萬祖德　李小勤　高啟平　帥　柯　陳佳楠　徐懷兵　冷向軍,3,4,5*

(1.上海海洋大學水產(chǎn)與生命學院，上海201306；2.通威股份有限公司，成都610041；

3.上海海洋大學農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點實驗室，上海201306；4.上海市水產(chǎn)養(yǎng)殖

工程技術(shù)研究中心，上海201306；5.水產(chǎn)動物遺傳育種中心

上海市協(xié)同創(chuàng)新中心，上海201306)

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萬祖德1李小勤1高啟平2帥柯2陳佳楠1徐懷兵1冷向軍1,3,4,5*

(1.上海海洋大學水產(chǎn)與生命學院，上海201306；2.通威股份有限公司，成都610041；

3.上海海洋大學農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點實驗室，上海201306；4.上海市水產(chǎn)養(yǎng)殖

工程技術(shù)研究中心，上海201306；5.水產(chǎn)動物遺傳育種中心

上海市協(xié)同創(chuàng)新中心，上海201306)

摘要：在實用飼料(含銅11.1 mg/kg)中分別添加0(對照)、5、10、20和40 mg/kg銅[以五水硫酸銅(CuSO4·5H2O)形式]，制成5種試驗飼料，投喂平均體重為(98.1±0.5) g的斑點叉尾42 d，研究實用飼料中補充銅對斑點叉尾生長和體色的影響。每種飼料設(shè)3個重復，每個重復放養(yǎng)20尾魚。結(jié)果表明：與對照組相比，飼料中添加10 mg/kg銅顯著提高了魚體的增重率(P<0.05)，顯著降低了飼料系數(shù)(P<0.05)；飼料銅添加量進一步增加到40 mg/kg，魚體的增重率則較添加量為10 mg/kg時顯著降低(P<0.05)，同時飼料系數(shù)顯著升高(P<0.05)。肝臟和骨骼銅含量隨著飼料中銅添加量的增加而上升，其中20、40 mg/kg銅添加組的肝臟銅含量顯著高于對照組和5 mg/kg銅添加組(P<0.05)，40 mg/kg銅添加組的骨骼銅含量也顯著高于對照組(P<0.05)，而肌肉銅含量保持基本不變(P>0.05)。飼料中添加0～40 mg/kg銅對斑點叉尾背部皮膚、肌肉色度值、總?cè)~黃素含量及背部皮膚酪氨酸酶活性均未產(chǎn)生顯著影響(P>0.05)。各組血清天門冬氨酸氨基轉(zhuǎn)移酶(AST)、丙氨酸氨基轉(zhuǎn)移酶(ALT)活性和總膽紅素(T-Bil)含量以及肌肉水分、粗蛋白質(zhì)、粗脂肪和粗灰分含量無顯著差異(P>0.05)。10 mg/kg銅添加組具有最高的血清銅鋅-超氧化物歧化酶(Cu，Zn-SOD)活性，顯著高于其他各組(P<0.05)，而其他各組間則無顯著差異(P>0.05)。綜上，在本試驗條件下，斑點叉尾實用飼料中銅的添加量建議為10 mg/kg(飼料銅含量實測值為20.2 mg/kg)。

關(guān)鍵詞：斑點叉尾；銅；生長；體色

1材料與方法

1.1試驗設(shè)計和試驗飼料

以魚粉、豆粕、菜籽粕、棉籽粕、次粉、麥麩等為主要原料配制粗蛋白質(zhì)水平為31%的實用型基礎(chǔ)飼料，在基礎(chǔ)飼料中分別添加0(對照)、5、10、20和40 mg/kg銅[以五水硫酸銅(CuSO4·5H2O)形式]，共制成5種試驗飼料，試驗飼料銅含量實測值分別為11.1、16.0、20.2、28.9和44.2 mg/kg。飼料原料經(jīng)粉碎過40目篩，充分混合混勻后，用膨化機(SLP-45，中國水產(chǎn)科學研究院漁業(yè)機械儀器研究所研制)制粒[制粒溫度為(110±5) ℃]形成直徑2 mm的浮性膨化飼料，晾干后置于4 ℃冰箱內(nèi)保存?zhèn)溆??；A(chǔ)飼料組成及營養(yǎng)水平見表1。

1.2試驗用魚及飼養(yǎng)管理

表1　基礎(chǔ)飼料組成及營養(yǎng)水平(風干基礎(chǔ))

1)配方中的飼料原料購自上海農(nóng)好飼料有限公司，其中魚粉(秘魯)、豆粕、菜籽粕和棉籽粕的粗蛋白質(zhì)含量分別為67.6%、46.5%、35.6%和45.0%。The ingredients in formula were purchased from theShanghaiNonghaoFeed Co., Ltd., and the crude protein content of fish meal (Peru), soybean meal, rapeseed meal and cottonseed meal was 67.6%, 46.5%, 35.6% and 45.0%, respectively.

