赤眼鳟精子入卵的扫描电镜观察

选取性成熟的赤眼鳟（Squaliobarbus curriculus）亲鱼，体质量800～2300g，经人工催产后，在27℃条件下进行干法人工授精。取成熟卵及相遇后不同时间段的精卵用扫描电镜对赤眼鳟成熟精、卵及精子入卵早期过程进行观察。结果显示，赤眼鳟成熟精子为鞭毛型形态，全长19．6～22．4μm。头部直径约1.65μm，鞭毛长17．8～20．6μm头径与鞭毛长度之比为（1：9．89）～（1：11．4）。赤眼鳟成熟卵呈圆形，具单一受精孔。卵膜厚度约为2.78μm，卵膜表面平滑光洁；无明显的沟或嵴，卵膜表面可见均匀分布有很多孔小管，直径约0.14μm。在动物极具一漏斗状凹陷卵膜孔，直径12．46～14．10μm，其底部为圆环状的受精孔，受精孔的周边平滑，向四周呈辐射状隆起，外孔径约5．21μm，内孔径约2．71μm。在27℃时，赤眼鳟精子18s内可完成入卵过程；约60s后受精孔外的精子全部解体。通过对精子、卵及精孔管的测量和扫描电镜观察，确定赤眼鳟受精方式为单精受精。     

黄颡鱼精子入卵的扫描电镜观察

应用扫描电镜观察了黄颡鱼(Pelteobagrus fulvidraco)成熟卵和精子的形态、卵子对精子的应答反应、受精孔的数量与形态、受精方式与过程。结果显示:黄颡鱼精子为鞭毛型,头部近圆球形,无顶体,单尾;卵为圆球形,卵膜表面多嵴,且有15～16条宽度不同的小沟;卵表面仅有1个受精孔,精孔管口内径约2.8μm。黄颡鱼为单精受精,受精后30 s内完成精子入卵过程。     

大菱鲆成熟精子、卵子及精子入卵早期过程的电镜观察

通过人工授精和电镜技术,观察和描述了大菱鲆Scophthalmus maximus成熟精卵的形态、精子入卵的早期过程及精子入卵过程发生的一系列形态结构变化。大菱鲆卵子表面均匀地布满纵横交错的网纹,并整齐地分布许多微小孔,在动物极有一个受精孔。大菱鲆精子为无顶体类精子,由头部、中片和尾部三部分组成,中片有9～14个圆形线粒体。大菱鲆精子入卵速度非常快,授精后0～5s已经有精子通过受精孔(Micropyle)进入卵子。精子入卵过程还伴随着其他一些结构的变化,如精孔管管壁边缘由锯齿状变为平滑的环状等。     

长江口纹缟虾虎鱼精子、卵子及受精过程扫描电镜观察

采用电镜对长江口纹缟虾虎鱼成熟精子、卵子及受精早期精子入卵过程进行了观察。结果显示:纹缟虾虎鱼成熟精子由头部和尾部两部分组成。头部呈圆形或近圆形,无顶体,细胞核长径为1.15±0.28μm,短径为0.97±0.22μm,尾部鞭毛长为8.17±1.12μm。核膜外具有双层质膜,质膜表面不平整,在核膜和双层质膜中间有较大空隙存在。中心粒复合体位于植入窝内。袖套两侧分布有2～3个较大的线粒体。鞭毛为典型的"9+2"结构,有侧鳍。成熟卵呈圆形,卵膜表面多沟和嵴,具单一受精孔,孔洞区外径约5μm,孔内壁呈螺旋嵴。在卵的植物极有一盘状突起,突起的中间为圆形,周边有呈网状结构的粘丝与卵壳膜连接。纹缟虾虎鱼精子入卵速度较快,受精过程较短,受精后10～18 s完成精子入卵过程。     

