雌性特异蛋白生物活性的研究

本实验采用蟾蜍离体跌卵法和鱼类腹腔注射法证明雌性特异蛋白有促进鱼类性腺发育的作用。     

史氏鲟及小体鲟不同组织中溶菌酶水平的比较

对史氏鲟及小体鲟血清和几种主要组织的溶菌酶含量进行了测定和比较后发现,同小体鲟相比,史氏鲟的血清、鳃、肝、胃、前肠及后肠的溶菌酶量均较高,血清之间有极显著的差异,鳃、肝及后肠均有显著性的差别,胃及前肠之间无差异;小体鲟的粘液、幽门垂及脾中的溶菌酶含量较史氏鲟高,但无显著性差异,由此可见,虽然同为鲟科鲟属,溶菌酶在两种鱼之间的差别还是很显著的。     

小体鲟血清卵黄蛋白原和 Ca2+浓度与卵巢发育的关系

采用ELISA方法、分光光度计法和组织学方法,系统研究了不同年龄小体鲟(Acipenser ruthenus)血清卵黄蛋白原(Vitellogenin Vg)、血清钙离子(Galcium Ca2+)含量与卵巢发育变化的关系.结果表明:血清Vg浓度、Ca2+浓度与卵巢的发育时期相关;在卵黄沉积期内,血清中Vg浓度与Ca2+浓度呈线性正相关(R2=0.926 4);卵黄生成前期和沉积初期,血清中Vg浓度和Ca2+含量较低;卵黄沉积期内,卵母细胞由沉积初期的0.56 mm 增大到卵黄沉积末期的2.0l mm,血清Vg质量浓度由110.65 μg/mL 上升到242.22 μg/mL,血清Ca2+浓度由2.34 mol/L增大到2.72 mol/L.对同年龄的雌、雄小体鲟血清Vg浓度和Ca2+浓度测定表明,根据血清中Vg和Ca2+含量差别,可以区分出沉积期小体鲟的性别.     

雌性特异蛋白复合物促鱼类性腺发育及提高孵化率机制的研究

本实验系用生物化学方法从鲤鱼性腺中提取出一种雌性特异蛋白复合物,氨基酸分析发现该复合物含有全部必需氨基酸;将该蛋白复合物注射到雌性大阪鲫鱼体内,发现鱼的血清中蛋白质含量增加,同时对大阪鲫鱼肝脏分析发现核糖核酸(RNA)含量也增加。     

水温对施氏鲟、小体鲟和西伯利亚鲟幼鱼生长的影响

在电子控温循环水系统中,研究不同水温(15℃、18℃、21℃、24℃和27℃)对56日龄的施氏鲟Acipenser schrenckii、小体鲟A.ruthenus和西伯利亚鲟A.baerii幼鱼生长的影响。结果表明,24℃组施氏鲟幼鱼的体长特定生长率(SGRL)显著高于其它温度组(P<0.05),而体质量特定生长率(SGRW)差异不显著(P>0.05);在18~27℃范围内,体长绝对增长率(AGRL)随温度的升高而降低,且显著高于15℃组(P<0.05)。18~24℃组的体质量绝对增长率(AGRW)显著高于15℃和27℃组(P<0.05)。在18~24℃范围内,施氏鲟幼鱼的体长相对增长率随温度的升高而增加,显著高于15℃和27℃组(P<0.05),最适生长温度为24℃。18℃组小体鲟幼鱼的SGRL显著高于其他温度(P<0.05),SGRW则差异不显著(P>0.05)。在15~27℃温度范围内,小体鲟幼鱼的AGRW升高后降低,其中18℃和24℃的SGRW分别为0.61g/d和0.64g/d,显著高于其他组。15℃组AGRL最低(1.6mm/d),显著低于其他组(P<0.05)。小体鲟幼鱼的最适生长水温范围为18~24℃,最适生长温度为18℃。在15~27℃范围内,21℃组西伯利亚鲟幼鱼的SGRL、AGRL和体长相对生长率均显著高于其他温度组(P<0.05),但27℃组存活率最低。西伯利亚鲟幼鱼的最适生长范围为15~24℃,最适生长水温为21℃。不同温度下,3种鲟幼鱼的肥满度变化不同,施氏鲟幼鱼的生长类型为强异速度生长,不同于小体鲟幼鱼与西伯利亚鲟幼鱼。在15~27℃范围内,3种鲟幼鱼的最适生长温度不同,在早期生长培育时应选用不同的培育温度。     

小体鲟染色体核型及倍性的细胞遗传学研究

采用体内注射PHA和秋水仙素,肾细胞短期培养,常规空气干燥法制备小体鲟(Acipenser dabryanus Dumerii)的染色体,对肾细胞染色体数目统计分析表明,小体鲟染色体组是由116±4条染色体组成,核型公式为40m+34sm+26st+16t±mc,NF 190±;采用德国Partec pasⅢ型流式细胞分析仪,以鸡红细胞为标准DNA(含量为2.3pg/N),测定小体鲟体细胞DNA含量为6.06 pg/N,与染色体分析结果比较倍性一致.综合以上两项结果可以得出,小体鲟为四倍体鲟鱼.     