2)維生素預混料為每千克飼料提供The vitamin premix provided the following per kg of the diet：VA 6 000 IU，VD 2 000 IU，VE 50 IU，VK 5 mg，VB115 mg，VB215 mg，VB330 mg，VB535 mg，VB66 mg，生物素 biotin 0.2 mg，葉酸 folic acid 3 mg，VB120.03 mg。

3)礦物質(zhì)預混料為每千克飼料提供The mineral premix provided the following per kg of the diet：Ca(IO3)20.04 g，CoCl2·6H2O 0.01 g，F(xiàn)eSO4·H2O 0.446 g，ZnSO4·H2O 0.232 g，MnSO4·H2O 0.063 g，Na2SeO3·5H2O 0.01 g，MgSO4·7H2O 0.645 g。

1.3測定指標和方法

1.3.1生長與形體指標計算

養(yǎng)殖試驗結(jié)束后，魚體饑餓24 h，統(tǒng)計每口網(wǎng)箱內(nèi)的魚尾數(shù)并稱總重，計算增重率(WGR)、飼料系數(shù)(FCR)、存活率(SR)；每個網(wǎng)箱隨機取出3尾魚，測量魚體長和體重，解剖后稱量其內(nèi)臟重、肝臟重，計算肝體指數(shù)(HSI)、臟體指數(shù)(VSI)及肥滿度(CF)。

增重率(%)=100×[(終末體重(g)-

初始體重(g)]/初始體重(g)；

飼料系數(shù)=總投喂量(g)/[終末體重(g)-

初始體重(g)]；

存活率(%)=100×終末魚總數(shù)(尾)/

初始魚總數(shù)(尾)；

肝體指數(shù)(%)=100×肝臟重(g)/體重(g)；

臟體指數(shù)(%)=100×內(nèi)臟重(g)/體重(g)；

肥滿度(g/cm3)=100×體重(g)/

體長(cm)3。

1.3.2飼料、肌肉常規(guī)成分分析

養(yǎng)殖試驗結(jié)束后，每口網(wǎng)箱隨機取3尾魚，取背鰭下方側(cè)線以上的背部肌肉，置于-20 ℃冰箱中凍存，用于肌肉常規(guī)成分分析。飼料、肌肉樣品水分含量的測定采用105 ℃烘干失水法(GB/T 5009.3—2003)；粗蛋白質(zhì)含量的測定采用凱氏定氮法(GB/T 5009.5—2003)；粗脂肪含量的測定參照Folch等[12]的氯仿甲醇抽提法；粗灰分含量測定采用馬福爐灰化法(GB/T 5009.4—2003)。

1.3.3皮膚、肌肉色度值測定

養(yǎng)殖試驗結(jié)束后，隨機從每口網(wǎng)箱取3尾魚，用吸水紙將魚體表面水分吸干，將色差計(WSC-S型色差計，上海精密科學儀器有限公司物理光學儀器廠)的探頭緊貼魚體側(cè)線以上的背部皮膚，測量背部皮膚色度值，之后剝?nèi)テつw，將探頭緊貼在側(cè)線以上的背部肌肉上，測量肌肉色度值，記錄亮度(L*)、紅綠度(a*)、黃藍度(b*)值。

1.3.4皮膚、肌肉總?cè)~黃素含量測定

上述皮膚、肌肉測定色度值后，各取2～3 g，參考Quackenbush等[13]的分析法進行總?cè)~黃素含量的測定，具體方法如下：將樣品剪碎，裝入25 mL棕色容量瓶中，加入7.5 mL提取液(正己烷∶丙酮∶無水乙醇∶甲苯=10∶7∶6∶7)，塞上塞子旋轉(zhuǎn)振搖1 min，加入1 mL 40%氫氧化鉀-甲醇溶液，旋轉(zhuǎn)搖勻1 min，于55.5 ℃水浴加熱20 min(注意冷卻容量瓶頸部以防止溶劑損失)，冷卻樣品，放置暗處1 h，加入7.5 mL正己烷，旋轉(zhuǎn)振搖1 min，以10%硫酸鈉溶液定容至25 mL，猛烈振搖1 min，于暗處放置1 h后，將上層液用分光光度計于474 nm處測定其吸光度值，根據(jù)標準曲線計算其總?cè)~黃素含量。

