Hepatotoxic effect of dietary phytoestrogens on juvenile cultured Russian sturgeon (Acipenser gueldenstaedtii).

In the last two decades, much controversy has grown over the use of soybean products in aquafeeds, especially for carnivorous fish like sturgeons. One point of discussion is the effect of soybean phytoestrogens on fish health. There are many aspects of phytoestrogen utilization in aquafeeds, therefore, the aim of this study is to verify if common legume phytoestrogens can affect juvenile cultured sturgeon erythrocyte and hepatocyte genotoxicity and cause liver pathology. Russian sturgeons were fed from 100 till 365 dph1 with daidzein, genistein, and coumestrol supplemented diets in concentrations: 10, 0.05 and 0.001 g kg-1 of feed, respectively. The SCGE2 method combined with qPCR of three genes involved in DNA repair and genome maintenance, namely cyp1a1, gaad45a and p53 were analyzed. The results were compared with histopathological evaluation of liver tissue. In fish fed with coumestrol supplemented diet, DNA strand damage was the highest in both erythrocytes and hepatocytes, however, simultaneously the lowest level of oxidative DNA damage was found. Additionally, slightly elevated expression of the p53 gene was observed along with a decreased number of apoptotic hepatocytes, which suggests that low concentration of coumestrol may support DNA repair mechanisms in the liver. Although, daidzein showed a preventive effect only against fibrosis. Isoflavones did not show a significant effect on DNA damage in studied cells. Genistein was found to increase macro- and microvesicular steatosis, portal hepatitis and fibrosis, indicating its negative role in the development of liver injuries. Daidzein alleviated some sturgeon liver damage, especially macrovesicular steatosis and interface hepatitis. However, it increased hepatocyte apoptosis, which may suggest daidzein potentially inducing liver injury, though not manifested by other histopathological lesions. Therefore, it can be concluded that at given concentrations, the tested phytoestrogens did not show clearly hepatoprotective effect in sturgeons.

Effects of sous vide cooking combined with ultrasound pretreatment on physicochemical properties and microbial communities of Russian sturgeon meat (Acipenser gueldenstaedti).

BACKGROUND: The production of Russian sturgeon is expanding rapidly in China but it is necessary to adopt measures to extend the shelf life of sturgeon meat. Previous studies found that sous vide cooking (SVC) at 60 °C increased the protein and lipid oxidation. The addition of antioxidant substances reduced the acceptance of the product. The effect of combination SVC and ultrasound pretreatment was therefore investigated. RESULTS: Results showed that SVC at 50 °C combined with ultrasound effectively restrained the growth of total viable counts (TVC) in samples. Meanwhile, the main dominant genera changed from Pseudomonas to Carnobacterium and the number of microbial species decreased. The odor profile of Russian sturgeon meat was more stable and the lipoxygenase (LOX) activity decreased more rapidly after treating with SVC and ultrasound. Importantly, more stable protein aggregates were formed in samples treated by SVC 50 °C together with ultrasound pretreatment, so the protein and lipid oxidation were slowed during storage. Higher springiness values were obtained and the color of sturgeon meat was lighter under these conditions. CONCLUSION: The combination of SVC 50 °C and ultrasound pretreatment effectively inhibited the microbial growth of Russian sturgeon meat at lower oxidation levels. These findings theoretically support the preservation and development of sturgeon meat, and the application of SVC technology. © 2022 Society of Chemical Industry.

The first outbreak of citrobacteriosis caused by Citrobacter gillenii in reared Russian sturgeon (Acipenser gueldenstaedtii) in Turkiye.

Russian sturgeon (Acipenser gueldenstaedtii) is an endangered fish species and also an important resource for the sturgeon aquaculture industry in Turkiye. Recently, a fatal and persistent bacterial disease occurred in the reared sturgeon kept in a trout farm in Turkiye. The disease outbreak has been with notable external signs including petechial hemorrhages and systemic anemia. This outbreak lasted for six weeks, and cumulative mortality reached around 35.00 - 40.00%. In this study, no parasitic and viral agents were observed in the sturgeons. Citrobacter gillenii was isolated from the diseased fish and identified by biochemical and molecular methods including API 20E and 20NE and 16S rRNA gene region sequencing, respectively. As a result, C. gillenii was identified for the first time in Russian sturgeon in Turkiye. The sequence was also deposited under the Genbank with MW057770 accession number. According to the result of disc diffusion method, bacteria were sensitive to enrofloxacin, streptomycin, amoxicillin and oxytetracycline and resistant to penicillin, trimethoprim/sulfamethoxazole, florfenicol and erythromycin. Also, ampC, sul1 and floR resistance genes were detected in the isolated bacteria. The results of this study provide important information for the diagnosis and treatment of this newly emerged disease of Russian sturgeon.

Comparation of the structural characteristics and biological activities of chondroitin sulfates extracted from notochord and backbone of Chinese sturgeon (Acipenser sinensis).

To compare the structural properties and biological activities of chondroitin sulfate (CS) in two different tissues of Chinese sturgeon (Acipenser sinensis) and Russian sturgeon (Acipenser gueldenstaedti), we extracted their backbone cartilage CS (Cart-CS) and notochord CS (Noto-CS), and analyzed the CS structural properties using chromatographic and spectroscopic methods. The molecular weights of Chinese sturgeon Cart-CS and Noto-CS were 54.7 and 25.4 kDa, respectively, and the molecular weights of Russian sturgeon were 50.0 and 38.4 kDa, respectively. The disaccharide composition results showed that Cart-CS was mainly composed of CS-C, while Noto-CS was almost composed of pure CS-A. The antioxidant activity of sturgeon CS and its effect on collagen fibril formation were discussed. Sturgeon CS exhibited higher antioxidant activity than shark and bovine CSs. Sturgeon CS inhibited the self-assemble of type I collagen into fibrils. The inhibition effect of Cart-CS was higher than that of Noto-CS. The high value-added utilization of Cart-CS and Noto-CS will increase the value of sturgeon by-products. Furthermore, the disaccharide composition of CS in sturgeon depends on tissues of origin, but not on species. It means that the CS of Chinese sturgeon can be substituted by the CS of other commercial sturgeon. That will contribute to the protection of endangered species of Chinese sturgeon from illegal fishing and increase the value of commercial sturgeon by-products.

Evaluation of Russian sturgeon (Acipenser gueldenstaedtii) Semen Quality and Semen Cryopreservation.

The alarming decline in sturgeon populations doubled by growing demands for sturgeon products, urge us to prevent the loss of these species. Fish stocking in natural habitats and developing fish farms are viable solutions, which can be successfully implemented with the help of reproductive biotechnologies. Despite the fact that semen cryopreservation is admittedly an important step for saving the Russian sturgeon, a reproducible standard method with good results has yet to be identified. Sperm quality assessment is essential for quantifying the impact of cryopreservation on spermatozoa. The purpose of our study was to provide additional information regarding semen cryopreservation and semen quality evaluation for the Russian sturgeon. Our study method is based on the use of two yolk-free extenders (with different cryoprotectants: DMSO and methanol) for freezing semen, using a simple freezing protocol. Parameters such as volume, concentration, motility, morphology and membrane integrity were evaluated. In conclusion, cryopreservation of Russian sturgeon spermatozoa using an extender containing methanol as cryoprotectant led to high egg fertilization percentages (72.67 ± 5.4%) even if the total motility values recorded for thawed semen were quite low (18-25%). Additionally, we identified two optimal stains for morphological studies and morphometry (Spermac stain kit and Trypan Blue Solution).

Antioxidant Activity and Cytotoxicity of Aromatic Oligosulfides.

Natural or synthetic antioxidants with biomimetic fragments protect the functional and structural integrity of biological molecules at a minimum concentration, and may be used as potential chemotherapeutic agents. This paper is devoted to in silico and in vitro evaluation of the antioxidant and cytotoxic properties of synthetic analogues of natural compounds-aromatic oligosulfides. The antiradical and SOD-protective activity of oligosulfides was demonstrated in the reaction with O2-• generated in enzymatic and non-enzymatic systems. It was found that phenol-containing disulfides significantly reduced the accumulation level of hydroperoxides and secondary carbonyl thiobarbituric acid reactive substances, which are primary products of oleic acid peroxidation. The antioxidant efficiency of bis(3,5-di-tert-butyl-4-hydroxyphenyl) disulfide increased over time due to the synergistic action of the 2,6-di-tert-butylphenol fragment and the disulfide linker. The highest cytotoxicity on the A-549 and HCT-116 cell lines was found for bis(3,4-dimethoxyphenyl) disulfide. Significant induction of apoptosis in HCT-116 cells in the presence of bis(3,4-dimethoxyphenyl) disulfide indicates the prospect of its use as an antitumor agent. The significant and moderate dependences revealed between various types of activities of the studied aromatic oligosulfides can be used in the development of a strategy for the synthesis and study of target-oriented compounds with predictable biological activity.

