Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm

A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N₂ in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.     

Beneficial co‐culture of jellyfish Rhopilema esculenta (Kishinouye) and sea cucumber Apostichopus japonicus (Selenka): implications for pelagic‐benthic coupling

This study investigated monthly changes of sedimentation and sediment properties in three different culture systems (ponds) – i.e. jellyfish Rhopilema esculenta monoculture (J), sea cucumber Apostichopus japonicus and jellyfish co‐culture (SJ) and sea cucumber monoculture (S) – to verify the feasibility of co‐culturing jellyfish and sea cucumbers. Results showed that jellyfish culture accelerated the settling velocity of total particulate matter (TPM). Average TPM settling velocities in the SJ (75.6 g m^?2 day^?1) and J (71.1 g m^?2 day^?1) ponds were significantly higher than that in the S pond (21.7 g m^?2 day^?1) from June to September during the jellyfish culture period. Average settling velocities of organic matter (OM), total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) in the SJ pond increased significantly by 3.0, 2.9, 3.3 and 3.8 times, respectively, compared with those in the S pond. Sediment contents of OM, TOC, TN and TP in the SJ and J ponds were significantly higher than those in the S pond during the jellyfish culture season. The specific growth rate of sea cucumbers feeding on SJ sediment was significantly higher than that of those feeding on S sediment. Co‐culturing sea cucumbers with jellyfish may help alleviate benthic nutrient loading due to the jellyfish and provide a secondary cash crop.     

刺参南方度夏试验

选择刺参南方池塘养殖收获时的小规格个体,采用鲍鱼养殖池流水度夏,时间超过6个月。2年刺参度夏存活率分别为70.6%和89.1%;失重率为56.1%和61.8%。度夏后刺参再养殖约150天,成活率分别为94.9%和81.1%;增长率为10.5倍和13.6倍。     

A new system for the culture and stock enhancement of sea cucumber,Apostichopus japonicus (Selenka),in cofferdams

A new multilayer, plate‐type system for the culture and stock enhancement of sea cucumbers in cofferdam was developed. To optimize and evaluate the system, four experimental designs were implemented using polyethylene (PE)‐corrugated sheets of various colours, interval spacing and shapes/styles. Results showed that a system equipped with black PE‐corrugated sheets attracted more animals than either blue, green, transparent or a selection of mixed sheets (six transparent sheets in the upper layer and five black sheets in the lower layer) (P<0.05). Also, more animals gathered in the system with oblique‐angled sheets (30° to the base plate) than either a wavy (the bottom and every second sheet was at an angle of 10° to the base plate) or parallel arrangement (P<0.05), and more animals assembled in the system with 2 cm between sheets than spacings of 3, 4 or 5 cm (P<0.05). As expected, the upper layers of the systems attracted more animals than lower layers in most cases except for those with transparent and mixed oblique‐angled sheets with a 3 cm spacing (P<0.05). Thus, a system with black, oblique‐angled‐corrugated sheets and 2 cm spacing is recommended for Apostichopus japonicus (Selenka) culture and stock enhancement in cofferdams or ponds.     

Effects of light intensity on daily activity rhythm of juvenile sea cucumber,Apostichopus japonicus (Selenka)

Eight light‐intensity treatments (natural light, continuous darkness, and 15, 30, 60, 125, 250 and 500 lx under LD 12:12 cycle) were used to investigate the effects of light intensity on the daily activity of 30.27±3.08 g sea cucumber Apostichopus japonicus (Selenka). Cyclic nocturnal activity patterns of behaviour were observed at different light intensities in the range of 15–500 lx under LD 12:12 cycle. And an ongoing nocturnal cycle persisted in DD cycle for up to 8 days, but longer feeding time and less marked rhythm occurred at continuous darkness. Under poor light conditions (I<5.18 lx), the daily activity rhythm of A. japonicus was governed by an innate biological clock and the effect of light intensity was not significant among different treatments. And more individuals tended to retreat to shelters (from 56.04% to 91.83%) with the increase of light intensity within the weak light condition (from 5.18 to 278 lx). However, the daily behaviours of A. japonicus were influenced under strong light conditions (>278 lx). Less than 8.17% individuals kept actively feeding and the proportion was not decreased with the increase of light intensity.     

