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Purpose. To optimize the conditions of sterlet sperm cryopreservation for works on egg fertilization and for producing viable offspring in the conditions of sturgeon farms. To analyze the effect of sperm freezing in different forms on spermatozoa survival and egg fertilization. To compare the results of cryoprotective solutions with compositions on the results of their use of sterlet sperm fertilization and to analyze their effect on the quality of the produced offspring. Methodology. The task aimed at optimizing the conditions of sterlet sperm cryopreservation for egg fertilization in the conditions of fish farms were carried out according to generally accepted methods in cryobiology and recommendations of Ye.F. Kopeika. The works with the produced sterlet offspring were carried out according to conventional sturgeon culture methods. Findings. The studies showed that the optimization of the conditions of sterlet sperm cryopreservation allowed preserving the properties of thawed spermatozoa at the level of native sperm. It was found that the functions of the sperm cryopreserved in liquid nitrogen vapors in granules in solutions with methanol or dimethyl sulfoxide (DMSO) were better preserved in granules when using methanol. The functions of the sperm after its freezing in cryopreservation solutions №№ 5 and 6 were evaluated in the conditions of a fish farm. It was found that the fertilizing capacity of the cryopreserved sperm in the solution № 5 (85.6 %) (with lower osmolality) did not virtually differ from the control (87.6 %) when using 25-60 g of eggs. In the cryopreservation solution № 5, this value was 17.6 % lower. The weight and length gain after egg fertilization with the sperm cryopreserved in both solutions in 3-month fish was higher than in the control. Originality. Introduction of creatine and fructose into the cryopreservation solution allowed increasing its protective properties and preserving spermatozoa activity after thawing at the level of the native sperm. Sperm cryopreservation in granules allowed optimizing the conditions of low temperature freezing of this sturgeon species and producing high quality viable offspring. Practical value. The obtained results can be used by sturgeon culturists in the conditions of fish farms for works aimed at producing viable sterlet offspring both for commercial purposes and for restocking into natural water bodies as well as by cryobiologists for works with other fish species.

Eggs of sterlet are discharged outside into ambient aquatic environment where egg activation and fertilization occur. Effects of different activation media including freshwater and clay suspension on protein abundances of egg were quantified in sterlet Acipenser ruthenus. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification in the eggs of five females. No significant (p > 0.05) difference was found between protein abundances in eggs activated with different media. However, results showed significant (p < 0.05, fold change ≥2) reduction in the abundances of nine proteins including six glycoproteins, enolase and heat shock protein in activated groups compared to freshly ovulated eggs as control. The fact that abundance of proteasome subunit alpha significantly reduced only in eggs which were activated by clay suspension suggests that activation medium can somehow intervene with protein regulation during fertilization. In conclusion, external fertilization in sturgeon egg is accompanied by huge release of proteins into the external environment that may participate in the construction of a transient microenvironment around egg for attraction and protection of spermatozoa to ensure ensuing fertilization. Data are available via ProteomeXchange with identifier PXD006232.

Artificial mitotic gynogenesis, a chromosome set manipulation, is applied to provide the homozygous progeny with only maternal inheritance. Here, gynogenetic development was induced in the sterlet Acipenser ruthenus L. (Acipenseridae) by activation of the eggs originating from albino females with the UV-irradiated spermatozoa from wild-coloured males, followed by the heat shock applied to suppress the first mitotic division in the haploid zygotes. All experimentally obtained gynogenetic offspring possessed recessive albino coloration. Moreover, the genetic verification, based on three microsatellite DNA markers, confirmed the only maternal inheritance in the albino progeny. Cytogenetic screening enabled identification of the aneuploids, haploids, diploids, triploids, tetraploids and mosaic individuals among the gynogenetic larvae that hatched from the eggs subjected to the heat shock. Furthermore, 40% of the larvae from the haploid variants of the research that were not exposed to the temperature shock showed the diploid chromosome number. A variation of the ploidy level observed in the gynogenetic sterlets may be the consequence of the spontaneous polyploidisation that occurred in the haploid zygotes. Moreover, observation during embryogenesis showed varied stages of eggs development and the asynchronous cell cleavages that may have resulted in the chromosomal disturbances observed in the gynogenetic sterlets here.

We studied the activity of the digestive enzymes, qualitative composition, and bacteria genera ratio in the intestinal chyme of carp Cyprinus carpio and sterlet Acipenser ruthenus grown on an artificial diet in cage culture. It was shown that the inclusion of live bacteria Bacillus subtilis in the probiotic ProStor had a positive impact on the size and the structure of the microbial community in intestinal chyme, on the activity of some digestive enzymes, and on the digestibility and the weight gain of carp and sterlet. The total amount of micro-organisms in the carp chyme was by 13% higher and in sterlet it was 3.5 times higher in the experimental groups compared to the control. In the studied species of fish, the increase in the activity of digestive enzymes was accompanied by the decrease in nitrogen, ammonia, and urea. Presumably, it happened because of better absorption of protein and more intensive use of nitrogen metabolites by intestinal microflora for the synthesis of their own biomass. The average daily weight gain of carp and sterlet was 25 and 35% higher, respectively, in the experimental groups than in the control.

Muscle extracts of some fish species, i.e. pike (Esox lucius), sterlet (Acipenser ruthenus), pink salmon (Oncorhynchus gorbuscha) and, to a lesser extent, perch (Perca fluviatilis) and Russian sturgeon, (Acipenser gueldenstaedtii) prevent the development of premature senescence of the human embryonic fibroblasts induced by the sublethal concentration of H2O2. Muscle extracts of other fish species tested, i.e. coho salmon (Oncorhynchus kisutch) and zander (Sander lucioperca), have not demonstrated this feature. Cell proliferation increased after the action of the senescence-inhibiting muscle extracts. Possible mechanisms of the action of nature biologically active compounds that interfere with the development of stress-induced cell senescence are discussed.Pokazano, chto ékstrakty iz myshts nekotorykh vidov ryb: shchuki (Esox lucius), sterliadi (Acipenser ruthenus), gorbushi (Oncorhynchus gorbuscha) i, v men'sheĭ stepeni, okunia (Perca fluviatilis) i russkogo osetra (Acipenser gueldenstaedtii) prepiatstvuiut razvitiiu prezhdevremennogo stareniia émbrional'nykh fibroblastov cheloveka, vyzvannomu subletal'noĭ kontsentratsieĭ N2O2. Myshechnye ékstrakty drugikh vidov ryb – kizhucha (Oncorhynchus kisutch) i sudaka (Sander lucioperca), takoĭ sposobnosti ne proiavliali. Proliferatsiia kletok posle vozdeĭstviia myshechnykh ékstraktov, ingibiruiushchikh starenie, usilivalas'. Obsuzhdaiutsia vozmozhnye mekhanizmy deĭstviia prirodnykh biologicheski-aktivnykh soedineniĭ, prepiatstvuiushchikh razvitiiu stress-indutsirovannogo kletochnogo stareniia.

The article reports data on the dynamics of blood parameters and parameters of protein, fat, and water-salt metabolism in female starlet × beluga hybrids kept under artificial conditions in the period of reproductive cycle. Gonad maturation in female hybrids in the artificial environment causes clear changes in the biochemistry of blood and of urine.

Sterlet (Acipenser ruthenus) is an important economic fish because of its nourishing caviar, isinglass and flesh. In order to facilitate the recovery of this species, the full understanding of its population genetic structure is necessary for taking appropriate management actions. However, genetic data on the use of nuclear loci in sterlet is still quite poor because microsatellite markers in sterlet that had been developed appeared to be polyploidy which add difficulties in studying the genetic of the sterlet. In this study, 24 tetranucleotide microsatellite markers were developed in sterlet from 160 microsatellite markers of the endangered Chinese sturgeon (Acipenser sinensis). Ten (ZHX76, ZHX64, Z194, Z217, Z184, Z242, Z250, Z258, Z268 and Z269) of the 24 loci showed disomic patterns while the rest loci showed tetrasomic patterns. In this paper, 24 microsatellite markers were characterized in 16 sterlet individuals and all of them were polymorphic with 2 to 7 alleles per locus. The Hardy-Weinberg departure value (d), polymorphic information content (PIC), the observed heterozygosity (HO), the Shannon-Wiener Diversity Indices (H') and the mean expected heterozygosity (HE) of all 24 polymorphic loci ranged from -0.334 to 0.484, 0.367 to 0.725, 0.438 to 1, 0.659 to 1.695, from 0.466 to 0.777, respectively. The markers described here will help in addressing practical problems such as the study of population genetics, conservation genetics and evolution in the polyploidy derivative nature of sterlet.

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Background: Sturgeons (Chondrostei: Acipenseridae) are a group of "living fossil" fishes at a basal position among Actinopteri. They have raised great public interest due to their special evolutionary position, species conservation challenges, as well as their highly-prized eggs (caviar). The sterlet, Acipenser ruthenus, is a relatively small-sized member of sturgeons and has been widely distributing in both Europe and Asia. In this study, we performed whole genome sequencing, de novo assembly and gene annotation of the tarlet to construct its draft genome. Findings: We finally obtained a 1.83-Gb genome assembly (BUSCO completeness of 81.6%) from a total of 316.8-Gb raw reads generated by an Illumina Hiseq 2500 platform. The scaffold N50 and contig N50 values reached 191.06 and 18.88 kb, respectively. The sterlet genome was predicted to be comprised of 42.84% repeated sequences and to contain 22,184 protein-coding genes, of which 21,112 (95.17%) have been functionally annotated with at least one hit in public databases. A genetic phylogeny demonstrated that the sterlet is situated in the basal position among ray-finned fishes and 4dTv analysis estimated that a recent whole genome duplication occurred 21.3 million years ago. Moreover, seven Hox clusters carrying 68 Hox genes were characterized in the sterlet. Phylogeny of HoxA clusters in the sterlet and American paddlefish divided these sturgeons into two groups, confirming the independence of each lineage-specific genome duplication in Acipenseridae and Polyodontidae. Conclusions: This draft genome makes up for the lack of genomic and molecular data of the sterlet and its Hox clusters. It also provides a genetic basis for further investigation of lineage-specific genome duplication and the early evolution of ray-finned fishes.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random., A genome assembly of the sterlet, Acipenser ruthenus, reveals a whole-genome duplication early in the evolution of the entire sturgeon lineage and provides details about the rediploidization of the genome.

Farming of sturgeon in worldwide has increased in recent years. The use of antibiotics to minimize the impact of disease outbreak in sturgeon aquaculture becomes very important.

Simple Summary Sturgeons, also called archaic giants, are critically endangered fish species due to overfishing for caviar and interference in their natural habitats. Some sturgeon species have life spans of over 100 years and sexual maturity is attained between 20 to 25 years. Sterlet (Acipenser ruthenus) has fastest reproductive cycle; thus, this species can be used for surrogate production in sturgeons. Primordial germ cells are the origin of all germ cells in developing embryos. Dnd1 is essential for formation and migration of primordial germ cells and its inactivation results in sterility in fish. In our study, we have used a cutting-edge genome editing technology known as CRISPR/Cas9 to knockout dnd1 and to prepare a sterile sterlet host. CRISPR/Cas9 knocked-out embryos lacked primordial germ cells and can be used as a sterile host for surrogate production in sturgeons. Abstract Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.

Fish species exhibit great diversity rating of aging (from negligible to rapid), which gives a unique possibility for the discovery of the molecular mechanisms that determine the differences in the rate of aging. A mass spectrometric metabolic profiling of skeletal muscle of fish with various aging rates was carried out by direct injection to a quadrupole time-of-flight mass spectrometer. The first group includes long-lived fish species (pike (Esox Lucius) and sterlet (Acipenser ruthenus), the second group&mdash, species with gradual senescence such as that observed in many mammalian species of similar size (zander (Sandra lucioperca) and perch (Perca fluviatilis)) and the third group&mdash, species with very short life cycle (chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha)). Multivariate analysis of metabolic profiles allowed the detecting of about 80 group-specific features associated with amino acids, lipids, biogenic amines, intermediates of glycolysis, glycogenolysis, and citric acid cycle. Possible roles in the aging process are hypothesized for the biochemical pathways of the metabolites that were altered in the different groups.

It is a first report on the structure of germline cells in ovaries of albino sterlet Acipenser ruthenus L. 1758. Ovarian nests, follicles, and germinal epithelium have been examined in gynogenetic and control specimens of this species. The structure of oogonia (named the cystoblasts) and of germline cysts in the nests has been described in detail. Also, the asymmetry in the cytoplasm and early growth of cystocytes in the cysts and of early previtellogenic oocytes has been described. In the cytoplasm of cystoblasts and in all cystocytes, a precursor of granular cytoplasm (Balbiani cytoplasm) is present and defines future vegetal region in the oocytes. Interestingly, the nuclei in cystoblasts comprise a large dense body that contains deoxyribonucleic acid (DNA). The role of this body in formation of multiple nucleoli has been explained. During the zygotene and pachytene stages, massive extrachromosomal amplification of DNA begins in the nucleoplasm of all cystocytes. As a result of the accumulation of extra DNA, an irregularly shaped DNA-body is formed. Multiple nucleoli arise in this DNA-body and around fragments of dense bodies. The asymmetry of the early previtellogenic oocyte cytoplasm is well marked by the presence of the granular cytoplasm. Moreover, the cisternae of the rough endoplasmic reticulum, dictyosomes, mitochondria, complexes of mitochondria with cement, nuage accumulations, and lipid droplets are located in specific zones in the granular cytoplasm. The follicular epithelium is composed of two subpopulations of somatic follicular cells (FCs): the main body cells and future micropylar cells.

The Danube River was originally inhabited by six native sturgeon species, but currently, the sterlet (Acipenser ruthenus L.) is the only native sturgeon species still occupying the Slovak–Hungarian stretch of the Middle Danube. All sturgeon species are facing extinction, suffering from overfishing, water pollution, illegal fishing, poaching or other negative impacts. Urgent and proper actions are needed to prevent their extinction, and evaluating its genetic diversity is one of the essential tools of conservation programs. Since the management actions are primarily local in nature, we first focused on comparing and analysing local sources of fish for population recovery and natural (wild) population in the adjacent stretch of the Danube River. We used 2 fragments of mitochondrial DNA and 12 microsatellites to analyse the genotype of the three groups of sterlets,i.e.wild, broodstock and stocked individuals from Slovak part of the Danube. Mitochondrial markers of all groups were diversified similarly to populations from other parts of the Danube. This confirmed that broodstock and stocked fish belong to the original Danube population. Microsatellites revealed very similar patterns among groups compared, but we detected possible negative trends reflected in losing polymorphism in a few loci in broodstock and stocked individuals.

Sturgeon aquaculture is economically important in many countries, for both meat and caviar production. Sturgeon is the common name for 27 species of fish belonging to the family Acipenseridae. Among them, only the sterlet (Acipenser ruthenus) and the Siberian sturgeon (Acipenser ba-erii) completes the lifecycle in fresh water. In Serbia, in the last few years, aquaculture enterprises have shown more interest in farming these fish species. Also, the importance of sturgeon aquaculture grows due to the rapid decrease of wild populations caused by overfishing, water pollution and destruction of habitat. The development of sturgeon aquaculture activities has been accompanied by the disease outbreaks, and possibility of the emergence and rapid dissemination of several infectious disease agents may represent serious problem in sturgeon aquaculture. Several viral, bacterial , fungal and parasitic diseases have been reported worldwide. Due to the limited knowledge about epizootiology and disease control methods, infectious diseases may represent a major challenge in sturgeon aquaculture. Moreover, none of the diseases reported in sturgeon are regulated in the World Organization for Animal Health (OIE) or European Union (EU) legislations. Due to the increasing interest in sturgeon aquaculture in Serbia present study is focused on the most important pathogens that may represent a threat to sturgeon aquaculture in Serbia. Akvakultura jesetre ima veliki ekonomski značaj u brojnim zemljama, kako za dobijanje mesa tako i za proizvodnju kavijara. Jesetra je uobičajeno ime za 27 vrsta riba koje pripadaju porodici Acipenseridae. Među njima, samo kečiga (Acipenser ruthenus) i sibirska jesetra (Acipenser baerii) pro-vode ceo životni ciklus u slatkoj vodi. Poslednjih godina, u Srbiji postoji veća zainteresovanost za uzgoj ovih vrsta riba. Značaj akvakulture jesetre raste zbog brzog opadanja njihovog broja u prirodi, izazvanog prekomernim izlivom, zagađenjem voda i uništavanjem staništa. Razvoj akvakulture jesetre je praćen pojavom bolesti, a mogućnost pojave i brzog širenja uzročnika zaraznih bolesti može predstavljati ozbiljan problem. Više virusnih, bakterijskih, gljivičnih i parazitskih bolesti je uočeno širom sveta. Zbog ograničenih podataka o epizootiologiji i metodama kontrole bolesti, zarazne bolesti mogu predstavljati veliki izazov u akvakulturi jesetri. Šta više, nijedna od bolesti utvrđenih kod jesetri nije regulisana od strane Svetske organizacije za zdravlje životinja (OIE) niti zakonodavstva Evropske unije (EU). Zbog sve većeg interesovanja za akvakulturu jesetre u Srbiji, u ovom radu je ukazano na najvažnije patogene koji mogu predstavljati pretnju za zapate jesetre u akvakulturi.

The sterlet (Acipenser ruthenus Linnaeus, 1758) is a relatively small sturgeon widely distributed in Eurasian rivers from the Danube to the Yenisei. During the twentieth century, all wild sterlet populations have declined due to anthropogenic factors including: overfishing, poaching, construction of dams, and pollution. Despite the necessity of characterization both wild and captive stocks, few studies of population genetics have been performed thus far. Here we studied the genetic diversity and geographic structure of sterlet populations across the eastern range – Ob-Irtysh and Yenisei basins – by sequencing a 628-bp fragment of mitochondrial DNA control region. We identified 98 new haplotypes, delineated 12 haplogroups and estimated the time of basal haplogroup divergence within the species as over 8 million years ago. Our data suggest that Ob-Irtysh and Yenisei populations are isolated from each other and much lower genetic diversity is present in the Yenisei population than in the Ob-Irtysh population. Our data imply that sterlet populations in Siberian rivers underwent bottleneck or fragmentation, followed by subsequent population expansion. The data obtained here are important for sterlet population monitoring and restocking management.

Приведены данные по зарыблению рек Тамбовской области молодью Acipenser ruthenus в 2015-2017 гг. и характеристика морфологических особенностей стерляди, выпущенной в реку Ворона на территории заповедника «Воронинский».

There are a number of different animals that belong to long- and short-lived species and show a various rate of ageing, providing an ideal model to investigate mechanisms of longevity. In this work, a metabolome profiling of blood plasma from fishes with various ageing rates—negligible (Pike Esox Lucius and Sterlet Acipenser ruthenus), gradual (Zander Sander lucioperca and Perch Perca fluviatilis) and rapid (Chum Salmon Oncorhynchus keta and Pink Salmon Oncorhynchus gorbuscha)—was assessed by means of direct infusion to quadrupole time-of-flight mass spectrometry. Of the 2056 distinct m/z features detected by a mass spectrometry metabolic profiling of blood plasma samples, fifteen metabolites in the classes of dipeptides, fatty acids, glycerolipids, phosphoethanolamines and phosphatidylcholines were significantly associated with ageing rate, independent of species differences. This is the first study of the metabolome of fishes with various ageing rate, and this untargeted approach highlighted the metabolic conditions that may serve to assess the ageing process.

The article provides data on the use of a complex preparation, which includes sodium selenite and tocopherol (vitamin E) in the form of injections for sterlet breeders (Acipenser ruthenus Linnaeus, 1758). Obtained data confirm the ability of E-selenium to regulate metabolic processes towards the formation of reproductive tissue by optimizing them. Positive dynamics is manifested during the gametogenesis, in particular, the number of individuals with reproductive cells at the IV stage of maturity is increased.

To increase the value of bester sturgeon (a hybrid of Huso huso × Acipenser ruthenus) and promote development of the sturgeon aquaculture industry, this study assessed the feasibility of using collagens from bester sturgeon by-products for industrial applications. We first found that individual stable characteristics in terms of yield, amino acid composition, thermal stability, and fibril-forming ability of bester skin collagen (SC), swim bladder collagen (SBC), and notochord collagen (NC) were comparable with those of the beluga and sterlet, the purebred parental species of bester sturgeon. In addition, seasonal variations in these factors were not evident, except for the fibril-forming ability of SC, which showed significant seasonal differences. In the summer, slower fibril formation with smaller individual variations was detected compared with winter for SC. Overall, the quality of products produced from bester sturgeon collagens was stable for industrial use, but batchwise certification of the fibril-forming ability of SC is essential if the products are to be used in fibril form, as in the case of collagen for tissue engineering scaffolds. Second, we determined that the optimal temperature for extracting SC, SBC, and NC was 4 °C, 4 °C, and 20 °C, respectively. We also found that the high endotoxin content of NC could be reduced by pretreatment with 80% ETOH-0.1 M NaOH and 0.1 M NaOH. This study suggests that industrial use of collagens from bester sturgeon is feasible.

Purpose.To iInvestigate and analyze the peculiarities of the accumulation of heavy metals (Cu, Fe, Zn, Cd, Pb, Co, Ni and Mn) in the organs (muscles, gills, liver, kidneys, fins, intestine) and tissues of sterlet (Acipenser ruthenus Linnaeus, 1758) for detecting the patterns of their accumulation and predicting the effect of their toxicological load on the organism of sturgeons as well as for evaluating the polymetallic load on this species in the Dnieper-Bug estuary. Methodology. The material for the research was represented by 3+ – 4+ sterlet caught in the spring of 2016 in the Dnieper-Bug estuary. Organs and tissue samples were homogenized and then burned in a mixture of concentrated nitric (HNO3) and hydrochloric acid (HCl). The determination of heavy metals in organs and tissues of fish were performed by flame atomic absorption spectroscopy using an atomic absorption spectrophotometer AAS-3 and AAS-3N company "Carl Zeiss" (Jena, Germany). Findings. The distribution of heavy metals in organs and tissues of sterlet (Acipenser ruthenus L.) of the Dnieper-Bug estuary in the spring of 2016 was characterized by heterogeneity and depended on their physico-chemical properties and functional characteristics of organs and tissues of the investigated fish. The highest quantities copper, zinc and iron are accumulated liver, while manganese and cobalt in gills. Toxic metals (lead and cadmium) are more concentrated in gills, skin and liver. The maximum contents in the tissues and organs of sterlet were observed for iron and zinc, while the minimals ones — for cadmium and cobalt. Originality. The paper describes the actual data on the contents and peculiarities of heavy metal accumulation in the body of sterlet inhabiting the Dnieper-Bug estuary. Practical value. The paper contains the newest information on the accumulation and content of heavy metals in organs and tissues of sterlet in the Dnieper-Bug estuary. The results of the work will be used for future comparative and biochemical studies of sterlet populations as well as serve as a basis for further monitoring of chemical contamination of water bodies in Ukraine.

Summary The purpose of this study was to mark endangered sterlet (Acipenser ruthenus) with visible implant elastomer (VIE) in order to assess mortality, compatibility, retention, persistence and histological reactions resulting from this tagging technique. It was hypothesized there would be only minor effects on the fishes' health, and assumed that acute effects would be more pronounced than long-term effects. On 11 September 2013, 20 specimens were tagged ventrally with visible implant elastomer, 20 received a subcutaneous injection with 0.9% NaCl solution, and another 20 served as untreated control. Mean total length was 28.0 ± 1.8 cm and mean body mass 64.1 ± 12.0 g. The sterlets were kept in four 4,000-L tanks filled with 2,400-L water. Acute effects were monitored for 95 days, where fish were held at temperatures between 2.4°C and 15.2°C, reflecting outdoor conditions. Chronic effects were examined 282 days post-tagging through histological sections of the tagging region in five sterlets. During the first 95 days of observation, tag retention was 100%. No signs of incompatibility were detected. Body mass did not significantly differ between VIE-tagged fish and controls. At day 282 post-tagging, however, distinct tissue reactions were visible at the tagging sites of nine fish. Histological examination of five fish revealed a variable degree of infiltration with leukocytes in the areas around the elastomer, which did not necessarily correspond with the externally visible degree of inflammation. After medical treatment, the lesions healed without complications, whereas the retention rate of the VIE tags was 5%. According to the findings, the tag location rather than the tag itself was responsible for the externally visible irritations, indicating that the ventral subcutis of sterlet is not a suitable site, even for small VIE tags in long-term studies. The results of this study also suggest that VIE marking should in general be critically evaluated before application in field studies.

In this study, the physiological response and growth performances of Acipenser ruthenus were investigated after a long-term background colour adaptation (12 weeks). Twelve groups of 10 individuals with initial mean body weight of 183 g were reared in black, dark blue, grey and white tanks (three replicates for each colour). At the end of the experiments, growth (initial body weight, final body weight, weight gain per cent, food efficiency ratio, protein efficiency ratio, specific growth rate), blood (cortisol, glucose, pO2, pCO2, pH, haematocrit, osmolality, triglycerides, cholesterol, total lipids) and liver (hepatosomatic index, total lipids, glycogen) parameters were analyzed. Plasma cortisol in the dark-adapted sterlet (21.95 ± 3.9 ng mL−1) was significantly lower than those in white-adapted fish (39.44 ± 6.5 ng mL−1), whereas there were no significant differences in cortisol levels between the grey-adapted fish (23.05 ± 4.1 ng mL−1) and dark blue-adapted fish (24.2 ± 3.6 ng mL−1). A remarkable increase in mean of body weight (%) was detected in dark-adapted sterlet (45.2 ± 3.2) being 27.67%, 12.1% and 11.8% higher than the white, grey and dark blue-adapted fish respectively. The obtained results verified that different background colours lead to different growth performances and physiological responses of starlet, depending on rearing conditions.

Purpose. The literature analysis of the results cryopreservation of different fish species highlights a variation of many parameters, in particular the sperm survival rate during the freezing and unfreezing process. The survival capability of spermatozoa may be called the main parameter, which indentifies the efficiency of the entire process of low temperature freezing of reproductive products. Therefore, the goal of this work was to investigate and find the causes of different degrees of fish sperm cryoimmunity, in particular that of starlet, which is a valuable of sturgeon (Acipenser) species. We also studies the possibility to find the optimum ways to improve the efficiency of the survival rate of the defrosted spermatozoa of different fish species for their further use to produce viable offspring. Methodology. The determination of sterlet sperm membrane permeability was performed after carrying out all necessary manipulations with brood males which included: prespawning incubation, hormonal stimulation, determination of sperm maturity degree, obtaining the sperm by stripping. The measurement of sperm membrane permeability for water molecules was performed based on the technique, which had been used earlier to measure carp sperm permeability, but taking account the specific peculiarities inherent to sterlet sperm. Findings. Based on the performed measurements, we determined the sterlet sperm membrane permeability for water molecules with the use of photometric method. The received experimental data show the highest degree of sterlet sperm membrane permeability for water molecules as compared to carp sperm membrane permeability. Originality. As a result of this experiment, we determined for the first time the absolute value of sterlet sperm membrane permeability for water molecules with the use of photometric method as well as compared the results with those obtained during our work with the carp sperm. Practical value. The data obtained during the experimental studies can be used in the practice of sperm cryopreservation for improving the efficiency of the sperm different of fish species during the process of low temperature freezing especially for such commercially valuable fish species as sturgens.

Summary The objective of this study was to provide information on changes in the metabolism and swimming ability of juvenile sterlet sturgeon, Acipenser ruthenus, caused by acutely low or high temperatures. Changes in critical swimming speed (Ucrit), oxygen consumption rate (MO2), tail beat frequency (TBF) and tail beat amplitude (TBA) were observed with a Steffensen-type swimming respirometer, an oxygen electrode and a camera at different swimming speeds at three temperatures: 5°C, 15°C, and 25°C. Fish tested at 5°C and 25°C were maintained at 15°C (near optimal) for one week to simulate conditions below a dam. The Ucrit value decreased significantly during acute temperature changes at 5°C and 25°C; Ucrit was highest near the optimal temperature. Oxygen consumption rate (MO2) increased with the swimming speed at 15°C; however, at 25°C and 5°C, the MO2 decreased with the swimming speed. Both TBA and TBF decreased at 5°C and 25°C compared to values at 15°C. The slopes of the regression lines (TBF/U) at 5°C and 25°C seemed lower compared to 15°C.

Reducing egg stickiness is a crucial step in artificial culture of sturgeon eggs, with commonly used methods being time consuming or interfering with hatching. Sodium hypochlorite (SH) at varying concentrations and exposure times was tested on sterlet Acipenser ruthenus eggs, with 0.03% SH for 40 s effectively eliminating stickiness without impairment to eggs, embryo development, or hatching when compared with other de-adhesion methods (clay; NaCl, urea, and tannic acid). Field tests using sterlet, Siberian sturgeon Acipenser baerii, and Russian sturgeon Acipenser gueldenstaedtii showed similar numbers of larvae hatched using SH and clay. Immunohistochemistry revealed a protein carbonyl group only on the surface of eggs treated with SH, evidence of oxidation as the mode of action and indicating that oxidation did not affect inner egg layers or cytoplasm. Treatment with SH is a rapid, simple, and inexpensive method for de-adhesion of sturgeon eggs. Statement of relevance Sturgeons represent a valuable group of fish in aquaculture, especially for caviar, but also for meat production. This study describes a novel approach to sturgeon egg de-adhesion involving oxidation of egg surface. The method requires little time, is low cost, and disinfects and softens the egg surface, which facilitates hatching.

Artificial propagation of sturgeons is becoming increasingly important for recovery efforts as well as for commercial production. Sterlet Acipenser ruthenus is a common Eurasian sturgeon with a small body size and one of the fastest reproductive cycles among the sturgeons. The practical question being addressed in this study was how long fertilization of ovulated eggs can be delayed without substantially reducing the hatching rate, and an ancillary question is under what' temperature conditions do eggs retain good quality. Broodstock were injected with homogenized carp pituitary extract (CPE); ovulated eggs from three females were allocated to various treatment groups for temperature storage (control, 7°C, 11°C, 15°C and 19°C) until fertilized. Storage times at the regulated temperatures prior to fertilization were for 2.5, 5.0, 7.5 and 10.0 h. After the selected storage times in ovarian fluid, eggs were fertilized and transferred to incubation cages and then they were counted. Three replicates were allocated to each storage period and temperature. Hatched larvae were counted at 7-day post-fertilization. We found that sterlet eggs do not need to be fertilized immediately after collection. Reasonably good quality was retained for several hours if temperature conditions are fairly cool and stable. Eggs retained good quality when stored at 7°C and 11°C for up to 10 h with 54.1 ± 2.9 to 69.9 ± 7.9% hatching success, but egg quality was significantly reduced after 5-h storage at 19°C (p < 0.01) and 7.5-h storage at 15°C (p < 0.05) compared to cooler temperatures. Uniform temperatures between 7°C and 11°C can be considered as appropriate for storage of eggs in ovarian fluid for up to 10 h. This information can have practical application to routine hatchery practice for acipenserids, as well as for certain research protocols.

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. KP050541) and HSP90 (GenBank accession no. KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98–99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P

Summary The objective of this study was to assess the response of three hepatic enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP)] in sturgeon broodstock females to hormonal treatment during the spawning season. Acetone-dried sturgeon pituitary was used for spawning induction in three cultured broodstocks (Acipenser baerii, bester, and the RsSs hybrid). Blood samples were collected from females during the spawning seasons between 2011 and 2013. Hepatic enzyme activities AST, ALT, and ALP were higher in the non-response of females to the hormone treatment. The differences were species-specific. The increased levels could indicate either an inappropriate timing of the hormone treatment or potential liver disorders in these treated females.

Summary The study aims were to determine the dynamic changes in oocyte characteristics in the ovaries of two hybrids of beluga and sterlet under controlled conditions. The study was carried out between 2010 and 2012, with fish approaching their maturation period in captivity. Duration of the inter-spawning period lasted from 10 to 12 months for the two hybrids raised at temperatures of 20–21°C. The oocyte envelopes and their characteristics were studied using histology. The oocyte envelope layers showed inter-hybrid differences in growth and structural characteristics. The bester eggs can be determined by their envelope thickness compared to most other sturgeon eggs. Duration of various stages of the individual oogenetic development and envelope formation is described in bester hybrids. These were similar to the oogenesis for both of the other hybrid forms and those observed in the pure sturgeon species.

This study was conducted to determine the minimum number of spermatozoa and optimal volume of activation water for effective fertilization of sterlet Acipenser ruthenus eggs. It was also to determine the minimum concentration of spermatozoa per ml of activation water during artificial propagation, when sperm with good motility and velocity (95 % motility and 150 μm s−1 curvilinear velocity) were used for fertilization. In three experiments, eggs (∼50) from three sterlet were fertilized by pooled sperm from four males in four replicates and incubated at 17 °C. The success of fertilization was evaluated at neurulation and hatching. Sterlet eggs were fertilized in proportion to the concentration of spermatozoa per ml in activation water. A total of 50,000 spermatozoa in 8 ml or 32 ml of activation water (concentrations of 6,250 and 1,563 spermatozoa per ml of activation water, respectively) was sufficient for successful fertilization based on neurulation and hatching rates. Neurulation and hatching gradually decreased with a decrease of the spermatozoa ÷ egg ratio from 6,250 to 114. A ratio 1÷4 between eggs and water was insufficient to provide adequate fertilization. Highest neurulation and hatching ratios were reported when the egg ÷ water ratio reached 1÷15, which is recommended for practical usage. Our results therefore showed that a very low concentration of 100 – 1,300 spermatozoa per ml with around 95 % motility and curvilinear velocity over 150 μm s−1 in activation water can provide effective fertilization. In studies of fishes with external fertilization, the volume of activation water during fertilization, total number of spermatozoa and eggs and/or concentration of sperm in activation water should be defined.

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.

This study was planned to investigate the effects of immersion of Sterlet sturgeon (Acipenser ruthenus) eggs by thyroxine hormone (T4) on the embryonic survival rate (ESR), hatching rate (HR), larval survival rate (LSR), and thyroid hormone levels. Hence, oocytes of six females were pooled, fertilized with pooled semen of three males and 45 min after fertilization process, the eggs were placed 120 min in four batches that were immersed in T4 solutions as follows: D1 (the control treatment: T4-free, hatchery water), D2 (0.01 mg L−1 of T4), D3 (0.05 mg L−1 of T4), D4 (0.1 mg L−1 of T4), and D5 (1 mg L−1 of T4). Next, the eggs were transferred to the incubator. The ESR was recorded 6 h after fertilization while HR, and LSR were measured at 1 day post hatching. In addition, the levels of thyroid hormones were measured using the competitive enzyme immunoassay method three hours and three days after fertilization and at the five-day pre-larval stage. The highest ESR was obtained following D5 treatment (P

Influence of in vitro temperature on sperm antioxidant enzyme activity, thiobarbituric acid-reactive substance (TBARS) content and motility parameters was evaluated in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss. Sperm activation was conducted at 4, 14 and 24 °C in both species. Duration of motility was significantly longer at 4 °C than at 14 and 24 °C in both species. At 60 s post-activation, the velocity of sterlet spermatozoa was highest at 24 °C. This trend continued to 420 s post-activation. In rainbow trout, at 10 s post-activation, the highest velocity was observed at 14 °C. Significantly higher catalase activity was seen at 4 °C in both species. No significant difference in spermatozoon superoxide dismutase activity among temperatures was observed. In sterlet, TBARS content was significantly higher at 24 °C compared to other temperatures, but, in rainbow trout, it was highest at 4 °C. The results presume species-specific level of antioxidant enzyme activity and TBARS content at studied temperatures.

Paper shows research data considering investigation and analysis of specific technological parameters due to external and internal marks of sterlet. Our research goal is to analyze influence of specific sterlet rearing conditions on the morphological, biochemical and hematological marks. Sterlet fry and fingerlings were the object of investigation. Sterlet fingerlings rearing was conducted in experimental ponds (2 ha square) of Dnieprovsky surgeon fish hatching facility. Three variances were formed: І – without fertilizers; ІІ – with organic (3 t/ha) and inorganic fertilizers (50 kg/ha); control – with organic fertilizers (5 t/ha). Stocking density in ponds ranged 95k-100k fishes/ha, stock bodymass ranged from 77,5 to 85,0 mg. Analysis of hydrochemical, morphometric and fishery marks was made according to well-known methods. Water temperature was ranged from 20°С to 27°С, average 24,2–25,4°С. Dissolved oxygen was about 4,6-10,4 mgО 2 /dm 3 . Phytoplankton biomass in I variance ponds ranged from 9,95 to 11,05 g/m 3 , in II variance ponds – 13,6-16,0 g/m 3 . The most abundant zooplankton and zoobenthos was registered in second variance, which was caused by using organic and inorganic fertilizers. Thus, zooplankton biomass was about 8,3–8,6 g/m 3 , zoobenthos –6,69–7,71 g/m 2 . As a result of rearing we received sterlet fingerlings with average individual bodymass 3,2±0,25 g (I variance), 4,17±0,30 g (II variance). The best survival rate (69.55%) and fish productivity (267kg/ha). We can see apparent difference in morphometric if we compare body length (M diff = 3,28-4,08.), pectoventral distance (M diff = 3,10-3,28) and bodymass (M diff = 3,22-3,96). Fulton’s condition factor was ranged from 0,70 in I variance (Cv = 28,21%) to 0,80 in II variance (Cv =27,73%). Analyzing chemical composition of sterlet fingerlings muscle tissue, we defined that moisture was about 80,0–82,2%, lipids – 1,8–2,0%, inorganic – 0,9–1,4%. Protein ranged from 12,6-12,9% (I variance) to 15,0-15,4% (II variance). Overall, biochemical marks of sterlet fingerlings corresponded to physiological standard for this age category. Sterlet fingerling muscle tissue contained less than 0,05% of ammonia, 35,6-50,0 mg/kg of nitrates, 0,6-1,8 mg/kg of nitrites, 190,0-220,0 mg/kg of phosphorus, 0,2-0,22% of NaCl. Acid value for lipids ranged from 0,1 to 0,3 mgКОН. Comparative hematology analysis defined that haemoglobin concentration in different variances ranged from 85 to 159 g/l. Red blood cells concentration was about 0,51-0,93 Т/l, leucocytes - 153-208 g/l, thrombocytes – 25-106 g/l. Relative standard deviation for red blood cells – from 11,8 to 36,4%. Mathematical analysis allowed us to build polynomial equations with coefficient of determination as much as 0,7892–0,9083. Also we investigated influence of rearing conditions in ponds of different fish farms on the separate blood marks. We defined reasonable correlation between level of intensification and main fishery indexes, such as bodymass, fish productivity and survival rate and biochemical marks. Obtained data complement existing technology of sterlet combined rearing in ponds of Southern Ukraine due to its later introduction into natural water reservoirs. Also obtained data may be used in modern aquaculture according to its rearing goals. Keywords: sterlet, fingerling, pond rearing, intensification, fertilizer, survival rate, average individual bodymass, fish productivity, red blood cells, protein, fat.

In this study, a cell line, designated as Acipenser ruthenus testis (ART), was successfully established from testis tissues of the sterlet Acipenser ruthenus and characterized by studying and comparing the expression of specific genes between the cell line and the parent gonad tissues. The results suggested that the developed ART cell line was composed of a mixture of germ cells and somatic cells. Ploidy analysis indicated that the cell line exhibited a high degree of genetic stability and that the cells remained in a good proliferating state after being subcultured to passage 80.

Genetic diversity of two natural populations of sterlet (Acipenser ruthenus L., Acipenseridae) was researched: in the lower part of the Sukhona river in Vologda Region and in themiddle part of the Kama river in Perm Krai; and also, two sterlet’s broodstocks Saratov branch of FSBSI GosNIORH and fish farm “Topol” in Perm Krai. ISSR (Inter–Simple Sequence Repeats) — method of DNA polymorphism analysis was used for identification of genetic diversity rate while using PCR. 89 ISSR–PCR markers in researched populations of A.ruthenus were identified. Thenumber of amplified ISSR–PCR markers depended on primer and ranged from 5 (primer (CT)8TG) to 15 (primer (CA)6GT), and its size — from 200 (ISSR–9 and X9) to 1500 (X9) bp. In total samples of A.ruthenus the rate of polymorphic loci is high amounted to 0.910, the expected heterozygosity is 0.296 and the number of effective alleles is — 1.518. The index of genetic diversity is higher in population from Kama river (HE = 0.224; ne=1.380) and lower in broodstock from “Topol” fish farm (HE= 0.170; ne =1,290). The highest number of rare ISSR–PCR markers were identified in natural population from Kama river (R=3), while in broodstock from “Topol” fish farm no rare ISSR-PCR markers were identified. The group of natural populations is more genetically heterogeneous in comparison with the group of broodstocks. Genetic structure of two natural populations (Gst=0.297) and two broodstocks of A.ruthenus (Gst=0.281) was established. Recommendations for genetic diversity data using of the studied populations and broodstocks have been provided for the gene pool conservation.

Somatic cell nuclear transfer (SCNT) is a very promising cloning technique for reconstruction of endangered animals. The aim of the present research is to implement the interspecific SCNT (iSCNT) technique to sturgeon; one fish family bearing some of the most critically endangered species. We transplanted single cells enzymatically isolated from a dissociated fin-fragment of the Russian sturgeon (Acipenser gueldenstaedtii) into non-enucleated eggs of the sterlet (Acipenser ruthenus), two species bearing different ploidy (4n and 2n, respectively). Up to 12% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced. Interestingly, although this transplant displayed tetraploidism (4n) as the donor species, the microsatellite and species-specific analysis showed recipient-exclusive homozygosis without any donor markers. Namely, with regards to this viable larva, host genome duplication occurred twice to form tetraploidism during its early development, probably due to iSCNT manipulation. The importance of this first attempt is to apply iSCNT in sturgeon species, establishing the crucial first steps by adjusting the cloning-methodology in sturgeon’s biology. Future improvements in sturgeon’s cloning are necessary for providing with great hope in sturgeon’s reproduction.