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Volume 45 Issue 10
Oct.  2021
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Effects of cottonseed protein source on growth, body composition and health of Micropterus salmoides

  • 1.

    Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, College of Fisheries, Southwest University, Chongqing 400716, China

  • 2.

    College of Fisheries, Guangdong Ocean University, Zhanjiang    524088, China

  • Corresponding author: LIN Shimei, linsm198@163.com
  • Received Date: 2021-04-12
    Accepted Date: 2021-05-25
    Available Online: 2021-08-30
  • To evaluate the feasibility of cottonseed protein in largemouth bass (Micropterus salmoides) diet, five isonitrogen-isolipid experimental diets (CPC0 as control group, CPC1, CPC2, CPC3, and CPC4) were prepared by replacing 30% fish meal with 4 cottonseed proteins of different quality. Micropterus salmoides [average body weight (12.20±0.11) g] were fed in indoor circulating culture system for 8 weeks. The results showed that the nutrient composition, contents of gossypol, raffinose and stysaccharide of the four kinds of cottonseed proteins were different, and CPC3 cottonseed protein quality was the best. The final body weight, weight gain rate and specific growth rate of CPC3 group were significantly higher than those of the other groups, but there were no significant difference in the routine nutrients and muscle amino acid nutritional composition among all groups. The activities of SOD and GSH-Px and the mRNA expressions of CAT and SOD in liver of CPC3 group were the highest, while the content of MDA was the lowest. The relative expressions of anti-inflammatory factors IL-10 and TGF-β were the highest in CPC3 group, while the relative expressions of pro-inflammatory factors IL-1β, IL-8 and TNF-α were the lowest in CPC3 group. In addition, cottonseed protein sources could significantly affect the liver protein metabolism of largemouth bass, and the activities of ALT and AST and the expression levels of P13K, Akt, m-TOR, S6K1 and 4E-BP in CPC3 group were the highest. Meanwhile, it was found that CPC3 group had the highest intestinal T-SOD enzyme activity, the lowest MDA content, and the lowest intestinal permeability indexes (diamine oxidase, D-lactic acid and endotoxin). Cottonseed protein sources also affected the expression of tight junction protein-related genes ZO-1, Claudin-1 and Occludin in the intestine. The results showed that different cottonseed protein quality had different effects on the growth and health of M. salmoides, among which cottonseed protein CPC3 showed the best effect and significantly improved the liver and intestinal health of M. salmoides, thereby promoting the growth of M. salmoides. Cottonseed protein CPC3 can be used as a good protein source for M. salmoides feed.
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Effects of cottonseed protein source on growth, body composition and health of Micropterus salmoides

    Corresponding author: LIN Shimei, linsm198@163.com
  • 1. Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, College of Fisheries, Southwest University, Chongqing 400716, China
  • 2. College of Fisheries, Guangdong Ocean University, Zhanjiang    524088, China
  • Received Date: 2021-04-12
  • Accepted Date: 2021-05-25
  • Available Online: 2021-10-01

Abstract: To evaluate the feasibility of cottonseed protein in largemouth bass (Micropterus salmoides) diet, five isonitrogen-isolipid experimental diets (CPC0 as control group, CPC1, CPC2, CPC3, and CPC4) were prepared by replacing 30% fish meal with 4 cottonseed proteins of different quality. Micropterus salmoides [average body weight (12.20±0.11) g] were fed in indoor circulating culture system for 8 weeks. The results showed that the nutrient composition, contents of gossypol, raffinose and stysaccharide of the four kinds of cottonseed proteins were different, and CPC3 cottonseed protein quality was the best. The final body weight, weight gain rate and specific growth rate of CPC3 group were significantly higher than those of the other groups, but there were no significant difference in the routine nutrients and muscle amino acid nutritional composition among all groups. The activities of SOD and GSH-Px and the mRNA expressions of CAT and SOD in liver of CPC3 group were the highest, while the content of MDA was the lowest. The relative expressions of anti-inflammatory factors IL-10 and TGF-β were the highest in CPC3 group, while the relative expressions of pro-inflammatory factors IL-1β, IL-8 and TNF-α were the lowest in CPC3 group. In addition, cottonseed protein sources could significantly affect the liver protein metabolism of largemouth bass, and the activities of ALT and AST and the expression levels of P13K, Akt, m-TOR, S6K1 and 4E-BP in CPC3 group were the highest. Meanwhile, it was found that CPC3 group had the highest intestinal T-SOD enzyme activity, the lowest MDA content, and the lowest intestinal permeability indexes (diamine oxidase, D-lactic acid and endotoxin). Cottonseed protein sources also affected the expression of tight junction protein-related genes ZO-1, Claudin-1 and Occludin in the intestine. The results showed that different cottonseed protein quality had different effects on the growth and health of M. salmoides, among which cottonseed protein CPC3 showed the best effect and significantly improved the liver and intestinal health of M. salmoides, thereby promoting the growth of M. salmoides. Cottonseed protein CPC3 can be used as a good protein source for M. salmoides feed.

