四种渔药对稀有(鱼句)鲫的急性毒性

用控温半静态换水式实验方法,测定了伊维菌素(0.4%)、辛硫磷(20%)、氯氰菊酯(4.5%)和精制马拉硫磷(20%)四种常用药物对稀有(鱼句)鲫(Gobiocypris rarus)幼鱼的急性毒性效应。分别在实验24、48、72 h和96 h后记录稀有(鱼句)鲫幼鱼的死亡数,并推算了实验药液的安全质量浓度。结果显示,四种渔药的96 h半致死浓度分别为7.01、24.22、9.37和460μg/L,安全浓度分别为0.70、2.42、0.94和46.00μg/L,这4种药物对稀有(鱼句)鲫的毒性大小依次为:伊维菌素>氯氰菊酯>辛硫磷>精制马拉硫磷。结果表明:稀有(鱼句)鲫可用于渔药对水体环境影响的监测。     

圆口铜鱼精子超低温冷冻保存

为建立圆口铜鱼(Coreius guichenoti)精子超低温冷冻保存方法,采用计算机辅助精子分析系统(CASA)评价了4种稀释液(D15、D20、L1、D1), 3种抗冻保护剂[二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、甲醇(METH)]在3种体积浓度(7.5%、10%、12.5%, V/V)下对圆口铜鱼精液的冷冻保存效果,并比较了150 mOsm/kg NaCl与超纯水作为激活剂对精子活力的影响。结果表明,D1组稀释液的精子活率(MOT)最高,为(74.64±13.17)%,与鲜精无显著差异(P0.05);以10%甲醇作为抗冻保护剂的圆口铜鱼精子经超纯水激活后,测得MOT最高,达到(78.11±14.74)%,与鲜精无显著差异(P0.05),精子运动速度达最大,平均曲线运动速度(VCL)、平均直线运动速度(VSL)、VAP(平均路径运动速度)分别达到(50.28±12.46)μm/s、(35.06±10.82)μm/s、(39.44±12.46)μm/s,精子快速运动时间和寿命分别为(8.67±1.15) s、(33.33±5.00) s,显著低于鲜精(P0.05); 7.5%甲醇组和7.5%二甲基甲酰胺组的MOT次之,分别为(77.71±17.74)%、(76.42±12.49)%,抗冻剂二甲基亚砜组的MOT显著低于甲醇组、二甲基甲酰胺组(P0.05)。12.5%的3种抗冻保护剂中,几乎无精子存活;在不同种类不同浓度的抗冻剂保护下,10%甲醇组,相较于使用超纯水激活,用150 mOsm/kg NaCl激活后的精子活率和寿命更高,说明Na~+有延长冻精寿命,提高精子活率的作用。研究表明, D1+10%METH (7.8 g/L NaCl+0.5 g/L KCl+15 g/L葡萄糖+10%METH)可用于圆口铜鱼精液超低温冷冻保存,为圆口铜鱼的繁育工作和种质资源保护奠定了基础。     

三倍体和二倍体银鲫精巢组织学的比较

通过光学显微镜对染色体数为150±的三倍体和染色体数为100的二倍体银鲫(Carassius ouratus gibclio Bloch)的精巢组织学结构进行了比较观察.三倍体和二倍体银鲫的精巢组织学结构基本相同,属于小叶型,都是由外膜和实质构成,各精小叶呈辐射状分布,一个小叶由数个精小囊组成.其精原细胞存在于精小叶内壁上,精母细胞和未成熟的精子细胞位于精小囊中,精子成熟后从精小囊进入小叶腔.三倍体银鲫成熟精子的体积为(11.8±2.8)μm3,二倍体平均为(6.8±1.8)μm3,二者的比例接近3:2.结果表明三倍体银鲫的精巢能够发育成熟,其精子发生过程正常,经过减数分裂,能产生正常的精子;因此三倍体的黑龙江银鲫是具有双倍性特征的多倍体群体.     

