刺参受精及早期胚胎发育过程的细胞学观察

采用HOECHST 33258染色荧光显微方法,对刺参成熟未受精卵以及受精过程中精子入卵、极体排放、雌雄原核的形成与结合、早期卵裂以及多精入卵等细胞学进行了研究。结果显示,刚产出的刺参成熟未受精卵呈圆形,核相处于第一次成熟分裂中期;在水温22~23 ℃、盐度29条件下进行受精,受精后12 min,完成第一次成熟分裂,释放第一极体;受精后20 min,大部分受精卵完成第二次成熟分裂,放出第二极体。受精后35 min,雌、雄原核开始在卵中央发生染色体联合;受精后80 min,部分受精卵完成第一次卵裂,受精后100 min,部分受精卵完成第二次卵裂。刺参在受精过程中存在极少数的多精入卵现象。     

Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas (Thunberg)

The robustness of Pacific oyster, Crassostrea gigas (Thunberg), sperm cryopreservation in the context of selective breeding based on family lines was investigated. Irrespective of egg density, high fertilization success was achieved with cryopreserved sperm when sperm:egg ratios of 1000:1 to 10 000:1 were used. Variation among replicate runs on the same oyster batches was minimal, indicating that cryopreservation and larval rearing procedures were repeatable. Twenty independent single male–female crosses were made to assess the utility of cryopreserved sperm in selective breeding. The fertility of unfrozen sperm was generally a poor predictor of cryopreserved sperm fertility. Based on D‐larval yields, 17 of the 20 crosses were likely to yield adequate spat for selective breeding (>10⁵ D‐larvae from 1 million eggs), two were marginal (5 × 10⁴ D‐larvae) and one was inadequate (4 × 10³ D‐larvae). An alternative fertilization strategy to improve D‐yield from a given number of sperm was then tested. Fertilizing 10 million eggs at a sperm:egg ratio of 200:1 increased the total D‐yield when compared with fertilizing 1 million eggs at a sperm:egg ratio of 2000:1 for the same male–female pair. We conclude that, despite wide variation in fertility, cryopreserved sperm is useful for family production.     

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.     

伊犁裂腹鱼胚胎及早期仔鱼发育的观察

2010年5月通过人工催产,人工授精方法获得伊犁裂腹鱼(Schizothorax pseudaksaiensis Herzenstein)受精卵,在人工培育的条件下对其胚胎和仔鱼发育进行观察.结果显示:伊犁裂腹鱼成熟卵为淡黄色,卵径为1.70～1.80 mm.受精40 min后,受精卵卵周隙接近最大化,此时卵径为2.9...     

缢蛏受精和早期卵裂过程的细胞学变化观察

采用普通光镜、荧光显微镜和石蜡切片技术,对缢蛏受精和早期卵裂过程中的精子入卵、减数分裂、雌雄原核形成与结合、早期卵裂以及多精入卵等细胞学事件进行了显微观察。结果表明,缢蛏成熟未受精卵多呈圆球形或卵圆形,少数呈梨形,卵径为82～88μm,核相处于第一次成熟分裂中期;精子为典型的原生型,全长55～58μm,头部呈保龄球形,顶体前端的顶体杆呈特别细长的花丝状;在水温21～22℃、盐度10的条件下进行人工授精,精、卵混合后,精子迅速附着于卵子表面,启动卵子发育;受精后4～6 min,精子的头部已进入卵内并明显膨胀,卵子外形变圆,卵外附着的精子量明显减少;在受精后12～15 min、20～25 min,受精卵先后排出第一极体、第二极体,完成两次成熟分裂;第二次成熟分裂结束以后,精、卵核体积迅速膨胀,雄原核的膨胀早于雌原核,核膜重建,在受精后约30 min形成雌、雄原核;雌、雄原核均向卵子中央移动,雄原核旁边的精子星光清晰可见,随后二者在卵子中央以染色体联合的方式结合,联合核的染色体共同排列在纺锤体的赤道板上,形成第一次有丝分裂的中期分裂相;受精后40 min左右,受精卵进行第一次卵裂,染色体在纺锤丝的牵引下向两极移动,结果形成2个大小不等的卵裂球;受精后45～50 min,卵子进行第二次卵裂,核相变化与第一次卵裂相同,在与第一次卵裂垂直的纵轴方向上发生不等全裂,最终形成3小1大4个卵裂球;受精后60～70 min,胚胎进行第三次卵裂,仍为不等全裂,但自此次卵裂起已开始进行螺旋分裂。此外,对实验中发现的缢蛏极少量多精入卵、多极分离等异常细胞学现象进行了分析,并探讨了海洋贝类卵子阻止多精入卵发生的机制。     

