Optimalizace chromozómových manipulací u jeseterovitých ryb

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required

Cryopreservation effects on a viable sperm sterlet (Acipenser ruthenus) subpopulation obtained by a Percoll density gradient method.

In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations

Ploidy Levels and Fitness-Related Traits in Purebreds and Hybrids Originating from Sterlet (Acipenser ruthenus) and Unusual Ploidy Levels of Siberian Sturgeon (A. baerii)

The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions

Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (<i>Acipenser ruthenus</i>)

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