刀鲚精子超微结构研究

在光学显微镜及透射电子显微镜下观察刀鲚精子超微结构,并对精子各部分长度进行测定。研究表明,刀鲚精子由头部、中段和尾部(鞭毛)组成。头部在光学显微镜下近梭形,透射电镜下纵切则近圆形。头部无顶体,由细胞核组成,核内染色质致密,空隙少,几乎无细胞质。头部后端偏一侧处有一植入窝,内有中心粒复合体。精子中段位于核后端,由中心粒复合体和袖套组成。中心粒复合体由近端中心粒和远端中心粒组成,两者基本位于一直线上。袖套肥厚的一侧有较多的线粒体和囊泡。尾部分为主段和末段,无侧鳍。主段具典型的9+2的轴丝结构,末段很短,无典型轴丝结构。通过光学显微镜测定,精子头部为(2.34±0.16)μm,中段(1.49±0.18)μm,头宽(1.29±0.21)μm,尾部长(34.07±4.31)μm,全长(37.77±4.21)μm。     

中华倒刺鲃、白甲鱼和岩原鲤精子的生理特性比较

对中华倒刺鲃(Spinibarbussinensis)、白甲鱼(Onychostomasimus)和岩原鲤(Procyprisrabaudi)3种鱼的鲜精在不同水溶液和不同浓度梯度的NaCl溶液中的活力以及精子形态、密度和精液pH值、浓度等进行了比较研究。结果表明:3种鱼的精液pH值都偏弱碱性,位于7·2~7·7之间;精液浓度依次为71·1%,76·1%和71%;精子头长依次为(3·89±0·53)μm,(2·98±0·08)μm和(6·42±0·59)μm,全长依次为(41·63±3·66)μm,(65·67±2·97)μm和(58·89±5·25)μm;精子密度依次为1·527×1010尾/mL,1·336×1010尾/mL和1·362×1010尾/mL。分析表明,3种鱼的精子大小与密度无相关性。在不同水溶液中,3种鱼的精子活力均以池塘水中最高;在不同浓度NaCl溶液中,3种鱼精子的最适浓度位于0·45%~0·55%之间,有效运动时间以中华倒刺鲃最高,为(85·23±12·02)s,其次是岩原鲤,为(50·45±6·89)s,白甲鱼最短,为(31·44±5·53)s。3种鱼的精子全长与精子活力存在负相关性,即精子越长,活力越低,精子越短,活力越高。     

棘头梅童鱼精子超低温冷冻保存研究

为保护和利用棘头梅童鱼种质资源,以常用无机盐及葡萄糖配制的5种溶液(依次编号A、B、C、D、E)作为稀释液,不同体积分数的DMSO作为抗冻剂,采用2 mL冻存管和两步降温的方式,对棘头梅童鱼精子的超低温冷冻保存技术进行了研究,并利用人工养殖黄姑鱼的成熟卵子对冻存3年的棘头梅童鱼冷冻精子的授精能力进行检验。结果表明,以E溶液为稀释液、10%DMSO为抗冻剂、两步降温方式冷冻保存的棘头梅童鱼精子在37℃水浴解冻后复活率较高,为(76.67±10.41)%^82.33±4.62%;以上述方法冻存3年的棘头梅童鱼冷冻精子与人工养殖黄姑鱼的成熟卵子杂交,受精率达到(20.26±4.12)%。     

