黃麴毒素之敏感性，並可供業界及未來相關研究之參考。

試驗材料與方法

一、試驗動物與飼養管理

        本試驗係選用中興大學畜牧系育成之BxDL品系臺灣土雞於出生日經性別鑑定與馬立克疫苗接種，經平飼育雛一週後，雞隻個別秤重並掛上識別翼號，依體重逢機分配至各處理組中供試。雞隻並於三日齡時施以新城雞瘟與傳染性支氣管炎(ND＋IB)疫苗接種，再於第二、四週齡時補強新城雞瘟疫苗。

        本試驗採完全逢機設計(CRD)，5種毒素含量處理，每處理二重覆，以每籠為一重覆之試驗單位，含24隻雞，計10籠，共240隻雞。

        試驗期間均飼以玉米－大豆粕為主之基礎日糧，其粗蛋白質與代謝能分別為22.5％、3100kcal／kg。日糧中添加黃麴毒素Ｂ₁之粗製品，使飼糧中黃麴毒素含量分別為0，100，200，400及800ppb，日糧組成列於表1。

Table  1.   Composition    of     basal    diet.

大豆粉

魚粉

大豆油

磷酸氫鈣

石灰石粉

食鹽

DL-甲硫胺酸

氯化膽鹼

維生素預伴料^a

礦物質預伴料^b

球蟲粉-60

合        計

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

粗蛋白質

代謝能

含硫胺基酸

鈣

有效磷

Crude protein, %

ME, kcal/kg

Sulfur amino acid, %

Calcium, %

Available phosphorus

22.5

3100

0.89

1.07

0.50

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

        試驗於雞隻5週齡時，自各處理組中逢機取32隻雞，經採血後備用，並以毛細吸管(9μL，32mm)吸取血液，經血球容積比測定儀(PCV meter，Minizentrifuge，Compar M1100)測定血球容積比。雞隻犧牲後，取肝臟、胰臟、脾臟秤重並記錄之。

        血液經遠心分離(3000RPM，1610xg，15分鐘)所得血清，貯存於－20℃冷凍櫃中；待作分析之用。並取肝臟右葉尖端約5gm，兩塊，一供肝臟總脂質(total lipids)濃度測定，另一個定於10％之福馬林溶液中供組織病理切片之用。

        試驗採籠飼，試驗期為1～5週齡，試驗期間之飼料與飲水自由攝取，並於試驗結束時，雞隻予以個別評量，同時記錄飼料消耗量。

二、黃麴毒素之製備及定性定量分析

        黃麴毒素粗製品之產製係綜合Shotwell et al. (1966)；Smith and Hamilton (1969)；West et al. (1973)之方法。

        粗製品經萃取與淨化處理後，再經silica gel管柱進行清淨化，以己烷；冰醋酸(9：1)流洗管柱，流洗液廢棄。以40ml二氯甲烷：丙酮(4：1)沖提管柱並用100ml茶色濃縮瓶收集之，40℃水浴加熱；減壓旋轉濃縮至乾，以丙酮洗至7ml茶色瓶，50℃加熱下，通氮氣吹乾。

        三氟乙酸(trifluoroacetic acid, TFA)衍生化處理：於茶色瓶加入200μl正己烷及50μl TFA，混合搖動30秒，於40℃烘箱加熱10分鐘，冷卻後加1ml水；乙氰(9：1)，混合搖動30秒靜置10分鐘後以高性能液相層析法(HPLC)分析(Tarter et al., 1984)。

四、生化分析

        血清中麩胺草醋酸轉移鋛(glutamate oxaloacetate transaminase, GOT)、麩胺丙酮酸轉移鋛(glutamate pyruvate transaminase, GPT)酵素活性及總膽紅質(total bilirubin, T-BIL)、總蛋白質(total protein, TP)、血清白蛋白(serum albumin, ABL)及球蛋白(globulin, GLO)濃度乃以全自動血液生化分析儀(Hitachi 7050 chemistry analyzer)輔以Wako公司生產之套組測定之。

五、統計分析

        試驗所得數據均先經變方分析，後以鄧肯氏新多變域測定法(Duncan's New Multiple Range Test)(Steel and Torrie, 1980)測定各處理間之差異顯著性。

結果與討論

        試驗雞隻生長性能之測定包括體增重、飼料採食量及飼料利用效率(表2)。日糧中黃麴毒素含量對1～5週齡台灣土雞各處理組之體增重、飼料採食量及飼料利用效率之影響，各處理間均無顯著差異(P＜0.05)。黃麴毒素對生長性能之影響，包括生長及發育受阻，飼料利用效率變差(Smith and Hamilton, 1970；Huff et al., 1986；Doerr et al., 1983)，此乃由於高量或純化AFB之添加所導致之毒害作用；Giambrone et al. (1985)指出800ppb會降低白肉雞之生長及飼料利用效率。Bryden et al. (1980)指出雞隻品系與黃麴毒素間具交感作用；且黃麴毒素顯著降低飼料轉換率，但採食量無影響。吳等(1990)指出黃麴毒素400ppb對台灣土雞，亦無生長抑制現象，認為台灣土雞對黃麴毒素受性頗高。本研究中由台灣土雞之結果顯示對800ppb處理時，仍未有體重顯著降低之現象。Donaldson et al. (1972)指出黃麴毒素抑制雞隻肝臟脂肪酸合成酵素及鏈加長鋛系統之作用。黃麴毒素症之典型病徵為脂肪肝，因肝中脂肪的蓄積乃為腸道吸收經門脈而來之脂肪酸蓄積，又因脂蛋白合成受黃麴毒素抑制，使脂肪自肝中移出少，而進入不斷增加所致。(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