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  • 棉籽蛋白作为非粮植物蛋白源，资源丰富，据统计2019年我国棉粕产量约320万t[1]，因其富含优质蛋白质而受到广泛关注。目前，棉籽蛋白已经在动物生产上广泛应用，包括猪[2]、鸡[3]、鱼[4]等，其添加量因动物品种以及棉籽蛋白质量而有所不同。然而，由于棉籽蛋白含有棉酚、纤维等抗营养因子限制了其在动物饲料中的使用，通常可通过生物降解[5]、加工[6]或遗传改良[7]来降低或去除抗营养物质—棉酚，进而提高其添加剂量。目前，在鱼类上研究较多的是单一棉籽蛋白(如发酵棉籽蛋白、脱酚棉籽蛋白)替代鱼粉或其他蛋白源的应用效果，未见不同加工工艺棉籽蛋白质量的比较研究。大口黑鲈 (Micropterus salmoides) 作为我国重要的经济养殖鱼类，因其肉质、体质量、贮存等优势而深受市场欢迎，但养殖效益受季节的影响而波动较大，降低其饲料养殖成本是目前关注的热点。为此，本实验以大口黑鲈为研究对象，从生长、体组成、肝功能和肠道健康角度，评价不同质量的棉籽蛋白在大口黑鲈饲料中应用的可行性，旨在为大口黑鲈饲料配方的优化提供技术资料。

1.   材料与方法

    1.1.   实验饲料

  • 以鱼粉、鸡肉粉、大豆浓缩蛋白和豆粕为蛋白源，以鱼油、豆油为脂肪源配制基础饲料，用4种棉籽蛋白(CPC1、CPC2、CPC3和CPC4，均来自新疆，加工工艺不同，根据GB 13086-91法测定其棉酚含量，根据高效液相色谱分析方法测定其棉籽糖和水苏糖含量[8]，具体组成见表1)分别替代30%的鱼粉配制成4种等氮等脂的实验饲料(表2)，饲料原料粉碎过80目筛，采取逐级稀释法混合均匀，制成粒径2.0 mm的颗粒饲料，自然风干后于4 °C冰柜中保存备用。

    项目
    items    		棉籽蛋白　cottonseed protein
    CPC1CPC2CPC3CPC4
    水分/%　moisture 7.20±0.08c 8.88±0.18d 5.20±0.05b 4.67±0.08a
    粗蛋白质/%　crude protein 49.27±0.57a 53.62±1.11b 62.42±0.58d 57.14±0.96c
    粗脂肪/%　crude lipid 3.26±0.04b 3.27±0.07b 1.24±0.01a 3.43±0.06b
    粗灰分/%　crude ash 6.80±0.08a 7.53±0.16b 8.06±0.07c 7.62±0.13bc
    棉酚/(mg/kg)　gossypol 970.93±11.21d 894.50±18.59c 169.36±1.56a 431.31±7.22b
    棉籽糖/(mg/g)　raffinose 52.23±0.60a 52.07±1.08a 60.12±0.56b 53.93±0.90a
    水苏糖/(mg/g)　stachyose 6.61±0.08a 7.37±0.15b 9.45±0.09d 7.84±0.13c
    注：同行中上标不同小写字母表示差异显著(P<0.05)，下同
    Notes: in the same row, values with different lowercase letter superscripts mean significant difference (P<0.05)，the same below