普安银鲫胚胎发育的初步研究

经人工催产、人工授精获得普安银鲫受精卵,对其胚胎发育过程和各发育时期外部形态特征进行了连续观察,并研究了不同温度条件下其胚胎发育的特点。研究结果表明,普安银鲫受精卵近似圆球形,淡灰色,沉性卵,强粘性;卵径1.50mm,吸水后1.88mm;受精后开始形成卵间隙,且植物极的卵间隙大于动物极的;卵间隙形成过程中卵黄收缩,略变小。普安银鲫胚胎发育过程可分为23个发育期,水温18℃时需98.72h出膜,水温24℃和26℃时分别需要56.48h、48.2h出膜。不同温度条件下,普安银鲫胚胎发育早期(胚盘～原肠胚)在18℃恒定水温条件下存活率最高,发育后期阶段(神经胚～出膜)在24℃恒定水温条件下存活率最高。     

Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818)

The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

Sperm vitrification of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>: effect of cryoprotectant solutions on sperm viability and spawning quality after artificial insemination

Vitrification is a fast freezing method with promising results for penaeids sperm cryopreservation. This study evaluated the efficiency of three cryoprotectant solutions for sperm vitrification and artificial insemination with cryopreserved spermatophores of Litopenaeus vannamei. The cryoprotectant solutions tested were 30% methanol, 2% soy lecithin, and 30% methanol?+?2% soy lecithin. Fully mature females were artificially inseminated with vitrified and fresh spermatophores as a control group. The vitrification method was efficient in maintaining high rates of sperm survival and membrane integrity. Although the egg fertilization was successfully attained by artificial insemination with cryopreserved spermatophores, low hatching rates suggested that possible DNA fragmentation of sperm cells should be further investigated. This is the first report of artificial insemination using vitrified sperm in penaeids.     

Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

The effects of sperm–egg ratios on polyspermy in the blood clam,Tegillarca granosa

Polyspermy in Tegillarca granosa, which leads to failure or abnormal development of early embryos, appears to be directly related to varying sperm–egg ratios. We investigated the correlation between sperm–egg ratios and conditions of embryonic development by setting six strict sperm–egg ratios and subsequently scoring fertilization rate, abnormal rate, rates of trochophore and D‐shaped larvae. When sperm–egg ratios rose from 2 × 10²:1 to 1 × 10³:1, D‐shaped larvae rate dropped significantly from 78.8 ± 13.3% to 47.8 ± 14.9% (analysis of variance, P< 0.05), and to 3.6 ± 2.8% in the 2 × 10⁴:1 group. Using fluorescence microscope observation, polyspermy rates rose from 2.6 ± 0.8% to 72.9 ± 4.8% with increases in sperm–egg ratios. Sperm–egg ratios were therefore correlated with polyspermy rate, which seriously affected larval survival. Under the premise of higher fertilization rate, the sperm–egg ratios of ≤2 × 10²:1 appeared to be more beneficial for embryonic development of T. granosa.     

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.     

Hormonally Induced Spawning, Embryonic Development, and Larval Rearing of the Southern Temperate Banded Morwong, Cheilodactylus spectabilis

Banded morwong (Cheilodactylus spectabilis) are of interest for marine finfish aquaculture in temperate southern Australia. To improve their ovulatory response, adult females were implanted during the autumn spawning season with slow‐release pellets containing 0–400 μg luteinizing‐hormone‐releasing hormone analogue (LHRHa)/kg body weight within 24 h of capture from the wild. Compared to the sham control group, animals treated with LHRHa produced significantly more eggs on each day after implantation for the following 7 d (91 ± 39 and 290 ± 38 mL) and a higher proportion ovulated (8/12 and 27/27). Of fish treated with LHRHa, 93% ovulated 2 d after implantation and 79% ovulated three times at 2‐d intervals, whereas control animals showed no cyclicity of ovulation and few ovulated more than once. Egg production was highest at the first ovulation after LHRHa treatment and declined at subsequent ovulations. In a second experiment investigating the range 100–400 μg LHRHa, there was no effect of dose rate on ovulation parameters, which additionally examined implantation either immediately after capture or after a 5‐d delay. Compared to immediate implantation, a delay resulted in a lower proportion of animals that could be stripped after implantation (100 and 50%, respectively) and the volume of eggs was lower (135 ± 15 and 107 ± 10 mL). The egg quality was poor following delayed implantation, resulting in no fertilization after artificial insemination compared with immediate implantation in which fertilization and hatch rates were higher for eggs collected on Day 2 after implantation (79 ± 8% and 58 ± 9%) than on Day 4 (23 ± 7% and 15 ± 6%). Thus, it is important to implant animals as soon as possible after capture to ensure optimum egg quality. Good‐quality eggs were buoyant and spherical and had a diameter of 1050 ± 25 μm with a single pigmented oil droplet of 190 ± 9 μm. When a separate large batch of eggs collected 2 d after implantation with 100 μg LHRHa was inseminated and cultured at 18 C, larvae hatched after 63 ± 2 h at a standard length of 2.6 ± 0.4 mm. Newly hatched larvae were buoyant and transparent with only a few melanophores, eyes were nonpigmented and jaws were nonfunctional. By the fourth day, jaws were functional and eyes were fully pigmented. Utilization of the endogenous yolk and oil was completed by Day 6, and swimming commenced with exogenous feeding. Larvae, initially fed lipid‐enriched rotifers followed by Artemia, reached 8.9 ± 0.7 mm length on Day 55, after which they metamorphosed to the postlarval paperfish stage of development, 22 ± 0.9 mm on Day 100, and 43 ± 1.0 mm at 6 mo of age. The results show that treatment of wild‐caught females with slow‐release pellets containing LHRHa is effective for the production of eggs for hatchery rearing.     

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。     

分光光度法测定俄罗斯鲟精子密度标准的研究

为了标准化精子超低温冷冻保存和人工授精程序,建立了分光光度法测定俄罗斯鲟精子密度的方法,比较了不同波长(380 nm、530 nm、780 nm)下吸光度(A)与精子密度(C)的关系。结果表明,分光光度法的检测下限为3×106cells/mL,且检测上限随波长的增加而上升,当精子密度为3×106~1.5×109cells/mL时,530 nm为最适检测波长,吸光度与精子密度呈对数回归关系,其回归方程为:A530=-8.560+1.323 lgC(R2=0.971)。     

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.     

带鱼人工授精和孵化

关于带鱼(Trichiuruslepturus)的早期生活史,Delsman[1]、Nair[2]、神谷尚志[3]、内田惠太郎[4]以及水户敏[5]分别描述了爪哇海、孟加拉湾和日本沿海带鱼早期发育阶段的形态;成庆泰等[6]对带鱼的早期发育阶段的形态作过简要的报道;姜言伟[7]对海州湾带鱼卵的发育状况和数量分布亦作过简要的报道;陈莲芳[8]对我国近海带鱼的产卵期、产卵场分布、产卵场的生态条件以及渤海带鱼早期发育阶段的形态作过较为详细的报道;沙学绅等[9]于1958年6月26日在渤海黄河口变化进行了讨论。1　材料和方法“北斗”调查船2002年6月执行山东半岛南部鱼产卵场…     

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。     

Development of embryos and larvae in the common Japanese conger Conger myriaster

Development of embryos and larvae in the common Japanese conger Conger myriaster was observed after artificial fertilization. Eggs were obtained from females matured artificially by hormone injections and milt was obtained from males matured naturally. Fertilized eggs were kept in seawater at 12–14°C. The first cleavage occurred at 4 h, epiboly began at 24 h, the embryonic body was formed at 38 h and hatching occurred at 84 h after insemination. Newly hatched larvae were approximately 2.5 mm (total length) and similar to those of Anguilla japonica in terms of external features. The mouth and anus opened on the 7th day after hatching. Pigments began to appear at the tip of the tail on the 10th day. The total length of the larvae reached approximately 8 mm on the 11th day. Eye pigmentation began on the 14th day. One larva lived for 19 days without food.     

Embryonic development of Ganga river prawn Macrobrachium gangeticum (Bate)

Macrobrachium gangeticum (Bate) are commercially valuable prawns that are broadly distributed in the Ganga river that flows from northern to eastern India. We studied embryonic development from cleavage (blastomeres), segmentation, formation of optic vesicle, eye pigment development to larva formation, as these data are largely unknown in this species. We conducted this study using mature M. gangeticum broodstock placed in freshwater tanks. Just after spawning, females were kept in separate aerated tanks, where fecundity was estimated from the total number of eggs. Thereafter, egg samples from berried females were used to study their embryonic stages. The correlation coefficient between size and prawn weight, egg weight, egg number, body weight and egg mass of different sizes of prawns was analysed. Our results show that an increase in prawn size led to an increase in egg weight and egg number while decreases in body weight:egg mass ratio was noticed. Further results indicate that an increase in prawn weight had a positive impact on egg weight and egg number. The embryo development stages were used in this study as a tool to evaluate the development process of the prawn in a hatchery environment.