Dead-end (dnd) protein in fish-a review.

Dead end (dnd) is a germ plasm-specific maternal RNA discovered in zebrafish and then in other vertebrates. Dnd protein is essential for migration and motility of primordial germ cells (PGCs), only cells destined to transfer genetic information to offspring. PGCs arise far from somatic cells of developing gonads and they must migrate to their site of function. Migration of PGCs follows complex path by various developing tissues as their disruption impacts on the fertility. Recently, it has been found that dnd is not required for survival of PGCs and dnd-deficient zebrafish PGCs transdifferentiate into the somatic cells. In fish, targeting dnd causes removal of PGCs that ultimately affects sex differentiation. Sterility in various fish species can be achieved by knockdown or knockout of dnd. In our review, we have discussed dnd as a germ cell-specific molecular marker in fish, its interaction with miRNAs, and its use in aquaculture and fish conservation.

Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging.

The aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning, and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing that regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species.

Polyspermy produces viable haploid/diploid mosaics in sturgeon.

Most of sturgeon species (Acipenseridae) are currently critically endangered. Attempts to revive these populations include artificial breeding in hatcheries. However, under artificial reproduction, sturgeon embryos occasionally develop atypically, showing 3, 5, 6, 7, 9, or 10 cells at the 2- to 4-cell stage. This study was undertaken with the objective of understanding the mechanism of the atypical division (AD) in embryos during artificial breeding. Using several sturgeon species, we tested two hypotheses: (1) polyspermy and (2) retention of the second polar body. We found that (1) AD embryos survive similar to controls, (2) the ratio of AD embryos is positively correlated with the amount of sperm used for fertilization, (3) the number of micropyles and the area covered by them in AD embryos is significantly greater when compared to controls, (4) numerous spermatozoa nuclei are in the cytoplasm after fertilization, (5) all AD embryos are mosaics, and (6) AD fishes with n/2n ploidy contain diploid cells from maternal and paternal genetic markers, while the haploid cells contained only paternal ones. These results clearly indicate that AD embryos arise from plasmogamy where the accessory spermatozoon/spermatozoa entry the egg and develop jointly with zygotic cells. This suggests that a well-controlled fertilization procedure is needed to prevent the production of sturgeon with irregular ploidy, which can have detrimental genetic effects on sturgeon populations. On the other hand, if AD fish can produce haploid-derived clonal gametes, induction of multiple-sperm mosaicism might be a useful tool for the rapid production of isogenic strains of sturgeons.

Elimination of primordial germ cells in sturgeon embryos by ultraviolet irradiation.

A technique for rescuing and propagating endangered species involves implanting germ line stem cells into surrogates of a host species whose primordial germ cells (PGCs) have been destroyed. We induced sterilization in sterlet (Acipenser ruthenus) embryos by means of ultraviolet (UV) irradiation at the vegetal pole, the source of early-stage PGCs of sturgeon eggs. The optimal cell stage and length of UV irradiation for the effective repression of the developing PGCs were determined by exposing embryos at the one- to four-cell stage to different doses of irradiation at a wavelength of 254 nm (the optimal absorbance spectrum for germplasm destruction). The vegetal pole region of the embryos was labeled immediately upon irradiation with GFP bucky ball mRNA to monitor the amount of germ plasm and FITC-dextran (M.W. 500,000) to obtain the number of PGCs in the embryos. The size of the germ plasm and number of surrounding mitochondria in the irradiated embryos and controls were observed using transmission electron microscopy, which revealed a drastic reduction in both on the surface of the vegetal pole in the treated embryos. Furthermore, the reduction in the number of PGCs was proportional to the dose of UV irradiation. Under the conditions tested, optimum irradiation for PGCs removal was seen at 360 mJ/cm2 at the one-cell stage. Although some PGCs were observed after the UV irradiation, they significantly reduced in number as the embryos grew. We conclude that UV irradiation is a useful and efficient technique to induce sterility in surrogate sturgeons.

Comparison of oocyte mRNA localization patterns in sterlet Acipenser ruthenus and African clawed frog Xenopus laevis.

In oocytes, RNA localization has critical implications, as assembly of proteins in particular subcellular domains is crucial to embryo development. The distribution of mRNA molecules can identify and characterize localized transcripts. The goal of this study was to clarify the origin of primordial germ cells in the oocyte body plan and to reveal the generation of cell lineages by localized RNAs. The distribution of 12 selected mRNAs in sterlet Acipenser ruthenus oocytes was investigated by qPCR tomography and compared with known patterns of mRNA localization in Xenopus laevis. We investigated the distribution of two gene clusters in the ooplasm along the animal-vegetal axis of the sturgeon oocyte, both of which showed clearly defined intracellular gradient pattern remarkably similar to their distribution in the frog oocyte. We elucidated the localization of sturgeon egg germplasm markers belonging to the vegetal group of mRNAs. The mRNAs coding otx1, wnt11, and veg1 found to be localized in the sturgeon animal hemisphere are, in contrast, distributed in the vegetal hemisphere in amphibian. Actinopterygii and Sarcopterygii, two major lineages of osteichthyan vertebrates, split about 476 Ma (Blair & Hedges, ), albeit basal lineages share conserved biological features. Acipenseriformes is one the most basal living lineages of Actinopterygii, having evolved about 200 Ma (Bemis, Birstein, & Waldman, ), contemporaneous with modern amphibians (Roelants et al., ).

