基于肝脂的草鱼肝脂过量蓄积评价指标构建

Indicators construction for excessive lipid accumulation in hepatopancreas of grass carp based on hepatopancreas lipid

[目的]高密度养殖及饲料营养素不平衡等易导致草鱼肝脂过量蓄积,有关草鱼肝脂过量蓄积评价工作相对较少,本文旨在构建草鱼肝脂过量蓄积评价指标。[方法]采用物理检测法、全自动生化分析仪、索氏提取法、石蜡切片法及荧光定量PCR分别检测了245尾草鱼（平均体质量1 317.60 g）的4项形体指标,23项血清生化指标、肝脏脂肪含量、脂肪空泡及不同肝脂蓄积程度草鱼肝胰脏脂质分解相关酶基因相对表达量。根据正态分布和3σ法则,取肝脂含量低于5%的草鱼各项指标在68.26%置信区间范围值为各指标正常参考值。将肝脂含量高于5%的草鱼各项指标与正常参考值进行比较,将平均值高于正常参考值上限的指标确定为草鱼肝脂过量蓄积间接评价指标,按3σ法则确定草鱼肝脂不同蓄积程度的间接评价指标范围值。[结果]正常草鱼肝脂含量为3.02%~4.60%,肝体指数为1.13~1.59,肝组织切片中无明显脂肪空泡;肝脂轻度蓄积草鱼肝脂含量为4.60%~7.56%,肝体指数为1.61~1.95,肝组织切片中有少量脂肪空泡;肝脂中度蓄积草鱼肝脂含量为7.56%~12.74%,肝体指数为1.95~3.13,肝组织切片中较多脂肪空泡;肝脂重度蓄积草鱼肝脂含量为12.74%以上,肝体指数大于3.13,肝组织切片中可见大量脂肪空泡。肝脂过量蓄积草鱼肝胰脏脂蛋白酯酶（LPL）mRNA相对表达量随肝脂蓄积程度增加而逐渐上调,而相对分子质量为14×10^3的载脂蛋白（APO-14）与激素敏感性脂肪酶（HSL）mRNA相对表达量则均随肝脂蓄积程度增加而逐渐下调。肝脂过量蓄积草鱼的肥满度、脏体指数、肠系脂肪指数,血清谷氨酸-丙酮酸转氨酶（ALT）、天冬氨酸氨基转移酶（AST）、总胆红素（TBIL）、间接胆红素（IBIL）、碱性磷酸酶（ALP）、总胆固醇（CHOL）、低密度脂蛋白（LDL）、空腹血糖（GLU）平均值均高于各指标正常参考值上限,初步确定为草鱼肝脂过量蓄积间接评价指标。[结论]肝脂含量、肝组织切片中脂肪空泡数量、肝体指数为评价草鱼肝脂蓄积程度的直接指标;3个形体指标、8个血清生化指标和脂质分解相关基因mRNA表达量可作为评价肝脂蓄积程度的间接指标。

[Objectives] Excessive lipid accumulation in hepatopancreas of grass carp due to high density cultivation and commercial feed has been the threat to the health of grass carp. However,the study on the evaluation of excessive lipid accumulation of grass carp is still very deficient.[Methods] To build evaluation indices of excess lipid accumulation in hepatopancreas of grass carp,two hundred and forty five adult grass carps （average mass 1 317.60 g） were used in this study. Body indicators,serum biochemical indices,hepatopancreas lipid,fat vacuoles in hepatopancreas and mRNA expression of genes about lipid decoposition were detected by physical detection,automatic biochemical analyzer,soxhlet methods,paraffin section and fluorescence quantitative PCR,respectively. Normal ranges of detected indicators from grass carp with hepatopancreas lipid lower than 5% were determimed by 68.26% confidence interval if they showed significant normal distribution. When the mean of a certain indicator from grass carp with hepatopancreas lipid higher than 5% was higher than the upper limit of normal range,then they would be selected out as indirect evaluation indicators of excessive lipid accumulation in hepatopancreas of grass carp.[Results] Results suggested that there was no obvious fat vacuole in normal grass carp hepatopancreas with hepatopancreas lipid 3.02% to 4.60% and hepatosomatic index 1.13 to 1.59. Grass carp could be evaluated to show mild,moderate and severe hepatopancreas lipid accumulation when the hepatopancreas lipid ranged from 4.60% to 7.56%,7.56% to 12.74%,and higher than 12.74%,respectively,and the hepatosomatic index ranged from 1.61 to 1.95,1.95 to 3.13 and higher than 3.13,respectively. With the increase of hepatopancreas,the expression of LPL mRNA and fat vacuoles increased,too. However,the mRNA levels of APO-14 and HSL genes declined as more and more lipid accumulated in hepatopancreas. Condition factor,viscerosomatic index,mesentericfat index,alanine aminotransferase,aspartate aminotransferase,total bilirubin,indirect bilirubin,alkaline phosphatase,cholesterol,low density lipoprotein and glucose were the indirect evaluation indicators of fatty liver due to their higher means than the upper limits of normal ranges.[Conclusions] It is concluded that the hepatopancreas lipid level,fat vacuoles in the paraffin section of heaptopancreas and hepatosomatic index can be directly used to evaluate the hepatopancreas lipid accumulation state. Meanwhile,other body indices,part of serum biochemical indexes,and expression levels of certain genes can be indirectly used to estimate the stage of lipid accumulation in hepatopancreas of grass carp.