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.     

Optimum dietary protein level for growth and coelomic fluid non‐specific immune enzymes of sea cucumber Apostichopus japonicus juvenile

A feeding trial was conducted to determine the optimum dietary protein level of sea cucumber Apostichopus japonicus juvenile focusing on growth performance and non‐specific immune response. Diets with seven crude protein levels (42.0, 108.9, 155.2, 216.7, 258.0, 313.3 and 357.5 g kg^?1) were fed to sea cucumber juveniles (1.05 ±0.01 g) once a day for 100 days. More than 70% survival was observed, and there was no significant difference among all treatments. The sea cucumbers fed diets containing 108.9 g kg^?1 crude protein showed significantly (P < 0.05) higher body weight gain than those of the sea cucumbers fed diets containing 42.0, 216.7, 258.0, 313.3 and 357.5 g kg^?1 crude protein. No significantly differences (P > 0.05) were observed in moisture, crude protein, crude lipid, ash and carbohydrate content of the body wall among all treatments. The coelomic fluid catalase activity of the sea cucumbers generally increased with increasing dietary protein levels. Therefore, the acid phosphatase, superoxide dismutase and lysozyme activity increased with increasing dietary protein levels at first and decreased subsequently. The relationship between dietary protein levels and body weight gain was analysed by a second‐order polynomial regression analysis model. The result indicates that the optimum dietary protein level for sea cucumber juveniles is 135.4 g kg^?1.     

Effects of dietary peptide intake on growth,energy budget,body composition and non‐specific immunity of the sea cucumber Apostichopus japonicus (Selenka)

In this study, a 60‐day feeding trial was conducted to determine the effects of feed supplementating the feed of Apostichopus japonicus with peptides on its growth, energy budget, body composition and immune responses. Sea cucumbers were fed with five experimental diets supplemented with different proportions of peptides: 0 (D1), 12.5 (D2), 25 (D3), 37.5 (D4) and 50 (D5) g/kg in basal diet. Our results suggested that specific growth rate (SGR) and ratio of viscera to body wall (RVBW) of sea cucumbers fed with D4 were significantly improved. Relative to D1, ingestion rate (IR) and faeces production rate (FPR) for D3 and D4 were reduced considerably. Notably, the energy intake increased when peptide level increased from 0 to 50 g/kg. Meanwhile, the energy deposited for growth increased, and the energy loss decreased when peptide level increased from 0 to 37.5 g/kg. The sea cucumbers in D4 had the highest level of crude fat and lowest crude ash. The activities of immunoenzyme, such as SOD, CAT, T‐AOC, ACP and AKP, increased with peptide increase. Results suggested that supplementation of the feed with 25–37.5 g/kg peptides could significantly improve the growth performance, body composition and immune capacity of Apostichopus japonicus.     

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。     

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.     

Effects of differently processed diets on growth,immunity and water quality of the sea cucumber,Apostichopus japonicus (Selenka, 1867)

The type of formulated diet used in aquaculture systems affects the feed efficiency and water quality. In this study, the water quality, growth performance and immunity of the sea cucumber, Apostichopus japonicus (Selenka, 1867), fed diets produced using four different processing methods (extruded pellet diet, cold‐bonded pellet diet, flake diet and mash diet) were quantified for 60 days to identify the optimal feed type for culturing of this species. Sea cucumbers grew faster, the feed conversion ratio was much lower, and immunity indicators were higher when fed the extruded pellet diet compared to the other diets. Sea cucumbers fed the extruded pellet diet had the highest specific growth rate (0.96), whereas A. japonicus fed the cold‐bonded pellet diet had the lowest specific growth rate (0.51). The lysozyme activity of sea cucumbers fed the extruded pellet diet was higher (57.52) than that of A. japonicus fed the cold‐bonded pellet diet (39.22), flake diet (37.32) and mash diet (32.68). When animals were fed the mash diet, the ammonia–nitrogen, nitrite–nitrogen and phosphate productions were higher than in animals fed the other diets. These results indicate that the extruded pellet diet is the optimum feed type for use in the culture of A. japonicus.     

