Effect of different methods for the induction of spermiation on semen quality in European eel

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g^?1 fish (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer‐assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium‐velocity spermatozoa, as well as different motility parameters. Sperm samples from A‐D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E‐treated males showed 0% motile cells. No significant differences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A–D groups.     

条纹锯精液超低温冷冻保存研究

为建立条纹锯精液超低温冷冻保存方法,实验采用计算机辅助精子分析系统（CASA）分析了采用6种抗冻保护剂（GLY[甘油]、DMSO[二甲基亚砜]、PG[丙二醇]、EG[乙二醇]、METH[甲醇]、DMA[二甲基乙酰胺]）在4种浓度（5%、10%、15%、20%,v/v）下对条纹锯精液的冷冻保存效果。结果发现,以HBSS为稀释液,采用程序降温仪分步降温冷冻保存条纹锯精液,37℃水浴解冻后的精子中,15% PG 作为抗冻保护剂的精子运动率最高,达到（93.1±0.9）%,与鲜精差异不显著（P>0.05）,15% PG 作为抗冻保护剂的精子水浴解冻后精子的运动速度最高,平均直线速度、平均曲线速度、平均路径速度分别达到了（88.3±0.3）μm/s、（76.2±0.5） μm/s、（86.7±0.7） μm/s,与鲜精差异不显著（P>0.05）。在不同种类及不同浓度抗冻保护剂保护下,15% PG 作为抗冻保护剂的精子解冻后 1 min内运动率变化与鲜精差异不显著（P>0.05）。研究表明,15% PG为条纹锯最佳抗冻保护剂,可用于条纹锯精液的超低温冷冻保存。     

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。     

欧洲鳗短钩拟指环虫病及其鳃组织病理

报道了患短钩拟指环虫病欧洲鳗的症状、流行状况和鳃显微组织病理.游动和呼吸频率异常、鳃丝浮肿和鳃上粘液增多为该病的主要症状.该病尽管在冬季也有发生,但水温在26℃以上的春末、夏季和秋初是最易发病的流行季节,流行出现明显的季节性变化.患病欧洲鳗鳃的组织病理变化主要表现出5种类型,其一是鳃小片上皮细胞增生,使邻近的鳃小片相连;其二是鳃小片粘液细胞增生,同样使鳃小片连成一片,增生的粘液细胞经阿利新蓝(Alclan blue)和过碘酸雪夫氏(Periodic acid schiff)二者联合染色后呈蓝紫色的染色反应,属于Ⅳ型的粘液细胞;其三是鳃小片肿大,但单层扁平上皮细胞无肿大现象,上皮细胞层与毛细血管相分离,鳃小片上皮细胞坏死,上皮细胞层破裂,血细胞外溢,鳃小片上皮细胞坏死脱落后,失去鳃小片原有的结构;其四是鳃小片肿大同时伴随单层扁平上皮细胞肿大,进一步发展,鳃小片上皮细胞坏死脱落后,鳃小片同样失去原有的结构;其五是鳃小片毛细血管严重充血扩张,比原毛细血管扩张几倍到十几倍,形成充满红细胞的棒状到球状的动脉瘤.结果表明鳃上皮细胞增生、粘液细胞增生、鳃小片肿大、上皮细胞坏死脱落和严重充血成动脉瘤等病理变化都可导致患病欧洲鳗呼吸困难,轻者影响其生长,重者最终因呼吸衰竭而死亡.     

Effects of feeding stimulants on the feed consumption,growth and survival at glass eel and elver stages in the European eel (Anguilla anguilla)

Elvers and glass eels of Anguilla anguilla were fed diets containing two types of feeding stimulants (FS) that were based on processed marine (MBFS) and yeast proteins (YBFS). Elvers (1.5 ± 0.3 g) were fed seven diets (MBFS and YBFS diets at 20 g kg^?1, 40 g kg^?1 and 60 g kg^?1 plus control) for 60 days. Glass eels (250 ± 100 mg) were weaned to 60 g kg^?1 MBFS, 60 g kg^?1 YBFS and control diets for 30 days. Diets containing 60 g kg^?1 FS had a beneficial effect in terms of growth, homogenous size distribution and feed intake in elvers. Elvers fed 60 g kg^?1 MBFS and YBFS diets grew 11.9% and 5.6% faster than the control group. No differences in growth and size distribution were detected in glass eels fed 60 g kg^?1 MBFS and YBFS diets. However, FS affected the digestive system maturation; fish fed the 60 g kg^?1 MBFS and YBFS diets showed higher and intermediate values in the specific enzyme activities in comparison with the control group. This study revealed that the incorporation of FS into a pelleted diet was beneficial on the overall performance of European glass eels and elvers. However, the observed results were different depending on the eel’s stage of development, as well as the type and inclusion level of the FS.     