Microsatellite genetic variation in wild and hatchery populations of the sea cucumber (Apostichopus japonicus Selenka) from northern China

Farming of the sea cucumber Apostichopus japonicus (Selenka) started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4–12.3 and 0.735–0.783 respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (F_st range: 0.008–0.036), and no obvious difference was detected between the wild populations (F_st=0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials.     

仿刺参超氧化物歧化酶基因cDNA全序列的克隆及其特征分析

以仿刺参Apostichopus japonicus为材料,利用cDNA末端快速扩增(RACE)方法首次得到仿刺参MnSOD的全长cDNA。该全长cDNA大小为955bp,其中5′端非编码区67bp,3′端非编码区216bp,开放阅读框(ORF)672bp,在3′端有多聚腺苷酸信号序列ATTTA,并有polyA加尾,符合有效翻译的基因全长cDNA的特征。MnSOD全长cDNA可编码223个氨基酸的前体蛋白,预测蛋白分子质量为24.64kD,理论等电点是6.66,为亲水性蛋白。该蛋白序列没有信号肽和跨膜结构,仅有一段线粒体导肽。其二级结构主要以α-螺旋为主,无规则卷曲和延伸链构成了蛋白中量最大的结构元件,不含β-折叠。仿刺参SOD的氨基酸序列与目前已报道的其他物种如人、牛、斑马鱼、原鸡、非洲爪蟾、中国对虾、皱纹盘鲍、海湾扇贝、果蝇等的MnSOD氨基酸序列进行序列比对的结果表明,相似性均在61%～69%之间,说明该基因在各物种间具有一定的保守性。该段氨基酸序列无N-糖基化位点,含有3个蛋白激酶C磷酸化位点,1个酪氨酸蛋白激酶Ⅱ磷酸化位点,5个肉豆蔻酰基化位点,还发现了1个SOD家族的特征肽段DVWEHAYY。对磷...     

Effects of diatom concentration in prepared feeds on growth and energy budget of the sea cucumber Apostichopus japonicus (Selenka)

Eight test diets were used in the experiment: seven fresh diatom (Cylindrotheca fusiformis) diets in which diatom occupied 100%, 83.3%, 50%, 33.3%, 20%, 14.3% and 11.1% in dry matter of the feeds (marked as D100, D83, D50, D33, D20, D14 and D11), and a formulated diet (70% Sargassum thunbergii powder, 20% sea mud and 10% fish meal, based on dry weight, marked as ST). The results showed that the specific growth rates (SGRs) of the animals fed diets D33, D20, D14 and D11 were not significantly different from the SGR of those fed diet ST. However, the energy allocated to growth for the animals fed with diet D14 was twice that of diet ST. For sea cucumbers fed diet D14 the largest expenditure part was allocated to respiration (55.4%), but for those fed diet ST, the largest part was allocated to faeces (62.7%). The organic content of diet D14 was also much lower than that of diet ST. Frozen diatom diets containing 14% (dry matter weight) fresh diatom could be an environmentally friendly feed as an alternative option for macroalgae powder.     

Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus

Shewanella marisflavi isolate AP629 is described as a novel pathogen of sea cucumber. The LD(50) values (14 days) in sea cucumber, mice and swordtail fish were 3.89 × 10(6) , 6.80 × 10(4) and 4.85 × 10(4) CFU g(-1) body weight, respectively. Studies on S. marisflavi were conducted, including morphology, physiological and biochemical characteristics, haemolysis, whole-cell protein and 16S rDNA gene sequence. Colonies of S. marisflavi appeared faint red on marine agar and green on thiosulphate-citrate-bile salt-sucrose media. Shewanella marisflavi had polar flagella. The cells were Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. The bacterium exhibited β-haemolysis on sheep blood agar and produced H(2) S. Shewanella marisflavi survived and grew at 4-35°C, pH 6.0-9.2 and in the presence of 0-8% NaCl. The whole-cell proteins included 13 discrete bands, and proteins of molecular weight 87, 44 and 39 kDa were found in all five strains of Shewanella spp. The difference in 16S rDNA gene sequences in S. marisflavi was at the 446 bp site: S. marisflavi (KCCM 41822) - G, isolate AP629 - A. This is the first report that Shewanella is pathogenic to sea cucumber.     