紫外辐射灭活大黄鱼和黄姑鱼精子及其激活大黄鱼卵子发育为胚胎的效果

为探讨大黄鱼(Larimichthys crocea)和黄姑鱼(Nibea albiflora)精子的紫外辐射灭活适宜剂量及其激活大黄鱼卵子发育为胚胎的效果,以Ringer氏液为稀释液,按1:30稀释大黄鱼和黄姑鱼精子,采用自制紫外灭活装置[紫外辐射强度2200μW/(cm~2·s),紫外波长254 nm]对这2种鱼的精子进行紫外照射处理及活力测定,然后与正常大黄鱼卵子进行人工授精,授精后一部分卵未作冷休克处理,另一部分卵进行了冷休克处理(受精2 min 30 s,3℃海水,冷休克10 min),并进行了早期胚胎成活率和仔鱼孵化率的测定与比较。结果显示:1)大黄鱼和黄姑鱼精子的激活率与紫外照射处理时间呈负相关,精子的快速运动时间变化呈典型的Hertwig效应。2)未冷休克组中大黄鱼和黄姑鱼诱导的早期胚胎成活率与精子的紫外照射时间总体呈负相关,而仔鱼孵化率呈Hertwig效应。3)冷休克组中大黄鱼和黄姑鱼精子诱导的早期胚胎成活率和仔鱼孵化率随紫外照射时间的增加呈Hertwig效应,分别于2 min 20 s和1min 30 s时达相对峰值,此时,大黄鱼早期胚胎成活率和仔鱼孵化率分别为(38.3±4.3)%和(66.5±5.1)%,黄姑鱼早期胚胎成活率和仔鱼孵化率分别为(43.3±3.3)%和(67.7±6.3)%。分析认为,大黄鱼和黄姑鱼精子遗传失活的紫外辐射剂量分别以308 m J/cm~2和198 m J/cm~2为佳。本研究旨在为大黄鱼雌核发育技术的改进提供依据。     

白梭吻鲈精子超微结构及K+、Ca2+、葡萄糖对其活力的影响

为了探究白梭吻鲈(Sander lucioperca)精子的结构及活力特点,采用扫描电镜观察其超微结构,并将其置于不同浓度的KCl、CaCl2和葡萄糖溶液中,在光镜下观察不同溶液对其活力的影响。结果显示:白梭吻鲈精子由头部、中段和尾部3个部分组成,其头部接近于圆形,长约1.8μm,宽约1.5μm,主要由细胞核构成,前端无顶体;中段位于细胞核外侧,长约0.4μm;尾部细长,属于典型的单鞭毛结构。适当浓度的KCl、CaCl2及葡萄糖溶液可以延长精子寿命,最适KCl浓度为75~100 mmol/L,精子快速运动持续时间的峰值为(51.54±7.04)s,精子寿命峰值为(300.34±16.69)s;最适Ca Cl2浓度为75 mmol/L,精子快速运动持续时间的峰值和寿命峰值分别为(41.07±0.83)、(104.24±4.00)s;最适葡萄糖浓度为150 mmol/L,精子快速运动持续时间的峰值和寿命峰值分别为(45.12±4.03)、(262.60±18.08)s。     

中华沙塘鳢(Odontobutis sinensis)精子入卵过程扫描电镜(SEM)观察

为探讨中华沙塘鳢(Odontobutis sinensis)单精入卵机制,指导中华沙塘鳢人工繁育技术,采用扫描电子显微镜(SEM)对中华沙塘鳢精子和成熟卵子的形态和精子入卵过程予以观察,较为准确地描述了中华沙塘鳢精子和成熟卵细胞的形态与结构。结果显示,中华沙塘鳢精子头部为椭圆形,长轴约为2.25±0.25μm,短轴1.00±0.20μm,颈部较短。成熟卵细胞呈鲜艳黄色,呈圆球形,直径为1.30±0.02 mm,黏性卵,具黏着丝。观察发现,中华沙塘鳢精子能够准确通过成熟卵细胞的黏着丝达到受精孔,整个授精过程平均需要60 s完成,相对于其他科鱼类时间较长。中华沙塘鳢授精15 s,精子开始通过黏着丝;20 s大量精子进入筛网;25 s精子位于筛网孔内部,部分穿过筛网;55 s大量精子通过卵细胞黏着丝;60 s精子入卵;120 s受精孔关闭。     

瓦氏黄颡鱼精子的生理特性及其超低温冷冻保存的初步研究

对瓦氏黄颡鱼(Pelteobagrus vachelli)精子在不同盐度和pH下的精子活力进行观察,同时研究了精子在4种不同稀释液与2种不同浓度抗冻剂组成的保存液中的超低温冷冻保存,并开展了冻精的授精实验。结果表明,瓦氏黄颡鱼精子浓度为(2.035±0.179)×1012cell·mL-1,在盐度为5.8、pH为7.17时,精子的活力都高达95%。以A液作为稀释液、10%甲醇作为抗冻剂时,冷冻保存精子效果最好,解冻后精子活力为(81.7±0.9)%。用解冻后的精子进行人工授精,获得的受精率为(88.4±2.1)%,孵化率为(74.0±0.8)%;而鲜精受精率为(91.0±0.8)%,孵化率(82±1.6)%,冻精与鲜精均无显著性差异。人工授精实验证明了解冻后的精子能正常用于该鱼的人工繁殖。     

Semen biology and stimulation of milt production in the European smelt (Osmerus eperlanus L.)