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

First evidence of viable progeny from three interspecific parents in sturgeon.

Polyspermy is the most commonly observed cause of embryonic abnormalities in fertilization, often resulting in death. In sterlet (Acipenser ruthenus), however, polyspermic embryos have high survival (similar to a control group) and morphological development is similar to monospermic larvae. Ploidy of these individuals is n/2n mosaic (whereas the normal state for A. ruthenus is a functional diploid). This study was undertaken to test whether sturgeon eggs can be fertilized by several spermatozoa from different species to produce viable offspring from three interspecific parents: A. ruthenus (2n), A. gueldenstaedtii (4n), and A. baerii (4n). Four trials were performed: (1) and (2) A. baerii eggs were fertilized with a mixture of A. ruthenus and A. gueldenstaedtii sperm; (3) A. gueldenstaedtii eggs were fertilized with a mixture of A. baerii and A. ruthenus sperm; and (4) A. gueldenstaedtii eggs were fertilized with a mixture of A. gueldenstaedtii and A. ruthenus sperm. Fertilized embryos with abnormal cleavage (3, 5, 6, 7, 9, and 10 cells) were collected and kept separately until 14 days post-fertilization. Ploidy level of 25 larvae (hatched from abnormal cleaved embryos) was evaluated by flow cytometry. Forty-four percent of observed hybrids had a mosaic 2n/3n ploidy. Five larva were processed further with microsatellite analysis and demonstrated that three specimens were heterospecific polyspermic larvae, containing alleles from three parents, and two specimens were conspesific polyspermic larvae from two parents. This astonishing phenomenon was emphasized by the fact that it was generated without any significant intervention.

Biotechnology applied to fish reproduction: tools for conservation.

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells.

Several sturgeon species are near extinction; therefore an efficient conservation strategy is required. Germ stem cells can be used for long-term storage and restoration of genetic information using surrogate reproduction. This study compared cryopreservation procedures of early stages of Siberian sturgeon Acipenser baerii testicular and ovarian cells. Whole gonad tissue or dissociated cells were frozen at a cooling rate of 1 °C/min in phosphate buffered saline with 0.5% bovine serum albumin, 50 mM glucose, and one of four different 1.5 M cryoprotectants: dimethyl sulfoxide, glycerol, ethylene glycol, or dimethyl sulfoxide with propanediol. The number of living cells obtained from 0.1 g of gonadal tissue after freeze/thaw of both whole tissue and dissociated cells was higher using ethylene glycol than with other cryoprotectants. Although there were no differences in the number of living cells in cryopreserved whole tissue vs. dissociated cells, the number of dead cells was lower with whole tissue cryopreservation, indicating that cells that died during freeze/thaw were digested during subsequent enzymatic dissociation. This resulted in more than 90% live cells after freeze/thaw and dissociation. The thawed tissue cryopreserved using ethylene glycol as protectant as well as fresh gonadal tissue were dissociated, and the cells were labelled by PKH26 and transplanted into larvae of sterlet Acipenser ruthenus. Ninety days post-transplant of both fresh and cryopreserved cells, introduced cells proliferated in more than half of the recipients.

Novel technique for visualizing primordial germ cells in sturgeons (Acipenser ruthenus, A. gueldenstaedtii, A. baerii, and Huso huso).

Primordial germ cells (PGCs) are the origin of all germ cells in developing embryos. In the sturgeon embryo, PGCs develop from the vegetal hemisphere, which mainly acts as an extraembryonic source of nutrition. Current methods for studying sturgeon PGCs require either killing the fish or using costly and time-consuming histological procedures. Here, we demonstrate that visualization of sterlet (Acipenser ruthenus>) PGCs in vivo is feasible by simply labeling the vegetal hemisphere with fluorescein isothiocyanate (FITC)-dextran. We injected FITC-dextrans, with molecular weights varying between 10 000 and 2 000 000, into the vegetal pole of 1- to 4-cell stage embryos. At the neurula to tail-bud developmental stages, FITC-positive PGC-like cells appeared ventrally around the developing tail bud in the experimental group that received a high-molecular-weight FITC-dextran. The highest average number of FITC-positive PGC-like cells was observed in embryos injected with FITC-dextran having a molecular weight of 500 000 (FD-500). The pattern of migration of the labeled cells was identical to that of PGCs, clearly indicating that the FITC-positive PGC-like cells were PGCs. Labeled vegetal cells, except for the PGCs, were digested and excreted before the embryos starting feeding. FITC-labeled PGCs were observed in the developing gonads of fish for at least 3 mo after injection. We also found that FD-500 could be used to visualize PGCs in other sturgeon species. To the best of our knowledge, this report is the first to demonstrate in any animal species that PGCs can be visualized in vivo for a long period by the injection of a simple reagent.

Sturgeon gut development: a unique yolk utilization strategy among vertebrates.

In vertebrates, maternally supplied yolk is typically used in one of two ways: either intracellularly by endodermal cells or extracellularly via the yolk sac. This study delves into the distinctive gut development in sturgeons, which are among the most ancient extant fish groups, contrasting it with that of other vertebrates. Our observations indicate that while sturgeon endodermal cells form the archenteron (i.e., the primitive gut) dorsally, the floor of the archenteron is uniquely composed of extraembryonic yolk cells (YCs). As development progresses, during neurulation, the archenteric cavity inflates, expands laterally, and roofs a semicircle of YCs. By the pharyngula stage, the cavity fully encompasses the YC mass, which begins to be digested at the hatching stage. This suggests a notable deviation in sturgeon gut development from that in other vertebrates, as their digestive tract initiates its function by processing endogenous nutrition even before external feeding begins. Our findings highlight the evolutionary diversity of gut development strategies among vertebrates and provide new insights into the developmental biology of sturgeons.

Germ cells are not the primary factor for sexual fate determination in goldfish.

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

Growth acceleration of Nile tilapia at 21 to 31 weeks of age with plasma-treated air-supplied water.

This study aimed to develop a technique to accelerate fish growth without using genetic modification or genome editing. We have prepared a reactor with four pairs of opposed electrodes and a high-voltage power supply for the discharge. An arc discharge generates a plasma-treated gas in the reactor. Plasma-treated gas containing active species such as nitric oxide (NO) was generated via an arc discharge in the atmosphere and inserted into an aquarium containing Nile tilapia. No ozone was detected in the plasma-treated gas. Plasma treatment gas was supplied to the 20 L tank at a flow rate of 10 L per minute for varying supply times. The supply duration of plasma-treated air to the water tank was 0.5, 2, 5, and 15 min. Tanks were prepared for each of these four conditions, and gas was supplied daily at the same time. We observed that on supplying plasma-treated gas to tilapia from the 16th week of age for 5 min daily, the average length of the fish at 31 weeks of age was ∼1.5 times longer than that of the control fish. All other supply time conditions were also found to grow acceleration over the control. In the 15-minute supply time condition, individual differences in body length were more significant. A sample had more growth suppression than controls. In other words, the results suggest that an excess supply of active species can cause growth inhibition. These results suggest that an optimal supply of plasma-treated gas has a growth-promoting effect on fish.Key policy highlightsThe fish growth was accelerated by supplying plasma-treated air to the tank.The amount of ozone in the plasma-treated air was below the detection limit, and a large amount of RNS, such as nitric oxide, was generated.After an experimental period of 16 to 31 weeks, the average length of fish in the most significant growth condition was 1.5 times that of the control fish.

Intracytoplasmic sperm injection in sturgeon species: A promising reproductive technology of selected genitors.

Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae.

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.

Author Correction: Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A state-of-the-art review of surrogate propagation in fish.

Surrogate propagation is a systematic approach to producing donor-derived gametes using germline chimeras. In fish, the use of germline chimeras to study the development of germ cells was first conducted in the 1990s in the model fish species medaka (Oryzias latipes) and zebrafish (Danio rerio). More recently, surrogate propagation has been actively investigated as a means of efficient gamete production not only in model fish species but also in aquaculture species and endangered species. Surrogate propagation has the following components: combination of the donor and host species, host sterilization, donor cell preparation, transplantation of germ cells, and gametogenesis and gamete production in surrogate fish. In this review, we first provide a general overview of previous studies related to germ cell transplantation and the methodologies developed for different species, and consider how these have been applied in practice. Second, we consider the development of primordial germ cells in fish embryos, particularly the molecular biological approaches used for the visualization of germ cells and sterilization of host embryos. Finally, we discuss sex control in germline chimeras, which may be a key component of the use of surrogate production in aquaculture. We focus on techniques to produce sterile fish, as these are crucial to the exclusive production of donor gametes in a surrogate host. The advantages and disadvantages of various aspects of surrogate propagation are discussed, the potential use of surrogate propagation as a seedling production system is considered, and future perspectives for aquaculture are suggested.

Production of Germ-Line Chimeras in Zebrafish.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Microinjection of Marine Fish Eggs.

Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.

A newly developed cloning technique in sturgeons; an important step towards recovering endangered species.

Several steps of sturgeon somatic cell nuclear transfer (SCNT) have been recently established, but improvements are needed to make it a feasible tool to preserve the natural populations of this group of endangered species. The donor cell position inside the recipient egg seems to be crucial for its reprogramming; therefore by injecting multiple donor somatic cells instead of a single cell with a single manipulation, we increased the potential for embryo development. Using the Russian sturgeon Acipenser gueldenstaedtii as a multiple cell donor and sterlet Acipenser ruthenus as the non-enucleated egg recipient, we obtained higher proportion of eggs developing into embryos than previously reported with single-SCNT. Molecular data showed the production of a specimen (0.8%) contained only the donor genome with no contribution from the recipient, while two specimens (1.6%) showed both recipient and donor genome. These findings are the first report of donor DNA integration into a sturgeon embryo after interspecific cloning. In all, we provide evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that the sturgeon cloning faces limitations, to date it is the most promising technique for their preservation.