鱼类生殖细胞移植技术的发展及应用前景

鱼类生殖细胞移植技术是通过诱导不同物种之间生殖系嵌合体来实现。原始生殖细胞和精原细胞(或卵原细胞)是诱导生殖细胞系嵌合体的关键材料。在过去的十多年中,通过采用不同的供体生殖细胞和不同发育阶段(囊胚期胚胎、初孵仔鱼和成鱼)的鱼为受体,开发出多种鱼类生殖细胞移植技术。这些成果的取得,为生殖细胞移植技术应用于诸多水产养殖新兴领域打下了坚实基础。该技术可以应用于:(1)生殖细胞生物学和转基因鱼等基础研究;(2)遗传资源和濒危鱼种的保护;(3)鱼类性别选择育种;(4)有效提高放流鱼苗的遗传多样性。     

Germ cell transplantation as a potential biotechnological approach to fish reproduction

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia—27%; grayling spermatogonia—28%; grayling oogonia—23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.     

Progress on larval and juvenile nutrition to improve the quality and health of seawater fish: a review

In seedling production of seawater fish, providing appropriate nutrition is a necessity for successful production of quality larvae and juveniles. Mass-produced live prey organisms, such as the rotifer Brachionus plicatilis species complex and brine shrimp Artemia spp., alone do not provide sufficient nutrition to the larvae and juveniles of seawater animals. This inadequacy has led to various problems related to fish quality and health, including increased incidence of morphological and behavioral abnormalities and mass mortalities. It is, therefore, important to identify the factors associated with these problems to improve seedling production techniques. This review collates the efforts made during the past two decades in larval nutrition–focusing on advances made in the use of certain nutrients, such as docosahexaenoic acid, vitamin A derivatives, and taurine that are important for the mass production of seawater fish larvae and juveniles–with an aim to improve the quality and health of fish.     

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology—transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.     

海水仔稚鱼的营养需求与微颗粒饲料研究进展

综述了近年来海水仔稚鱼对蛋白质、脂肪和维生素营养需求的研究状况，同时对其微颗粒饲料的特性和制作工艺进行探讨，以期为研制海水仔稚鱼的微颗粒饲料提供参考。     

Development,characterization, conservation and storage of fish cell lines: a review

Cell lines provide an important biological tool for carrying out investigations into physiology, virology, toxicology, carcinogenesis and transgenics. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain, skin. One hundred and twenty-four new fish cell lines from different fish species ranging from grouper to eel have been reported since the last review by Fryer and Lannan (J Tissue Culture Methods 16: 87–94, 1994). Among the cell lines listed, more than 60% were established from species from Asia, which contributes more than 80% of total fish production. This includes 59 cell lines from 19 freshwater, 54 from 22 marine and 11 from 3 brackish water fishes. Presently, about 283 cell lines have been established from finfish around the world. In addition to the listing and a scientific update on new cell lines, the importance of authentication, applications, cross-contamination and implications of overpassaged cell lines has also been discussed in this comprehensive review. The authors feel that the review will serve an updated database for beginners and established researchers in the field of fish cell line research and development.     

Developing native fish species for aquaculture: the interacting demands of biodiversity, sustainable aquaculture and livelihoods

Aquaculture continues to be the fastest growing animal production industry and this rate of expansion must continue if aquaculture is to satisfy global demand for fish products in the face of dwindling capture fisheries. The relationship between aquaculture and biodiversity is complex, with examples of positive and negative impacts having been reported. To enable this expansion while avoiding negative impacts from introductions of exotic species, the investigation of indigenous species becomes important and worthwhile.This paper establishes the background to development of new species for culture and describes the example of the Mexican silverside Menidia estor, which has for centuries been the principal species in an artesanal fishery in Lake Pátzcuaro, Mexico. The species is geographically isolated and is unique but is now endangered because of a range of factors including overfishing, environmental degradation and introduction of exotic species. Considerable advances have been made recently in developing a closed reproductive cycle, understanding feeding and small‐scale on‐growing technology for the species. Based on this, a Darwin Initiative programme was developed focused on technology transfer to implement small‐scale pilot on‐growing thus helping to conserve the species and to improve livelihoods. This has allowed successful pilot scale development of aquaculture for the species while at the same time addressing the objectives of the international Convention on Biodiversity.     

Molecular identification of laser‐dissected gut contents from hatchery‐reared larval cod,Gadus morhua: a new approach to diet analysis

The transition between endogenous and exogenous feeding in hatchery production of fish larvae has long been a bottleneck to increased production. Identification of alternative prey species with a wider array of nutritional profiles is essential for further expansion and diversification within the aquaculture industry. Traditional morphological methods to identify dietary composition are limited. In this study, we present a novel DNA‐based methodology for identifying the gut contents of larval fish that is independent of prey retaining identifiable characteristics. Laser capture microdissection is used to collect ingested material for DNA extraction, thus limiting contaminating DNA originating from the larval fish. PCR is then conducted using universal eukaryotic primers that have the potential to detect a wide diversity of prey items. Using this approach, we identified ingested Artemia salina from hatchery‐reared Gadus morhua larvae. Differential length amplification PCR was used to evaluate the effects of DNA degradation on the sensitivity of A. salina detection. Although it was possible to detect A. salina with relatively long (626 bp) fragments, targeting smaller fragments (264 bp) resulted in the highest return of prey sequences. This combined LCM and molecular approach to diet analysis could offer a more complete assessment of the diets of naturally feeding larvae.     

Angling for endangered fish: conservation problem or conservation action?

Recreational angling has been implicated in population declines of some marine and freshwater fish, but this activity is rarely considered as a threat or even halted when endangered species are targeted. Indeed, in some cases, anglers are drawn to fish for rare or endangered species. Conservation‐oriented behaviours such as catch‐and‐release are often practiced voluntarily due to the ethics of anglers, yet even in these cases, some fishing mortality occurs. Nonetheless, there are many indirect conservation benefits associated with recreational angling. Here, we present a series of case‐studies and consider whether catch‐and‐release angling for endangered fish is a conservation problem or a conservation action. If recreational angling activities contribute to population‐level consequences that are contrary to recovery strategies, then angling for endangered species would seem to be a poor option. However, as revealed by several case‐studies, there is much evidence that anglers are vocal and effective proponents of fish and habitat conservation, and for endangered species, they are often the only voice when other stakeholders are not engaged. Anglers may contribute directly to conservation actions via user fees (e.g. licences), philanthropic donations or by volunteering in research, education and restoration activities. However, it is important to quantify post‐release mortality as well as understand the full suite of factors influencing a given population or species to know the potential risks. A risk assessment approach outlined in the paper may be used by managers to determine when the benefits of angling for endangered species outweigh the risks.     

环境DNA技术及在鱼类监测中的应用

随着人类活动的影响,鱼类资源正在急剧下降,开展鱼类监测对于鱼类多样性的保护具有非常重要的意义。传统的鱼类监测方法因具有破坏性、调查者也要专业形态学知识、调查工作量大等弊端,故亟需一种新的技术方法进行辅助补充。环境DNA （eDNA）技术的发展使得鱼类调查更加的省时省力、节约成本、无损伤,目前在鱼类的监测中广泛使用,然而eDNA技术也存在着一定的缺陷所以不能完全取代传统方法。eDNA技术包含了样品的采集及处理、eDNA提取、eDNA扩增、测序和生物信息学分析几个主要步骤,在整个流程中每一个步骤都非常必要,对单个步骤又有不同的实验方案,选择不同对鱼类eDNA的检测效率也会产生重要的影响。目前国外对于eDNA技术应用于鱼类监测所研究的问题较为丰富而全面,如生物多样性监测、入侵物种检测、濒危物种检测和生物量的估算等,虽然国内的起步不晚,但国内对此研究方向较为单一,需要进一步重视。     

Hormones and Egg/Larval Quality in Fish

Recent evidence suggests that hormones are passed on to eggs by broodfish. This store of maternal hormones may fill the regulatory needs of fish larvae for growth, development, osmoregulation, stress response and other physiological functions prior to the functional development of their own endocrine glands. Thus the hormonal levels in eggs/larvae may be an important determinant of egg/larval quality.
Thyroid hormones have been best studied in this regard. Both thyroxine (T₄) and triiodothyronine (T₃) are present in fish eggs and in most cases, the levels decrease as development proceeds until the onset of endogenous thyroid hormone production, which usually occurs before or at around yolk-sac resorption. Enhancement of T₄/T₃ levels in newly-hatched larvae through immersion or maternal injection has been shown to promote larval growth, development and/or survival in several fish species, although overdose causes thyrotoxicosis.
Other hormones such as corticosteroids, sex steroids, growth hormone and prolactin are also being studied, but to a much lesser extent.
The paper reviews the evidence and discusses the practical implications of the various findings for larval rearing in aquaculture.     

