美洲鲥Ⅳ、Ⅴ期卵巢营养成分、卵黄蛋白原、组织蛋白酶D及其mRNA表达的比较分析

Comparison and analysis of nutrient components,vitellogenin,cathepsin D and its mRNA expression in stageⅣandⅤovaries of Alosa sapidissima

为探明美洲鲥(Alosa sapidissima)雌鱼发育后期卵巢内营养物质的变化规律,提高卵子受精率,采用生化分析、酶联免疫吸附及荧光定量PCR等方法,对性腺发育期Ⅳ期和Ⅴ期的卵巢内氨基酸、脂肪酸、卵黄蛋白原(VTG)和组织蛋白酶D(CTSD)含量及其mRNA表达量进行对比分析。结果显示,Ⅳ期卵巢中水分含量(75.73%)显著低于Ⅴ期(90.64%),而粗蛋白含量(16.07%)、粗脂肪含量(2.86%)和粗灰分含量(1.28%)均显著高于Ⅴ期(5.83%、1.25%、0.81%)。在Ⅳ、Ⅴ期卵巢内各检测出18种氨基酸,其中谷氨酸含量最高(8.64%和8.13%);8种必需氨基酸中,Ⅳ期苏氨酸、苯丙氨酸、赖氨酸和色氨酸含量均显著高于Ⅴ期,且二者第一限制氨基酸均为色氨酸;Ⅳ期氨基酸总量(TAA)、半必需氨基酸(HEAA)和非必需氨基酸总量(NEAA)均显著高于Ⅴ期。两期卵巢内共检测出19种脂肪酸,Ⅳ期C17∶0、C20∶1n9、C20∶4n6和C20∶5n3含量显著高于Ⅴ期;其中ΣMUFA、ΣPUFA、ΣUFA、Σn3PUFA和EPA+DHA含量均大于Ⅴ期,ΣUFA(70.54%)达到显著性差异。Ⅳ期卵巢中VTG含量高于Ⅴ期,但不显著;而Ⅳ期卵巢中CTSD含量明显比Ⅴ期低。Ⅴ期卵巢VTG基因表达量是Ⅳ期的13.88倍;而Ⅴ期肝脏内VTG基因表达明显低于Ⅳ期,是Ⅳ期的25%。从Ⅳ期至Ⅴ期,卵巢中CTSD mRNA表达出现下调,肝脏中CTSD mRNA表达出现上调,但均不明显。因此,当前养殖条件下,美洲鲥雌鱼亲本后期发育过程中,卵巢内营养物质积累不均衡,建议适当调整饲料配方,提高限制性氨基酸色氨酸、蛋氨酸、苯丙氨酸及含量较低的赖氨酸、苏氨酸比例和调高多不饱和脂肪酸C20∶3n6、C20∶4n6(AA)、C22∶6n3(DHA)和C20∶5n3(EPA)比例,降低C18∶2n6c的比例,保证卵巢后期发育所需营养的均衡。

In order to study the change rule of nutrient substance in ovaries of female Alosa sapidissima at the late stage of development and improve the fertilization rate of eggs,the contents of amino acids,fatty acids,vitellogenin(VTG)and cathepsin D(CTSD)and their mRNA expression in the ovaries of female Alosa sapidissima at the stagesⅣandⅤof gonadal development were compared and analyzed by biochemical analysis,enzyme-linked immunosorbent assay and fluorescence quantitative PCR.The results showed that the water content(75.73%)in stageⅣovary was significantly lower than that(90.64%)in stage V ovary,while the crude protein content(16.07%),crude fat content(2.86%)and crude ash content(1.28%)were significantly greater than those(5.83%,1.25%,0.81%)in stageⅤovary.Among the 18 amino acids detected in the ovaries of stagesⅣandⅤ,the content of glutamic acid was the highest(8.64%and 8.13%).Among the 8 essential amino acids,the contents of threonine,phenylalanine,lysine and tryptophan in stageⅣwere significantly higher than those in stageⅤ,and the first limiting amino acid of both was tryptophan.The total amino acid(TAA),semi-essential amino acid(HEAA)and total non-essential amino acid(NEAA)in stageⅣwere significantly higher than those in stageⅤ.A total of 19 fatty acids were detected in the ovaries of the two stages,and the contents of C17∶0,C20∶1n9,C20∶4n6 and C20∶5n3 in stageⅣwere significantly higher than those in stageⅤ;the contents ofΣMUFA,ΣPUFA,ΣUFA,Σn3PUFA and EPA+DHA in stageⅣwere greater than those in stageⅤ,andΣUFA(70.54%)reached significant differences.The content of VTG in stageⅣovary was higher than that in stageⅤovary,but it was not significant,while the content of CTSD in stageⅣovary was significantly lower than that in stageⅤovary.The expression of VTG gene in stageⅤovary was significantly higher than that in stageⅣ,13.88 times of that in stageⅣ,while the expression of VTG gene in liver was significantly lower than that in stageⅣ,accounting for 25%higher than that in stageⅣ.From stageⅣto stageⅤ,CTSD mRNA expression showed down-regulation in the ovary and up-regulation in the liver,but none of them was significant.Therefore,under the current aquaculture conditions,the accumulation of nutrients in the ovary is uneven during the late development of the Alosa sapidissima,and it is recommended to appropriately adjust the feed formula,increase the ratio of limiting amino acids tryptophan,methionine,L-Phenylalanine and lysine and threonine with lower content,increase the ratio of polyunsaturated fatty acids C20∶3n6,C20∶4n6(AA),C22∶6n3(DHA)and C20∶5n3(EPA),and decrease the ratio of C18∶2n6c in order to ensure the balance of nutrients required for the late development of the ovary.