Basic characteristics of the European smelt (Osmerus eperlanus) sperm are reported here for the first time. Smelt spermatozoa had a bullet-shaped head (1.42 μm length), a short midpiece and a long flagellum (27.72 μm). Two mitochondria were located along the flagella. The volume of smelt sperm was small (30-60 μl) and the duration of sperm motility was short (22 s in distilled water and 41 s in 20 mM sodium bicarbonate solution). Sodium chloride at concentrations ranging from 0-120 mM did not influence the percentage of motile spermatozoa but caused a steady increase in the duration of sperm movement. Potassium ions clearly reduced the percentage of motile sperm at a concentration of 10 mM. Spermatozoa were motile through a broad range of pH with an optimum from 7.5 to 8.5. Testicular spermatozoa had a different motility pattern compared to stripped spermatozoa (the latter exhibiting a reduction of motile spermatozoa by 30%, lower ALH and VCL and higher LIN and VSL). These results indicate that maturation of smelt spermatozoa occurring in sperm ducts is related not only to an increase of the percentage of motile spermatozoa but also to changes in the sperm motility pattern. Maintaining males with females resulted in stimulation of milt production. Our results indicate that European smelt sperm characteristics are similar to those of ayu (Osmeridae).     

Spermatological research of experimentally farmed Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopsidae) in Chile

Spermatological research of the Patagonian blennie was carried, specifically biometric parameters, sperm density, sperm count and motility in different activation mediums (815, 716, 590 and 0 mOSm kg^?1), at different temperatures (5, 10 and 15°C) and pH levels (5, 7 and 9). The results indicate that Patagonian blennie spermatozoa have a primitive form, with a total length of 44.09 ± 3.36 μm, with a head length of 2.15 ± 0.28 μm and head width of 2.5 ± 0.31 μm. The mid‐piece had a length of 0.72 ± 0.12 μm, and its tail measures 41.21 ± 3.21 μm long. The motility pattern indicates that the spermatozoa are found immobile in the seminal plasma and only initiates its movement in a hypertonic medium from 590 to 815 mOsm kg^?1. The longest motility time that was registered at 10°C in 716 mOSm kg^?1 was of 245 ± 39 s and an optimum pH of 7 was observed.     

白点鲑(Salvelinus leucomaenis)精子活力的观察

在水和BSS激活剂的激活下,白点鲑(Salvelinus leucomaenis)精子的A、B级快速运动时间分别为34.83±2.15 s和36.70±3.26 s;精子寿命分别为120.26±5.75s和117.45±4.33s,激活比例高于98%,BSS的激活效果更好。在4℃和15℃条件下,白点鲑精子活力、激活比例和精子寿命与保存时间呈负相关性,保存1～2h以内,经保存的精子活力和精液质量较好,不影响人工授精效果。     

日本黄姑鱼精子生理特性及超低温冷冻保存研究

对日本黄姑鱼精子部分生理特性及其超低温冷冻保存技术进行了研究。结果表明,日本黄姑鱼精液pH值为7.0~7.5,精子密度为(11.23±2.78)×109/m l,精子寿命(229.33±17.16)s。在盐度为30~35,pH为7.5~8.5时,精子的活力最高,分别达到(88.33±2.89)%和(88±4.33)%。精子在室温(25℃)条件下,可存活24 h;在低温(4℃)条件下可以存活36 h。以D液作为稀释液,20%的乙二醇作为抗冻剂,利用快速降温法对精子进行超低温冷冻保存,38℃水浴快速解冻,解冻后的精子获得最高的活力为(47±5.78)%。     

Application of Computer‐assisted Sperm Analysis in Selecting the Suitable Solution for Common Carp,Cyprinus carpio L., Sperm Motility

The common carp, Cyprinus carpio L., sperm motility parameters were analyzed by using computer‐assisted sperm analysis system. The percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, µm/sec), average path velocity (VAP, µm/sec), the wobbling index (WOB, %), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and amplitude of lateral head displacement (ALH, µm) were determined. Five activation solutions (As) were used to activate sperm movement. As 1 solution: 68 mM NaCl, 50 mM urea, 0.5% bovine serum albumin (BSA), pH: 7.7, 181 mOsm/kg; As 2 buffer: 100 mM NaCl, 10 mM Tris, 0.5% BSA, pH: 9.0, 199 mOsm/kg; As 3 solution: 86 mM NaCl, 0.5% BSA, pH: 7.4, 167 mOsm/kg; As 4 buffer: 5 mM KCl, 45 mM NaCl, 30 mM Tris, 0.5%, pH: 8.0, 160 mOsm/kg; and As 5 solution: distilled water with the addition of 0.5% BSA, pH: 7.3, <3 mOsm/kg. Among five tested solutions, a buffer with a pH of 9.0 and osmolality of approximately 200 mOsm/kg (As 2) was the most suitable. After its activation, a significant increase in MOT and ALH values was observed, which can be of importance to the effectiveness of egg fertilization .     

Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga,Brycon nattereri

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS^? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO₃ (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.     

环境因子对胭脂鱼精子活力影响的研究

通过观察精子的快速运动时间和寿命,研究了6种环境因子对胭脂鱼(Myxocyprinus asiaticus Bleeker)精子活力的影响。结果表明:胭脂鱼精子对酸碱的耐受能力较强,在pH值为4~10时,精子均可以快速运动,且在pH值为8时精子的活力最强。不同浓度的NaCl、KCl、葡萄糖、CaCl2、MaCl2溶液对胭脂鱼精子运动的诱导效果不同。5种溶液最适于胭脂鱼精子运动的浓度分别为:NaCl 102 mmol/L;KCl 75 mmol/L;葡萄糖100 mmol/L;CaCl275.6 mmol/L;MgCl275.6 mmol/L。且在5种溶液低浓度时精子活力较强,超过一定浓度精子的运动开始被抑制。     

Effect of two commercial preparations containing different GnRH analogues with dopamine antagonists on barbel Barbus barbus (L.) sperm quantity and quality

The effectiveness of applying Ovaprim [(D-Arg⁶, Pro⁹NEt)-sGnRH + domperidone] and Ovopel [(D-Ala⁶, Pro⁹NEt)-mGnRH + metoclopramide] to male barbel Barbus barbus (L.) 6, 12 and 24 h after hormonal stimulation was analyzed. The control group (Control) during each time interval was stimulated with 0.9 % NaCl. Milt was collected from seven fish only once (n = 7) for Ovopel, Ovaprim and Control group in order to determine total volume of milt, volume of milt per kg of body weight, sperm concentration, total sperm production, seminal plasma osmotic pressure, pH of milt and pH of seminal plasma. Woynarovich’s solution (68 mM NaCl + 50 mM urea) with the addition of 0.5 % BSA (pH 7.7; 181 mOsm kg^?1) was used as the activating liquid. Selected parameters of sperm motility (MOT %) and progressively motile sperm (%), curvilinear velocity (VCL, μm s^?1), straight-line velocity (μm s^?1), movement linearity (%), wobbling index (%), amplitude of lateral head displacement (μm) and beat cross frequency (Hz) were determined using the Computer-assisted sperm analysis system. A time of 6 h proved to be too short to obtain milt from barbel following hormonal stimulation with Ovaprim and Ovopel. Extending the time to 12 h, however, resulted in 100 % spermiation in males, regardless of hormonal preparation used for stimulation. The stimulation of spermiation in barbel is best performed using Ovopel 12 h upon application. Extending the latency period to 24 h following the application of this preparation results in a significant decrease in the volume of milt obtained, sperm count and motility parameters, including MOT and VCL, which may influence sperm fertilization ability.     

Sperm cryopreservation of tiete tetra Brycon insignis (Characiformes): effects of cryoprotectants,extenders, thawing temperatures and activating agents on motility features

The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution^? and Merck III^?), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at ?170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO₃ (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO₃ (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.