单环刺螠受精过程的细胞学观察

利用组织学和荧光显微镜对单环刺螠(Urechis unicinctus)受精过程中精子入卵以及核相变化进行了观察.结果表明:单环刺螠成熟卵呈卵圆形,核相处于生发泡尚未破裂的第1次成熟分裂前期.在(21±1)℃时,精卵混合后约5 min,精子由卵偏向植物极一侧入卵,受精膜开举起,继而精核发生第1次膨胀.直至受精后约25 min,受精膜完全举起;受精后10～50 min,卵核相继完成二次成熟分裂,分别形成第一极体和第二极体;受精后50～70 min,精核卵核分别膨胀,形成雄原核和雌原核,进而相向迁移,最后在卵的中央联合形成合子核.受精后70～90 min,受精卵完成第1次卵裂,形成2个大小相等的卵裂球.单环刺螠受精全过程历时约70 min,略长于多数海洋无脊椎动物的受精持续时间.本研究旨在为今后单环刺螠人工育苗和新品种的培育提供基础资料.     

Effects of stocking density on the growth performance,physiological response and intestinal microbiota of juvenile Echiura worms (Urechis unicinctus)

Aquaculture of the Echiura worm Urechis unicinctus has developed rapidly in recent years. However, the artificial cultivation technology remains to be perfected. In this study, the effects of stocking density on the growth performance, physiological responses and intestinal microbiota of U. unicinctus juveniles were evaluated. Juvenile worms were randomly stocked at densities of 200 U (U. unicinctus/m³), 500 U, 1,000 U and 2,000 U, in aquariums. The results indicated that the contents of triglycerides, glucose, total cholesterol, lactic acid, alkaline phosphatase, acid phosphatase and aspartate transaminase in the coelomic fluid of U. unicinctus juveniles were significantly enhanced with increasing stocking density. The digestive enzyme activities increased with increasing stocking density from 200 U to 1,000 U, before exhibiting a significant decrease in the 2,000 U group. In contrast, the antioxidant capacity of U. unicinctus juveniles was suppressed under high stocking density conditions. In case of the intestinal microbiota of U. unicinctus juveniles, Actinobacteria were more abundant in the 200 U group; SJA15, Gluconobacter and OP81 were higher in the 1,000 U group; Burkholderiales and Limnohabitans were enriched in the 2,000 U group. The highest and lowest survival rates of the U. unicinctus juveniles were recorded in the 500 U and 200 U groups respectively, and high stocking densities negatively affected the individual growth performance. In summary, due to the highest survival rate and appropriate growth performance and physiological responses, 500 U was considered to be the optimal stocking density for the cultivation of U. unicinctus juveniles.     

单环刺螠消化道的发生和分化

用组织学、组织化学和电镜技术对单环刺螠（Urechis unicinctus）消化道的发生及分化进行研究。结果表明，单环刺螠消化道起源于原肠胚期内陷而成的原肠腔；至早期担轮幼虫时期，消化道连通，开始摄食，此时据管腔直径不同消化道可分为口、食道、胃、中肠及后肠。附着变态后，其消化道在结构及功能上都有了明显的发育与分化。至蠕虫状幼虫时期。食道、胃及后肠已出现黏膜下层及肌层。嗉囊与砂囊可明显区分。中肠已产生肌纤维的分化，呼吸肠由中肠分化而来，其胞体细长，且核质比较小，可与中肠明显区分。至幼螠时期，食道前端产生了咽部的分化，至此消化道各部位在结构及功能上都与成体有了很大的相似性。即由口、咽、食道、嗉囊、砂囊、胃、中肠、呼吸肠、后肠和肛门组成。组织化学研究结果表明，随着发育，消化道各部位分别呈现不同的碱性磷酸酶、酸性磷酸酶、脂酶、ATP酶、非特异性酯酶及多糖类物质活性。     

单环刺螠精巢年周期发育及精子发生

采用组织学方法观察了单环刺螠(Urechis unicinctus)精巢的形态结构及其年周期发育,并利用超微技术观察了精子的发生过程。结果表明,单环刺螠的精巢呈条带状,位于虫体尾部,两端通过肌束分别与呼吸肠管壁外侧和体壁内侧相连。精巢由生精细胞团与结缔组织组成,结缔组织位于精巢中央,精原细胞团分布于结缔组织外周,精母细胞团散布于结缔组织内,各级精母细胞脱离精巢落入体腔液中,历经精母细胞、精细胞,最后形成精子脱离细胞团进入肾管。根据精巢的组织学特征,将单环刺螠的精巢年周期发育划分为增殖期、生长期、成熟期、排放期和休止期5个时期。光镜和电镜下观察了精巢和体腔液中精子发生各时期的结构变化,包括细胞核形态的变化、顶体发生以及尾部的形成。本研究揭示了单环刺螠的精巢发育特点、精子发生历经的场所和分化特征。     

单环刺螠增养殖生物学的研究

单环刺螠个体鲜出肉率大致为冬季39～47％,春季17～27％。食性为滤食性,以水中悬浮性颗粒为食。气温7℃,干露时间持续72h．均能成活。生活水温是0～30℃,临界高温32．5℃,临界低温-4℃。生活的盐度范围是15～36,适宜盐度范围24．94～35．77。pH值的耐受范围是4．46～9．5,适宜范围7.5～9．42。在溶解氧含量达1．14mg／L时,活动正常。     

单环刺虫益人工育苗技术的研究

在单环刺自然繁殖期，以诱导法或剖取法获取精、卵，在水温１５～２２℃下，进行授精、孵化、幼虫培育。对幼培育过程的设施、理化条件、管理技术等进行了研究试验。结果：１９９４年，单位水体平均培育幼１６８４个／；１９９５年，单位水体平均培育幼４０６万个／。经专家鉴定，这项成果达国际先进水平     

氨氮及盐度对单环刺螠耗氧率的影响研究

在实验室条件下测定了盐度、氨氮对单环刺螠耗氧率的影响。单环刺螠耗氧率随时间的延长逐渐降低,两者之间呈二次多项式关系：OR=ax^2＋bx＋c;盐度对单环刺螠耗氧率的影响非常明显（p〈0．01）,盐度在31时,各种规格单环刺螠的耗氧率达到最大值;单环刺螠的耗氧率与NH4^＋浓度呈明显幂函数关系：OR=ax^-b,经F检验总氨氮与体重对单环刺螠耗氧率的影响均达极显著水平（p〈0．01）。     

单环刺螠体壁氨基酸组分与含量的分析

单环刺螠体壁肌含有１８种氨基酸（其总量占体壁肌干重的５７．３９％）。其中,８种人体必需氨基酸含量 １７．７９％。５种鲜味氨基酸（Ｇｉｎ,Ａｓｐ,Ａｒｇ,Ａｌａ,Ｇｌｙ）含量都比较高。必需氨基酸含量组成模式与人体需要模式较接 近。支／芳值高达２．５９,接近正常人的支／芳值     

单环刺螠生活史研究

有关单环刺发育周期的研究，国内外迄今未见专门报道。本文报道了烟台沿岸单环刺早期胚胎发育、各期幼虫形态标志、幼和成的形态特征的研究结果。     

pH、温度和盐度对单环刺螠消化酶和溶菌酶活力的影响

采用L25(56)正交试验法测定了不同pH(6、7、8、9、10)、温度(10、15、20、25、30℃)和盐度(15、20、25、30、35)对体质量为(71.2±8.5)g的单环刺螠肠蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响,以及不同盐度对单环刺螠血液溶菌酶活力的影响。试验结果表明,单环刺螠肠消化酶活力的最适环境条件分别为,蛋白酶:pH 8、温度30℃、盐度35;淀粉酶:pH 9、温度25℃、盐度25;纤维素酶:pH6、温度30℃、盐度35;脂肪酶:pH 6、温度25℃、盐度30。盐度15和20两组单环刺螠血液溶菌酶活力均先降后升再降,而盐度30和35两组则先升后降。至处理第4d,各试验组溶菌酶活力依次为:盐度3025352015,盐度15和20两组溶菌酶活力显著低于盐度较高试验组(P0.05)。盐度25~35、pH 6~9为单环刺螠适宜的环境条件,高温(25~30℃)下其消化酶活力较高,而低盐度(15~20)下其消化酶活力和免疫能力明显降低。     

单环刺螠虫精子生物学特性和环境因子的关系

解剖成熟的单环刺虫(Urechis uniconctus)成体,直接从肾管中取出新鲜精子进行实验。结果显示,单环刺虫精子为鞭毛型,头部最前端为顶体,呈奶嘴状;细胞核近似杯状,核内有核泡。中段由一个大环状线粒体组成。尾部轴丝为典型的“9+2”型结构。精液的pH值为6.5±0.2;精子密度为(4.2±0.2)×109/mL;新鲜精子经过滤海水(10 cm厚的脱脂棉过滤)的激活率为86.4%±6.3%,涡动时间为(17.0±6.9)min,寿命为(24.4±7.8)min。室温(20±1)℃下,精子保存12 h活力无显著变化,但至24 h活力明显下降;低温(4℃)可明显延长精子活力的保存时间,可保存21 d;盐度和酸碱度对精子的激活率、涡动时间和寿命都有较大的影响,精子适宜的盐度为20~30,最佳盐度为25;适宜的酸碱度为7~9,最佳为8。因此,低温可以较长时间的保存单环刺虫的精子,在盐度稍低或偏碱性的海水中精子活力较高。     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

A morphological classification proposal for curimba (Prochilodus lineatus) sperm damages after cryopreservation