    Table 1.  Nutrient composition of different cottonseed proteins

    项目
    items    		棉籽蛋白　cottonseed protein
    CPC0CPC1CPC2CPC3CPC4
    原料/%　ingredients
    　蒸汽鱼粉　steam fish meal 40.00 28.00 28.00 28.00 28.00
    　国产鸡肉粉　domestic chicken powder 10.00 10.00 10.00 10.00 10.00
    　棉籽蛋白1　cottonseed protein 1 18.61
    　棉籽蛋白2　cottonseed protein 2 17.12
    　棉籽蛋白3　cottonseed protein 3 13.48
    　棉籽蛋白4　cottonseed protein 4 14.27
    　豆粕　soybean meal 7.00 7.00 7.00 7.00 7.00
    　大豆浓缩蛋白　soy protein concentrate 11.00 11.00 11.00 11.00 11.00
    　小麦面筋蛋白　wheat gluten protein 3.00 3.00 3.00 3.00 3.00
    　木薯淀粉　cassava starch 9.00 9.00 9.00 9.00 9.00
    　鱼油　fish oil 1.15 2.10 2.03 1.86 2.05
    　大豆卵磷脂　soybean lecithin 1.50 1.50 1.50 1.50 1.50
    　豆油　soybean oil 4.00 4.00 4.00 4.00 4.00
    　维生素预混料　vitamin premix 1.00 1.00 1.00 1.00 1.00
    　矿物质预混料　mineral premix 1.50 1.50 1.50 1.50 1.50
    　氯化胆碱　choline chloride 0.50 0.50 0.50 0.50 0.50
    　磷酸二氢钙　Ca(H2PO4)2 1.40 2.58 2.64 2.65 2.85
    　维生素C　vitamin C 0.05 0.05 0.05 0.05 0.05
    　乙氧基喹啉　ethoxyquin 0.05 0.05 0.05 0.05 0.05
    　微晶纤维素　microcrystalline cellulose 8.85 0.00 1.59 5.41 4.00
    　赖氨酸　lysine 0.00 0.16 0.17 0.11 0.22
    　蛋氨酸　methionine 0.00 0.07 0.04 0.05 0.05
    营养成分　nutritional levels
    　粗蛋白质/%　crude protein 48.21 48.16 48.12 48.15 48.17
    　粗脂肪/%　crude lipid 11.29 11.31 11.30 11.31 11.29
    　粗灰分/%　crude ash 10.25 11.80 11.66 11.70 11.69
    　棉酚/(mg/kg)　gossypol 0.00 182.69 156.14 23.85 61.75

    Table 2.  Composition and nutrient levels of the test diets (air-dry basis)

    • 1.2.   饲养管理

  • 实验用大口黑鲈购于重庆长寿区鱼种场，用商品饲料驯化10 d，禁食24 h，用0.01% MS-222麻醉后称重，将体质健壮、规格整齐、初始体质量为(12.20±0.11) g的大口黑鲈幼鱼随机分为5个处理，每个处理3个重复，每个重复30尾鱼，在室内淡水循环养殖系统(有效容积为200 L)中饲养8周，实验期间，每日投喂3次(08:30、12:30、18:00)，投饲率3%~5%，养殖水源为充分曝气的自来水，养殖期间水温为23.2~28.5 °C，溶解氧含量≥6.5 mg/L，氨氮含量<0.1 mg/L，亚硝酸盐含量<0.01 mg/L，pH为7.2~7.6。

    • 1.3.   样品采集及分析方法

    样品采集

  • 饲养实验结束后，禁食24 h，用0.01% MS-222麻醉后称重并计数，每个重复随机选取3尾鱼用于全鱼常规成分的测定；每个重复随机取5尾鱼，测量体质量、体长，在冰盘上解剖并分离出内脏团、肝脏和肠道后称重，用于形体指标的测定，并收集肝脏和肠道组织用液氮速冻后于−80 °C冰箱中保存备用；每个重复随机取5尾鱼，用一次性注射器于尾静脉取血，置于4 °C冰箱过夜后4 000 r/min离心10 min，收集血清，−80 °C冰箱中保存备用；每个重复随机取6尾鱼，于无菌操作台上处死并使用预先去除 RNA 酶并灭菌的解剖器材解剖，取出大口黑鲈的肝脏及肠道，在相同部位取 3 mm3 左右大小的肝脏及肠道组织样品，置于含有500 μL RNA 保护剂(Takara，Japan)的去RNA 酶的无菌离心管中，液氮速冻后−80 °C冰箱中保存，用于基因相对表达量的测定。

    • 饲料、全鱼常规营养成分的测定

  • 采用标准方法对饲料、全鱼常规营养成分进行测定。水分采用105 °C烘干恒重法测定；粗蛋白质含量采用凯式定氮法测定；粗脂肪含量采用索式抽提法测定；粗灰分含量采用550 °C马弗炉煅烧法测定；肝脏脂肪含量采用氯仿−甲醇法测定；肌肉氨基酸组成采用GB/T 18654.11—2008法测定。