黄姑鱼精子的超低温冻存及细胞结构损伤的检测

以HBSS溶液为稀释液,DMSO为抗冻剂,0.25 mL麦细管为冻存管,两步降温法超低温冻存黄姑鱼精子,并用单细胞凝胶电泳(SCGE)技术检测了冻精的DNA损伤,荧光双染色流式细胞仪技术(FCM)检测了冻精的细胞膜性结构损伤。结果表明,DMSO质量分数在5%~20%时,冻精的活力与鲜精相比无显著差异;其中DMSO质量分数在10%时,冻精的激活率、运动时间及寿命分别为85.25%±3.95%、(3.23±0.27) min及(3.83±0.33) min。DMSO质量分数在25%、30%时,冻精的活力显著下降。SCGE检测显示,DMSO质量分数在5%~15%时、冻精的DNA损伤与鲜精相比差异不显著,DMSO质量分数为20%、25%、30%时,冻精的DNA损伤明显加重,冻精的DNA损伤与抗冻剂DMSO的质量分数成正相关。FCM检测显示,DMSO质量分数在5%~20%时,冻精中线粒体及细胞膜结构保持完整性的精子比例与鲜精相比无显著差异,DMSO质量分数在25%、30%时,冻精中的线粒体及细胞膜结构保持完整性的精子比例明显下降。分析认为,较高质量分数的DMSO是引起冻精活力下降,DNA、线粒体及细胞膜结构损伤加重的主要原因。     

二株鳗鲡益生菌对欧洲鳗鲡免疫应答的初步研究

由养殖鳗鲡肠道分离、筛选出的益生菌A40209CDC4和A31009NA,添加鳗鲡饲料,细菌浓度达107cfu·g-1,连续投喂30d,测定试验鳗鲡白细胞吞噬活性、溶菌酶活力和血清抗体滴度.结果显示:与未添加细菌的对照组相比,添加益生菌试验组的白细胞吞噬百分比、吞噬指数、溶菌酶活力和血清抗体滴度无显著变化,说明筛选菌株作为饲料添加剂应用于欧洲鳗鲡未产生特异性刺激.     

长期超低温冷冻保存对鞍带石斑鱼精子超微结构及酶活性的影响

【目的】本文旨在探究长期超低温冷冻保存中鞍带石斑鱼精子质膜、活力、超微结构及酶活性的变化,为阐明影响鞍带石斑鱼精子冷冻保存质量的相关机制提供理论依据。【方法】采集2022年鞍带石斑鱼鲜精及储存时间分别为23、49、61个月冷冻保存精液,用伊红-苯胺黑染色方法检测精子质膜完整性;用计算机辅助精子分析仪（CASA）检测精子运动参数;测量精浆和精子中琥珀酸脱氢酶（SDH）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）、总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-PX）和肌酸激酶（CK）共六种酶活性的变化及三磷酸腺苷（ATP）浓度变化;用扫描电镜和透射电镜观察鲜精和冻精超微结构。【结果】伊红-苯胺黑染色检测结果发现,鲜精质膜完整性最高为83.43±2.73 %,经过超低温冷冻后,精子质膜完整性显著降低（P<0.05）,且随着冷冻保存时间的延长而逐渐降低。CASA结果显示鲜精活力最高为90.47±3.34 %,经过超低温冷冻后精子活力显著降低（P<0.05）,但长期保存23-61个月精子活力无显著性差异,精子活力保持在63.95±3.66 %-68.58±2.73 %,具有稳定的活力,且鲜精与冻精之间精子平均直线运动速度（VSL）、平均曲线运动速度（VCL）和平均路径速度（VAP）均没有显著差异（P>0.05）。精子超微结构显示,鲜精形态结构正常、线粒体排列结构规则、形态大小正常。经过超低温冷冻保存后,精子形态结构损伤明显,表现为精子头部质膜破损、细胞质外漏、细胞核膜破损、尾部鞭毛断裂或脱落等损伤;鞍带石斑鱼精浆和精子超低温冷冻前后六种酶活性的变化及ATP含量结果显示,经过超低温冷冻后,精子内SOD、GSH-PX和CAT三种酶及ATP含量均有显著性降低（P<0.05）。精浆中酶活力升高,除GR和CAT外,其余酶活性均差异显著（P<0.05）。【结论】长期超低温冷冻对鞍带石斑鱼精浆和精子酶活性、精子活力及精子超微结构均具有较显著影响,【意义】研究结果为鱼类精子冷冻损伤机理研究积累了丰富的数据,为鱼类精子长期冷冻保存提供了技术参考和评价指标。     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.     