飼料消耗量

Feed intake, g

增重／飼料

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／體重

Liver wt.／Body

肝臟總脂質，g

Liver total lipid, g

脾臟重，g

Spleen wt., g

胰臟重，g

Pancrease wt., g

血球容積比，％

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)顯示肝臟總脂質含量各處理組均無顯著差異，故無脂肪肝情形。800ppb造成肝重顯著降低應為初期黃麴毒素症，使雞隻生長受抑制，肝重亦呈同步減少，故肝重／體重比值時，各處理組呈無顯著差異。此與Merkley et al. (1987)及(Huff et al. (1986)所指稱初期黃麴毒素症，肝重降低之結果一致。

        一般用GOT與GPT活性以測知肝臟疾患之指標，日糧中黃麴毒素對臺灣土雞血液生化值之影響(表4)、GOT活性並不受黃麴毒素之影響，但GPT於800ppb處理組時活性則顯著地較其他處理組為高，由於GPT活性具有肝臟專一性，此與肝臟重量(表3)可相印證。Avidar et al. (1986)指出肝臟受黃麴毒素影響使功能減弱，肝細胞之滲透性增加致GPT活性提高。且以血液生化值為指標，評估雞隻對黃麴毒素之敏感性較生長性能高(表2)為敏感。

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

麩胺丙酮酸轉移，U／l

Glutamate pyruvate transaminase, U／l

總膽紅質，mg／dl

Total bilirubin, mg／dl

總蛋白質，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)。

        台灣土雞之T-BIL，TP，ABL，GLO之濃度均未受黃麴毒素處理之影響，顯見肝臟功能喪失僅屬早期，故對上述之功能均能維持正常；但若攻毒時間再加長，則上述濃度均可能因肝臟脂肪蓄積之影響而使濃度降低。Lanza et al. (1980)及Tung et al. (1972)試驗顯示TP及ALB乃偵測白肉雞對黃麴毒素最敏感之指標。但此一論點顯然並不適用於台灣土雞。

        黃麴毒素餵飼台灣土雞1～5週齡後，經切片檢查發現0～800ppb處理組之台灣土雞均未見有關肝臟病變之發生(故此不刊示)。顯示台灣土雞之肝臟組織形態上未受破壞。林(1984)指出致癌物之致癌作用取決於代謝活化及代謝去毒兩勢力之消長。由於黃麴毒素為強力之致癌物質，屬非極性的脂溶性物，為利於排出體外；遂行代謝活化(metabolic activation)，產物主要為黃麴毒素環氧化物(aflatoxin epoxide)，續由粒線體混合功能氧化鋛(mixed funcation oxidase)進行代謝去毒(metabolic detoxification)將環氧化物之致癌性消除。試驗結果發現臺灣土雞將毒素行代謝去毒之速率大於代謝活化，致黃麴毒素對肝臟組織不致有重大的破壞，但因GPT活性的提高(表4)顯然肝細胞表面己受毒素之損傷。

        由上述之結果歸納得知，血液生化及肝臟重量在做為黃麴毒素中毒指標上為最敏感。故若現場疑有雞隻黃麴毒素中毒時，測定血液中GOT、GPT活性可做一客觀之診斷，配合飼料中黃麴毒素含量之檢測，可做為預防黃麴毒素症發生之重要措施。

誌   謝

        本試驗之完成，蒙台灣養豬科學研究所陳美雲及吳敏娟小姐之協助黃麴毒素分析與中興大學獸醫系陳三多老師之指導肝臟組織切片，和畜產系李淵白老師慨贈試驗雞隻謹此誌謝。

參考文獻

        1.沈添富、嚴家清、郭忠政，1987。飼料中含低濃度黃麴毒素對童子雞的影響。中畜會誌。16 (3-4)：63-71頁。

        2.沈添富、嚴家清、郭忠政，1988。飼料中含低濃度黃麴毒素對土番鴨之影響。中華農學會報。142:67-77頁。

        3.吳學華、嚴家清、郭忠政、沈添富，1990。飼料中含低量黃麴毒素對小型有色肉雞的影響。中華農學會報。150：66-73頁。

        4.林仁混，1984。化學致癌作用之分子機制。國科會生物科學研究中心生物技術專題演講論文專刊。11:181-196頁。

        5.Avider, Y., E. Bogin and S. Soback, 1986. Fatty liver syndrome in farm animalsbiochemical and pharmacological aspects. lsr. J. Vet. Med. 42:318-323.

        6.Bryden, W. L., R. B. Cumming and A. B. Lloyd, 1980. Sex and Strain responses to aflatoxin B₁ in the chicken. Avain Pathol. 9:539-550.

        7.Campbell, M. E., J. D. May, W. E. Huff and J. A. Doerr, 1983. Evaluation of immunity of young brolier chickens during simultaneous aflatoxicosis and ochratoxicosis. Poult. Sci. 62:2138-2144.

        8.Doerr, J. A. and P. B. Hamilton, 1981. Aflatoxicosis and intrinsic coagulation function in broiler chickens, Poult, Sci. 60;1406-1411.

        9.Doerr, J. A., W. E. Huff, C. J. Wabeck, G. W. Chaloupka, J. D. May and J. W. Merkley, 1983. Effect of low level chronic aflatoxicosis in broiler chickens, Poult. Sci. 62: 1971-1977.

        10.Donaldson, W. E., H. T. Tung and P. B. Hamilton, 1972. Depression of fatty acid synthesis in chick liver (Gallus Domesticus) by aflatoxin. Comp. Biochem. Physiol. 41:843-847.

        11.Giambrone, J. J., D. L. Ewert, R. D. Wyatt, C. S. Eidson, 1978. Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken. Am. J. Vet. Res. 39: 305-307.

        12.Giamborne, J. J., U. L. Diener, N. D. Davis, V. S. Panangala, F. J. Hoerr, 1985. Effect of aflatoxin on young turkeys and broiler chickens. Poult. Sci. 64:1678-1684.

        13.Hamilton, P. B. and J. D. Garlich, 1971. Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poult. Sci. 50:800-804.

        14.Huff, W. E., R. D. Wyatt and P. B. Hamilton, 1975. Effects of dietary aflatoxin on certain egg yolk parameters. Poult. Sci. 54: 2014-2018.

        15.Huff, W. E., L. F. Kubena, R. B. Harvey, D. E. Corrier and H. H. Mollenhauer, 1986. Progression of aflatoxicosis in broiler chickens. Poult. Sci. 65: 1891-1899.

        16.Lanza, G. M., K. W. Washburn, R. D. Wyatt, 1980. Strain variation in hematological response of broilers to dietary aflatoxin. Poult. Sci. 59: 2686-2691.

        17.Merkley, J. W., R. J. Maxwell, J. G. Phillips and W. E. Huff, 1987. Hepatic fatty acid profiles in aflatoxin exposed broiler chickens. Poult. Sci. 66: 59-67.

        18.Osborne, D. J. and P. B. Hamilton, 1981a. Steatorrhea during aflatoxicosis in chickens. Poult. Sci. 60: 1398-1402.

        19.Osborne, D. J. and P. B. Hamilton, 1981b. Decreased pancreatic digestive enzymes during aflatoxicosis. Poult. Sci. 60: 1818-1821.

        20.Raj, H. G. and T. A. Venkitasubramanian, 1974. Carbohydrate metabolism in aflatoxin B₁ toxicity. Envirom. Physiol. Biochem. 4: 181-187.

        21.Rajion, M. A. and D. J. Farrell, 1976. Energy and nitrogen metabolism of diseased chicks: aflatoxicosis. Br. Poult. Sci. 17:79-92.

        22.Sargeant, K., A. Sheridan, J. O. Kelly and R. B. A. Carnaghan, 1961. Toxicity associated with certain samples of ground nuts. Nature. 192: 1096-1097.

        23.Schaeffer, J. L., J. K. Tyczkowski, J. E. Rivere and P. B. Hamiltion, 1988a. Aflatoxin-impaired ability during restoration in young chickens. Poult. Sci. 67: 619-625.

        24.Schaeffer, J. L., J. K. Tyczkowski, J. E. Rivere and P. B. Hamiltion, 1988b. Depletion of oxycarotenoid pigments in chickens and failure of aflatoxin to alter it. Poult. Sci. 67:1080-1088.

        25.Shotwell, O. L., C. W. Hasseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol, 14:425-428.

        26.Smith, J. W. and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask culture. Appl. Microbiol. 17:317-321.

        27.Smith, J. W. and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poult. Sci. 49:207-215.

        28.Steel, R. G. D., and J. H. Torrie, 1960. Principles and procedures of statistics. p.107-109. MaGraw-Hill Book Comp. lnc. N. Y.

        29.Tarter, E. J., J. P. Hanchay, and P. M. Scott, 1984. Improved liquid chromatographic method for determination of aflatoxins in peanut buter and other commodities. J. Assoc. Off. Anal. Chem. 67: 597-600.

        30.Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22: 97-104.

        31.Tyczkowski, J. K. and P. B. Hamilton, 1987. Altered metabolism of carotenoids during aflatoxicosis in young chickens. Poult. Sci. 66: 1184-1188.

        32.Virdi, J. S., R. P. Tiwari, M. Saxena, V. Khanna, G. Singh, S. S. Saini and D. V. Vadehra, 1989. Effects of aflatoxin on the immune system of the chick. J. Appl. Toxicol. 9: 271-275.

        33.West, S., R. D. Wyatt and P. B. Hamilton, 1973. Improved yield of aflatoxinby incremental increases of temperature Appl. Microbiol. 25: 1081-1019.

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)