条纹锯鮨精子超微结构及其入卵过程的电镜观察

采用扫描和透射电镜技术对自然成熟的条纹锯精子、卵子及精子入卵过程进行观察。观察结果显示,其精子由头部、中段和尾部三部分组成:头部主要由细胞核构成,无顶体结构;中段由线粒体、中心粒复合体(近端中心粒和基体)、袖套组成;尾部主要由轴丝组成,外部包裹质膜,轴丝为典型的"9+2"结构。卵子表面分布纵横交错的网纹,均匀分布着大小不一的微小孔,在卵壳的动物极精孔区的中央有一个受精孔。在授精后10 s即可见到精子通过受精孔进入卵子,刺激卵子发生形态变化封闭受精孔,阻止其他精子入卵,60 s可见受精孔完全封闭。     

条纹锯精子超微结构及其入卵过程的电镜观察

采用扫描和透射电镜技术对自然成熟的条纹锯精子、卵子及精子入卵过程进行观察。观察结果显示,其精子由头部、中段和尾部三部分组成:头部主要由细胞核构成,无顶体结构;中段由线粒体、中心粒复合体(近端中心粒和基体)、袖套组成;尾部主要由轴丝组成,外部包裹质膜,轴丝为典型的"9+2"结构。卵子表面分布纵横交错的网纹,均匀分布着大小不一的微小孔,在卵壳的动物极精孔区的中央有一个受精孔。在授精后10 s即可见到精子通过受精孔进入卵子,刺激卵子发生形态变化封闭受精孔,阻止其他精子入卵,60 s可见受精孔完全封闭。     

诱导鲤雌核发育时精子入卵的扫描电镜观察

以雌核发育性成熟鲤(Cyprinuscarpio)卵子和经紫外线照射处理遗传失活的鲫鱼(Carassiusauratus)精子为材料,在再次诱导其雌核发育时,通过扫描电镜和光学显微镜对比观察诱导组和正常受精组精子入卵的情况及鲤鱼成熟卵受精过程中精孔区的外部形态变化。结果表明,无论试验组或对照组在受精后2s精子就能迅速到达精孔区。精子入孔后,受精孔便被絮状物质堵塞;受精后35～40s壳膜表面开始模糊;60s后,精孔区絮状物及精子均消失。     

鸭绿沙塘鳢胚胎壳膜结构的电镜观察

扫描电镜观察发现,鸭绿沙塘鳢(Odontobutis yaluensis)卵壳膜由卵柄和卵体两部分构成.卵柄基部为丝状纤维围成的直径200μm、高12.5μm的"圆盘状"结构,使卵子黏附在附着物上.卵柄基部正中有一直径为20 μm的凹陷受精孔.卵柄丝为圆柱体.未受精卵子的系丝表面光滑,呈发散状向外扩散.随着胚胎发育,受精卵的系丝逐步拉长,直径变小,脆性增加.卵壳主要分为两层:内层具有卵膜孔,肌节出现后内膜孔直径为0.2～0.5μm,眼点出现后达到0.9～1.22μm,破膜前期为0.4μm,平均每100μm2有卵孔38个;卵膜外层规则地排布有直径为0.30～0.63μm的孔径.文中讨论了卵壳膜和受精孔的结构与受精卵孵化生态环境的统一.     

青蛤Cyclina Sinensis(Gmelin)胚胎发育生物学

本文对青蛤成熟的精卵进行授精和胚胎发育观察。受精膜形成、极体释放、卵细胞分裂，简述了青蛤受精和胚胎发育过程。     

栉孔扇贝（♀）×虾夷扇贝（♂）精子入卵过程的电镜观察

采用扫描电镜和透射电镜技术对栉孔扇贝(Chlamys farreri,)×虾夷扇贝(Patinopecten yessoensis,)的精子入卵过程进行观察。结果表明,这2种扇贝杂交与亲本自交的精子入卵过程没有本质的区别。成熟的精卵相遇时,相互激活,产生一系列胞间反应,卵子的激活集中表现为皮层反应、受精膜举起、成熟分裂重新启动;精子激活集中表现为顶体反应和受精锥形成。在16～18℃条件下,精子入卵的时间集中在精卵混合后的第6 min。8 min后,精子的线粒体随精核一起进入卵子中。精子入卵后精核略微膨胀,卵细胞中的线粒体在精核附近出现聚集现象。杂交中有多精入卵现象。本研究目的旨为优化扇贝种间杂交技术及探讨种间受精生物学过程提供基础依据。     

