Ancient vertebrate dermal armor evolved from trunk neural crest.

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost.

BACKGROUND: Surrogate production by germline stem cell transplantation is a powerful method to produce donor-derived gametes via a host, a practice known as surrogacy. The gametes produced by surrogates are often analysed on the basis of their morphology and species-specific genotyping, which enables conclusion to be drawn about the donor's characteristics. However, in-depth information, such as data on epigenetic changes, is rarely acquired. Germ cells develop in close contact with supporting somatic cells during gametogenesis in vertebrates, and we hypothesize that the recipient's gonadal environment may cause epigenetic changes in produced gametes and progeny. Here, we extensively characterize the DNA methylome of donor-derived sperm and their intergenerational effects in both inter- and intraspecific surrogates. RESULTS: We found more than 3000 differentially methylated regions in both the sperm and progeny derived from inter- and intraspecific surrogates. Hypermethylation in the promoter regions of the protocadherin gamma gene in the intraspecific surrogates was found to be associated with germline transmission. On the contrary, gene expression level and the embryonic development of the offspring remained unaffected. We also discovered MAPK/p53 pathway disruption in interspecific surrogates due to promoter hypermethylation and identified that the inefficient removal of meiotic-arrested endogenous germ cells in hybrid gonads led to the production of infertile spermatozoa. CONCLUSIONS: Donor-derived sperm and progeny from inter- and intraspecific surrogates were more globally hypermethylated than those of the donors. The observed changes in DNA methylation marks in the surrogates had no significant phenotypic effects in the offspring.

Characterization of potential spermatogonia biomarker genes in the European eel (Anguilla anguilla).

Identification of specific molecular markers for spermatogonial stem cells in teleost is crucial for enhancing the efficacy of reproductive biotechnologies in aquaculture, such as transplantation and surrogate production in fishes. Since it is not yet possible to distinguish spermatogonial stem cells of European eel (Anguilla anguilla) using specific molecular markers, we isolated spermatogonial cells from immature European eels to find these potential markers. We attempted this by studying three candidate genes: vasa, nanos2, and dnd1. Two vasa (vasa1 and vasa2) genes, nanos2, and dnd1 were identified, characterized, and studied in the muscle, testis, and isolated spermatogonia. Our results showed that vasa1 and vasa2 had the highest levels of expression when measured by qPCR. In situ hybridization and immunochemistry assays showed that the four genes were localized explicitly in type A spermatogonia. However, vasa1 and vasa2 exhibited stronger signals in the immature testicular tissue than the other two potential markers. According to this, vasa1 and vasa2 were found to be the most effective markers for spermatogonial cells in the European eel.

Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq.

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized transcripts is still limited to a few models. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four vertebrate models, two amphibians (Xenopus laevis, Ambystoma mexicanum) and two fishes (Acipenser ruthenus, Danio rerio) using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important transcripts such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. It was also observed that there was a poor correlation between the vegetally localized transcripts but a relatively good correlation between the animally localized transcripts. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

Pre-oral gut contributes to facial structures in non-teleost fishes.

Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto-endoderm interface, the primary mouth, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.

The remarkable dynamics in the establishment, rearrangement, and loss of dentition during the ontogeny of the sterlet sturgeon.

BACKGROUND: Sturgeons belong to an early-branching lineage often used as a proxy of ancestor-like traits of ray-finned fishes. However, many features of this lineage, such as the transitory presence and the eventual loss of dentition, exemplify specializations that, in fact, provide important information on lineage-specific evolutionary dynamics. RESULTS: Here, we introduce a detailed overview of the dentition during the development of the sterlet sturgeon. The dentition is composed of tooth fields at oral, palatal, and anterior pharyngeal regions. Oral fields are single-rowed, non-renewed and are shed early. Palatal and pharyngeal fields are multi-rowed and renewed from the adjacent superficial epithelium without the presence of the successional dental lamina. The early loss of oral fields and subsequent establishment of palatal and pharyngeal fields leads to a translocation of the functional dentition from the front to the rear of the oropharyngeal cavity until the eventual loss of all teeth. CONCLUSIONS: Our survey shows the sterlet dentition as a dynamic organ system displaying differential composition at different time points in the lifetime of this fish. These dynamics represent a conspicuous feature of sturgeons, unparalleled among extant vertebrates, and appropriate to scrutinize developmental and evolutionary underpinnings of vertebrate odontogenesis.

Clonal gametogenesis is triggered by intrinsic stimuli in the hybrid's germ cells but is dependent on sex differentiation†.

Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrid's reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrid's gonial cells or whether it is regulated by the surrounding gonadal tissue. We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrid's SSCs lead to sterility when transplanted into sexual males but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females. This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals.

Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (Acipenser ruthenus).

DNA damage during early life stages may have a negative effect on embryo development, inducing mortality and malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. The survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus were analyzed. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress response, which could be applied in toxicological studies on fish embryos. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

Migratory patterns and evolutionary plasticity of cranial neural crest cells in ray-finned fishes.

The cranial neural crest (CNC) arises within the developing central nervous system, but then migrates away from the neural tube in three consecutive streams termed mandibular, hyoid and branchial, respectively, according to the order along the anteroposterior axis. While the process of neural crest emigration generally follows a conserved anterior to posterior sequence across vertebrates, we find that ray-finned fishes (bichir, sterlet, gar, and pike) exhibit several heterochronies in the timing and order of CNC emergence that influences their subsequent migratory patterns. First, emigration of the cranial neural crest in these fishes occurs prematurely compared to other vertebrates, already initiating during early neurulation and well before neural tube closure. Second, delamination of the hyoid stream occurs prior to the more anterior mandibular stream; this is associated with early morphogenesis of key hyoid structures like external gills (bichir), a large opercular flap (gar) or first forming cartilage (pike). In sterlet, the hyoid and branchial CNC cells form a single hyobranchial sheet, which later segregates in concert with second pharyngeal pouch morphogenesis. Taken together, the results show that despite generally conserved migratory patterns, heterochronic alterations in the timing of emigration and pattern of migration of CNC cells accompanies morphological diversity of ray-finned fishes.

Brief communication: rapid elimination of egg stickiness using sodium hypochlorite in wels catfish Silurus glanis.

The wels catfish Silurus glanis is valuable fish for aquaculture. Its production relies mainly on artificial reproduction. One of the crucial steps determining success of the reproduction is elimination of egg stickiness after fertilization. To date, the catfish egg de-adhesion is usually carried out using proteolytic enzymes. Here, we prove a novel method based on oxidation of the egg surface by means of sodium hypochlorite. An effect of different exposure times and concentrations on the egg adhesiveness and damage was tested in the first trial. The selected concentration of sodium hypochlorite 0.3 mg · l-1 with exposure time 40 s was used for comparison with the conventical de-adhesion method using alcalase treatment. The fertilization and hatching rates reached very satisfactory outcome in both treatments (98.3 ± 0.7% vs 97.5 ± 2.2% and 86.6 ± 8.3% vs 91.3 ± 8.5% in alcalase- and sodium hypochlorite-treated embryos, respectively) without any statistical differences. Thus, the de-adhesion method using sodium hypochlorite can be recommended as a suitable method for wels catfish eggs. The method is simple, cheap, very fast, and the treated eggs are disinfected.

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.

Ancient Sturgeons Possess Effective DNA Repair Mechanisms: Influence of Model Genotoxicants on Embryo Development of Sterlet, Acipenser ruthenus.

DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 µM benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 µM etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.

Preservation of female genetic resources of common carp through oogonial stem cell manipulation.

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

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.

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.

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., ).

Rapid de-adhesion of northern pike Esox lucius eggs using sodium hypochlorite.

The elimination of egg stickiness is required for effective artificial reproduction of northern pike, but until now, available methods have required at least 40 min. Sodium hypochlorite was tested under laboratory conditions, and exposure to aqueous concentrations of 0.025-0.05% for 40 s effectively eliminated stickiness without adverse effects. Fertilization and hatching rates in laboratory trials were similar to those observed in eggs treated with traditional methods using clay or milk for 40 or 60 min, respectively, as well as those without treatment. Testing using conventional hatchery incubation techniques did not reveal differences in fertilization rates, while the number of hatched larva was significantly higher in eggs treated with sodium hypochlorite vs. clay. Eggs treated with sodium hypochlorite retained transparency, which facilitated monitoring of embryo development.

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.