鱼类精子超低温冷冻保存技术及其应用

鱼类精子的超低温冷冻保存对鱼类种质资源保护、低温生物学、遗传育种和水产养殖业都具有重要的意义。鱼类精子超低温冷冻保存技术主要包括冷冻保存方法、抗冻保护液、降温、解冻速率、冷冻保存温度、抗冻剂去除等过程。本文就鱼类精子超低温冷冻保存技术及其应用进行了综述。     

Cryopreservation of sperm in annual fish Austrolebias minuano (Cyprinodontiformes; Rivulidae)

The effects of the cryoprotectants dimethyl sulfoxide, glycerol and methyl glycol at different concentrations on Austrolebias minuano sperm quality parameters were evaluated in this study. The cellular kinetic parameters, determined using flow cytometry, indicated the best results with the samples cryopreserved with 7.5% methyl glycol. Dimethyl sulfoxide concentrations of 7.5% and glycerol concentrations of 10%, 12.5% and 15% demonstrated the least benefit across all evaluated sperm kinetic parameters. When assessed using flow cytometry, a concentration of 7.5% methyl glycol similarly showed better sperm kinetic parameters than 12.5% dimethyl sulfoxide. We conclude that cryoprotectants, especially methyl glycol, are effective for the preservation of sperm quality in A. minuano. However, high sensitivity of spermatozoa against glycerol was observed in these studies; thus, it is not recommended for cryopreservation purposes.     

Cryopreservation of carp sperm

About 70% of eggs of Cyprinus carpio reached the eyed stage in fertility tests with 1 ml of cryopreserved semen added to 1 g of eggs. When 1 ml of semen was added to 100 g of eggs, few eggs reached the eyed stage. In two fertility tests with sperm preserved for 342 days, using 5 g of eggs inseminated with 1 ml of semen, percentages of eyed eggs were 31.5 and 25.5, respectively. In a series of fertility tests, the chemical constituency of the medium in which fertilization was attempted influenced the fertility of cryopreserved sperm.     

Cryopreservation of common carp sperm

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.     

Cryopreservation of sperm from spotted wolffish

The effects of different concentrations of cryoprotectant (dimethyl sulfoxide; DMSO), cooling rate and straw size on the post-thaw motility of frozen sperm from spotted wolffish, Anarhichas minor, were studied. There was no significant difference in the post-thaw motility of sperm treated with three different concentrations of DMSO (10, 20 and 30%). Similarly, there was no significant difference in the post-thaw motility of spermatozoa when using different freezing rates (i.e. distance of straws from the surface of liquid N₂, 4.7, 5.5 and 7.1°C min⁻¹) and the straw size (0.5 and 1.0 ml) did not affect survival. The cryopreservation of sperm can be used to make up for the frequent lack of sperm and/or the unsynchronised timing of sperm production in spotted wolffish males and the ovulation time in females. The results show that sperm from spotted wolffish can be frozen to secure access to viable sperm, but further experiments are needed in order to reveal the effect of different parameters on the post-thawing mortality and define the optimum conditions for cryopreservation.     

Osmoregulation in fish sperm

Fish Physiology and Biochemistry - In most fish exhibiting external fertilization, spermatozoa become motile after release into water, triggered by differences between intracellular and...     

Vitrification of sperm from marine fish: effect on motility and membrane integrity

Our goal was to develop a standardized approach for sperm vitrification of marine fish that can be applied generally in aquatic species. The objectives were to: (i) estimate acute toxicity of cryoprotectants over a range of concentrations; (ii) evaluate the properties of vitrification solutions (VS); (iii) evaluate different thawing solutions and (iv) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10‐μL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post‐thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X‐1000^? + 1% Z‐1000^? had an average post‐thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to higher osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.     

半滑舌鳎精子冷冻保存

半滑舌鳎精子冷冻保存对于人工繁殖育苗、杂交育种、雌核发育及其性别控制研究具有重要的意义,为此,本文对半滑舌鳎精子冷冻保存方法进行了研究。分别利用2．8mol／L的二甲基亚砜（DMSO）、甘油（Gly）和1,2-丙二醇（PG）冷冻保存该鱼精子。结果显示,DMSO冷冻保存精子的活力较高。利用MPRS＋2．8mol／L DMSO以1：0．5、1：1、1：1．5和1：2的比例稀释并冷冻精子,1：1比例在冻前能够抑制精子的运动,冻后活力可达82．50±3．54％,显著高于其他稀释比例（P〈0．05）。分别利用冷冻保存液A（MPRS＋2．8mol／L DMSO）和B（TS-2＋2．8mol／LDMSO）稀释平衡精子,精子在A中的冻前快速运动时间、寿命分别为37．75±6．45S和145．00±78．98S,与鲜精无显著差异（P〉0．05）。利用以上两种冷冻稀释液冷冻保存精子,精子在A液中的冻后活力和寿命分别可达53．50±6．69％和98．00±13．51s,冷冻效果优于B液（P〈0．05）。冷冻后精子的受精率和孵化率分别为55．00±5．00％和35．00±13．23％,受精率与鲜精无显著性差异（P〉0．05）,因此认为MPRS＋2．8mol／L DMSO可用于半滑舌鳎精子的冷冻保存。     

海洋贝类配子及胚胎的低温冷冻保存

综述了海产贝类配子及胚胎的低温冷冻保存技术的发展现状,对目前的文献进行了汇总和分析,讨论了冷冻保存的降温程序、抗冻剂配方以及评价保存效果的指标等方面的问题,并对目前在海产贝类配子和胚胎的低温冷冻研究方面存在的问题和解决的途径进行了探讨。     

鱼类精子质量评价研究进展

在鱼类人工繁育中,研究者主要关心的是卵子质量,长期以来对精子质量未引起足够重视.而精子质量同样会影响繁育效果的重要因素.鱼类精子质量的评价指标有多种,如精子活力、运动时间、密度、形态、受精率和生理功能等.其中最传统的评价指标是精子活力,其测定方便,能较准确地预测受精率.将精子运动时间和活力综合考虑可更好地反映精子的运动能力.而精子受精率则是精子质量的直接反映,但会受到卵质等因素的影响.质膜完整性、线粒体功能、染色质结构完整性等可体现精子的质量,但测定方法较繁琐.近年来,鱼类精子质量检测技术迅速发展,计算机辅助精子分析(CASA)、流式细胞术(FCM)分析、低渗肿胀(HOS)、单细胞凝胶单泳(SCGE)等技术的建立,使得测定指标更多样、客观、准确.本文逐一介绍了评价精子质量的各种指标,并对各指标的测定方法、测定原理、国内外研究情况进行详细叙述,旨为我国鱼类精子质量评价研究提供背景资料.     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

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.     

Commercial marine fish farming in Singapore

Commercial marine fish farming in Singapore is mainly the culture of economically important foodfish species in floating cage nets. There are 84 licensed fish farms occupying 46.5 hectares (ha) of coastal waters. Production from these farms accounts for the bulk of aquaculture production in Singapore, being 3554 tonnes (‘metric tons’, t) in 1995, or 98% of total production of 3625t. The commonly cultured species are the green mussels, Perna viridis L., which form the bulk of production (70.4%), finfish like the groupers, Epinephelus tauvina Forsskal and E. malabaricus Schneider, Asian sea bass, Lates cakarifer Bloch, and snappers, Lutjanus johni Bloch and L. argentimaculatus Forsskal, and crustaceans like the mangrove crab, Scylla serrata Forsskal and spiny lobster, Panilurus polyphagus Herbst. The basic farm structure for fish and mussel culture is the floating wooden raft. In finfish farming, polyethylene cage nets are attached to the raft in which popular foodfishes are cultured. The mussel raft is a structure to which polyethylene ropes are attached to collect and grow out green mussels from natural spatfall. Fish seeds for farming are mostly wild-caught. Only the Asian sea bass and the banana shrimp (Penaeus merguiensis de Mann) are produced by commercial hatcheries in Singapore and the region. The fry of pompano (Trachinotus blochii Lacepede, and T. falcatus Klausewitz & Nielsen) are imported from Taiwan. Trash fish is still the main feed used for the farming of finfish and crustaceans like the mangrove crab and lobster because it is cheap and readily available. This paper also reviews the economics of commercial finfish and mussel farming in Singapore today.     

Cryopreservation of sperm of common carp, Cyprinus carpio L.

Milt of common carp, Cyprinus carpio L. was cryopreserved in pellet form with the use of 12 extenders. Most efficient were: BE2 original extender (containing 85 mM NaCl, 50 mM KCl, 3 mm CaCl₂, 1 mm MgCl₂ with 10% dimethyl-acetamide (DMA) and 10% addition of hen's egg yolk) and Kurokura et al.'s extender with 15% DMA and 10% yolk (about 73% and 69% of eyed eggs, about 61% and 52% of swim-up larvae, respectively). Within the most effective treatments, survival from the eyed-egg stage to the swim-up stage was similar to that observed in the control group. Survival from the eyed-egg stage to the swim-up stage (percentage of eyed eggs was considered as 100%) was highly significantly and positively correlated with the actual rate of swim-up larvae.     

A review of mycobacteriosis in marine fish

Mycobacteriosis is a serious and often lethal disease of fish, affecting a wide range of species globally both in culture and wild settings. Caused by several species of the genus Mycobacterium , the disease has received considerable attention in recent years because of the discovery of new species in piscine hosts, epizootics in wild fisheries, and the ability of a few species to infect humans. The impact of this disease in aquaculture and the aquaria trade has been well reported and there is currently no widely accepted cure other than depopulation and facility disinfection. However, the impact on wild fisheries is poorly understood and may relate to species-specific interactions (host–pathogen) and possibly environmental stressors. In this review, much of what is known about mycobacteriosis in marine fish is summarized with particular attention to an epizootic in striped bass, Morone saxatilis , (Walbaum), in Chesapeake Bay, USA.     