Artificial fertilization in turbot, Scophthalmus maximus (L.): different methods and determination of the optimal sperm–egg ratio

With the aim of improving artificial fertilization (AF) in turbot, Scophthalmus maximus (L.), a series of fertilization experiments was carried out under dry conditions and different wet conditions (eggs/sea water: 2V/V and V/V). Another series of fertilization experiments was carried out with different quantities of sperm pool to determine the optimal ratio of spermatozoa to eggs for each AF method. Sperm pool from two males and eggs from spawns with a viability rate of > 70% were used. The sperm pool’s density (0.4–5.18 × 10⁹ sperm mL^–1) and motility (1–5) had been assessed previously. Significantly different fertilization rates were found when comparing 2V/V and V/V wet conditions. Significantly higher fertilization rates were found in dry fertilization when the sperm–egg ratio was > 9000 spermatozoa per egg and, under wet condition V/V, at 3000–4000 spermatozoa per egg.     

黄鳝精子活力检测和精子入卵早期过程观察

采用Olympus3×51相差系统显微镜和SQIAS—1000彩色精液质量图文分析系统检测黄鳝精子活力。结果表明,在NaCl溶液浓度为0～0.3%时,黄鳝精子激活比例随溶液浓度升高而极显著增加(P<0.01);当NaCl浓度达到0.7%时,精子激活比例、直线运动速度和鞭毛摆动频率均显著(P<0.05)或极显著(P<0.01)降低。扫描电镜观察显示:黄鳝成熟卵卵壳膜上的精孔区呈漏斗状凹陷,其底部中央可见一精孔管外孔,口径约4.22±0.66μm;黄鳝精子入卵速度缓慢,受精过程较长,从精子附着于卵球表面到精孔管完全堵塞,约30s～5min。     

High fry production rates using post-thaw silver carp (Hypophthalmichthys molitrix) spermatozoa under farming conditions

Silver carp (Hypophthalmichthys molitrix) is not endemic to Cuba, and egg fertilization is totally artificial; males produce spermatozoa only between March and October. Cryopreservation of silver carp spermatozoa would reduce the number of males needed, minimize handling stress through less frequent stripping, and facilitate artificial propagation when eggs are available. The effects on motility and fry production from eggs fertilized with thawed sperm under farm-conditions were examined in this study. Five, seven and ten percent of dimethyl sulfoxide (DMSO), glycerol and methanol were tested as cryoprotectants. DMSO was a more suitable cryoprotectant than methanol or glycerol. The effect of equilibration time on the motility rate at 10% DMSO was evaluated. Hatching rates equal to the control (P>0.01) were obtained under farm conditions with frozen spermatozoa, stored even for a year in liquid nitrogen, with a final DMSO concentration of 10%. Cryopreservation offers a useful routine method for sperm storage and silver carp handling. To our knowledge, this is the first report on the production of fry from post-thaw silver carp spermatozoa under farming conditions.     

Fertilization efficiency of cryopreserved sperm from striped catfish, Pangasius hypophthalmus (Sauvage)

The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 10⁷ to 6.94 × 10¹⁰ to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 10⁸. Increasing the sperm dose to 3.47 × 10⁹ did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 10⁶ and 3.47 × 10⁷ sperm per egg. Both highest (6.94 × 10¹⁰) and the lowest (6.94 × 10⁷) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 10⁶ fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 10⁶ (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.     