Different response of Acipenser gueldenstaedtii CRP/SAP and SAA to bacterial challenge and chronic thermal stress sheds light on the innate immune system of sturgeons.

Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation of natural habitat and overfishing for meat and caviar production. Consequently, sturgeon aquaculture has extensively developed lately, being Russian sturgeon (Acipenser gueldenstaedtii) the second most important species reared for caviar production. However, Russian sturgeon aquaculture in subtropical countries, such as Uruguay, confronts difficulties because fish have to endure excessive summertime warm temperatures, which weaken their innate defences facilitating opportunistic infections. To address this problem, we look for identifying putative acute phase proteins (APPs), which might be robust serum biomarkers of both infection and chronic thermal stress, applied to monitoring Russian sturgeon health status in farms. We focused on the C-Reactive Protein/Serum Amyloid P (CRP/SAP) pentraxin since the pentraxin family includes well-known APPs, better characterised in mammals than fish. We identified A.gueldenstaedtii CRP/SAP (AgCRP/SAP), as a member of the universal CRP/SAP pentraxin sub-family, and studied AgCRP/SAP involvement in sturgeon response to bacterial challenge and chronic thermal stress, in comparison with A. gueldenstaedtii Serum Amyloid A (AgSAA), a previously described positive APP. Results showed that AgCRP/SAP is a constitutive serum component that remained constant upon Aeromonas hydrophila challenge and chronic thermal stress. Contrastingly, serum AgSAA was subjected to regulation by bacterial and thermal stress challenges, showing a 50-fold increase and 3-fold decline in serum levels, respectively. Overall, results highlight the potential value of AgSAA, but not of AgCRP/SAP, as a biomarker of bacterial infection and the need to continue searching for robust chronic thermal stress biomarkers in sturgeons.

Cryoprotective activity of phosphorus-containing phenol.

The antioxidant and cryoprotective efficiencies of a 3,5-di-tert-butyl-4-hydroxyphenyl)methylenediphosphonic acid (MDPA) differ significantly for sperm cells of various species of sturgeon fish (Russian sturgeon, beluga and Stellate sturgeon). The ability of phosphorus-containing phenol MDPA to decrease the level of lipid peroxidation of sperm, beneficial effect on the activity indicators of the sperm of native sturgeon and of the defrosted one after deep freezing as well as on the fertility of sperm cells was shown.

Comparative transcriptome analysis reveals expression signatures of albino Russian sturgeon, Acipenseriformes gueldenstaedtii.

Albinism is a genetically inherited condition that is caused by a series of genetic abnormalities leading to a reduction in melanin production. Russian sturgeon is one of the most valuable freshwater fish species worldwide, and albino individuals have been found in fish farms. Due to its complicated genome and scarce genome-wide genetic resources, the underlying molecular basis of albinism in Russian sturgeon is unknown. In the present study, we first generated transcriptome profile of Acipenser gueldenstaedtii using pooled tissues, which provided reliable reference sequences for future molecular genetic studies. A total of 369,441 contigs were assembled, corresponding to 32,965 unique genes. A comparative analysis of the transcripts from the skin of albino and wildtype individuals was conducted afterwards. A total of 785 unique genes were differentially expressed, including the upregulation of 385 genes and the downregulation of 400 genes in albino individuals. The expression pattern of 16 selected differentially expressed genes was validated using qRT-PCR. Additional annotation, GO enrichment analysis and gene pathway analysis indicated that the melanogenesis pathway may be interrupted in albinism. Eight potential causative genes that were highly likely to be responsible for sturgeon albinism were identified, including Dct, Tyrp1b, Slc45a2, Ctns, Pmela, Pmelb, Cd63, and Bloc1s3, which were found to be significantly down-regulated in albino Russian sturgeon. Moreover, a sliding window analysis of the ratio of nonsynonymous to synonymous nucleotide substitution rates (Ka/Ks) ratios indicated that seven out of the eight genes underwent positive selection during evolution. Our results provide a valuable basis for understanding the molecular mechanism of albinism in fish species and will facilitate future genetic selection and breeding of sturgeon with market-favored traits in aquaculture.

Some Hematological and Non-Specific Immune Responses of Rosehip (Rosa canina) - Fed Russian Sturgeon (Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833) to Mycobacterium salmoniphilum

ABSTRACT In the study, the immunostimulant effects of rosehip (Rosa canina) on the health indicator hematological and non-specific immune parameters of Mycobacterium salmoniphilum-infected sturgeon were investigated. The rosehip was applied in the ratios of R5 (5%), R10 (10%), and R15 (15%) in three repetitions. After a 35-day feeding period, the fish were infected with M. salmoniphilum and analyzed after day 7, when symptoms were observed at elevated levels. Erythrocyte, erythrocyte indices, hemoglobin, hematocrit, leukocyte levels and the non-specific immune parameters including lymphocyte, monocyte, eosinophil, neutrophil, cytokines, phagocytic activity levels were comparatively examined using positive and negative control groups. The evaluations revealed that the RBC, Hb, Hct and WBC counts in the R15 group were significantly higher than those of the C+ group. Moreover, depending on the level of the immune response of the fish, among the immune parameters, significant increases in the lymphocyte, monocyte, cytokine, and phagocytic activity levels were observed in the R15 group. The results showed that the hematological and immune response to M. salmoniphilum infection was stimulated significantly in the 15% rosehip-fed fish. Hence, the effective dose of rosehip in fish was determined to be 15% and rosehip is suggested as an alternative to currently recommended immunostimulants.

Russian sturgeon cultured in a subtropical climate shows weaken innate defences and a chronic stress response.

Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past ten years. However, during the Uruguayan summer fish endure high water temperatures and increased bacterial infections that threaten aquaculture. Our understanding of sturgeon's immune system and its interplay with environmental factors like temperature is almost unknown. This study analysed the way in which seasonal variations affect enzymatic blood components of Russian sturgeon's innate defences, including the serum alternative complement pathway (ACP), ceruloplasmin (Cp) and lysozyme activities. Results showed that summertime conditions in the farm altered these defences in different ways, inducing a significant decrease in ACP and Cp, and an increase in lysozyme. In addition, serum levels of total protein and cortisol decreased in summer, suggesting a chronic stress response was induced in parallel. Subsequently, we analysed whether the increase in water river temperature during summer could account for the observed results. To that end, we acclimated juvenile sturgeons to mild (18 °C) or warm (24 °C) temperatures for 37 days. Like in summer, sturgeons exposed to 24 °C showed lower levels of serum ACP, Cp and total proteins, together with a progressive decrease in body weight and increased fish mortality. Administration of an immunostimulant containing Se and Zn slightly reverted the temperature-induced effects on sturgeon's defences. Altogether, our study provides novel data on various physiological parameters of the Russian sturgeon and highlights the impact warm temperature has on stress and innate immunity in this chondrostean fish.

Synbiotic (Biomin imbo) alters gut bacterial microflora of Russian sturgeon, Acipenser guldenstadti (Brandt & Ratzeburg, 1833) in a time-dependent pattern.

We investigated the efficiency of Synbiotic (Biomin imbo) in alternation and stabilization of gut microflora of Russian sturgeon, Acipenser guldenstadti. For this purpose, five experimental treatments and one control group with three replicates were considered. In treatment groups, fish were fed with diet containing 1 (T1), 1.5 (T2), 2 (T3), 2.5 (T4) and 3 (T5) g Synbiotic/kg diet. In control group, fish were fed without supplement of Synbiotic. Sampling for bacterial studies was conducted at days 20, 40 and 60 after the beginning of the experiment. According to results, there were no significant differences in total bacterial count between Synbiotic treatments and also in comparison with control group 20 days after the beginning of the experiment (P > 0.05). At days 40 and 60 of experiment, the highest total bacterial counts were observed in control and T1 group respectively. At day 20, the lactic acid bacteria count was higher significantly in Synbiotic treatments compared to control group (P < 0.05). In all sampling times (i.e. days 20, 40 and 60), the highest lactic acid bacteria count was observed in fish fed with 3 g Synbiotic/kg diet i.e. treatment 5 (P < 0.05). In each experimental group, total bacteria and lactic acid bacteria counts increased significantly as the duration of experiment expanded. In this regard, these parameters were significantly higher at day 60 compared to day 40 and 20 (P < 0.05). In conclusion, our results showed that Synbiotic (Biomin imbo) alters and stabilizes efficiently the microflora of Russian sturgeon gut towards lactic acid bacteria in a time-dependent pattern.

Discovery and identification of candidate sex-related genes based on transcriptome sequencing of Russian sturgeon (Acipenser gueldenstaedtii) gonads.

As the Russian sturgeon (Acipenser gueldenstaedtii) is an important food and is the main source of caviar, it is necessary to discover the genes associated with its sex differentiation. However, the complicated life and maturity cycles of the Russian sturgeon restrict the accurate identification of sex in early development. To generate a first look at specific sex-related genes, we sequenced the transcriptome of gonads in different development stages (1, 2, and 5 yr old stages) with next-generation RNA sequencing. We generated >60 million raw reads, and the filtered reads were assembled into 263,341 contigs, which produced 38,505 unigenes. Genes involved in signal transduction mechanisms were the most abundant, suggesting that development of sturgeon gonads is under control of signal transduction mechanisms. Differentially expressed gene analysis suggests that more genes for protein synthesis, cytochrome c oxidase subunits, and ribosomal proteins were expressed in female gonads than in male. Meanwhile, male gonads expressed more transposable element transposase, reverse transcriptase, and transposase-related genes than female. In total, 342, 782, and 7,845 genes were detected in intersex, male, and female transcriptomes, respectively. The female gonad expressed more genes than the male gonad, and more genes were involved in female gonadal development. Genes (sox9, foxl2) are differentially expressed in different sexes and may be important sex-related genes in Russian sturgeon. Sox9 genes are responsible for the development of male gonads and foxl2 for female gonads.

Application of new phenolic antioxidants for cryopreservation of sturgeon sperm.

Heterocyclic derivatives of butylated hydroxytoluene (BHT) were studied as cryoprotectants of the basic media for cryopreservation of the Russian sturgeon sperm. Rates of lipid peroxidation of sturgeon sperm before and after cryopreservation were reduced in the presence of the studied compounds, exceeding the effects of BHT and water-soluble analogue of vitamin E, trolox. The most efficient antioxidant has the effective concentration of 0.1 mM. Novel antioxidant agents as cryomedium supplements not only reduced the level of lipid peroxidation, but also enhanced the translational motility of the sperm of the Russian sturgeon after defrosting.

Evolutionary relations and population differentiation of Acipenser gueldenstaedtii Brandt, Acipenser persicus Borodin, and Acipenser baerii Brandt.

Russian ( Acipenser gueldenstaedtii), Persian ( A. persicus) and Siberian ( A. baerii) sturgeons are closely related 'Ponto-Caspian' species. Investigation of their population structure is an important problem, the solution of which determines measures for conservation of these species. According to previous studies, 'baerii-like' mitochondrial genotypes were found in the Caspian Sea among 35% of Russian sturgeon specimens, but were not found in Persian sturgeons. This confirms genetic isolation of the Persian sturgeon from the Russian sturgeon in the Caspian Sea. However, in order to clarify the relationships of these species it is necessary to analyze nuclear DNA markers. The amplified fragment length polymorphism (method) allows estimating interpopulation and interspecific genetic distances using nuclear DNA markers. In the present study, four samples were compared: Persian sturgeons from the South Caspian Sea, Russian sturgeons from the Caspian Sea and the Sea of Azov, and Siberian sturgeons from the Ob' River, which are close to the latter two species, but are also clearly morphologically and genetically distinct from them. For the amplified fragment length polymorphism (AFLP) method, eight pairs of selective primers were used. The analysis revealed that the Siberian sturgeon has formed a separate branch from the overall Persian-Russian sturgeons cluster, which was an expected result. In addition, the results showed that the Caspian Russian sturgeon is closer to the Persian sturgeon from the Caspian Sea than to the Russian Sturgeon from the Sea of Azov. The present DNA marker data confirm that despite the genetic isolation of the Persian sturgeon from the Russian sturgeon in the Caspian Sea, the Persian sturgeon is a young species.

Characterization and inhibition studies of carbonic anhydrase from gill of Russian Sturgeon Fish (Acipenser gueldenstaedtii).

An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from gill of Acipenser gueldenstaedtii as an endangered sturgeon species. The carbonic anhydrase was purified 66-folds with yield 20.7% by Sepharose-4B-l-tyrosine-sulfanilamide affinity column and the specific activity was determined as 222.2 EU/mg protein. Km and Vmax kinetic values for gill carbonic anhydrase were calculated by a Lineweaver-Burk graph using p-nitrophenol acetate (p-NPA) as a substrate, and was defined as 2.5 mM and 5 × 10(6 )µM/min, respectively. It was observed that CA from the sturgeon gill in the presence of the sulfanilamide and acetazolamide as an inhibitor had very low IC50 values such as 13.0 and 0.1 µM, respectively. In addition, it was determined that the enzyme was inhibited by Fe(2+,) Co(2+,) Ni(2+), and Zn(2+)-Ba(2+) with the IC50 values of 0.2, 1.7, 1.2, and 1.1 mM, respectively.