The present study evaluated some cryoprotectants and concentrations for curimba (Prochilodus lineatus) semen preservation from a simple methodology of analysis of sperm morphology. Semen of nine males was diluted (1:4) into eight cryosolutions, all of them containing 5% of BTS^? Minitüb, still comprising methanol or dimethyl sulphoxide (DMSO) at four concentrations: 5%, 7.5%, 10% and 12.5%. Morphological sperm analysis was performed on an optical microscope at × 1000 magnification. Damages on the head, midpiece and tail rest (the main and terminal portions) were investigated and classified as primary or secondary, according to the assumed influence on fertilization. There was a linear reduction in the total damages with an increase in the cryoprotectant concentration. Samples with DMSO showed the lowest percentage of fractured tails and tail stumps. The cryopreservation protocol retained the fertilizing potential of the sperm after freezing and both methanol 5% and DMSO 7.5% conferred adequate protection to the curimba sperm cells. Sperm morphology, as pointed out here, must be incorporated into routine fish sperm (cryopreserved or not) analysis once sperm defects influence fertilization directly. To the best of our knowledge, this was the first report on curimba sperm damages after cryopreservation by optical microscopy. The classification model presented in this study can be adequate to tackle fish sperm damages.     

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.     

Effect of storage time and cryoprotectant concentrations on the fertilization rate and hatching rate of cryopreserved sperm in red seabream (Pagrus major Temminck & Schlegel, 1843)

This study examined the effects of storage time and cryoprotectant concentrations on the post‐thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for <60 days and fresh sperm (98.8±0.8%, 96.4±1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6±3.0% to 7.0±1.9%) and hatching rates (79.4±7.2% to 3.3±0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P<0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long‐term sperm cryopreservation of red seabream.     

Evaluation of the potential source of bacterial contamination during cryopreservation process of silver barb (Barbodes gonionotus) sperm

Application of good sanitation practice in cryopreservation process is the key issue to improve the quality of cryopreserved fish sperm. This study implemented standard sanitation protocol with minimal contamination and evaluated the source of bacterial contamination associated with laboratory equipment and related materials across a series of cryopreservation process of silver barb (Barbodes gonionotus) semen. The use of 16s rRNA sequencing and traditional biochemical methods were performed for bacterial identification. Animal origin (anal fin, culture water and semen contaminated with faeces and urine) and non‐animal origin (liquid nitrogen from liquid nitrogen dewar, outer surface of straw, air circulation in cryopreservation laboratory and latex gloves used during cryopreservation procedure) were determined for bacterial contamination. Aeromonas punctata subsp. caviae was the most abundant species in anal fin, latex groves and semen contaminated with faeces and urine. Bacillus safensis and Bacillus sp. were found as frequently recovered species from liquid nitrogen dewar. Aeromonas hydrophila subsp. hydrophila and Pseudomonas fluorescens, fish pathogenic bacteria, were isolated from all animal origin samples. This was the first report indicating that standard sanitation and hygiene methodologies are recommended for sperm cryopreservation of B. gonionotus to prevent bacterial contamination.     

Development of a simple method for sperm cryopreservation of Scombridae fishes in outdoor environments

We developed a simple dry shipper method for cryopreserving the sperm of Scombridae fish in outdoor environments. First, we undertook a preliminary study to optimize the sperm cryopreservation conditions using bullet tuna, Auxis rochei (Risso, 1810) sperm. We found that the optimum cryomedium contained 90% foetal bovine serum (FBS) or 300 mM trehalose as an external cryoprotectant and 10% dimethyl sulfoxide (DMSO) as an internal cryoprotectant. Under these optimized conditions, the post‐thaw sperm had a duration of motility of 500 s and a motility rate of >70%. We then performed practical trials of the optimized protocol in various outdoor environments (e.g., fishing boats and ports) using the sperm of five Scombridae species: chub mackerel, Scomber japonicus (Houttuyn, 1782); blue mackerel, S. australasicus (Cuvier, 1832); skipjack tuna, Katsuwonus pelamis (Linnaeus, 1758); longtail tuna Thunnus tonggol, (Bleeker, 1851) and Pacific bluefin tuna, T. orientalis (Temminck & Schlegel, 1844). The post‐thaw sperm of all five of these species had a duration of motility of 650 s and a motility rate of >70%, indicating that this simple method can be used to obtain high‐quality cryopreserved sperm of various Scombridae species in outdoor environments.     

Cryopreservation of filefish (Thamnaconus septentrionalis Gunther, 1877) sperm

The present study examined the possibility of long‐term storage, by cryopreservation in liquid nitrogen, of the sperm of filefish (Thamnaconus septentrionalis). Changes in motility, survival rate, ultrastructure and fertilization rate of the sperm after freezing and thawing were tested. For selection of the immobilizing solution, artificial seawater (ASW) of 250, 350 and 450 mOsmol kg^?1 were tested. Sperm motility was significantly inhibited in 350 mOsmol kg^?1 ASW, and restored entirely after 100% ASW (1200 mOsmol kg^?1) was added. Two cryoprotectants, dimethyl sulphoxide and glycerol, were employed. The sperm was diluted at the ratio of 1:6 with the extenders, and frozen at a freezing rate of ?40°C min^?1 to ?100°C after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The highest post‐thawed sperm motility and survival rate were obtained with 5% glycerol. Afterwards, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of ?30°C min^?1 to ?100°C showed the best result.