1.3.5背部皮膚酪氨酸酶活性測定

取1 g左右背部皮膚，按1∶5比例用67 mmol/L pH 6.8的磷酸緩沖液勻漿(冰水浴)，在4 ℃下離心25 min (8 000 r/min)，取上清液參照丁玉庭等[14]的方法測定其中酪氨酸酶活性，具體方法如下：取3 mg/mLL-多巴0.5 mL，加入28 ℃預熱的2 mL上述上清液，總反應(yīng)體積為2.5 mL，混合后立即室溫下用分光光度計于475 nm處測定其吸光度值(OD0)，10 min后再次測定吸光度值(OD10)。

酪氨酸酶活性按照下述公式計算：

式中：△OD475為2次測定吸光度值的差值，即△OD475=OD10-OD0；V為樣品體積；T為2次測定間隔時間。

1.3.6血清生化指標測定

養(yǎng)殖試驗結(jié)束后，每口網(wǎng)箱隨機取3尾魚，尾靜脈取血，離心(3 000 r/min，15 min)，取血清于-80 ℃冷凍保存。分別測定血清天門冬氨酸氨基轉(zhuǎn)移酶(AST)、丙氨酸氨基轉(zhuǎn)移酶(ALT)活性及總膽紅素(T-Bil)含量，均采用邁瑞B(yǎng)S-200全自動生化分析儀測定。血清Cu,Zn-SOD活性采用南京建成生物工程研究所提供的試劑盒進行測定。

1.3.7組織(肌肉、肝臟、脊椎骨)銅含量測定

組織銅含量的測定參考張韻華[15]的方法，采用原子吸收法，具體方法如下：準確稱取樣品1 g，置于坩堝中，先在電爐上炭化至不再冒煙為止，然后移入550 ℃的馬福爐中灰化6 h；取出，冷卻至室溫，加入硝酸與高氯酸的混合酸(4∶1)10 mL；放置5 h以上；然后在電爐上小心加熱，使灰化樣品溶解(黑色炭粒消失)，直到溶液接近蒸干為止；用1%的鹽酸溶液溶解析出的晶體，再轉(zhuǎn)移到25 mL的容量瓶定容、待測。以不加樣品，用同樣方法獲得的試液作為空白對照。采用TAS-900原子吸收分光光度計(北京普析通用公司)測定銅含量。

1.4數(shù)據(jù)分析

試驗結(jié)果采用SPSS 17.0統(tǒng)計軟件進行處理分析，數(shù)據(jù)以平均值±標準差(mean±SD)表示，采用單因素方差分析(one-way ANOVA)，用Duncan氏法進行多重比較，P<0.05為差異顯著。

2結(jié)果

2.1生長與形體指標

表2　飼料中銅添加量對斑點叉尾生長與形體指標的影響

Table 2　Effects of dietary copper supplemental level on growth and morphological in

dices of channel catfish

表2　飼料中銅添加量對斑點叉尾生長與形體指標的影響

項目Items銅添加量Coppersupplementallevel/(mg/kg)05102040初始體重IBW/g98.0±0.898.1±0.398.6±1.298.1±0.497.8±0.6終末體重FBW/g217.7±4.7a218.5±2.1a229.8±4.2b223.3±3.9ab210.3±9.0a增重率WGR/%119.5±6.3a122.5±3.1a133.2±4.8b128.3±4.8ab115.0±8.3a飼料系數(shù)FCR1.37±0.06b1.33±0.03b1.22±0.04a1.31±0.04b1.43±0.11b存活率SR/%100100100100100肝體指數(shù)HSI/%1.82±0.171.89±0.211.96±0.221.93±0.151.76±0.18臟體指數(shù)VSI/%6.01±0.546.10±0.586.22±0.486.24±0.496.20±0.59肥滿度CF/%1.47±0.051.50±0.091.51±0.101.52±0.131.42±0.10

同行數(shù)據(jù)肩標無字母或相同字母表示差異不顯著(P>0.05)，不同小寫字母表示差異顯著(P<0.05)。下表同。

In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.2肌肉常規(guī)成分

由表3可見，肌肉水分、粗蛋白質(zhì)、粗脂肪及粗灰分含量在各組間無顯著差異(P>0.05)。

2.3背部皮膚、肌肉色度值和總?cè)~黃素含量及背部皮膚酪氨酸酶活性

由表4可見，各組背部皮膚、肌肉的色度值和總?cè)~黃素含量及背部皮膚酪氨酸酶活性均無顯著差異(P>0.05)。

2.4血清生化指標

由表5可見，各組血清AST、ALT活性和T-Bil含量無顯著差異(P>0.05)；10 mg/kg銅添加組具有最高的血清Cu，Zn-SOD活性，與其他各組差異顯著(P<0.05)，而其他組間則無顯著差異(P>0.05)。