中国对虾精子入卵过程的观察

利用光镜和电镜对中国对虾精子入卵过程进行了观察。结果表明，精子的核物质籍胞吐作用穿过卵黄膜到达卵膜，其余部分则留在卵黄膜外。只有在卵子激活前到达卵膜的精子才能参与受精作用，其余的精子将被卵子激活所形成的胶质层推离卵子。中国对虾精子激活过程，没有发现顶体丝的形成。精子的入卵开始于卵子的皮层反应后期，此时精卵的接触逐渐密切，首先接触部位的卵膜稍有突起，然后开始下陷，同时这一部位的卵膜变得粗糙不平，随后卵膜解体，精子由此进入卵子。中国对虾精子的入卵位置是随机的，有多精入卵的现象。     

扇贝精子及卵子的受精生物学特性

采用扫描电子显微镜和普通光学显微镜观察方法,结合全光谱扫描分析方法,对栉孔扇贝和虾夷扇贝的精子及卵子的受精生物学特性进行了详细的观察,包括精卵产出后形态学变化、卵水的特性、精子顶体反应、卵子皮层反应以及精子分级分离组分对卵子的作用5个方面.结果表明,这两种扇贝精卵生物学特性没有本质的区别,扇贝精子排入海水中10min以内,外形没有太大的变化,但30min以后约有1/4的精子出现头部膨大变成圆球形的现象,受精能力明显下降;扇贝卵子产入海水中1h以内受精能力没有变化,但2h以后,约1/3的卵子出现卵裂现象,未见极体的排放,且卵裂多停止在2细胞期,少数达到4细胞期,基本属于均等卵裂,并失去受精能力;滴入卵水中的精子极易发生自溶解体,未解体的精子发生凝集,10min之后卵水中基本检测不到完整的精子,卵水和钙离子载体A23187均能诱导精子发生顶体反应;精子分级分离组分分别为精浆、精子头部和精子尾部,这三部分除了少数精子头部能够附着在卵子表面外,其他都不具备激活卵子的作用.对扇贝精子和卵子的生物学特性的研究将为解释不同种扇贝的精卵相互识别并受精的现象提供基础资料.     

Spawning of ocean pout (Macrozoarces americanus L.): evidence in favour of internal fertilization of eggs

Sperm physiology, in vivo artificial insemination and spawning of the ocean pout (Macrozoarces americanus L.), a marine bottom fish, were studied. Milt was collected from the reproductive tract of mature males by suction using a catheter. The uncontaminated milt, having a very low sperm concentration, contains highly motile spermatozoa and sperm motility was retained in vitro at 4 °C for at least 24 h in both seminal plasma and ovarian slime collected from the oviduct of pre-spawning females. Instead of activating sperm, dilution in sea water instantly immobilized the spermatozoa of ocean pout. Osmolarity and pH of ocean pout seminal plasma were in the ranges 365–406 mOsM and 7.2–7.5, respectively. A study of the ionic composition of ocean pout seminal plasma demonstrated the presence of various ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻, with a remarkably lower K⁺ concentration compared to that from other fish species. Since injections of milt containing motile sperm into the ovaries of pre-spawning females, which spawned in the absence of males, yielded fertilized ocean pout eggs, it is concluded that the ocean pout exhibits internal fertilization. The larvae hatched after 3 months of egg incubation in ambient sea water (9–10 °C). With proper timing of in vivo artificial insemination of mature females, fertilized ocean pout eggs can be obtained from fish reared in captivity.     

Evaluation of fertilizing capacity of cryopreserved rainbow trout sperm

ABSTRACT:   Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%.     

Cryopreservation of sea bass (Dicentrarchus labrax) spermatozoa in experimental and production simulating conditions

A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min⁻¹ allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·10³ spermatozoa to egg ratio was applied. When 70·10³ and 200·10³ spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·10³ and 50·10³ eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·10³ spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.