西伯利亚鲟和小体鲟摄食前后耗氧率和窒息点变化与体重的关系

在水温22~25℃下,测定了不同体质量的西伯利亚鲟Acipenser baerii Brandt和小体鲟Acipenser ruthenus linnaeus在饱食和空腹情况下的耗氧率和窒息点。结果表明:西伯利亚鲟和小体鲟摄食后耗氧率与体质量关系的变化趋势与空腹情况时相同,均随体质量的增加而下降,饱食耗氧率与空腹耗氧率的比值随体质量的增加也下降,饱食与空腹西伯利亚鲟平均耗氧率与平均体质量的相关关系式分别为:Q=0.809*W-0.2414(0.179相似文献   

恩诺沙星对小体鲟和史氏鲟体内SOD活力影响的比较研究

在水温23℃、pH值6．5、溶氧10mg／L、氨氮含量小于0．1mg／L和亚硝酸盐含量小于0．001mg／L的条件下,将恩诺沙星按照0、20、40、60、80和100mg／kg浓度,连续口服给平均体重50．0±5．0g的小体鲟（Acipenser ruthenus Linnaeus）及史氏鲟（Acipenser schrenckii Brandt）5d,停药2d后测定其血浆及肝脏组织中过氧化物岐化酶SOD的活力,以期掌握不同恩诺沙星给药浓度下,两种鲟两种组织中SOD活力变化趋势,探讨分析该酶在药物代谢过程中的作用机制。结果表明：两种鲟两种组织内均含有一定量的SOD酶,且在对照组及所有给药组肝脏中酶活力均高于血浆中。不同给药浓度下,两种鲟两种组织中SOD活力均先受诱导升高,而后被抑制降低的变化趋势,且在40mg／kg浓度组达到最大值。血浆中SOD活力受给药浓度影响较小,起伏较平稳,而小体鲟SOD活力始终高于史氏鲟。肝脏中SOD活力变化较剧烈,在低浓度组（〈40mg／kg）史氏鲟肝脏中SOD活力明显高于小体鲟,而在高给药浓度组（〉40mg／kg）则小体鲟略高于史氏鲟。     

大阪鲫鱼血清雌性特异蛋白的鉴定

鱼类是在卵母细胞外合成卵黄的动物，具体说是在肝脏中合成的，经过血液循环输送到卵母细胞，首先在肝脏中合成的是卵黄蛋白的前体——卵黄蛋白原(Vitellogenin)，卵黄蛋白原在雌激素作用下，雌体血清中就会出现一种雌性特异蛋白成分，K．Aida(1973)称此蛋白为血清雌性特异蛋白。近年来，作者在鱼类血液分析方面的工作证明，雌雄鱼血清存在差异是普遍的，并利用醋酸纤维薄膜电泳对雌性特异蛋白化学特性进行了鉴定。     

人工养殖施氏鲟和小体鲟精浆微量元素分析

采用原子吸收分光光度法测定了施氏鲟(Acipenserschrenckii)和小体鲟(Acipenserruthenus)的精浆中7种微量元素(K、Na、Mg、Zn、Cu、Fe、Mn)的含量。施氏鲟分别取自宜昌和荆州,小体鲟取自宜昌。分析结果显示,不同种类和产地的鲟精浆中微量元素含量有差异,其中2条不同产地施氏鲟精浆Zn含量有显著性差异(P<0.05);施氏鲟和小体鲟精浆Na、K含量与我国主要淡水养殖鱼类有很大差异,在2种鲟精浆中Na与K浓度之比为10～14。     

Induced spermiation in 3-year-old sterlet, Acipenser ruthenus L.

Spermiation in 3‐year‐old sterlet, Acipenser ruthenus (L.), males maintained under warm water conditions was induced by intramuscular injection of either (i) (D‐Ala⁶)‐GnRH‐ProNHEt (Kobarelin); (ii) mammalian GnRH analogue+metoclopramide (Ovopel); or (iii) human chorionic gonadotropin (Biogonadyl). The volume of milt, sperm concentration and motility were measured. A higher percentage of spermiating males was obtained after Kobarelin or Ovopel treatment (81.8% and 77.7% respectively) in comparison with fish treated with Biogonadyl (40.0%). However, only stimulation with Ovopel guaranteed motile spermatozoa in all spermiating males. Moreover, treatment with Ovopel resulted in the highest average milt volume, sperm concentration and motility. The average total number of motile spermatozoa (milt volume×sperm concentration×sperm motility) per individual was 0.99×10⁹, 5.31×10⁹ and 0.02×10⁹ after stimulation with Kobarelin, Ovopel or Biogonadyl respectively. Our data indicate that Ovopel is a good stimulator of spermation in sturgeons. Moreover, the time of sexual maturation could be significantly reduced in sturgeons maintained in cages under warm water conditions.     

Studies on the semen biology and sperm cryopreservation in the sterlet, Acipenser ruthenus L.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg^?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg^?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg^?1, completely and irreversibly. In 50 mosmol kg^?1 solutions with 2.5–5 mM L^?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L^?1 NaCl, 5 mM L^?1 KCl, 10 mM L^?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.     

小体鲟AR基因克隆、序列分析及其组织mRNA表达

采用RT-PCR 和RACE技术, 克隆了小体鲟(Acipenser ruthenus)雄激素受体(AR)基因cDNA全长序列, 应用real-time qPCR法对雌、雄个体不同组织中AR mRNA 的表达进行检测。序列分析表明, AR基因cDNA全长为2 953 bp, 包括5′端68 bp的非编码区(untranslation region, UTR), 2 528 bp的开放阅读框ORF(open reading frame)和3′端327 bp的非编码区(不包括ploy A 的尾巴)。AR前体蛋白由843个氨基酸组成, 理论分子质量为94.24 kD。同源比较结果表明, 小体鲟AR氨基酸序列与其他鱼类的AR氨基酸序列同源性较高, 与杂交鲟(A. ruthenus × Huso huso)、西伯利亚鲟(A. baerii)、虹鳟(Oncorhynchus mykiss)和日本鳗鲡(Anguilla japonica)的雄激素受体同源性分别为99%、98%、83%和83%。real-time qPCR分析小体鲟雌雄个体不同组织中的表达结果显示, AR基因在雌雄个体中相对表达基本相同, 都是在性腺、肌肉和肾中高量表达, 而在其他组织中少量表达。AR基因在小体鲟体内广泛表达, 表明该基因可能对其生长和繁殖有重要作用。

     

中华鲟幼鲟蛋白质营养最适需要量

为确定中华鲟 (Acipensersinensis)幼鲟最佳生长的饲料蛋白质适宜含量 ,按单因子梯度设计原理 ,设计 5种不同蛋白质水平配方的饲料 ,分别投喂中华鲟幼鲟 ,测定中华鲟幼鲟体重、体成分 ,同时用外源性指示剂 (Cr2 O3)测定饲料中干物质和粗蛋白的表观消化率 ,比较其测定结果。结果表明 ,饲料蛋白质质量分数为 4 4 .18%时 ,中华鲟的生长效果最好 ;以增重率为指标 ,通过直线回归和抛物线回归推算出中华鲟蛋白质需要的适宜质量分数为4 0 .0 0 %～ 4 3 .0 1%。     

Short‐term physiological role of thyroxine administration on plasma metabolites and growth performance of Sterlet sturgeon Acipenser ruthenus

Thyroid hormones (THs) play important roles in regulating growth, development and physiological functions in vertebrates. To study the role of short‐term effects of thyroxine (T4) on metabolism and growth in female Sterlet sturgeon, thirty fish with a weight of 707.97 ± 37.15 g were divided into six tanks. Fish were injected intraperitoneal with (a) coconut oil alone (control), (b) 1 mg T4 kg per BW in coconut oil (T₁) and (c) 10 mg T4 kg per BW in coconut oil (T₁₀). Blood samples were collected at 0, 4, 7, 14, 28 and 60 days for plasma biochemical analysis, and body length and mass were determined at day 60. Based on results, higher growth was indicated in T₁₀ group. The highest T4 levels were observed on day 4 in the T₁ and T₁₀ treatments and gradually declined during the trial. Plasma T3 levels were highest on days 4 and 7 in the T₁ and T₁₀ groups, respectively. Plasma triglyceride levels were significantly elevated on day 28 in the T₁ group and on day 60 in the T₁₀ group. Plasma glucose concentrations were significantly elevated on days 7 and 14 in T₁₀ group. Overall, the present results suggest that a single injection of T4 can improve growth via changes in the metabolic profile of Sterlet sturgeon.     

