超低温冷冻对俄罗斯鲟精子形态结构的影响

选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,运用扫描电镜和透射电镜,观察了超低温冷冻前后精子的形态结构。结果显示,经过超低温冷冻后精子形态结构发生了较大变化,其中冻精顶体长度、头中部宽度及中段宽度与鲜精相比显著增加(P0.05);中段长度及后外侧延伸物与鲜精相比显著减少(P0.05)。冻后有38.6%的精子在形态结构上受到不同程度的损伤。精子的结构损伤主要表现在精子顶体不明显或与核发生糅合,后外侧延伸物消失,甚至顶体脱落;精子核膜皱褶,泡状化,膜与核之间空隙加大,有的部分甚至出现膜断裂,核内内切沟模糊;精子中段发生膨大和变形,线粒体外膜破损,线粒体条状嵴结构弥散,线粒体脱落;鞭毛外鳍褶与鞭毛脱离,鞭毛与中段脱离,少数鞭毛在中段断开。结果表明,超低温冷冻对精子的损伤主要集中在膜系统,中心粒等微管系统基本完好。     

虾夷扇贝精子形态结构和超低温冷冻损伤的电镜观察

应用透射电镜和扫描电镜技术,研究了超低温冷冻保存前后虾夷扇贝精子超微结构和形态的变化。结果显示,虾夷扇贝精子由头部、中段和尾部3部分组成,外被光滑质膜;顶体位于头部最前端,呈倒"v"形,细胞核近似圆柱状,电子密度较高;4个线粒体和两个相互垂直的中心粒构成了精子的中段;鞭毛细长,轴丝为典型的"9 2"结构。经超低温冷冻保存后,冷冻损伤的精子表现为:精子被膜肿胀、被膜与核膜分离、丢失;顶体破裂、内容物流出;线粒体解体、线粒体嵴变形;鞭毛被膜肿胀、部分精子鞭毛脱落。可以推测,超低温冷冻保存对精子膜、顶体、线粒体和鞭毛的损伤可导致冻精活力和受精能力的下降。     

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。     

南美白对虾精子超低温保存前后形态和超微结构的研究

应用光学显微镜和电子显微镜对超低温保存前后南美白对虾精子的形态和超微结构进行了观察。结果发现,正常精子由圆球形的主体部和棘突组成,主体部由顶体、亚顶体区、细胞核和环核细胞质带组成。超低温冷冻处理及升温复苏后受损精子棘突脱落,顶体囊泡化,精子细胞膜皱缩、肿胀,膜间腔增大,损伤严重者,顶体脱落,核变形,核膜破裂,核出现空泡化。结果表明,超低温冷冻处理及升温复苏过程主要造成南美白对虾精子的顶体和膜结构的损伤,致使精子成活率降低。     

西施舌精子发生过程的超微结构观察

利用透射电镜研究西施舌精子的发生和结构 ,揭示了从精原细胞逐渐发育为精子过程中细胞形态结构的变化和细胞器的演变规律。精细胞的分化分为 6期 ,主要包括 :核形态由扁圆到圆再到椭圆形 ;核染色质以颗粒状形式凝集 ;高尔基体分泌的前顶体颗粒聚合发育为前顶体囊 ,参与顶体的形成 ;线粒体逐渐融合与发达 ;中心体的移动及鞭毛的形成 ;胞质的逐步减少。成熟精子为原生型 ,由头部、中段和尾部组成。顶体圆锥状 ,高密度的顶体物质集中分布于基部四周 ,呈灯罩状 ;亚顶体腔呈尖锥状 ,内含密度较低的均匀物。细胞核近椭圆形。中段由 4个椭圆形的线粒体和 2个相互垂直的中心粒组成。尾部鞭毛为典型的“9+2”型结构。     

西施舌精子发生过程的超微绘结构观察

利用透射电镜研究西施舌精子的发生和结构，揭示了从精原细胞逐渐发育为精子过程中细胞形态结构的变化和细胞器的演变规律。精细胞的分化分为6期，主要包括：核形态由扁圆到圆再到椭圆形；核染色质以颗粒状形式凝集；高尔基体分泌的前顶体颗粒聚合发育为前顶体囊，参与顶体的形成；线粒体逐渐融合与发达；中心体的移动及鞭毛的形成，胞质的逐步减小，成熟精子为原生型，由头部、中段和尾部组成。顶体圆锥状，高密度的顶体物质集中分布于基部四周，呈灯罩状；亚顶体腔呈尖锥状，内含密度较低的均匀物。细胞核近椭圆形。中段由4个椭圆形的线粒体和2个相互垂直的中心粒组成，尾部鞭毛为典型的“9 2”型结构。     

人工养殖西伯利亚鲟精子超低温冷冻保存研究

研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。     

史氏鲟精子超微结构

利用扫描和透射电镜观察了史氏鲟精子形态和超微结构。精子具有顶体、头部、中段和单鞭毛等部分。顶体长0.99±0.06μm,宽0.87±0.09μm。细胞核从后往前逐渐变细,前端宽度为0.88±0.04μm,后端宽度为1.26±0.06μm,细胞核长7.29±0.32μm。核内含有3条核管(E),核管上行至顶体下行至植入窝而止。中段紧接头后部,长为0.51±0.12μm,宽0.91±0.05μm。中段含有线粒体、中心粒复合体和鞭毛的起始部分。线粒体分2~3层排列,线粒体中可见髓样嵴结构。在细胞核与中段接合部细胞核向内凹陷形成植入窝,纤维体位于植入窝内,其后是中心粒复合体。中段后缘延长为袖套,袖套腔中含有线粒体、脂质空泡。鞭毛从袖套中伸出,由轴丝组成,为典型的"9+2"微管结构。     

褐牙鲆精子超微结构观察

利用扫描电镜和透射电镜对自然成熟的褐牙鲆精子超微结构进行了观察。褐牙鲆精子的头部近似圆形,直径1.49±0.17μm,尾部鞭毛较长,达40.17±0.65μm。褐牙鲆精子由头部、中段和尾部(鞭毛)构成。头部的顶端无顶体,细胞核为圆形,染色质致密。核膜分由内核膜和外核膜构成,其间有核周腔。核膜与质膜间有较大间隙,其间分布有许多细胞质、囊泡和溶酶体,在细胞核后端,核膜与质膜之间除细胞质外,还分布有5~6个呈环形单层排列的线粒体。植入窝位于细胞核后端的凹陷处,中心粒复合体位于植入窝内。基体的头端由电子致密物质构成,基体的末端与鞭毛起始端相连,鞭毛从袖套腔中伸出。鞭毛中心结构是轴丝,轴丝为"9+2"微管结构。轴丝外侧有侧鳍。     

云斑尖塘鳢精子的超微结构

采用扫描电镜和透射电镜观察了云斑尖塘鳢(Oxyeleotris marmoratus)的精子结构。结果显示:扫描电镜下云斑尖塘鳢精子由头部和尾部(鞭毛)组成。头部近似圆形,直径1.98～2.12μm,尾部鞭毛长23.22～32.92μm。透射电镜下云斑尖塘鳢精子由头部、中段和尾部三部分组成。头部细胞核呈扁卵圆形,前端不具顶体,染色质致密,细胞核内偶见小空隙;核膜与质膜间有间隙,其间分布有细胞质、囊泡、溶酶体,在细胞核后端,还分布有5～7个呈环形单层排列的线粒体;植入窝较浅,中段不明显,包括中心粒复合体和袖套;精子尾部的鞭毛细长,轴丝为典型的"9+2"型结构;轴丝外侧具有较发达的侧鳍。     

Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa

This paper provides data concerning biochemical composition of milt of two sturgeon species, Siberian sturgeon bred in aquaculture facility of Inland Fisheries Institute in North Poland and sterlet (from two different populations from Danube and Odra). Milt plasma of Siberian sturgeon and sterlet, when compared to teleost fish, is characterized by much lower osmolality (up 70 to 96 mOsm kg⁻¹) and protein concentration (0.24–0.58 g l⁻¹). In spite of the presence of an acrosome and acrosin the antiproteinase activity of seminal plasma was low (12.79–25.40 U l⁻¹). Activities of arylsulfatase and β-N-acetylglucosaminidase were found in spermatozoa. This agrees with the presence of an acrosome in sturgeons sperm. Similarly to mammals, these enzymes are also present in milt plasma. We determined a range of enzymatic activities from the minimal (seminal plasma) to the maximal damage (supernatants obtained after freezing-thawing without cryoprotectant). Activities of arylsulfatase, β-N-acetylglucosaminidase, lactic dehydrogenase and acid phosphatase were released from spermatozoa after freezing-thawing. For this reason they are good potential candidates as a markers of cryoinjury to sperm acrosome and midpiece. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Changes in the flagellum morphology of intact and frozen/thawed Siberian sturgeon Acipenser baerii (Brandt) sperm during motility

Morphological investigations on the changes in flagellar beating was carried out on native (taken from the milt) and thawed sperm of the Siberian sturgeon Acipenser baerii (Brandt). Immediately after activation, the pattern of flagellar wave formation and distribution was the same in native and thawed sperm but, after 27–42 s, depending on the samples, the thawed flagella showed asymmetric and poorly developed waves. The swimming trajectories recorded during 1‐s exposure were much shorter in thawed than in native sperm after 26–28 s motility. In native sperm, the flagella remained in the same axis as the head during the entire motility course, while the head of thawed sperm showed a right angle after 47 s. It is concluded that the freezing/thawing procedure induces some alteration in the dynamics of flagellar beating in many sperm, but these sperm still show progressive displacement. Therefore, the change in morphology of the flagellum during motion is a parameter that should be taken into account in the evaluation of the impact of various treatments on sperm motility.     

Structural abnormalities of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> spermatozoa

Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or “double head” in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation.