Vitrification of the ovarian tissue in sturgeons.

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

Developmental changes of opsin gene expression in ray-finned fishes (Actinopterygii).

Fish often change their habitat and trophic preferences during development. Dramatic functional differences between embryos, larvae, juveniles and adults also concern sensory systems, including vision. Here, we focus on the photoreceptors (rod and cone cells) in the retina and their gene expression profiles during development. Using comparative transcriptomics on 63 species, belonging to 23 actinopterygian orders, we report general developmental patterns of opsin expression, mostly suggesting an increased importance of the rod opsin (RH1) gene and the long-wavelength-sensitive cone opsin, and a decreasing importance of the shorter wavelength-sensitive cone opsin throughout development. Furthermore, we investigate in detail ontogenetic changes in 14 selected species (from Polypteriformes, Acipenseriformes, Cypriniformes, Aulopiformes and Cichliformes), and we report examples of expanded cone opsin repertoires, cone opsin switches (mostly within RH2) and increasing rod : cone ratio as evidenced by the opsin and phototransduction cascade genes. Our findings provide molecular support for developmental stage-specific visual palettes of ray-finned fishes and shifts between, which most likely arose in response to ecological, behavioural and physiological factors.

Optimization of in vitro culture conditions of common carp germ cells for purpose of surrogate production.

Common carp (Cyprinus carpio) is the fourth most-produced fish species in aquaculture and frequently used model species with significant effort invested in development of biotechnological applications. In present study, we attempted to establish an in vitro germ cell culture condition for short term cell culture, which could facilitate further applications such as surrogacy or gene manipulation. Basal media and different types of feeder cells were investigated to optimize carp germ cell culture condition to favor maintenance of mitotic proliferation. Results indicated that germ cells cultured with hESC media and RTG2 cell line as feeder possessed significantly higher proliferation and survival rate compared to that cultured with StemPro media and Sertoli cell line as feeder. In addition, we compared two dissection strategies to compare risk of cell culture contamination and body cavity was open from dorsal part or from ventral part. As a result, carp open from the dorsal side can minimize the risk of contamination. In summary, this is the first study to optimize the cultivation of germ cells in common carp. This opens up new opportunities for the application of specific techniques in the breeding of those species with high commercial value and frequent use as a model fish. Results obtained in this study are important for implementation of new strategies in common carp breeding, conservation of genetic resources, restoration of lines or development of clonal and isogenic carp lines.

How do suboptimal temperatures affect polyploid sterlet Acipenser ruthenus during early development?

Sturgeons are ancient fish exhibiting unique genome plasticity and a high tendency to produce spontaneously autopolyploid genome states. The temperature profiles of the rivers in which sturgeon live and reproduce have been severely altered by human intervention, and the effect of global warming is expected to cause further temperature shifts, which may be detrimental for early developmental stages with narrow windows of thermal tolerance. The comparison of the performance of diploid and autopolyploid sturgeon kept at unfavourable temperatures contributes to scientific knowledge of the effects of polyploid genome states on organisms and can shed light on the ability of polyploids to cope with human-induced alterations to natural conditions. Using the sterlet Acipenser ruthenus as a model species, we carried out conventional artificial fertilization, as well as the induction of the second polar body retention (SPBR), of the first mitotic division suppression (FMDS) and of the second polar body retention followed by the first mitotic division suppression (SPBR+FMDS). Two experiments were conducted to evaluate the effect of polyploidy on two basic performance parameters, survival and growth. In Experiment 1, fish belonging to untreated, SPBR-, FMDS- and SPBR+FMDS-induced groups were kept at 10, 16 and 20°C from the neurula stage until the end of endogenous feeding. In Experiment 2, larvae from the untreated and SPBR-induced groups were reared at 10, 16 and 20°C after their endogenous feeding transition for 3 weeks. Based on our findings, we report that the embryos, prelarvae and larvae of triploid A. ruthenus do not differ from diploids in their ability to survive, grow and develop under suboptimal temperature conditions, while the survival of tetraploids was significantly reduced even at the optimal temperature and even more so at temperatures far from the optimum. This was also the case in the 2n/4n mosaics observed in FMDS-induced group. Thus, we assume that in tetraploid and 2n/4n individuals, the limits of thermal tolerance are closer to the optimum than in diploids. We also conclude that the hexaploid genome state is probably lethal in A. ruthenus since none of the hexaploids or 3n/6n mosaics arising from the SPBR+FMDS induction survived the prelarval period.

Intraspecific Hybrids Versus Purebred: A Study of Hatchery-Reared Populations of Sterlet Acipenser ruthenus.