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.     

Novel methodologies in marine fish larval nutrition

Major gaps in knowledge on fish larval nutritional requirements still remain. Small larval size, and difficulties in acceptance of inert microdiets, makes progress slow and cumbersome. This lack of knowledge in fish larval nutritional requirements is one of the causes of high mortalities and quality problems commonly observed in marine larviculture. In recent years, several novel methodologies have contributed to significant progress in fish larval nutrition. Others are emerging and are likely to bring further insight into larval nutritional physiology and requirements. This paper reviews a range of new tools and some examples of their present use, as well as potential future applications in the study of fish larvae nutrition. Tube-feeding and incorporation into Artemia of ¹⁴C-amino acids and lipids allowed studying Artemia intake, digestion and absorption and utilisation of these nutrients. Diet selection by fish larvae has been studied with diets containing different natural stable isotope signatures or diets where different rare metal oxides were added. Mechanistic modelling has been used as a tool to integrate existing knowledge and reveal gaps, and also to better understand results obtained in tracer studies. Population genomics may assist in assessing genotype effects on nutritional requirements, by using progeny testing in fish reared in the same tanks, and also in identifying QTLs for larval stages. Functional genomics and proteomics enable the study of gene and protein expression under various dietary conditions, and thereby identify the metabolic pathways which are affected by a given nutrient. Promising results were obtained using the metabolic programming concept in early life to facilitate utilisation of certain nutrients at later stages. All together, these methodologies have made decisive contributions, and are expected to do even more in the near future, to build a knowledge basis for development of optimised diets and feeding regimes for different species of larval fish.     

鱼类营养级在海洋生态系统研究中的应用

营养级是生态系统营养动力学的核心概念,最早用于反映食物网中生命体的位置.近年来,营养级广泛应用于群落和生态系统研究中.作者在广泛收集国内外相关文献资料基础上,结合实际研究经验,分析了国内外营养级的研究现状.研究结果显示,营养级耦合了生态系统的结构和功能,在海洋生态学研究中优势明显.随着研究的深入,营养级概念的缺陷也逐渐显露,但新方法、新技术(稳定同位素方法)的产生可能为解决此问题带来帮助.在传统营养级研究基础上,近年来群落平均营养级概念的提出,扩充了营养级概念的内涵.群落平均营养级不仅可以揭示系统或群落的营养格局和结构组成特征,也能用于评估生态系统的资源利用状况和外界干扰程度,应作为群落营养动力学的主要发展方向.基于营养级的生态系统营养控制机制是生态系统动力学研究内容,是揭示系统动态变化规律的重要途径.此外,将群落大小结构和营养级相关联的新思路有效避免了营养级划分过程的局限性,是营养动力学的重要进展.     

Patterns of population variability in marine fish stocks

Exploited marine fish and invertebrate stocks fluctuate in a myriad of complex patterns, exhibiting variability on interannual, decadal, and longer time scales. To characterize various patterns of variation, time series of catch, catch per unit effort, or biomass from 30 stocks were examined with a variety of statistical methods including autocorrelation analysis and Lowess smoothing. A hierarchical cluster analysis classified the stocks into six identifiable groups: steady-state; low-variation, low-frequency; cyclic; irregular; high-variation, high-frequency; and spasmodic. The observed patterns are consistent with life history traits; for example, stocks with high variability are generally small, pelagic species whereas low-variability stocks are generally slow-growing, demersal fish. Each of the six general patterns of variability can be produced from a simple multiple-equilibrium population model by varying the intrinsic rate of population growth, and the time scale and amplitude of environmental variability. Suitable management policies depend on the type of variation observed, and the vast majority of stocks examined do not correspond to the steady-state assumptions of classical fisheries models. For example, management of spasmodic stocks may alternate between periods of active exploitation and periods of rebuilding, a process enhanced by the existence of alternative fisheries.     

海水养殖鱼类消化酶研究进展

综述了海水养殖鱼类消化酶活性研究的以下三方面：（1）消化酶活性的研究,内容包括消化酶的活性分布,温度、pH、外界环境及季节变化对消化酶活性的影响;（2）不同生长阶段消化酶活性的变化,内容包括与仔鱼生长发育相对应的消化酶形成的时间,稚鱼、幼鱼在生长进程中,消化酶的变化规律;（3）鱼类的食物组成对消化酶活性的影响,内容包括饲料的种类和性质,以及饲料不同成分对鱼类消化酶的分泌、活性的影响,进而对饲料利用率的影响。本本对海水养殖鱼类消化酶的活性进行了较为系统的综述,为海水养殖鱼类饲料的开发提供参考数据。