赣昌鲫(日本白鲫♀×兴国红鲤♂)及其双亲同工酶的研究

采用不连续聚丙酰胺凝胶电泳法,研究了赣昌鲫(Carassius cuvieri♀×Cyprinus carpio var. singuonensis ♂)及其双亲的肌肉、肝脏、肾脏、心脏、脑、晶状体6种组织在超氧化物歧化酶(SOD)、苹果酸脱氢酶同工酶(MDH)、乳酸脱氢酶同工酶(LDH)、酯酶(EST)四种同工酶酶谱表达上的遗传差异。结果显示:赣昌鲫的SOD酶谱表达在6种组织中与母本一致;心脏、肝脏、肾脏的MDH表达酶带数与母本相同,且具偏母遗传特性,肌肉的MDH表达谱带数与父本肌肉相同,具偏父遗传特性;LDH、EST在各组织中的酶带表达形式则多数特征偏向于父本,部分与母本相似;LDH同工酶的表达显示出新杂种酶带特异性,EST同工酶在肾脏中也表达了自身特有的杂种酶带。结果表明,属间杂交的后代遗传物质上也存在着变异,存在可选育的性状。     

Effects of graded levels of dietary myo‐inositol on non‐specific immune and specific immune parameters in juvenile Jian carp (Cyprinus carpio var. Jian)

The aim of this study was to evaluate the effects of myo‐inositol (MI) on non‐specific immune and specific immune defence in fish. A total of 1050 Jian carp (Cyprinus carpio var. Jian) (22.28±0.07 g) were randomly distributed into seven groups, of three replicates each, of feeding diets containing graded levels of MI (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg MI kg^?1 diet). After a 60‐day growth trial, an infectious trial was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that the red blood cell (RBC) and the white blood cell count were significantly increased with increasing MI levels up to 535.8 mg kg^?1 diet (P<0.05). The spleen index showed a tendency similar to RBC, whereas the head kidney index showed the inverse pattern (P<0.05). The phagocytic activity of leucocytes, haemagglutination titre, lysozyme activity, anti‐A. hydrophila antibody titre and immunoglobulin M, after being injected with A. hydrophila, were all improved with an increase in the MI levels up to 232.7–687.3 mg kg^?1 diet respectively (P<0.05). Myo‐inositol did not influence serum acid phosphatase activity and total iron‐binding capacity (P>0.05). These results suggested that MI could enhance non‐specific immune and specific immune responses in fish.     

鲤疱疹病毒2型和3型三重PCR检测方法的建立与应用

鲤疱疹病毒2型和3型主要感染鲫(金)鱼(Carassius auratus)、鲤鱼(Cyprinus carpio)和锦鲤(Cyprinus carpio haematopterus)及其杂交种,具有高度的传染性和致病性,对养殖鲤科鱼类危害严重。为了建立快捷、高效且能同时检测这2种类型鲤疱疹病毒的检测技术,本研究根据鲤疱疹病毒的DNA聚合酶基因(2、3型)、解旋酶基因(2型)和胸苷激酶基因(3型)的保守序列,设计了3对特异性引物,通过对三重PCR的反应条件进行优化,建立起鲤疱疹病毒2型和3型三重PCR检测方法,并运用该方法对实验室保存的鲫鱼和鲤鱼组织样品进行检测。结果显示,该三重PCR检测方法仅在鲤疱疹病毒阳性样品中扩增出3条特异性条带,表现出良好的特异性;以克隆的目的基因质粒为模板,进行10倍梯度稀释,该检测方法能检出的极限值为2.85×102 copies/μL,表现出较高的灵敏性;运用该方法检测122份鲫鱼和60份鲤鱼样品,其结果与运用国家标准(GB/T 36194-2018)和行业标准(SC/T 7212.1-2011)方法检测的结果一致。本研究表明,构建的三重PCR检测方法不仅具有较高的准确性和灵敏度,还能同时检测2种类型的鲤疱疹病毒,有效提高检测效率。     