In vitro oocyte maturation and blood‐circulating steroid hormones in Sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baeri)

The oocyte of Sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baeri) was evaluated after incubation with 17α, 20β‐Dihydroxyprogesterone (DHP) in artificial media. Concentration of testosterone (T), progesterone (P4) and 17‐β estradiol (E2) in blood and their relation to fertilization rate were measured during experimental period. The oocytes at the same developmental stages were incubated in SIS (based on sturgeon blood ionic composition), RM2 (Ringer solution modified for sturgeons) and L‐15 (Leibovitz medium) artificial mediums in the presence of 1 μg/ml of 17α, 20β‐Dihydroxyprogesterone for 12, 18 and 24 hr. The result of this study demonstrated that fertilization rate decreased with the increasing duration of incubation in all mediums. In the Siberian and Sterlet sturgeon, the highest fertilization rate was observed in the oocytes incubated in RM2 (0.81 ± 0.4) and SIS (0.44 ± 0.8) mediums for 12 hr respectively. In the Siberian sturgeon, fertilization rate decreased significantly (p < .05) with incubation time in all three mediums. In Starlet, fertilization rate decreased significantly (p < .05) with time and reached a minimum after 24 hr (0.16 ± 0.1) in L‐15 medium. No significant (p > .05) differences were observed between blood plasma hormones and fertilization rate. The results of this study indicated that 17α, 20β‐Dihydroxyprogesterone is essential for induction of in vitro oocyte maturation in these species.     

中华鲟神经内分泌多肽7B2基因克隆及组织特异性表达

以中华鲟(Acipenser sinensis)脑垂体总RNA为模板,采用RT-PCR和RACE方法,获得中华鲟神经内分泌多肽(7B2)基因的3个重叠片段,测序后拼接得到986 bp全长基因序列,其中包括5'端非翻译区(5′-UTR)14 bp、3′端非翻译区(3-′UTR)261 bp和开放阅读框711 bp。翻译编码236个氨基酸。其中前43个氨基酸为7B2的信号肽。经BLAST比对发现中华鲟7B2蛋白的同源性与斑马鱼(Danio rerio)的相似性最高为82%。系统发育分析表明,中华鲟与斑马鱼亲缘关系最近。半定量RT-PCR分析表明:在脑中7B2 mRNA表达量最高,心脏、性腺、胰等组织中表达次之,肠、肾、皮肤等组织中少量表达,肝和鳃几乎不表达。     

鲟源致病性鲁氏耶尔森菌的分离、鉴定及药敏研究

2017年,新疆阜康市某鲟鱼养殖场的西伯利亚鲟出现大批死亡,主要症状为生殖孔红肿外突,腹部有点状出血。自患病西伯利亚鲟肝脏、脾脏分离到一株优势菌株XB2,通过革兰氏染色、生理生化试验、药敏试验,并对其16S rRNA基因序列进行测定,用Mega 6.0对序列进行分析。生理生化试验结果显示,菌株XB2为革兰氏阴性短杆菌,单独或成对出现;产酸不产气,V-P反应呈阳性,能利用蔗糖、果糖、葡萄糖、明胶、枸橼酸;在含1%(m/V)氯化钠中能生长,高于3%(m/V)氯化钠中不生长;在4℃不生长,20～30℃均能生长,但在37℃不生长;不利用乳糖、水杨苷、纤维二糖、硫化氢、酒石酸、阿拉伯糖。进化树显示,菌株XB2与鲁氏耶尔森菌KJ812974同源性达99%。通过回感试验确定其半致死密度为2.37×104 cfu/mL。结合生理生化鉴定结果和16S rRNA基因序列分析结果,确定菌株XB2为鲁氏耶尔森菌。药敏结果显示,鲁氏耶尔森菌XB2对氟苯尼考、左氧氟沙星、哌拉西林、阿洛西林等9种药物敏感,对麦迪霉素、大观霉素、链霉素、氨苄西林等15种药物耐药。     

俄罗斯鲟鱼卵与西伯利亚鲟鱼卵的营养成分比较

采用生化方法分析了俄罗斯鲟(Acipenser gueldenstaedti)和西伯利亚鲟(Acipenser baerii)鱼卵的营养成分,并对两者的营养品质进行了比较。结果表明:这两种鱼卵的水分含量平均值为62.83%～64.88%,粗蛋白平均含量为20.38%～20.77%,粗脂平均含量为10.41%～13.58%。统计分析表明,俄罗斯鲟鱼卵的粗脂肪含量显著低于西伯利亚鲟鱼卵,而粗蛋白、水分和灰分含量略高于西伯利亚鲟鱼卵。俄罗斯鲟鱼卵和西伯利亚鲟鱼卵的氨基酸组成一致,均含有17种氨基酸,必需氨基酸指数(EAAI)分别为50.66和44.43。两种鲟鱼卵的脂肪酸中,均以C16∶0、C18∶2n6c和DHA为主要脂肪酸,三者总的质量分数达50%左右;而二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的总量俄罗斯鲟鱼卵高于西伯利亚鲟鱼卵,在两种鱼卵中含量分别为19.47%和16.83%。分析结果表明,两种鲟鱼卵均含有丰富的各种营养成分,但从粗蛋白、必需氨基酸(EAA)、DHA和n3PUFA含量来看,俄罗斯鲟鱼卵的营养价值优于西伯利亚鲟鱼卵。