Is there a role of ocean environment in American and European eel decline?

American eel (Anguilla rostrata) recruitment has declined dramatically, in parallel with that of European eel (Anguilla anguilla). Since both species spawn in the Sargasso Sea and migrate as larvae to continental waters, the coincidence in recruitment failure implies an Atlantic-wide cause, due perhaps to ocean climate. There is indirect evidence that the Gulf Stream has weakened in the 1980s. A slower Gulf Stream could interfere with larval transport and generate observed patterns of declining abundance of American eel only in northern North America and relatively uniform declines of European eel throughout Europe. While specific causes are still unclear, these data indicate a threat to both species and to their commercial fisheries.     

Did lack of spawners cause the collapse of the European eel,Anguilla anguilla?

Since the 1980s, a 90% decline in recruitment of European eel, Anguilla anguilla (L.), has occurred across most of Europe. Whether the continental stock has equally declined is uncertain. This study compiles available landings statistics since the beginning of the 20th century and identifies trends over time and space, using a statistical model that takes varying levels of reporting into account. Landings in the pre‐1940s reached over 40 000 tonnes yr^?1, declined during World War II, rose to a peak of 40 000 tonnes yr^?1 in the 1960s (coincident with a peak in re‐stocking) and dropped to an all time low of <20 000 tonnes yr^?1 in the 1990s. The decline in recruitment since the early 1980s was preceded by a decline in landings two or more decades earlier, indicating a decline of the continental stock. Considering the continental stock and the spawning stock must have declined in parallel, insufficient spawning stock biomass might have caused the recruitment collapse currently observed.     

Differential distribution and response to experimental sexual maturation of two forms of brain gonadotropin-releasing hormone (GnRH) in the European eel,Anguilla anguilla

Using specific radioimmunoassays for the two GnRH molecular forms present in the European eel, Anguilla anguilla, (mGnRH and cGnRH II), we compared their distributions in the pituitary and different parts of the brain of female silver eels, as well as the modifications of their levels in experimentally matured female eels (treated with carp pituitary extract). In control eels, mGnRH levels were higher than cGnRH II levels in the pituitary, olfactory lobes and telencephalon, di- and mesencephalon, while the opposite was found in the posterior part of the brain (met- and myelencephalon). Experimental sexual maturation of the gonads significantly increased mGnRH levels in the pituitary and anterior parts of the brain; such a positive effect was not observed on the low cGnRH II levels, which were, in contrast, reduced. These data indicate that the positive feedback of gonadal hormones on GnRH, that we previously demonstrated, would specifically affect the mGnRH form. The differential distribution and control of mGnRH and cGnRH II suggest that these two forms have different physiological roles in the eel. The large increase in mGnRH during sexual maturation suggests the prime implication of this form in the neuroendocrine control of reproduction.     

计算机辅助对几种鲟鱼冻精激活液的比较

首次在我国采用鱼类彩色精子图文分析系统(FSQAS-2000)对中华鲟、史氏鲟、西伯利亚鲟和匙吻鲟经超低温冷冻保存后的精子,在不同激活液条件下,平均曲线运动速度(VCL)、平均直线运动速度(VSL)、平均路径运动速度(VAP)、平均移动角度(MAD)、以及冷冻前后精子活率的变化等多项参数进行了统计学比较研究。研究分析了不同渗透压、pH以及含Mg~(2 )的激活液对这些参数的影响。研究结果表明:几种鲟鱼激活液的渗透压均较低,其中西伯利亚鲟最低,为10 mOsm·kg~(-1);中华鲟和史氏鲟为20 mOsm·kg~(-1);匙吻鲟最高,为30 mOsm·kg~(-1)。不同的渗透压对冷冻精子的VCL、VSL、VAP、以及精子活率均产生显著(P≤0.05)或极显著(P≤0.01)影响。激活液的pH除了对冷冻精子的上述参数产生影响外,最显著的是对精子的运动轨迹,即平均移动角度(MAD°·s~(-1))产生影响(P≤0.01)。pH越低,精子的MAD越小。几种鲟鱼的最适pH分别为西伯利亚鲟pH 7.5,中华鲟、史氏鲟和匙吻鲟pH 8.5。当4种鲟鱼的冷冻精子激活液中加入5 mmol·L~(-1)MgCl_2时,冷冻精子的3种运动速度显著提高(P≤0.01)。综合分析后认为:西伯利亚鲟的冷冻精子激活液应为10 mmol·L~(-1)Tris-HCl 5 mmol·L~(-1)MgCl_2,pH 7.5;中华鲟和史氏鲟的冷冻精子激活液为20 mmol·L~(-1)Tris-HCl 5 mmol·L~(-1)MgCl_2,pH 8.5;匙吻鲟的冷冻精子激活液为30 mmol·L~(-1)Tris-HCl 5 mmol·L~(-1) MgCl_2,pH 8.5。用此激活液对冷冻前后4种鲟鱼精子活力的相关性进行线性回归分析发现,西伯利亚鲟的R~2=0.8296(P<0.01);中华鲟的R~2=0.9860(P<0.01);史氏鲟的R~2= 0.9622(P<0.01);匙吻鲟R~2=0.9477(P<0.01)。说明冷冻前后4种鲟鱼精子的平均活率存在着极显著的线性正相关性。     