鲤trim25多拷贝基因进化和表达调控初探

天然免疫系统是硬骨鱼类抵抗病毒感染的主要防御系统,三重基序(tripartite motif, TRIM)蛋白家族作为天然免疫系统的重要组成部分,参与病毒感染的免疫网络调控,其中,TRIM25已被证实在多种鱼类的免疫反应中发挥重要作用。本研究对鲤(Cyprinus carpio) trim25基因的16个拷贝进行了序列进化分析、共线性分析和功能域结构分析,并比较了各拷贝在组织中的表达和顺式调控位点的差异。序列比对和系统进化分析结果均显示,位于鲤11和12号染色上、结构完整的TRIM25的2个拷贝与金线鲃(Sinocyclocheilus grahami)和斑马鱼(Danio rerio)的TRIM25蛋白结构高度相似,与鲤科鱼类以外的其他物种的结构差异较大。基因共线性结果显示,trim25基因上下游基因在不同物种的进化过程中相对保守。鲤TRIM25蛋白的结构分析显示,在鲤TRIM25的16个拷贝中,有6个拷贝具有完整功能结构域,其中,各有5个拷贝在鲤的肝与脑组织中高表达。在构建的表达数量性状基因座(eQTL)调控网络中,在肝和脑组织中分别筛选到5个和17个顺式调控trim25基因表达的单核苷酸多态性(SNP)位点。本研究对鲤trim25基因多个拷贝的序列差异进行了比较,并对鲤与其他物种TRIM25的序列、进化关系和共线性相似度进行了比较,揭示了鲤trim25各拷贝间的结构多样性和在组织中的表达情况,筛选出了可能调控trim25基因表达的SNP位点,为今后研究鲤TRIM25相关的调控和抗病研究提供了理论依据。     

Koi herpesvirus and carp edema virus threaten common carp aquaculture in Croatia

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.     

Effects of dietary zinc sources and levels on growth performance,tissue zinc retention and antioxidant response of juvenile common carp (Cyprinus carpio var. Jian) fed diets containing phytic acid

Plant protein‐based diets are widely used for common carp (Cyprinus carpio var. Jian). The plant source antagonist, such as phytate, negatively affects the bioavailability of trace minerals, but this aspect was not considered in previous studies on this species. A 10‐week growth trial was conducted to investigate the effects of inorganic Zinc (ZnSO₄·7H₂O, Zn‐S) and organic Zn (2‐hydroxy‐4‐(methylthio) butanoic Zinc, Zn‐M) on growth performance, Zn retention and antioxidant responses of common carp (initial weight 10.0 ± 0.03 g). Eleven semi‐purified diets containing 7 mg/kg phytic acid were prepared with 0, 5, 20, 35, 50 and 65 mg/kg Zn from Zn‐S and Zn‐M (12.8 mg/kg Zn in basal level), respectively. The results showed that either Zn‐S or Zn‐M inclusion improved growth performance, vertebral Zn deposition and antioxidant responses of fish. The Zn requirement based on WGR, vertebrae Zn and plasma SOD were 47.0, >77.8 and 48.5 mg/kg, and 63.3, 42.2 and 36.8 mg/kg, respectively, with Zn‐S and Zn‐M as Zn sources. The relative bioavailability of Zn‐M was 2.44‐ (vertebrae Zn deposition) and 1.74‐fold (plasma SOD activity) the availability of Zn‐S in the diet of common carp. The lower growth performance and poor bioavailability in vertebrae of Zn‐S group were related to the chelating of zinc with phytic acid.     

Study on the adaptability of common carp (Cyprinus carpio) to diets from the perspective of digestion and absorption

As an ecologically sustainable aquaculture mode, the rice‐fish approach has been paid more attention in recent years. In rice paddies, there are plant and animal diet items available to fish, but it is not clear how common carp adapt to different diets. In view of this, common carp (initial weight 492.6 ± 30 g, n = 270) were randomly divided into three groups and fed with earthworms (group A), earthworms + duckweed (group M) and duckweed (group P) respectively. After 8 weeks under these feeding regimes, the intestinal digestive enzymes activities were assayed, and intestinal tissue sections stained with HE and AB‐PAS were used to observe the morphology. The results showed that the activities of trypsin and lipase were highest in group A, followed by group M and group P. Group A had greater intestinal fold height, fold width and fold absorption area than the other groups (p < .05), especially in the foregut. Meanwhile, the total number of mucous cells was largest in group P, followed by the groups M and A. Totally, activities and distributions of digestive enzymes, fold height, fold width, fold absorption area and number and distribution of mucous cells of common carp were affected by the type of diet. Based on these results, it was found that common carp has strong adaptability to diets, and it was speculated that the majority of digestion and absorption of protein was concentrated in the foregut, while for starch and cellulose, it was spread out along the whole intestine.     