    • 抗氧化及代谢指标测定

  • 超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、谷丙转氨酶(ALT)、谷草转氨酶(AST)以及丙二醛(MDA)含量均采用南京建成生物工程研究所试剂盒测定，测定方法参照试剂盒说明书进行。

    • 抗氧化、免疫及代谢相关基因表达分析

  • 采用实时荧光定量PCR法测定基因mRNA表达水平。肝脏及肠道总RNA使用RNAiso Plus试剂(Takara，Japan)提取，使用NanoDrop 2000 超微量分光光度计(Thermo Fisher，USA)测定总RNA浓度。随后使用反转录试剂盒 FastKing-RT SuperMix(天根，北京)将提取的总RNA反转录为cDNA，并用DEPC水进行稀释后置于−20 °C保存备用。实时荧光定量PCR的体系为20 μL，其中上下游引物各1 μL、cDNA 2 μL、10 μl 2×NovoStart® SYBR qPCR SuperMix Plus (Novoprotein，上海)和 DEPC水6 μL。定量仪器为实时定量PCR仪(Bio-rad- CFX96)。实时定量PCR的程序为95 °C持续1 min 1个循环；95 °C持续20 s，60 °C持续1 min，共计40个循环。以β-actin为内参，采用2−ΔΔGt法计算基因的相对表达量，相关引物序列见表3。

    基因
    gene    		上游引物(5′-3′)
    forward primer (5′-3′)    		下游引物(5′-3′)
    reverse primer (5′-3′)    		登录号
    GenBank
    CAT TGGTGTTCACGGATGAGATGG GGAGAAGCGGACAGCAATAGG XM_038704976.1
    SOD CCACCAGAGGTCTCACAGCA CCACTGAACCGAAGAAGGACT XM_038713969.1
    GSH-Px ATACCAAGTCTCCTTCCCTCTGT CGTCCACCACTTTGCCATT XM_038715030.1
    IL-10 CGGCACAGAAATCCCAGAGC CAGCAGGCTCACAAAATAAACATCT XM_038696252.1
    TGF-β GCTCAAAGAGAGCGAGGATG TCCTCTACCATTCGCAATCC XM_038693206.1
    IL-1β CGTGACTGACAGCAAAAAGAGG GATGCCCAGAGCCACAGTTC XM_038733429.1
    IL-8 CGTTGAACAGACTGGGAGAGATG AGTGGGATGGCTTCATTATCTTGT XM_038704088.1
    TNF-α CTTCGTCTACAGCCAGGCATCG TTTGGCACACCGACCTCACC XM_038710731.1
    PI3K AAGACCTTCCTCATCACGAC CCTTCCACTACAACACTGCA XM_038733024.1
    AKT ATGGACTCCTCTCCAGACCC TTCATGGCGTAGTAGCGTCC XM_038729217.1
    m-TOR GCGTATCGTAGAGGACTGGC GTCAACACCGAGAAGGAGCA XM_038723321.1
    S6K1 TCGTTCGAGCCAAAGGTCC TCGATACCTCCATGGGTTGC XM_038708508.1
    4E-BP ACGAGGTCTGCCCAACATTC CAGCGTTGCTGCTATCAGGT XM_038703879.1
    Claudin-1 CCAGGGAAGGGGAGCAATG GCTCTTTGAACCAGTGCGAC XM_038713307.1
    Occludin GATATGGTGGCAGCTACGGT TCCTACTGCGGACAGTGTTG XM_038715419.1
    ZO-1 ATCTCAGCAGGGATTCGACG CTTTTGCGGTGGCGTTGG XM_038701018.1
    β-actin AAAGGGAAATCGTGCGTGAC AAGGAAGGCTGGAAGAGGG XM_038695351.1

    Table 3.  Primers pair sequences for real-time PCR

    • 1.4.   计算公式

  • 特定生长率(specific growth rate，SGR，%/d)=(lnWt−lnW0)/t×100%

    增重率(weight gain rate，WGR，%)=(Wt−W0)/W0×100%

    摄食率(feeding ratio，FR，% /d)= Wf /[(Wt + W0)/2×t]×100%

    蛋白质效率(protein efficiency ratio，PER，%)=(Wt−W0)/(Wf×Wp) ×100%

    饲料系数(feed conversion ratio，FCR)=Wf/(Wt−W0)