A preliminary telemetry study of the migration of silver European eel (Anguilla anguilla L.) in the River Mosel, Germany

Abstract  – To study the behaviour of silver eels ( Anguilla anguilla L.) during their downstream migration, particularly near a hydroelectric power dam, we tagged nine eels with ultrasonic transmitters and tracked their paths in the River Mosel, Germany. The onset of migration coincided with the first flood event that followed the full moon but was independent of daytime, because migration and turbine passage occurred during both day and night. During migration eels swam actively downstream with a velocity of 0.3–1.2 m · s⁻¹. When migrating eels arrived at the dam, they either passed through the turbines immediately or stayed upstream of the powerhouse for up to 8 days, showing a characteristic circling behaviour. Circling eels repeatedly approached the trashrack, sprinted upstream, and finally passed through the turbines with the next high water discharge. These observations are discussed with regard to the design of appropriate downstream passage facilities.     

Virus infections of the European Eel in North Rhine Westphalian rivers

Viral infections have been suggested to play a role in the decline of the panmictic population of the European eel (Anguilla anguilla). However, despite the importance of knowledge about pathogenic eel viruses, little is known about their spread in the wild European eel population and only a few eel pathogenic viruses have been described so far. In this study, we aimed to investigate the health status of the A. anguilla stock in North Rhine Westphalia (NRW) State of Germany. For this purpose, we examined tissue samples of 16 elvers, 100 yellow eels and 6 silver eels, sampled from the rivers Rhine, Lippe and Ems. Virus detection was performed via a combination of cell culture and PCR. Next to the detection of frequently encountered pathogenic eel viruses (anguillid herpesvirus 1 and eel virus European X (EVEX)), we isolated the eel picornavirus 1 (EPV-1) from tissue of yellow eels and elvers and demonstrate the distribution of EPV-1 in wild eel population in NRW.     

Detection of Herpesvirus anguillae during two mortality investigations of wild European eel in England: implications for fishery management

Herpesvirus anguillae (HVA) was detected during disease investigations of European eel, Anguilla anguilla L. at two stillwater fisheries in central England. These represent the first records of HVA from UK eels. Both mortalities were eel‐specific and took place during August 2009 and July 2010 at water temperatures between 17 and 19.4 °C. Pathological changes consistent with HVA infection included haemorrhaging in the fins, skin lesions and necrosis within the gills and liver. Transmission electron microscopy revealed active virion replication within the gill tissue. An initial assessment of risk is presented, indicating that HVA represents a high disease risk to UK eel stocks. However, further studies are required to establish the distribution of HVA before a reliable assessment of impact may be obtained. Until then, the detection of HVA holds important implications for eel conservation and management, in particular eel stocking activity.     

A Strategy for Sperm Cryopreservation of Atlantic Salmon,Salmo salar,for Remote Commercial‐scale High‐throughput Processing

Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 10⁷, 3 × 10⁸, and 1 × 10⁹ cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.     

3种免疫途径下迟缓爱德华菌菌蜕疫苗对欧洲鳗的免疫保护效果

为了探讨迟缓爱德华菌菌蜕疫苗预防鳗鲡爱德华菌病的可行性,实验采用腹腔注射、口服、浸泡3种途径探讨了迟缓爱德华菌菌蜕疫苗对欧洲鳗的免疫保护效果。结果表明,免疫后欧洲鳗血清抗体效价均明显升高,但菌蜕疫苗(ETG)组与福尔马林灭活疫苗(FKC)组间血清抗体效价无显著性差异。欧洲鳗注射、口服、浸泡ETG组的相对免疫保护率分别为75.0%、52.5%、37.5%,分别高于FKC组的55.0%、40.0%、32.5%,且ETG组欧洲鳗死亡时间明显推后,表明菌蜕疫苗的免疫保护效果优于福尔马林灭活疫苗。易操作、对鱼体应激少的ETG疫苗口服免疫欧洲鳗获得了52.5%的免疫保护率,在预防鳗鲡爱德华菌病中具有良好的应用前景。