表3　飼料銅添加量對斑點叉尾肌肉常規(guī)成分的影響(鮮重基礎(chǔ))

Table 3　Effects of dietary copper supplemental level on muscle conventional

components of channel catfish (wet weight basis)　%

表3　飼料銅添加量對斑點叉尾肌肉常規(guī)成分的影響(鮮重基礎(chǔ))

項目Items銅添加量Coppersupplementallevel/(mg/kg)05102040水分Moisture76.74±0.9177.10±0.6776.68±0.8176.27±0.9576.84±0.93粗蛋白質(zhì)CP18.62±0.3418.98±0.4118.70±0.3518.61±0.5118.64±0.41粗脂肪EE2.53±0.392.62±0.222.79±0.272.63±0.282.53±0.25粗灰分Ash1.21±0.041.22±0.011.21±0.041.21±0.011.20±0.04

表4　飼料中銅添加量對斑點叉尾背部皮膚、肌肉色度值和總?cè)~黃素含量及背部皮膚酪氨酸酶活性的影響

Table 4　Effects of dietary copper supplemental level on chroma values and total xanthophylls

content of dorsal skin, muscle and tyrosinase activity of dorsal skin of channel catfish

表4　飼料中銅添加量對斑點叉尾背部皮膚、肌肉色度值和總?cè)~黃素含量及背部皮膚酪氨酸酶活性的影響

項目Items銅添加量Coppersupplementallevel/(mg/kg)05102040背部皮膚Dorsalskin亮度L*20.48±2.6019.35±1.9421.38±2.2621.28±1.9921.43±1.66紅綠度a*3.53±1.203.10±1.312.67±0.712.50±0.832.11±0.63黃藍度b*2.48±0.732.02±1.092.72±1.212.44±0.502.02±0.91總?cè)~黃素Totalxanthophylls2.75±0.052.81±0.573.02±0.982.86±0.462.86±0.18酪氨酸酶Tyrosinase0.99±0.271.10±0.081.14±0.061.03±0.131.14±0.10背部肌肉Dorsalmuscle亮度L*50.76±2.6850.72±2.8151.68±2.5951.61±2.7349.37±2.44紅綠度a*3.59±0.894.54±0.964.22±0.933.96±1.013.42±1.13黃藍度b*3.50±0.994.08±1.484.49±0.683.91±1.164.13±0.98總?cè)~黃素Totalxanthophylls/(mg/kg)1.06±0.261.18±0.331.18±0.351.24±0.111.24±0.28

表5　飼料中銅添加量對斑點叉尾血清生化指標的影響

Table 5　Effects of dietary copper supplemental level on serum biochemical indices of channel catfish

表5　飼料中銅添加量對斑點叉尾血清生化指標的影響

項目Items銅添加量Coppersupplementallevel/(mg/kg)05102040天門冬氨酸氨基轉(zhuǎn)移酶AST/(U/L)24.85±3.5624.25±2.8525.60±3.2624.72±3.9724.06±1.62丙氨酸氨基轉(zhuǎn)移酶ALT/(U/L)11.07±1.8510.98±2.3311.20±2.2510.89±1.9710.38±0.49總膽紅素T-Bil/(μmol/L)2.54±0.422.43±0.532.29±0.362.20±0.642.43±0.51銅鋅-超氧化物歧化酶Cu,Zn-SOD/(U/mL)58.02±1.83a56.00±2.14a64.01±3.72b55.56±2.97a56.37±2.71a

2.5組織銅含量

由表6可見，各組肌肉銅含量無顯著差異(P>0.05)；肝臟、骨骼銅含量隨飼料銅添加量的增加而增加，其中20、40 mg/kg銅添加組的肝臟銅含量顯著高于對照組和5 mg/kg銅添加組(P<0.05)，40 mg/kg銅添加組的骨骼銅含量也顯著高于對照組(P<0.05)。

表6　飼料中銅添加量對斑點叉尾組織銅含量的影響

Table 6　Effects of dietary copper supplemental level on tissue copper content of channel catfish　mg/kg