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.     

Effect of replacing dietary fish meal with silkworm (Bombyx mori L) caterpillar meal on growth and non‐specific immunity of sea cucumber Apostichopus japonicus (Selenka)

The effect of dietary substitution of silkworm (Bombyx mori L) meal (SM) for fishmeal (FM) on the growth performance and non‐specific immunity of sea cucumber (Apostichopus japonicus) (initial weight: 12.8 ± 0.16 g) was determined. Four isonitrogenous and isocaloric diets were formulated: Diet 1, which served as the control diet, contained 5% FM; Diet 2 contained 3.75% FM and 1.25% SM; Diet 3 contained 2.5% FM and 2.5% SM; and Diet 4 contained 5% SM. Other ingredients in each of the four diets were kept in the same proportion. After 8 weeks of feeding, the results showed that sea cucumbers fed Diet 2 had 18.7% increases in weight over those fed the control diet, but no significant difference was observed. No obvious difference in body wall composition was detected among the sea cucumbers fed the four different diets. Immunity analysis indicated that phagocytosis and serum alkaline phosphatase activity were not significantly (P > 0.05) affected when FM was partially or completely replaced with SM. Serum lysozyme activity of sea cucumbers fed Diet 4 showed a significant (P < 0.05) growth increase compared with those fed control diet. The results revealed that SM could be an effective substitute for FM in sea cucumber diet.     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

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.     

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.     

Intra‐specific effects of sea cucumber (Apostichopus japonicus) with reference to stocking density and body size

The present studies deal with the intra‐specific effects of sea cucumber Apostichopus japonicus with unlimited food resources, especially the effects of stocking density on growth variation of the animal and energetic changes of small individuals under the stress of large individuals. The results showed that with the initial body weight of 5.12±0.09–6.11±0.26 g of sea cucumber among the densities of 5, 10, 20, 30, 40 and 50 ind./100 L, the density of 20 ind./100 L was the optimum stocking density because of its highest specific growth rate, crude protein content and crude lipid content in tissue. Individual growth variation of A. japonicus increased with the increase of stocking densities, whereas no significant differences in variation were found when the density was over 30 ind./100 L (P>0.05). The low‐weight individuals under the stress of heavy‐weight individuals exhibited obvious changes in energetics, such as lower ingestion rate, lower energy deposited as growth but higher respiration and excretion. The coefficient of variation in growth of the animals was over 70% due to the simultaneous action of aggression and maybe a factor of chemical mediator, and led to significant changes in the energetics of small‐sized individuals.     

Dietary supplements of guanosine improve the growth,non‐specific immunity of sea cucumber,Apostichopus japonicus Selenka,and its resistance against Vibrio splendidus

The growth response, non‐specific immune activities and disease resistance were measured in sea cucumber, Apostichopus japonicus Selenka (initial average weight 6.80 ± 0.10 g), when fed diets supplemented with graded levels of guanosine from the guanosine‐5’‐monophosphate disodium (GMP) at 0 (control), 0.3, 0.6 and 1.2 g/kg for 8 weeks. The results showed that GMP supplemented at 0.6 and 1.2 g/kg significantly enhanced the growth of sea cucumber. Sea cucumber fed a diet with 0.6 g/kg of GMP had significantly higher intracellular superoxide anion production, nitric oxide synthase activity, lysozyme activity and the total superoxide dismutase (T‐SOD) activity than those in control group (p < .05). Increased lysozyme activity and T‐SOD activity were also found in sea cucumber fed GMP at 1.2 g/kg. Moreover, there was significantly lower cumulative mortality after the disease challenge in sea cucumber fed the diets with 0.6 and 1.2 g/kg GMP than that in control and 0.3 g/kg GMP groups (p < .05), and no significant difference was observed between 0.6 and 1.2 g/kg GMP groups. These results suggested that feeding GMP at a dose of 0.6 g/kg could enhance growth, non‐specific immunity of sea cucumber as well as its resistance against Vibrio splendidus.     

Effect of dietary carbohydrate levels on growth performance,non‐specific immune enzymes and acute response to low salinity and high temperature of juvenile sea cucumber Apostichopus japonicus

This study was performed to determine the optimum dietary carbohydrate (CHO) levels of sea cucumber, based on the parameters of growth, digestive enzymes, digestibility, non‐specific immune enzymes and acute low‐salinity (20 g/L) stress and high‐temperature (30°C) stress tolerance. Diets with eight different CHO (dextrin) levels (32.9, 107.6, 192.5, 257.2, 316.8, 428.0, 482.4 and 572.8 g/kg) were fed to sea cucumber juveniles (0.49 ± 0.01 g) for 60 days. Significant higher amylase activity was observed in sea cucumbers fed diet with CHO ranging between 32.9 and 192.5 g/kg than that of other treatments (p < .05). The sea cucumbers fed with 192.5 g/kg CHO showed significantly higher acid phosphatase activity than the treatments of 482.4 and 572.8 g/kg CHO (p < .05), and significantly higher alkaline phosphatase activity than other treatments (p < .05, except 257.2 g/kg). The treatments of 428.0–572.8 g/kg were found significantly lower values than other treatments in apparent digestibility coefficients for dry matter and crude protein (p < .05). The sea cucumbers fed with 192.5, 257.2 and 316.8 g/kg CHO showed better tolerance to high‐temperature (30°C) and low‐salinity (20 g/L) stress than other treatments. In brief, the optimal dietary CHO level for the growth of juvenile sea cucumber is 177.96 g/kg. However, excessive CHO will inhibit amylase enzyme activity and decrease digestibility, resulting in low growth of sea cucumber.     

Effect of dietary chitosan oligosaccharide complex with Ce (IV) on growth,immunity and disease resistance against Vibrio splendidus of sea cucumber,Apostichopus japonicas

An 8‐week feeding trial was conducted to investigate the effects of dietary chitosan oligosaccharide complex with cerium (Ce IV) (COS‐Ce) on growth performance, nonspecific immunity and disease resistance of sea cucumber, Apostichopus japonicas. Five isonitrogenous (18.6%) and isolipidic (1.1%) practical diets were formulated with graded level of COS‐Ce (0, 150, 300, 600 and 1200 mg kg^?1 dry feed), which were named as COS‐Ce/0, COS‐Ce/150, COS‐Ce/300, COS‐Ce/600, COS‐Ce/1200 respectively. Each diet was allocated to four replicates of sea cucumbers (Initial weight: 6.72 ± 0.02 g). Sea cucumbers were fed to apparent satiation once daily (19:00 hours) for 56 days. During the experiment, water temperature was kept at 16 ± 0.5°C, pH 7.8–8.2, dissolved oxygen beyond 5 mg L^?1, ammonia nitrogen below 0.5 mg L^?1 and salinity from 30‰ to 31‰. Results showed that the specific growth rate of sea cucumbers was significantly higher in COS‐Ce/600 than that in other four treatments. Activities of phagocytosis, respiratory burst, acid phosphatase and alkaline phosphatase in COS‐Ce/600 were significantly higher than that in COS‐Ce/0 (P < 0.05) respectively. On the contrary, cumulative mortality was the lowest in COS‐Ce/600 following 14 days exposure to Vibrio splendidus (P < 0.05). In conclusion, these results confirmed that dietary COS‐Ce had beneficial effects on growth performance, nonspecific immunity and disease resistance of sea cucumber.     

Dietary supplement of fructooligosaccharides and Bacillus subtilis enhances the growth rate and disease resistance of the sea cucumber Apostichopus japonicus (Selenka)

The effects of two dietary supplements, entrapped fructooligosaccharide (FOS) and microencapsulated Bacillus subtilis, on the growth and disease resistance of the sea cucumber Apostichopus japonicus were investigated. Sea cucumbers (a total of 810 animals, with initial weight of 3.72 ± 0.16 g each) were fed with basal diet only or basal diet supplemented with three different doses of B. subtilis (0.2, 0.4 and 0.8%), three different doses of FOS (0.4, 0.8 and 1.6%), 0.2% B. subtilis plus 0.4% or 0.8% FOS. Growth and lysozyme activity were measured after 50 days feeding. The results showed that dietary 0.2% and 0.4% B. subtilis alone or 0.2% B. subtilis plus 0.8% FOS significantly increased the specific growth rate (SGR) of sea cucumbers compared with control (P < 0.05). The addition of B. subtilis with or without FOS had no significant effect on lysozyme activity. The animals were also challenged with Vibrio splendidus to assess the level of disease resistance provided by the two dietary supplements. Sea cucumbers fed with 0.8% B. subtilis or 0.2% B. subtilis plus 0.8% FOS exhibited the highest survival (P > 0.05). Under these experimental conditions, dietary B. subtilis and FOS had no synergistic effect on enhancing SGR and disease resistance of sea cucumber.     

地衣芽孢杆菌对仿刺参生长、消化和免疫功能的影响

为探讨益生菌制剂对仿刺参生长、消化和免疫功能的影响,选用分离自仿刺参肠道的地衣芽孢杆菌作为潜在的益生菌株进行仿刺参投喂试验.选择初始体质量为(7.17±0.86)g的仿刺参为试验对象,设计空白对照组及益生菌处理组进行投喂.益生菌在饲料中的添加密度分别为105、107、109、1011 cfu/g,每10 d测定相关指标...     

An artificial oyster‐shell reef for the culture and stock enhancement of sea cucumber,Apostichopus japonicus,in shallow seawater

Because of its high nutritional value and health benefits, aquaculture production of Apostichopus japonicus in China is the largest of any single species. Therefore, the development of new farming methods is of considerable significance. In this study, discarded oyster shells have been used to create an artificial reef for the culture of this species. The results have shown that from 6th March 2009 to 26th November 2009, the wet weight of sea cucumber increased from 49.57 ± 1.16 to 79.87 ± 1.46 g ind^?1. Between 16th July and 18th October, the specific growth rate and daily weight gain of A. japonicus differed significantly from other periods. Population density was higher within the reef compared with outside the reef area, and the difference was significant (P < 0.01). The maximum distance between A. japonicus individuals within the reef area on 3rd March and 16th July was 65.0 ± 3.3 and 62.9 ± 4.4 cm, respectively, but the difference was not significant (P > 0.05). The diatom species attached to the oyster shells were similar to those found in the stomach content of A. japonicus. In conclusion, the oyster‐shell reef provides a suitable habitat and shelter for the culture of the sea cucumber, A. japonicus.     

Effect of stocking density on key growth traits of a fast‐growing and heat‐resistant strain of sea cucumber (Apostichopus japonicus)

We evaluated the effect of rearing density on the yield of a fast‐growing and heat‐resistant strain of Apostichopus japonicus using a full‐sib family. We analysed the relationship between density and key growth parameters during factory seedling of A. japonicus and developed a model to explain the variation in growth. From 130 to 220 days, the rearing density of A. japonicus had a significant effect on body weight, specific growth rate, coefficient of variation and survival rate (P < 0.05). We obtained predicted integral regression equations for yield and growth rate, density fluctuation, variable coefficient fluctuation and survival rate. Specific growth rate had a significant effect on the yield of A. japonicus and, therefore, can be used as a primary indicator of yield. We recommend that stocking density should be adjusted from age 130–166 days to avoid frequent seedling separation to different pools of different seedling sizes during the fast growth period of A. japonicus seedlings, a procedure that causes yield decline. The optimal initial stocking density was 24.4 g m^?2 (0.2 g L^?1).