Fin cell cryopreservation and fish reconstruction by nuclear transfer stand as promising technologies for preservation of finfish genetic resources

Domestication is a long-term process during which wild fish are acclimated to farming conditions and hopefully are reproduced over several generations, possibly using selective breeding. Preservation of the genetic diversity of the original population, together with that of the ongoing selection steps, is important for ecological and economical purposes. Cryobanking of reproductive cells is one answer to meet this need. In fish, however, only sperm can be cryopreserved as neither oocytes nor embryos are capable of handling the freeze-thawing stress. In this review, we explore to what extent diploid cells obtained from fin pieces can be used for the preservation of both parental genomes. The main parameters, which should be under control to ensure proper production of fin cells in culture and to enable cryopreservation of the material are described. After cryobanking of such non-reproductive cells, fish can be reconstructed using the nuclear transfer technology whose potentials and difficulties are discussed. The gametes produced by the fish reconstructed after somatic cells nuclear transfer are different to some extent from the gametes obtained after the direct transplantation of primordial germ cell or spermatogonial germ cells into host embryos or larvae. However, in some cases, only somatic cells can be obtained in quantities which would be compatible with strain restoration purposes. From the knowledge available today, it is reasonable to expect that nuclear transfer becomes available for fish reconstruction, even if restricted to high-tech biotechnology facilities. Therefore, cryobanking of fin somatic cells can be farsightedly considered for high-throughput diploid genome conservation.     

Essential fatty acid requirements of cultured marine fish larvae

Feeding of marine fish larvae is, in most cases, limited to the administration of two species of live prey. This reduction in the range of food available for the cultured larvae may occasionally lead to nutritional imbalances or deficiencies. A large amount of research has been recently devoted to the study of the essential fatty acid requirements of marine fish larvae. Studies on the biochemical composition of developing eggs and larvae, as well as the comparison of the patterns of loss and conservation during starvation, pointed out the importance of n-3 HUFA and arachidonic acid as essential fatty acids for larvae of marine fish. The biochemical composition of marine fish larvae, in terms of lipid content and fatty acid composition of total and polar lipids, is modified by dietary levels of essential fatty acids. Larval growth, survival and activity have also been reported to be affected by dietary levels of essential fatty acids. In addition, some pathological signs, such as hydrops or abnormal pigmentation, have been related to essential fatty acid deficiency in these fish. Based on these effects, the essential fatty acid requirements of marine larval fish have been reported to range between 0.3 and 55 g kg^?1 n-3 HUFA on a dry weight basis, suggesting that quantitative requirements of fish larvae may differ from those of juveniles or adults. But quantitative requirements for larvae of the same species reported by various authors are often contradictory. These differences are discussed in relation to the dietary lipid content, ratio 20:5n-3/22:6n-3 and culture conditions used.     

Advances in remote sensing of freshwater fish habitat: A systematic review to identify current approaches,strengths and challenges

Remote sensing technology offers the ability to derive information on freshwater fish habitats across broad geographic areas and has the potential to transform approaches to monitoring. However, the numerous platforms, sensors and analytical software that are available may overwhelm those interested in utilizing this important technology and thus limit its broad application and uptake. Our review is intended to shed light on the capacity of this technology to transform freshwater fish habitat monitoring by examining the fundamental characteristics of the major remote sensing technologies that have been used for characterizing freshwater habitats, conducting a systematic literature review of studies that have used remote sensing technologies to characterize freshwater fish habitats and, highlighting some of the key habitat features, fish species and regions, that have been examined. Lastly, we identify the relative strengths and weaknesses of the various remote sensing technologies that can be used, recommend future research that could help improve the use of these technologies, and, provide a series of important considerations for those who are interested in utilizing these technologies for freshwater fish habitat characterization.     

三峡库区丰都江段鱼类早期资源现状研究

探究三峡库区丰都江段鱼类早期资源现状,评估长江上游梯级水库群联合调度对鱼类早期资源的影响,可为三峡库区渔业资源管理和保护措施制定提供科学依据。2018年5-7月,在三峡库区丰都江段利用圆锥网在左岸、江心和右岸进行鱼卵、鱼苗逐日取样调查。结果显示,调查期间共采集鱼卵7426粒、鱼苗15461尾,隶属于5目、10科、40种(鱼卵16种,仔鱼36种),其中产漂流性卵鱼类19种。估算通过丰都断面的卵苗径流量为290.55×10⁸粒(尾),其中鱼卵89.84×10⁸粒,鱼苗200.71×10⁸尾;四大家的卵苗总径流量为2.24×10⁸粒(尾),其中鱼卵0.95×10⁸粒,鱼苗1.29×10⁸尾。调查期间出现3次产卵高峰,集中在5月下旬和6月下旬。各类仔鱼数量呈现出不同的时间动态,太湖新银鱼(Neosalanx taihuensis)、吻鰕虎鱼属(Rhinogobius sp.)、鲤(Cyprinus carpio)主要出现在5月,四大家鱼以及宜昌鳅鮀(Gobiobotia filifer)主要出现在6月。研究表明,三峡库区靠近库尾段的鱼类早期资源丰富,库尾江段仍存在四大家鱼产卵场。     

Application of sex reversal technology in ornamental fish culture

Many species of ornamental fish, includingboth egg-layers and livebearers, exhibit a marked sexualdimorphism due to the more pigmented bodies and larger finsusually observed in males. This results in male fish commandingup to four times the price of females. Because of this pricediscrepancy, the culture of monosex (all-male) stocks ofornamental fish could be of a significant economic advantage.This paper outlines the current sex reversal technology alreadydeveloped for several fish species important for aquaculture andexamines its potential applicability to ornamental fish culture.The methods to obtain all-male stocks include direct androgentreatment and the production of YY males. The direct approach iseasy to implement and straightforward. However, in somelivebearers such as the guppy, treatment of newly born fry iscomplicated because they already have sexually differentiatinggonads and the masculinization may be temporary. In this regard,the paper stresses the importance of correctly timing treatmentin relation to gonadal development to achieve permanentmasculinization     

Possible applications of modern fish larviculture technology to ornamental fish production

There has been rapid development in the marine foodfishlarviculture technology in Europe since the early eighties,especially in the flat fish, turbot and halibut, and the bass andbream species. The most significant improvements in the eightieswere the introduction of light control, artificial reproductiontechniques, appropriate water treatment and the use of rotifersand Artemia nauplii of specific sizes and in the late eightiesand early nineties the quality enhancement of live food organismsusing specific enrichment techniques. Present research is focusedon the implementation of several microbial techniques to improvethe hygiene of live prey and fish.Many of the modern larviculture techniques being used in marine foodfish could be adapted for application in the ornamental fish industry. For examples, research in the Onamental Fish Section, Primary Production Department, Singapore has demonstrated that the use of freshwater rotifers and Artemia nauplii would enable artificial rearing of Discus in the absence of the parent fish and improve the larval performance of Gouramis and Tetra larvae. The use of such small live food organisms is likely to facilitate breeding of new fish species with small larvae. Research has also indicated that the use of diets containing vitamin C and certain immuno-stimulants improved the stress resistance of guppy. Such techniques would have important application in the fish transport, an important aspect in the ornamental fish industry     

Nutrition and feeding research in the spotted rose snapper (<Emphasis Type="Italic">Lutjanus guttatus</Emphasis>) and bullseye puffer (<Emphasis Type="Italic">Sphoeroides annulatus</Emphasis>), new species for marine aquaculture

The spotted rose snapper (Lutjanus guttatus) and bullseye puffer (Sphoeroides annulatus) are fish species from the tropical Eastern Pacific for which controlled production of larvae and juveniles has been accomplished in recent years. Diverse topics relating to their biology and aquaculture production are currently under study, in particular the nutrition and feeding aspects required to formulate practical feeds and rearing protocols. Improvements in larval growth and survival are possible by feeding live food organisms with natural or enhanced essential fatty acids content and highly digestible artificial microdiets. The ontogeny of the digestive tract and the expression and activity of digestive enzymes have been described for S. annulatus larvae. The effect of various protein and lipid levels on growth and feed utilization has been studied in juvenile and on-growing fish. Both species have carnivorous feeding habits and require high levels of protein in their diets, from 40% to 45% (dry weight) in spotted rose snapper and above 50% in bullseye puffer, with the younger stages requiring the highest protein levels. Encouraging results have been obtained in feeding experiments with different sources of dietary protein from animal and plant origin to evaluate their suitability as feed ingredients in practical diets. Optimization of fish culture practices through feeding management has also been investigated. Trials with various fish densities and feeding frequencies in intensive culture systems are providing information to improve feed utilization and growth in on-growing fish. Further research is underway to evaluate factors in broodstock nutrition which have an impact on egg and larval quality, and into the use of various commercially available oil sources in on-growing diets. In this paper, the results on nutrition and feeding research with both species are reviewed and research needs to support their commercial production in the region are discussed.