表6　飼料中銅添加量對斑點叉尾組織銅含量的影響

項目Items銅添加量Coppersupplementallevel/(mg/kg)05102040肌肉銅Musclecopper0.80±0.060.74±0.060.71±0.070.86±0.010.83±0.11肝臟銅Livercopper3.46±0.04a3.46±0.15a3.56±0.21ab3.95±0.22b4.99±0.20c骨骼銅Bonecopper2.04±0.29a2.42±0.25ab2.45±0.14ab2.45±0.03ab2.60±0.04b

3討論

Lorentze等[30]認為，肝臟中銅的蓄積量是評價機體銅狀況最敏感的指標。分別用含銅5、35、700 mg/kg飼料飼喂大西洋鮭4周，發(fā)現(xiàn)肝臟銅含量顯著升高[22]；在鯉魚的研究中，肝臟銅含量隨飼料銅含量(0～1 000 mg/kg)的增加而增加，而肌肉銅含量只有在飼料銅含量達到500 mg/kg后才顯著升高[29]；De Boeck等[5]在鯉魚上也有類似報道；此外，喬永剛[31]對軍曹魚的研究發(fā)現(xiàn)，銅在骨骼中的沉積隨飼料銅含量的增加而增加。本試驗中，肝臟、骨骼銅含量隨飼料銅添加量的增加而增加，但肌肉銅含量則保持基本穩(wěn)定。

4結(jié)論

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(責任編輯菅景穎)

Effects of Practical Diet with Copper on Growth and Body Color of Channel Catfish

WAN Zude1LI Xiaoqin1GAO Qiping2SHUAI Ke2CHEN Jianan1XU Huaibing1LENG Xiangjun1,3,4,5*

(1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; 2. Tongwei Co., Ltd., Chengdu 610041, China; 3. Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Aquaculture, Shanghai 201306, China; 4. Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China; 5. Shanghai Collaborative Innovation Collaborative Innovation Center for Aquatic Animal Genetics and Breeding, Shanghai 201306, China)

Abstract:In the present study, copper (as the form of CuSO4·5H2O) with the levels of 0 (control), 5, 10, 15 or 20 mg/kg were supplemented in a practical diet which contained 11.1 mg/kg copper to formulate five experimental diets to evaluate the effects of practical diet with copper on growth and body color of channel catfish (Ictalurus punctatus). The five experimental diets were fed to channel catfish with the average body weight of (98.1±0.5) g for 42 d. Each diet had 3 replicates, and each replicate had 20 fish. The results showed that weight gain rate was significantly increased and feed conversion ratio was significantly decreased by the supplementation of 10 mg/kg copper when compared with control group (P<0.05), but with the copper supplemental level increased to 40 mg/kg, the weight gain rate was significantly decreased and the feed conversion ratio was significantly increased compared with 10 mg/kg group (P<0.05). The copper content in liver and bone was increased with the increase of dietary copper supplemental level, the liver copper content in 20 and 40 mg/kg groups was significantly higher than that in control group and 5 mg/kg group (P<0.05), and the bone copper content in 40 mg/kg group was significantly higher than that in control group (P<0.05), while the muscle copper content showed no significant difference among all groups (P>0.05). The chroma values and total xanthophylls content of dorsal skin, muscle and tyrosinase activity of dorsal skin of channel catfish were not significantly affected by 0 to 40 mg/kg copper supplementation (P>0.05). Serum aspartate aminotransferase (AST), alannine aminotransferase (ALT) activities and total bilirubin (T-Bil) content, and muscle moisture, crude protein, ether extract and ash contents were not significantly different among all groups (P>0.05). The activity of serum copper, zinc-superoxide dismutase in 10 mg/kg group was the highest, and significantly higher than that in other groups (P<0.05), but no significant difference was found among other groups (P>0.05). Base on results above, the optimal copper supplementation in the practical diet is suggested to be 10 mg/kg for channel catfish under this experimental condition, and the measured value of dietary copper content is 20.2 mg/kg.[Chinese Journal of Animal Nutrition, 2016, 28(1)：265-273]

Key words:channel catfish; copper; growth; body color

*Corresponding author, professor, E-mail: xjleng@shou.edu.cn

中圖分類號：S963

文獻標識碼：A

文章編號：1006-267X(2016)01-0265-09

作者簡介：萬祖德(1991—)，男，湖北武漢人，碩士研究生，研究方向為水產(chǎn)動物營養(yǎng)與飼料。E-mail： wanzude1991@163.com*通信作者：冷向軍，教授，博士生導師，E-mail： xjleng@shou.edu.cn

基金項目：通威集團產(chǎn)學研合作項目(TW2014A004)

收稿日期：2015-07-16

doi：10.3969/j.issn.1006-267x.2016.01.034