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Abstract：
This study aimed to evaluate the effects of arginine (0.5%, 1%, 1.5%, 2%, and 2.5% arginine supplementation levels were selected) on the ovarian development of Pacific white shrimp (Litopenaeus vannamei). The analyzed arginine supplementation levels in each diet were 2.90%, 3.58%, 4.08%, 4.53%, 5.04%, and 5.55%, respectively. A total of 540 shrimp (an initial weight of approximately 14 g) with good vitality were randomly distributed into six treatments, each of which had three tanks (300 L in volume filled with 200 L of water), with 30 shrimp per duplicate. Shrimp were fed three times a day (6:00 a.m., 11:00 a.m., and 6:00 p.m.). The results showed that after the 12-week raring cycle, shrimp fed with 4.08% and 4.53% Arg achieved better ovary development, which was identified by ovarian stage statistics, ovarian morphology observation, serum hormone levels (methylfarneside (MF); 5-hydroxytryptamine (5-HT); estradiol (E2); and gonadotropin-releasing hormone (GnRH)), gene expression (DNA meiotic recombinase 1 (dmc1), proliferating cell nuclear antigen (pcna), drosophila steroid hormone 1 (cyp18a), retinoid X receptor (rxra), and ecdysone receptor (ecr)). Further in-depth analysis showed that 4.08% and 4.53% Arg supplementation increased the concentration of vitellogenin in hepatopancreas and serum (p < 0.05) and upregulated the expression level of hepatopancreatic vg and vgr (p < 0.05), which promoted the synthesis of hepatopancreas exogenous vitellogenin and then transported it into the ovary through the vitellogenin receptor and further promoted ovarian maturation in L. vannamei. Meanwhile, compared with the control group, the expression level of vg in the ovary of the 4.53% Arg group was significantly upregulated (p < 0.05), which indicated endogenous vitellogenin synthesis in ovarian maturation in L. vannamei. Moreover, the expression of genes related to the mechanistic target of the rapamycin complex 1 (mTORC1) pathway and protein levels was regulated by dietary arginine supplementation levels. Arginine metabolism-related products, including nitric oxide synthase (NOS), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), were also affected. RNA interference was applied here to study the molecular regulation mechanism of arginine on ovarian development in L. vannamei. A green fluorescent protein (GFP)-derived double-stranded RNA (dsGFP) is currently commonly used as a control, while TOR-derived dsRNA (dsTOR) and NOS-derived dsRNA (dsNOS) were designed to build the TOR and NOS in vivo knockdown model. The results showed that the mTORC1 and NO-sGC-cGMP pathways were inhibited, while the vitellogenin receptor and vitellogenin gene expression levels were downregulated significantly in the hepatopancreas and ovary. Overall, dietary arginine supplementation could enhance endogenous and exogenous vitellogenin synthesis to promote ovary development in L. vannamei, and the appropriate dosages were 4.08% and 4.53%. The NO-sGC-cGMP and mTORC1 signaling pathways mediated arginine in the regulation of ovary development in L. vannamei.
摘要：
本研究旨在评估精氨酸(0.5%，1%,1.5%,2%,并选择了2.5％的精氨酸补充水平）对太平洋白对虾（凡纳滨对虾）的卵巢发育。每个饮食中分析的精氨酸补充水平为2.90%，3.58%,4.08%,4.53%,5.04%,5.55%，分别。共有540只具有良好活力的虾(初始重量约为14克)随机分配到6个处理中,每个都有三个水箱（容积为300升，装有200升的水），每个重复30只虾。每天喂虾三次(早上6点，上午11:00,下午6:00）。结果显示，经过12周的轮回，饲喂4.08%和4.53%Arg的虾卵巢发育较好,这是通过卵巢分期统计确定的，卵巢形态学观察,血清激素水平（甲基farneside（MF）;5-羟色胺（5-HT）;雌二醇（E2）;和促性腺激素释放激素（GnRH），基因表达(DNA减数分裂重组酶1(dmc1)，增殖细胞核抗原（pcna），果蝇类固醇激素1（cyp18a），类视黄醇X受体（rxra），和蜕皮激素受体（ecr））。进一步深入分析显示，添加4.08%和4.53%Arg增加了肝胰腺和血清中卵黄蛋白原的浓度（p<0.05），并上调了肝胰腺vg和vgr的表达水平（p<0.05）。促进了肝胰腺外源性卵黄蛋白原的合成，然后通过卵黄蛋白原受体将其转运到卵巢中，并进一步促进了凡纳滨对虾的卵巢成熟。同时,与对照组相比，4.53%Arg组卵巢中vg的表达水平明显上调(p<0.05),这表明凡纳滨对虾卵巢成熟过程中内源性卵黄蛋白原的合成。此外，与雷帕霉素复合物1（mTORC1）途径的机制靶标和蛋白质水平相关的基因表达受饲粮精氨酸添加水平的调控。精氨酸代谢相关产品，包括一氧化氮合酶（NOS），一氧化氮（NO），和环磷酸鸟苷（cGMP），也受到了影响。利用RNA干扰技术研究精氨酸对凡纳滨对虾卵巢发育的分子调控机制。绿色荧光蛋白（GFP）衍生的双链RNA（dsGFP）目前通常用作对照，而TOR来源的dsRNA（dsTOR）和NOS来源的dsRNA（dsNOS）被设计用于构建TOR和NOS体内敲低模型。结果表明,mTORC1和NO-sGC-cGMP通路受到抑制,而卵黄蛋白原受体和卵黄蛋白原基因表达水平在肝胰腺和卵巢中显著下调。总的来说,日粮补充精氨酸可促进南美白对虾内源和外源卵黄蛋白原的合成，促进卵巢发育，适宜剂量分别为4.08%和4.53%。NO-sGC-cGMP和mTORC1信号通路介导精氨酸调控凡纳滨对虾卵巢发育.