    成活率(survival rate，SR，%)=Nt/N0×100%

    脏体比(viscerosomatic index，VSI，%)=Wv/W×100%

    肝体比(hepatosomatic index，HSI，%)=Wh/W×100%

    肥满度(condition factor，CF，g/cm3)=W/L3×100

    式中，Wt和W0分别为终末鱼体质量和初始鱼体质量(g)；t为养殖实验天数(d)；Wf为总摄食量(g)；Nt和N0分别为终末尾数和初始尾数(尾)；W为鱼质量(g)；L为鱼体长(cm)；Wv为内脏重(g)；Wh为肝脏重(g)；Wp为饲料蛋白质含量(%)。

    • 1.5.   数据处理与分析

  • 实验数据均以平均值+标准误(mean+SE)表示，采用SPSS 22.0软件进行方差性检验和单因素方差分析(One-Way ANOVA)，若达到显著水平，则进行Tukey氏多重比较，显著水平为P<0.05。

2.   结果

    2.1.   棉籽蛋白对大口黑鲈生长性能的影响

  • CPC3组大口黑鲈的FBW、WGR和SGR显著高于其他实验组(P<0.05)，CPC0、CPC1、CPC2和CPC4组之间无显著差异。PER值也是CPC3组最高。各实验组FR无显著差异，成活率SR均为100% (P>0.05)(表4)。

    项目
    items    		棉籽蛋白　cottonseed protein
    CPC0CPC1CPC2CPC3CPC4
    初始体质量/g　IBW 12.21±0.06 12.13±0.02 12.23±0.02 12.13±0.05 12.23±0.03
    终末体质量/g　FBW 83.19±0.38ab 82.17±1.47a 82.14±1.59a 87.49±0.32b 83.92±1.07ab
    增重率/%　WGR 581.35±5.86ab 577.21±11.04a 571.73±13.99a 621.04±1.03b 586.29±10.28ab
    特定生长率/%　SGR 3.427±0.02ab 3.415±0.03ab 3.401±0.04a 3.528±0.01b 3.439±0.03ab
    摄食率/(%/d)　FR 2.75±0.01 2.86±0.07 2.89±0.01 2.76±0.02 2.74±0.05
    蛋白质效率/%　PER 2.01±0.01ab 1.94±0.04ab 1.91±0.02a 2.04±0.01b 2.03±0.04b
    饲料系数　FCR 1.03±0.00ab 1.08±0.02ab 1.09±0.01b 1.02±0.01a 1.03±0.02a
    存活率/%　SR 100.00 100.00 100.00 100.00 100.00

    Table 4.  Effects of dietary cottonseed protein on growth performance and feed utilization of M. salmoides

    • 2.2.   棉籽蛋白对大口黑鲈形体指标和体组成的影响

  • CPC3组HSI显著低于对照组(CPC0)(P<0.05)，各实验组VSI以及全鱼营养含量(水分、粗蛋白质、粗脂肪、粗灰分)无显著差异。尽管各实验组肝脂含量无显著差异，但对照组肝脂含量比其他实验组低10.8%~29.0%，以CPC2组最高(表5)。CPC3组肌肉非必需氨基酸总量 (ΣNEAA)、必需氨基酸总量(ΣEAA)和氨基酸总量(ΣTAA)含量均高于其他实验组(表6)。

    项目
    items    		棉籽蛋白　cottonseed protein
    CPC0CPC1CPC2CPC3CPC4
    形态指标　morphological measurements
    　脏体比　VSI 8.50±0.43 8.60±0.20 8.45±0.31 7.49±0.25 7.71±0.11
    　肝体比　HSI 2.18±0.03b 2.14±0.06ab 1.98±0.05ab 1.93±0.04a 2.05±0.06ab
    营养组成　nutrition composition
    　水分　moisture 70.07±0.81 70.44±0.53 70.90±0.81 70.09±0.64 70.19±1.21
    　粗蛋白质　crude protein 17.70±0.20 17.47±0.24 17.13±0.14 18.08±0.21 17.89±0.19
    　粗脂肪　crude lipid 6.27±0.12 6.29±0.07 6.33±0.10 6.12±0.08 6.15±0.10
    　粗灰分　crude ash 4.48±0.05 4.55±0.12 4.52±0.10 4.47±0.07 4.39±0.09
    　肝脂含量　liver lipid content 3.79±0.18 4.53±0.02 4.89±0.09 4.20±0.61 4.65±0.19

    Table 5.  Effects of dietary cottonseed protein on morphological measurements and nutrition composition of M. salmoides (fresh weight) %