Hatchery-reared sterlet originating from the Danube and Volga river basins that showed population-discriminatory alleles on at least one microsatellite locus were used to produce purebred (within-population) and hybrid crosses to evaluate intraspecific hybridization with respect to the genetic polymorphism and physiological fitness of fish for commercial aquaculture and, conservation programs. Reciprocal crossing assessed the effect of parent position. The fish were reared in indoor and outdoor tanks and monitored over 504 days for growth traits. The highest final mean body weight (144.9 ± 59.5 g) was recorded in the Danube (♀) × Volga (♂) hybrid and the highest survival in the Volga (♀) × Danube (♂) hybrid. The Volga purebred exhibited the lowest mean body weight (124.8 ± 57.6 g). A set of six microsatellites was used to evaluate the heterozygosity. The mean number of alleles was highest in the Danube (♀) × Volga (♂) hybrid and lowest in the Volga purebred, suggesting an influence of the parent position in the hybridization matrix. The higher level of genetic polymorphism, as in the Danube (♀) × Volga (♂) hybrid, may confer greater fitness in a novel environment. Our analysis revealed that the intraspecific hybrids performed better than the purebred fish in the controlled and suboptimal rearing conditions.

Optimization of sterlet (Acipenser ruthenus) egg incubation.

Sterlet Acipenser ruthenus was used to assess egg and embryo development when incubated at 17 °C in Petri dishes placed in a hatchery tank (300 L recirculating dechlorinated water) with incubation occurring in a static tabletop system in an air-conditioned laboratory, or in a 700 L Q-cell incubator. Eggs in each dish were placed in a plastic box with 300 mL dechlorinated water. Separated eggs from three individual females were fertilized using pooled sperm from four males with there being four replicates. There were no differences (P > 0.05) in mean percentages of neurulation and embryos undergoing cleavage for eggs incubated in the hatchery tank and with use of the static tabletop system. Furthermore, there were no differences (P >  0.05) in percentage of embryos undergoing cleavage, neurulation and hatching for each female when eggs were incubated using the two systems. Results indicate a Petri dish placed in a small plastic box with 300 mL of dechlorinated water was adequate for incubation of sterlet eggs. Results of the study also indicate that with the static system: 1) eggs should be fertilized from each female to retain individual identity; 2) eggs should be dispersed in Petri dishes to avoid clumping; 3) water should be changed at 24 h, but not at 48 h (neurulation) post-fertilization; and 4) embryos that do not optimally develop should be removed the day after neurulation (72 h of post-fertilization period) and water should be exchanged every day subsequent to the 48 h time-point post-fertilization.

Cryopreservation and transplantation of common carp spermatogonia.

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

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.

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., ).

Ovarian fluid impacts flagellar beating and biomechanical metrics of sperm between alternative reproductive tactics.

Alternative reproductive tactics (ARTs) are prevalent in nature, where smaller parasitic males typically have better sperm quality than larger territorial guard males. At present, it is unclear what is causing this phenomenon. Our objective was to gain insights into sperm form and function by examining flagellar beating patterns (beat frequency, wave amplitude, bend length, bend angle, wave velocity) and biomechanical sperm metrics (velocity, hydrodynamic power output, propulsive efficiency) of wild spawning Chinook salmon ARTs. Ovarian fluid and milt were collected to form a series of eight experimental blocks, each composed of ovarian fluid from a unique female and sperm from a unique pair of parasitic jack and guard hooknose males. Sperm from each ART were activated in river water and ovarian fluid. Flagellar parameters were evaluated from recordings using high-speed video microscopy and biomechanical metrics were quantified. We show that ART has an impact on flagellar beating, where jacks had a higher bend length and bend angle than hooknoses. Activation media also impacted the pattern of flagellar parameters, such that beat frequency, wave velocity and bend angle declined, while wave amplitude of flagella increased when ovarian fluid was incorporated into activation media. Furthermore, we found that sperm from jacks swam faster than those from hooknoses and required less hydrodynamic power output to propel themselves in river water and ovarian fluid. Jack sperm were also more efficient at swimming than hooknose sperm, and propulsive efficiency increased when cells were activated in ovarian fluid. The results demonstrate that sperm biomechanics may be driving divergence in competitive reproductive success between ARTs.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-µM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables.

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 10(8) cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 µg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 µg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 µg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments.

Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants.

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (DAla(6)-Pro(9)-LHRHa) at 25 and 75 µg kg(-1) b.w. and compared with those males treated with 4 mg kg(-1) b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg(-1) b.w., which contains DAla(6)-Pro(9)NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72 h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7 days after treatments in all hormonally treated groups. Spermiation rates were 100 % in the CPE and Ovopel groups and 25-50 % in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72 h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75 µg) groups compared to the CPE and GnRHa (25 µg) groups and decreased 72 h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75 µg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75 µg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.

Structural abnormalities of common carp Cyprinus carpio spermatozoa.

Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or "double head" in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation.