莱氏拟乌贼纳精囊超显微结构

本研究采用组织切片和透射电镜技术，对莱氏拟乌贼(Sepioteuthis lessoniana)纳精囊进行超显微结构分析，并通过扫描电镜和投射电镜技术观察纳精囊中精子的形态构造。结果显示，莱氏拟乌贼口膜具有1个椭圆形的纳精囊，呈乳白色，交配后雌体的口膜内侧散布大量精子囊。纳精囊由囊壁组织、中心管腔和储精小囊组成，储精小囊数量较多，少数呈圆形或椭圆形，2个或多个储精小囊形成中心管腔，二者的囊腔中均分布大量精子。储精小囊和中心管腔由上皮细胞组成，细胞间排列紧密，其细胞核较大，细胞质中含有多种细胞器，包括线粒体、内质网、高尔基复合体和囊泡等；中心管腔中精子大多排列紧密有序，精子头部朝向纤毛，鞭毛位于囊腔中间。莱氏拟乌贼的精子头部细长，由圆屋顶形的顶体和细长圆柱形的精核组成，线粒体距位于精核后端，尾部为细长鞭毛。研究表明，莱氏拟乌贼纳精囊具有储存精子的功能。其中，储精小囊的上皮细胞具备一定分泌功能，其分泌物质能吸引精子进入纳精囊。     

拟目乌贼繁殖行为学的初步研究

拟目乌贼在繁殖期间的行为较为复杂,包括求偶、雄性争斗、交配、雄性陪护、产卵等行为。拟目乌贼在生殖策略上并非"一夫一妻"制,交配时,雄性用第1、2对腕固定雌性的头部,然后用茎化腕将精荚输送至雌性纳精囊内。雌性受精后不久便产卵,拟目乌贼雌性轮流在产卵区产卵。卵白色,半透明,卵群葡萄状,分枝,刚产出的卵短径约1.2cm,长径约2.4cm,拟目乌贼平均产卵量为354枚。     

某种雌性乌贼以雄性精子为食

一项新的研究显示,生活在澳大利亚南部斯潘塞湾的澳洲后耳乌贼（Sepiadarium austrinum）具有独特的繁育习性。在交配期间,雄性澳洲后耳乌贼会将精子注入雌性的口腔洞中。雌性会以这些精子为食促进未受精卵子的发育。     

头足类生殖系统及其在分类学上的应用

头足类被认为是未来重要蛋白质来源之一。生殖系统是头足类的重要系统之一,不仅是生物学研究的基础,也是繁殖生物学研究的重点,且在分类学上也具有重要作用。分析认为,雌性个体的输卵管和缠卵腺在柔鱼类、枪乌贼类、乌贼类和蛸类间存在明显差别;十腕类的纳精囊位于口球内,而蛸类位于输卵管腺上;交配囊是耳乌贼亚科的特殊生殖结构,在小乌贼属的分类上具有一定意义。茎化腕是雄性头足类的重要生殖器官,也是重要的分类性状,各大类的茎化腕部位和特征有所差异;交配器为耳乌贼亚科茎化腕上的特殊结构,在分类上具有一定作用。绝大多数雄性个体的精囊结构复杂,其放射导管、连接管以及精团在帆乌贼科的分类上具有一定意义。     

半滑舌鳎性腺的组织学研究

采用组织切片,对半滑舌鳎性腺的组织学结构进行了观察研究。结果表明,半滑舌鳎的精巢为小叶型,精巢内的生殖细胞分为五种。其精原细胞存在于精小叶的内壁上,精母细胞和成熟的精子细胞位于精小囊中。精子成熟后,精小囊破裂释放精子进入小叶腔,完成发育过程。半滑舌鳎卵巢外有被膜包裹,其被膜的结缔组织可明显地分为内外两层。内层的结缔组织、血管与原始生殖细胞一同伸进卵巢内部形成许多产卵板。产卵板以卵巢腔为中心向四周呈辐射状排列,上有原始生殖细胞分化形成卵原细胞和不同时相的卵母细胞。半滑舌鳎卵巢中的卵母细胞的发育可以分为5个时相。本文描述了各时相卵母细胞的形态学特征。并阐述了半滑舌鳎的产卵类型。     

脉红螺交配后性选择机制

交配后性选择是雌性混交动物中的一种普遍现象,其选择机制较为复杂,是性选择研究领域中的重点和难点问题.脉红螺是我国重要的大型经济贝类之一,繁殖形式为多雌多雄混交模式.为了研究该模式繁殖机制,本研究利用6个微卫星标记及行为学观察对脉红螺3个家系进行父权分析.结果表明,3个家系均存在多父性现象,证实脉红螺繁殖属于真正遗传意义上的雌性混交模式.3个家系中父权比例与父本的6个形态学参数及交配持续时间不存在关联性,但均表现出最先与母本交配的父本其后代比例最高,而最后交配的父本其后代比例最低,表明脉红螺交配后性选择存在最先雄性精子优先受精现象,这一现象符合“topping off”假说,即最先交配的雄性排放了大量精子占据了受精囊的主要空间,导致其子代的比例最高;而最后交配的雄性由于受精囊空间的限制,其精子占据空间最少.本研究为海洋无脊椎动物的交配后性选择机制研究提供重要参考资料.     

室内养殖条件下中华绒螯蟹雄性个体的生殖潜力

为评判中华绒螯蟹育苗工作亲本投放性比(♀∶♂=2~3∶1)的合理性,通过室内连续交配实验,对雄性个体的生殖潜力进行了研究。结果显示,雄蟹具备完成3次连续交配活动的生殖潜力;个体丧失交配能力前,贡献了超过60%的精子(≥6×109个)等雄性产物;多次交配活动并没有对雌蟹的个体繁殖力产生影响;雌蟹抱卵后,纳精囊亦没有出现明显的偏差;实验条件和基地育苗池个体纳精囊内均还存有一定数量的精子,表明雄蟹提供的雄性产物可以满足雌蟹受精的需要,因此,该性比在实际育苗生产中具有一定合理性,而作为亲本雄蟹的大规格及自身的生殖策略则可能是中华绒螯蟹避免精子限制发生的重要原因,其潜在的精卵比达(1.59~10.86)×10~3∶1。此外,研究成果揭示这个被广泛采用的亲本性比存在进一步优化的可能,可以提升至4∶1。     

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。     

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.     

冬眠中华鳖雌、雄生殖道的精子储存

应用光镜和电镜技术,系统观察冬眠中华鳖(Trionyx sinensis)雌、雄生殖道的精子储存情况,显示与精子储存相关的形态结构和细胞特征。结果表明,中华鳖在冬眠期生精活动处于相对静止状态,仅有1～3层精原细胞排列在睾丸曲细精管基膜附近,其余生精细胞排列松散而紊乱,生精上皮中未见有各级细胞规律性排列,不能形成完整的管壁。附睾管腔增大,腔中储存有大量精子。附睾上皮及附睾管腔中见有PAS反应阳性物质。附睾上皮主要由主细胞、亮细胞和基细胞组成。超微结构显示,附睾上皮细胞中含有大量分泌颗粒,内质网膨大。储存在附睾中的精子结构完整,精子中段由35～40个同心圆状线粒体构成线粒体鞘,中段胞质中含有大量糖原颗粒。雌性输卵管分布着数量不等的精子,尤其狭部最多。狭部黏膜皱襞高度融合,形成大量纵行于输卵管的储精细管。在细管中含有大量精子,精子头部嵌入上皮细胞纤毛中。蛋白部和子宫部也有少量精子存在,但在这些部位不能形成明显的储精细管。PAS反应显示,输卵管狭部储精细管上皮分泌有糖蛋白类物质。储精细管上皮下固有层中分布有大量的管状腺,通过腺导管与输卵管管腔相通。雌、雄中华鳖隔离4个月(12月初至来年3月底)后,在雌性输卵管峡部的储精细管中仍可观察到大量结构完整的精子,表明中华鳖精子在输卵管中至少可以储存120 d。以上结果显示,在冬眠期,雄性附睾和雌性输卵管中有大量精子储存,储存的精子能够渡过漫长的冬季用于来年春天交配或受精,这一特殊的生殖策略对其成功繁殖具有重要意义。本研究还对中华鳖的繁殖特性进行了讨论。     

蟹类精荚贮存和裂解研究进展

甲壳动物在海洋中分布广泛,有多种精子传输方式,大部分产生精荚,粘于雌体腹部或通过交接器输送到纳精囊中。研究表明,在蟹类交配,精子转移,贮存和受精等过程中,存在如精子竞争和亲体保护,多次受精等众多复杂事件。这些过程主要是以蟹类纳精囊为核心异型,为此本文综述了纳精囊的结构和功能,精子活力的维持,精荚形态,精子竞争和转移等近年研究进展,旨为相关研究提供参考。     

Proteins of seminal fluid and spermatozoa in the trout (Oncorhynchus mykiss): Partial characterization and variations

The protein composition of seminal fluid, blood serum, sperm plasma membrane and flagellum of rainbow trout were analysed by SDS-polyacrylamide gel electrophoresis. Immunological identity between proteins of the 2 fluids and sperm components was studied using crossed immunoelectrophoresis, rocket immunoelectrophoresis and immunoblotting. Results indicate that many seminal proteins are antigenically-related to serum proteins, proteins of sperm origin are present in seminal fluid in varying amounts, depending on the animals and sampling time, and several serum-like seminal proteins are bound to spermatozoa. Lipoproteins were isolated from seminal fluid (mean level: 33 μg/ml) and characterized. They were identified as being HDL-like lipoproteins. A possible physiological role is proposed for these seminal lipoproteins.     

The antioxidant system of seminal fluid during in vitro storage of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> sperm

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.     

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane,acrosome and DNA of black tiger prawn (Penaeus monodon) spermatozoa

To explore the impact of moulting and short‐term chilled storage of spermatophores on the sperm quality for a commercially important penaeid prawn, spermatozoa of Penaeus monodon from early (B‐C), middle (D0‐1) and late (D2‐3) moult stages were compared for sperm quality parameters relating to the structural integrity of plasma membrane (Viab%), acrosome (AR%) and DNA (SDF%) after being stored at 4°C for 0–26 days. The three different sperm extenders used for chilled storage included artificial lobster haemolymph (AH), calcium free saline (CS) and filtered seawater (FS); two storage conditions were applied either as a free sperm suspension or retained within the intact spermatophore. Results showed that (a) the lowest natural AR% was shown for B‐C spermatozoa in CS whereas the highest levels were for B‐C, D0‐1 and D2‐3 spermatozoa in FS and D2‐3 spermatozoa in AH; (b) the calcium ionophore A23187 agent used in this study was able to increase the mean AR% by 6.22%; (c) the Viab% was significantly lower in CS than that in FS; (d) the SDF% significantly increased over the period of chilled storage for B‐C and D0‐1 spermatozoa, while the SDF% of D2‐3 spermatozoa was initially elevated and did not change significantly over time; and (e) there was no difference in sperm quality between two storage conditions. This study has successfully demonstrated the moult‐related variance in the percentages of acrosome‐reacted and DNA‐damaged spermatozoa, providing evidence of moult‐related spermatophore renewal cycle in this species.     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

Changes in Sperm Production,Sperm Motility,and Composition of Seminal Fluid in Caspian Brown Trout,Salmo trutta caspius,Over the Course of a Spawning Season

This study was carried out to evaluate milt quality in male Caspian brown trout (Salmo trutta caspius) over the course of the winter spawning season. Milt samples were collected biweekly during December and January. Chemical composition of seminal fluid, sperm production (milt volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were measured. Milt volume, sperm density, osmolality, seminal minerals (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl^?), and total protein gradually decreased over the spawning season. Glucose and triglyceride content of milt did not show significant changes over the spawning season. Milt pH and the percentage and duration of motility were comparatively stable, declining only at the end of the season. Significant positive correlations were found between sperm density and seminal minerals, total protein and spermatocrit; percentage of motile spermatozoa and seminal minerals, total protein; and duration of motility and K⁺, Cl^?, total protein, and pH. Results show that season has a significant influence on milt quality in male Caspian brown trout, with the best milt being available at the beginning of spawning season.     

Changes in Atlantic cod (Gadus morhua L.) sperm quality during the spawning season

The biology of cod reproduction is well described in the scientific literature. However, sperm biology and spermatozoa management are poorly studied in this species. Because of its recent farming expansion, a better knowledge of cod gametes is becoming especially useful. This work aimed at establishing tools to study sperm biology in cod, and also investigated the existence of changes in cod sperm quality during the spawning period. We showed that sperm concentration could be assessed using spectrophotometry at 260 nm. Sperm motility significantly decreased after a 168‐h storage at 4 °C. A 1:9 dilution of sperm in a non‐activating medium (1/3 seawater and 2/3 freshwater, osmotic pressure: 360 mOsm kg^?1) improved sperm storage. Sperm concentration, sperm velocity and storage capacity at 4 °C peaked during the medium period of the spawning season and then decreased to values close to those observed at the beginning of the reproductive period. The measured values of osmotic pressure, pH, protein, Na⁺, Cl^? and Ca²⁺ concentrations of the seminal fluid were modified along the spawning period. Cell damage was noted at the end of the spawning period: local blebs were observed on the flagellum but also loops at its distal part. On the other hand, spermatocrit did not vary with the sampling date. In conclusion, cod sperm quality is modified during the spawning period, the highest‐quality samples being collected during the medium part of this season.     

Chemical composition and osmolality of seminal fluid of Acipenser persicus; their physiological relationship with sperm motility

Determination of semen quality is necessary to understand the basic biochemical processes occurring during motility of sperm and during fertilization to evaluate the reproductive ability of different fish species and to create an optimal environment for storage of spermatozoa; in this regard less information is available for Acipenseridae compared with Cyprinidae and Salmonidae. The aim of the present study is to determine chemical composition and osmolality of seminal fluid and their relationship with sperm motility in Acipenser persicus. The results obtained show that sodium (Na⁺), chloride (Cl^?) and potassium (K⁺) were predominant ions in the seminal plasma and the average of osmolality of seminal plasma was 82.56 mOsm kg^?1. The higher chemical contents and osmolality compared with other sturgeon species reveal species‐specific characteristics and high secretory activity of spermatic duct in A. persicus. Significant positive correlations were observed between osmolality‐Cl^?, Na⁺‐osmolality and Na⁺–Cl^? (P<0.05, P<0.001 and P<0.05 respectively). But statistically significant correlation was not observed between seminal plasma parameters and sperm motility. Probably, the Na⁺ and Cl^? are the main electrolytes playing a major role in maintaining the osmolality of the seminal plasma and the viability of the spermatozoa in vivo.