    项目　　
    items　　    		棉籽蛋白　cottonseed protein
    CPC0CPC1CPC2CPC3CPC4
    非必需氨基酸 (NEAA)
    　天门冬氨酸　Asp 9.10±0.10 9.04±0.18 8.99±0.16 9.22±0.09 9.09±0.09
    　谷氨酸　Glu 14.00±0.16 13.81±0.28 13.82±0.24 14.16±0.13 13.97±0.14
    　丝氨酸　Ser 3.41±0.04 3.33±0.07 3.39±0.06 3.42±0.03 3.38±0.03
    　甘氨酸　Gly 4.08±0.05 3.99±0.08 3.97±0.07 4.06±0.03 3.99±0.04
    　丙氨酸　Ala 5.15±0.06 5.10±0.10 5.10±0.09 5.22±0.05 5.11±0.05
    　脯氨酸　Pro 2.93±0.03 2.87±0.06 2.90±0.05 2.95±0.03 2.95±0.03
    　酪氨酸　Tyr 3.10±0.03 3.05±0.06 3.04±0.05 3.13±0.03 3.10±0.03
    　非必需氨基酸总量　ΣNEAA 41.77±0.48 41.19±0.83 41.22±0.72 42.16±0.39 41.57±0.43
    必需氨基酸 (EAA)
    　组氨酸　His 2.17±0.03 2.16±0.04 2.13±0.04 2.19±0.02 2.13±0.02
    　精氨酸　Arg 6.41±0.08 6.31±0.13 6.29±0.11 6.39±0.06 6.27±0.07
    　苏氨酸　Thr 3.88±0.04 3.80±0.08 3.84±0.07 3.88±0.04 3.79±0.04
    　缬氨酸　Val 4.30±0.05 4.19±0.09 4.20±0.07 4.35±0.04 4.22±0.04
    　蛋氨酸　Met 2.65±0.03 2.61±0.05 2.63±0.04 2.70±0.02 2.64±0.03
    　异亮氨酸　Ile 4.09±0.05 3.97±0.08 3.99±0.07 4.15±0.04 3.98±0.04
    　亮氨酸　Leu 6.73±0.08 6.59±0.13 6.63±0.11 6.80±0.06 6.62±0.07
    　苯丙氨酸　Phe 3.71±0.04 3.67±0.08 3.65±0.06 3.76±0.03 3.66±0.04
    　赖氨酸　Lys 7.67±0.09 7.55±0.15 7.55±0.13 7.78±0.07 7.59±0.08
    　必需氨基酸总量　ΣEAA 41.60±0.48 40.85±0.83 40.89±0.71 42.00±0.39 40.91±0.42
    　氨基酸总量　ΣTAA 83.37±0.96 82.04±1.66 82.11±1.42 84.15±0.78 82.48±0.86
    　ΣEAA/ƩTAA 49.90±0.58 49.79±1.00 49.80±0.86 49.91±0.46 49.60±0.52
    　ΣNEAA/ΣTAA 50.10±0.58 50.21±1.02 50.20±0.87 50.10±0.46 50.40±0.52

    Table 6.  Effects of dietary cottonseed protein on amino acid content in muscle of M. salmoides

    • 2.3.   棉籽蛋白对大口黑鲈肝脏抗氧化及免疫的影响

  • CPC3组肝脏SOD、GSH-Px活性以及CAT和SOD mRNA表达水平显著高于其他实验组(表7，图1)，而MDA含量最低 (P<0.05)。CPC3组抗炎因子IL-10和TGF-β表达量最高，促炎因子IL-1β、IL-8和TNF-α表达量最低 (P<0.05)(图2)。

    项目　　　
    items　　　    		棉籽蛋白　cottonseed protein
    CPC0CPC1CPC2CPC3CPC4
    过氧化氢酶/(U/mg prot)　CAT 18.77±0.77ab 18.27±0.21ab 17.74±0.07a 19.92±0.33b 19.32±0.11ab
    总超氧化物歧化酶/(U/mg prot)　SOD 451.57±5.70a 458.27±2.77a 442.63±12.89a 498.38±9.82b 466.49±8.67a
    谷胱甘肽过氧化物/(U/mg prot)　GSH-Px 40.23±1.29a 39.88±0.33a 38.98±0.61a 46.56±0.94b 41.30±0.17a
    丙二醛/(nmol/mg prot)　MDA 0.81±0.02b 0.81±0.02b 0.82±0.03b 0.67±0.02a 0.80±0.03b