饲料中n-3/n-6多不饱和脂肪酸水平对黄河鲤幼鱼生长性能及生长相关基因mRNA表达的影响

为了分析饲料n-3/n-6多不饱和脂肪酸(PUFAs)水平对黄河鲤幼鱼生长性能和生长相关基因的影响,实验以鱼油和混合植物油(花生油和紫苏籽油)为脂肪源配制5组等氮等能饲料。对照组(T1)以鱼油为唯一脂肪源,18:3 n-3/18:2 n-6为0.97,第二组(T2)以花生油为唯一脂肪源,18:3 n-3/18:2 n-6为0.02,其他3组实验饲料以花生油和紫苏籽油为脂肪源,且n-3/n-6比值分别为0.46(T3)、1.09(T4)和1.53(T5)。10周养殖实验结束后,分析各组鱼体的生长性能、血清中生长激素(GH)含量及不同部位肌肉中生长相关基因的表达水平。结果显示,与对照组相比,n-3/n-6对鱼体的脏体比(VSI)、饲料系数(FCR)、摄食率(FI)无显著影响,增重率(WGR)随n-3/n-6比值的增加先升后降,n-3/n-6比值等于1.09时,WGR达到峰值,为218.53%±24.32%。T2组血清GH含量显著高于其他处理组,随着n-3/n-6比值增大GH含量逐渐降低。背部肌肉中生长相关基因gh、ghr、igf-1和myod均表现为先升后降的趋势,igf-1r为先降后升,mstn无显著差异。红肌中gh、myod含量逐渐降低,ghr、mstn不存在显著差异,igf-1与背部肌肉相似,先升后降,igf-1r先降后升。腹部肌肉中gh、ghr和igf-1表达情况与背部肌肉一致,igf-1r和myod的表达不存在显著差异。T2组和T4组背部肌肉肌纤维数目存在显著差异,且T4组肌纤维较T2组粗。研究表明,n-3/n-6不仅在一定程度上提高鱼体的WGR,且不同比例的n-3/n-6水平会影响血清GH的含量,不同部位肌肉组织中GH/IGF轴生长相关基因的水平也受饲料中n-3/n-6比例的影响。     

Use of supplemental amino acids on the success of reducing dietary protein levels for Jian carp (Cyprinus carpio var. Jian)

A feeding trial was conducted to investigate the effects of essential amino acids (EAA) supplementation on the success of dietary protein reduction for Jian carp (Cyprinus carpio var. Jian). Five diets were formulated including a positive control (Diet 1, 378.4 g/kg protein), a negative control (Diet 5, 289.8 g/kg protein) and three test diets (Diet 2‐Diet 4, 340.4–299.0 g/kg protein supplemented with EAA). No significant decline of growth performance in fish fed decreased dietary protein with EAA supplementation, which were significantly higher than the negative control. The apparent digestibility coefficients (ADC) of protein and most EAAs were not significantly affected by the dietary treatments. Dietary treatments did not affect whole‐body composition, but influenced protein and EAAs retentions. Total ammonia nitrogen (TAN) excretion decreased with the decreasing dietary protein, and the highest plasma ammonia content was observed in fish fed Diet 5. Plasma glucose concentration increased with the decreasing dietary protein reduction, while other plasma parameters were not affected by dietary treatments. These results indicated that low‐protein diets (299.0 g/kg) supplemented with EAA can maintain protein, EAA digestibility and retention in Jian carp, and also has benefit in the reduction of TAN excretion.     

Effects of antimicrobial peptides on growth,morphology of foregut villi and related genes mRNA expression in the common carp (Cyprinus carpio)

A 60‐day feeding trial was conducted to examine the effects of different levels (0, 100, 200, 400 and 600 mg/kg) of antimicrobial peptides on growth, protease activity of foregut, the morphology of foregut villi and related genes mRNA expression level in the common carp (Cyprinus carpio). The results showed that the feed of antimicrobial peptides promote common carp growth, and the optimal dosage of antimicrobial peptides is 200–333 mg/kg in the common carp feed. The protease activity of 200 and 400 mg/kg groups were significantly higher than the control and other groups (p < 0.05). The foregut villus height with 100, 200 and 400 mg/kg antimicrobial peptide groups were significantly higher than control group (p < 0.05). The crypt depth of 200 and 400 mg/kg antimicrobial peptide groups were significantly lower than control group (p < 0.05). The ratio of villus height and crypt depth of 100, 200 and 400 mg/kg antimicrobial peptide groups were significantly higher than control group (p < 0.05). The ratio with 600 mg/kg group was significantly lower than the control group (p < 0.05). The IGF‐I gene expression level of 200 mg/kg and 400 mg/kg groups were significantly higher than the control group and 600 mg/kg group (p < 0.05). The IL‐1β gene expression level of 100 mg/kg and 200 mg/kg groups were significantly higher than the control group (p < 0.05). These results indicated up‐regulation of growth and immune related genes in antimicrobial peptides fed common carp. Correlation analysis showed that IGF‐I mRNA and IL‐1β mRNA were positively correlated with SGR. IL‐1β mRNA and FCR were significantly negative correlated. It indicated that growth and immune gene common regulated the growth of the carp under antimicrobial peptides intervention. In conclusion, antimicrobial peptides can improve growth and related genes mRNA expression in the common carp. Further studies using molecular biological technique or immunologic methods are required to conclude that antimicrobial peptides are beneficial in common carp.