Bottom culture of the sea cucumber Apostichopus japonicus Selenka (Echinodermata: Holothuroidea) in a fish farm,southern China

The bottom culture of southward‐transplanted sea cucumber Apostichopus japonicus in a subtropical fish farm was investigated in a field study at Dapeng Cove, Daya Bay, from January 5–August 5 2011, with the aim of finding the ideal period for culturing A. japonicus in fish farms, and developing an integrated multi‐trophic aquaculture (IMTA) in southern China. Results showed that the bottom‐cultured sea cucumbers survived well (100%) before summer, survival rates decreased to 65.00 ± 21.21% in July, and all animals had died at the end of the study. Specific growth rates of the sea cucumbers were high during winter (1.05 ± 0.03% d^?1), decreased in early spring (0.44 ± 0.11% d^?1) and became negative in the following months. Growth rate was mainly influenced by water temperature, dissolved oxygen and sulphide content; the anoxia caused by water column stratification at the seafloor in the summer were the main causes of mass mortality. Our results indicate that bottom culture in the temperate season (winter and spring, optimally from late November to early April) is a viable way to rear the deposit feeder A. japonicus underneath a subtropical fish farm.     

Vitamin E requirement of sea cucumber (Apostichopus japonicus) and its' effects on nonspecific immune responses

This feeding trial was conducted to determine the vitamin E requirement of sea cucumber (Apostichopus japonicus) and to study the effects of vitamin E on nonspecific immune parameters. Commercial sea cucumber diets supplemented with 0, 50, 100, 200, 500 or 1000 mg kg^?1 vitamin E were fed to juvenile sea cucumber (7.96 ± 0.01 g) for 60 days. Specific growth rate and total coelomocyte counts were maximized in sea cucumber fed diets supplemented with 100 mg kg^?1 vitamin E. Reactive oxygen species production was maximized at 50 mg kg^?1 vitamin E, whereas superoxide dismutase activity was the highest in sea cucumber fed diets supplemented with 100 mg kg^?1 vitamin E. The vitamin E concentration in the body wall of the sea cucumber increased as the vitamin E level in the diet increased. Analysis of specific growth rate and total coelomocyte counts of the sea cucumber indicated that 88–92 mg kg^?1 vitamin E is required for maximal growth and nonspecific immune responses of A. japonicus and that 114.7 mg kg^?1 vitamin E is required to minimize lipid peroxidation levels.     

Energy budget adjustment of sea cucumber Apostichopus japonicus during breeding period

Reproductive success is unpredictable for many cultured echinoderms. And cost of reproduction plays an important role in animal life‐history. Therefore, understanding cost of reproduction contributes to improving breeding techniques during broodstock rearing in aquaculture. In this study, energetic costs during breeding were explored in the sea cucumber, Apostichopus japonicus, an important aquaculture species in China, Japan and South Korea. The food intake, digestive functions and energy budget patterns were estimated at different breeding stages. Unexpectedly, in the growing gonad phase of the sea cucumber lifecycle, animals showed a decline in feed consumption (from 4.49 to 2.6 g ind^?1 day^?1), ingestion rate (from 0.4 to 0.13 g g^?1 day^?1) and apparent digestive rate (from 14.73% to 10.92%), resulting in a reduced energy input. In addition, the increased energy investment in reproduction (from 1.76% to 15.61%) resulted in a decrease in energy allocated to soma growth (from 17.1% to 5.64%) and self‐maintenance (from 47.82% to 34.67%). These results suggest that reproduction impairs energy acquisition ability in breeding A. japonicus. And broodstock can adapt energy shortage by internal adjustment of energy allocation strategy.     

The concentrating method of benthic diatom affects the growth of juvenile sea cucumber (Apostichopus japonicus) and water quality

Concentrating methods generally affect the vitality of the microalgae and may alter its feeding and environmental effect. This article studied the concentrating methods (centrifugation and settlement) on primary productivity of the microalgae Cylindrotheca fusiformis. The effects of free settled C. fusiformis, centrifuged C. fusiformis and macroalgae Sargassum polycystum on growth of the sea cucumber, farming water and sediment quality under two light intensities (1500 lx and 500 lx) were studied by a six‐week rearing experiment. The results showed that the primary productivity of free settled C. fusiformis was 3–4 times higher than that of those centrifuged, indicating centrifugation inhibited the photosynthesis process. In the rearing experiment, feed, light intensities and their interactions significantly affected the specific growth rate (SGR) of the animals (P < 0.01). The animals reared in higher light intensity also had higher SGR, but only those fed free settled C. fusiformis reached a significant level (P < 0.05). The group also had the highest SGR (P < 0.05), suggesting that the photosynthesis ability of C. fusiformis directly contributed to the growth of the animals. During the experiment, the soluble inorganic nitrogen, reactive phosphorus in farming water, total nitrogen and total organic carbon in the sediment gradually increased in all treatments. The S. polycystum group had slightly higher level of nutrients in water and sediment at the end of the experiment for its high organic content. These results provide evidence that the harvesting methods of C. fusiformis directly affect its vitality, which consequentially alter the feeding and environmental effect, especially under higher light densities.     

Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus

We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post‐thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre‐cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to ?50°C at 10.4 ± 0.4°C/min, and immediately immersed in liquid nitrogen. Although this method showed the highest post‐thaw motility in all the conditions we examined, its post‐thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post‐thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post‐thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post‐thaw motility and lowering the cryoprotectant concentration is necessary for commercial‐scale artificial fertilization.     

Effects of body size and water temperature on food consumption and growth in the sea cucumber Apostichopus japonicus (Selenka) with special reference to aestivation

To investigate the effects of body size and water temperature on feeding and growth in the sea cucumber Apostichopus japonicus (Selenka), the maximum rate of food consumption in terms of energy (C_maxe; J day^?1) and the specific growth rate in terms of energy (SGRe; % day^?1) in animals of three body sizes (mean±SE) – large (134.0±3.5 g), medium (73.6±2.2 g) and small (36.5±1.2 g) – were determined at water temperatures of 10, 15, 20, 25 and 30°C. Maximum rate of food consumption in terms of energy increased and SGRe decreased with increasing body weight at 10, 15 and 20°C. This trend, however, was not apparent at 25 and 30°C, which could be influenced by aestivation. High water temperatures (above 20°C) were disadvantageous to feeding and growth of this animal; SGRe of A. japonicus during aestivation was negative. The optimum temperatures for food consumption and for growth were similar and were between 14 and 15°C, and body size seemed to have a slight effect on the optimal temperature for food consumption or growth. Because aestivation of A. japonicus was temperature dependent, the present paper also documented the threshold temperatures to aestivation as indicated by feeding cessation. Deduced from daily food consumption of individuals, the threshold temperature to aestivation for large and medium animals (73.3–139.3 g) was 24.5?25.5°C, while that for small animals (28.9–40.7 g) was between 25.5 and 30.5°C. These values are higher than previous reports; differences in sign of aestivation, experimental condition and dwelling district of test animals could be the reasons.     

5种海藻在刺参幼参饲料中的应用研究

本研究通过分析刺参(Apostichopus japonicus)幼参[(10.02±0.03) g]生长性能、体组成、肠道消化酶活性及非特异性免疫性能的变化,评价鼠尾藻(Sargassum thunbergii)、铜藻(Sargassaum horneri)、海带(Saccharina japonica)、海带渣、石莼(Ulva lactuca L.)及混合藻粉在刺参幼参养殖中的应用效果,在室内循环水系统中进行了为期56 d的养殖实验。结果显示,1) 鼠尾藻和混合藻粉组刺参的增重率(WGR)和特定生长率(SGR)极显著高于其他各藻粉组(P<0.01),海带渣组刺参的WGR和SGR最低,肠体比(IBR)和脏体比(VBR)均显著低于其他各组(P<0.05),藻粉对刺参存活率(SR)影响不显著(P>0.05);2) 不同藻粉对刺参体壁水分、粗灰分、粗蛋白和粗脂肪影响不显著(P>0.05);3) 鼠尾藻和混合藻粉组肠道胰蛋白酶活性极显著高于海带、海带渣和石莼组,海带渣组最低(P<0.01);藻粉对α-淀粉酶和脂肪酶活性无显著影响(P>0.05),但对刺参粪便的酸不溶性灰分具有极显著影响(P<0.01);4) 鼠尾藻组和混合藻粉组刺参肠道超氧化物歧化酶(T-SOD)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活性极显著高于其他各组(P<0.01),海带渣组丙二醛(MDA)含量极显著高于其他各组(P<0.01)。研究表明,在本实验条件下,综合考虑刺参的生长性能及非特异性免疫性能,添加任一海藻均未达到替代鼠尾藻的饲喂效果,但多种海藻混合添加,其生长性能及非特异性免疫性能与单独添加鼠尾藻效果一致。