    Table 7.  Effects of dietary cottonseed protein on hepatic antioxidant capacity of M. salmoides

    Figure 1.  Effects of dietary cottonseed protein on mRNA expression of antioxidant related genes in liver of M. salmoides

    Figure 2.  Effects of dietary cottonseed protein on mRNA expression of inflammation related genes in liver of M. salmoides

    • 2.4.   棉籽蛋白对大口黑鲈肝脏蛋白质代谢酶及相关基因表达的影响

  • 实验发现，棉籽蛋白源也显著影响大口黑鲈肝脏的蛋白质代谢。CPC3组ALT、AST活性以及基因P13K、AKT、m-TOR、S6K1、4E-BP表达水平最高 (图3)。

    Figure 3.  Effects of dietary cottonseed protein on protein metabolism enzymes (a) and related gene expression (b) in the liver of M. salmoides

    • 2.5.   棉籽蛋白对大口黑鲈肠道健康的影响

  • CPC3组大口黑鲈肠道SOD活性及SOD基因表达水平显著高于其他实验组(P<0.05)，而MDA含量最低，各实验组肠道CAT活性及CAT基因表达水平无显著差异 (P>0.05) (图4)。CPC1和CPC2组血清二胺氧化酶活性、D-乳酸和内毒素含量显著高于其他实验组 (P<0.05) (图5)。肠道基因Occludin和ZO-1 mRNA的表达水平组间无显著差异，但CPC4组基因Claudin-1表达水平显著高于CPC2组(图6)。

    Figure 4.  Effects of dietary cottonseed protein on intestinal antioxidant capacity (a) and related gene expression (b) of M. salmoides

    Figure 5.  Effects of dietary cottonseed protein on intestinal permeability indexes of M. salmoides

    Figure 6.  Effects of dietary cottonseed protein on mRNA expression of intestinal tight junction protein-related genes in M. salmoides

3.   讨论

  • 大量研究表明，棉籽中含有棉酚、粗纤维等抗营养因子会对鸡[3]、鹅[9]、猪[2]等畜禽动物的生长、肝脏功能、生殖产生不利影响。畜禽动物对游离棉酚的耐受剂量因品种、日龄、棉籽蛋白质量等因素而有较大差异，如我国饲料卫生标准限定蛋鸡饲料游离棉酚含量≤20 mg/kg (GB 13078-2017)，蛋鸡对棉酚的耐受能力不如肉鸡[10]。研究同样发现，饲料中棉酚含量会影响鱼类生长和健康，不同鱼类对棉酚的耐受程度不同，如异育银鲫(Carassius auratus gibelio)可以耐受饲料642 mg/kg棉酚[11]，斑点叉尾鮰(Ictalurus punctatus)可以耐受300 ~1 200 mg/kg 棉酚[12]，而条纹锯鮨(Centropristis striata)饲料含150 mg/kg 棉酚就会降低其生长性能[13]。本实验中4种棉籽蛋白以CPC3质量最优，棉酚含量最低(169.36 mg/kg，表1)，相应大口黑鲈的生长效果也最好，即CPC3组鱼的生长速度最快，饲料中棉酚含量仅有23.85 mg/kg，CPC4组鱼生长速度相较CPC3组略有下降，其饲料中棉酚含量为61.75 mg/kg (表2)，这些结果表明大口黑鲈对棉酚的敏感性强于其他鱼类，且棉酚是影响其生长的一个重要因子[14]。有研究指出，对棉籽蛋白进行适当的工艺处理[15-16]，可以有效降低棉酚的含量(脱毒率达到70.0%以上)，提高棉籽蛋白在鱼饲料中的用量(从10.0%[17]提高至30.5%[18])。此外，棉酚与赖氨酸结合会引起赖氨酸缺乏或不足，进而导致鱼类的生长下降[19-20]。本实验各处理饲料中赖氨酸含量一致，而高棉酚组(CPC1、CPC2)大口黑鲈的生长效果显著低于低棉酚组(CPC3)的实验结果支持这个推断。可见，在富含棉酚的棉籽蛋白饲料中易致其可利用赖氨酸水平降低，不能满足大口黑鲈生长的需要。目前还不明确大口黑鲈能够耐受多少剂量的棉酚，需要进一步通过长期的生长实验来证实。

    本研究发现饲料中添加不同质量的棉籽蛋白不会影响大口黑鲈的摄食量，类似的结果在珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×E. lanceolatu♂)[21]、卵形鲳鲹(Trachinotus ovatus)[22]上也有报道。但有研究发现，棉籽蛋白会影响饲料的适口性，如饲料中添加46.0%脱酚棉籽蛋白会降低花鲈(Lateolabrax japonicus)的摄食量，进而导致其生长不良[23]。然而，条纹锯鮨[24]饲料中添加30.2%棉籽浓缩蛋白(鱼粉含量仅7%)既不影响鱼的摄食也不影响鱼的生长。本研究中，棉籽蛋白的实际添加量为13.0%~19.0%，其中CPC1组添加水平最高为18.61%，这些添加量都远远低于上述研究结果。这些结果表明，不同的鱼类对棉籽蛋白的敏感程度不一样，涉及鱼类的摄食生理，情况比较复杂。

    肝脏是鱼类重要的代谢器官，因此维持正常的肝功能对鱼类的生长有非常重要的作用。以往研究表明添加适量的植物蛋白对鱼体的抗氧化能力及免疫功能有一定的改善作用[25]，但是过量的植物蛋白会损伤鱼类的肝脏组织，导致其蛋白质代谢功能受损[26]。本研究发现饲料中添加棉籽蛋白CPC3能够显著提高大口黑鲈肝脏抗氧化能力(CAT和SOD mRNA表达水平增强，CAT、SOD、GSH-Px活性升高)，同时上调肝脏抗炎因子(IL-10、TGF-β) mRNA的表达，下调促炎因子(IL-1β、IL-8和TNF-α) mRNA的表达。此外，还可以提高肝脏转氨酶(AST、ALT)活性以及蛋白质代谢基因(P13K、AKT、m-TOR、S6K1、4E-BP)表达水平。这说明适宜的棉籽蛋白可以通过提高肝脏的免疫功能、抗氧化能力，改善肝脏健康，进而提高蛋白质的利用效率，促进大口黑鲈的生长。CPC3组SGR和PER优于其他实验组，也证实和支持这个结论。这与在卵形鲳鲹[22]、草鱼(Ctenopharyngodon idella)[27]上的研究结果一致。这可能是因为棉籽蛋白中含有棉籽糖、水苏糖等益生元所致。研究证实在饲料中添加棉籽糖和水苏糖能够提高尼罗罗非鱼(Oreochromis niloticus)[28]、大菱鲆(Scophthalmus maximus) [29]的肝脏抗氧化能力，促进巨噬细胞活化，增强机体免疫功能，从而促进生长。

    肠道是从外界获取营养和能量的主要场所，并与体内各种免疫、应激和新陈代谢息息相关，因此保护肠道屏障对鱼体的生长和健康极为重要。研究证实添加适量的植物蛋白能够提高鱼类的肠道抗氧化酶活性，维持良好的肠道黏膜屏障功能[30]。本实验中不同质量的棉籽蛋白源显著影响大口黑鲈肠道健康，饲料中添加棉籽蛋白CPC3对大口黑鲈的肠道抗氧化能力(CAT和SOD活性提高)及通透性(二胺氧化酶活性、D-乳酸和内毒素含量降低)有改善作用，而棉籽蛋白CPC1和CPC2显著增加肠道的通透性，同时下调紧密连接蛋白基因Claudin-1的表达水平。这可能是棉籽蛋白CPC3抗营养因子含量少，降低了肠道的氧化应激，维持相对正常的肠道生理功能。众所周知，棉籽蛋白含有一定量的益生元(如棉籽糖、水苏糖)，它可以促进有益菌的生长，减少有毒代谢物的产生，从而维持正常的肠道功能[31]。本实验中CPC1和CPC2组含有较高的棉酚，但与对照组相比，并没有降低大口黑鲈的生长性能，这可能是棉籽蛋白中益生元的作用效果抵消了或减弱了棉酚的危害作用所致，这个推论需要进一步证实。在团头鲂(Megalobrama amblycephala)[32]上的研究也发现，饲料中添加适宜的植物蛋白可以改善肠道健康。建议今后可以选择肠道抗氧化和通透性指标详细评估蛋白质的质量。

    综上所述，饲料中棉籽蛋白源会影响大口黑鲈的肝脏和肠道健康，进而影响其生长性能。本研究中棉籽蛋白CPC3有助于提高肝脏抗氧化能力及免疫功能，改善肠道健康。因此，棉籽蛋白CPC3可以作为鱼类的饲料蛋白源，降低肉食性鱼类对鱼粉的依赖性。

Reference (32)

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