Recording of Processes of Hemoglobin Oxidation and Cryoprotective Effect of Glycerol

It was shown earlier that functional properties of hemoglobin changed during storage. Our goal was to evaluate processes of hemoglobin autooxidation and to develop simple methods for conservation of hemoglobin samples. It is revealed that cryoprotective effect of glycerol rapidly rises with increase of its concentration up to 5% for hemoglobins of all 4 studied species of sturgeon fish, sterlet, Russian sturgeon, starred sturgeon, and great sturgeon. The cryoprotective effect of glycerol is the highest on hemoglobin of the starred sturgeon, the lowest, of the sterlet. The results of this study allow proposing the method for cryoconservation of the sturgeon fish hemoglobin with addition of glycerol (up to 5% ) to the frozen solution of hemoglobin.     

Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in sterlet was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.     

Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in stellate sturgeon was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.     

Osmotic and ion regulation in different species of acipenserids (acipenseriformes, acipenseridae)

Species specificity of osmotic and ion regulation in five species of acipenserids connected in varying degrees with sea conditions is investigated. The objects of study were freshwater middle-Volga sterlet Acipenser ruthenus; Siberian sturgeon A. baerii, inhabiting the Lena River and migrating to the sea near the river mouth; and the following migratory acipenserids of the Volga-Caspian Basin (regular diadromous migrations): the Russian sturgeon A. gueldenstaedtii, the great sturgeon Huso huso, and the starred sturgeon A. stellatus. The study was carried out on immature specimens (of age 2+). The level of osmolarity and concentrations of cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) were determined in blood serum, urine, and liquid from the cavity of spiral valve in fish from the fresh water (control) and after their adaptation (period of seven to ten days) to sea water with salinity 12.5–12.7 and 18‰. The increase was found in the functional activity of the mechanism of osmotic and ion homeostasis in sturgeons according to the increase of the environmental salinity of a particular species. The freshwater sterlet adapted to the sea environment as osmoconformer. The other studied species were capable of maintaining a relative stability of osmolarity and ion concentration in blood serum at different levels while adapting to the sea environment; they changed from hyperosmotic (in fresh water) to hypoosmotic type of regulation. The kidney plays a more significant role in the removal of excessive Na⁺ in sturgeons than in bony fish. An increase in the sodium-reabsorption and magnesium-secretion functions of the kidney as well as the sodium-absorption function of the intestines was observed in sturgeons, relative to the increase of environmental salinity.     

Meiotic gynogenesis in the stellate,Russian sturgeon and sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

Sturgeon production trials in the waste-heat effluents of a pulp and paper plant in Archangelsk, Russia

Sturgeons were cultured at the Solombalsky cellulose and paper plant (CPP) in Arkhangelsk over a 3 year period. The warm-water effluents of the biological treatment unit were utilized to rear the Siberian sturgeon (Acipenser baerii) and its hybrids with the sterlet (Acipenser ruthenus). Another culture trial included the Russian sturgeon (Acipenser gueldenstädtii) and its hybrids with the beluga (Huso huso). The most profitable culture (in terms of growth rates) was with the Siberian sturgeon and its hybrids with the sterlet. After the second year of culture, Siberian sturgeon and its hybrid averaged about 2.3 kg in wet weight while Russian sturgeons gained less (1.5 kg). One- and two-year old sturgeons grown at the Solombalsky CPP met practically all requirements needed for a successful culture.     

Discrete phenotypical variability of electrophoretical specters of one of system of myogens in the Acipenseridae family

Analysis of electrophoregrams of water-soluble proteins of white skeletal muscle of 10 species belonging to three genera of the Acipenseridae family has allowed revealing an earlier not described system that can serve as a biochemical marker. This protein system represents a set of five fractions (A, B, C, D, E). A part of the studied species is monomorphous for one of these fractions (sterlet, giant sturgeon, Caspian sturgeon, and Amu-Darya shovelnose had phenotype A, barbel sturgeon—phenotype C, Amur sturgeon—phenotype D). In all individuals of the Amur sturgeon the identical three-component specter (phenotype ACE) was revealed. In the Sakhalin, Siberian, and Russian sturgeons a clearly expressed polymorphism was observed. Among the studied species the Russian sturgeon was characterized by the highest degree of heterogeneity and polymorphism. In the studied sample of this species all five protein variants are found, whereas in the rest of them—only a part of this set.     

Meiotic Gynogenesis in the Stellate and Russian Sturgeons and Sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon, A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

Genetic monitoring of progeny derived from natural spawning of Acipenser ruthenus Linnaeus, 1758 and Acipenser stellatus Pallas, 1771 as well as their hybrids in the Lower Volga river during the period 2017 to 2019

The state of natural spawning of sturgeons in the Lower Volga River was investigated through genetic monitoring of juveniles (larvae, fingerlings, subadults) captured at seven traditionally used monitoring locations. Sampling was performed during the period 2017–2019. In total, 460 individuals were caught and genotyped using a set of six microsatellite loci (Afug41, Afug51, Afug135, An20, AoxD161, AoxD165) and mtDNA control region sequencing. The observed species-specific microsatellite alleles revealed the presence of juvenile sturgeons originating from natural spawning also included some hybrids between Acipenser stellatus and A. ruthenus. Thus, the majority of sterlet breeding takes place in natural spawning grounds but also sterlet hybridizes with a small number of stellate sturgeons. Juveniles of other sturgeon species were not identified, and it is concluded that natural spawning activities of Russian sturgeon, beluga and ship sturgeon were insignificant or even absent in the Lower Volga during the observation period.     

鳙团移核鱼LDH,MDH同工酶的研究

对二龄鳙鱼细胞核和团头鲂细胞质配合的核质杂种鱼－－鳙团移核鱼及其亲本,供核体鳙鱼和受核体团头鲂肌组织ＬＤＨ、ＭＤＨ同工酶进行了研究试验。鳙团移核鱼和供核体鳙鱼肌组织ＬＤＨ同工酶均具有ＬｄｈＡ２Ｂ２一条谱带;受核体团头鲂的则具有ＬｄｈＡ２、ＬｄｈＡ２Ｂ１、ＬｄｈＡ２Ｂ２、ＬｄｈＡ１Ｂ３、ＬｄｈＢ４等五条谱带。移核鱼和供核体鳙鱼肌组织的ＭＤＨ同工酶都各具有二条谱带：Ｓ－ｍｄｈＡ２、Ｓ－ｍｄｈＡＢ;受核     

New strategies for brood stock management of sturgeon in the Sea of Azov basin in response to changes in patterns of spawning migration

The sturgeon stock in the Sea of Azov is presently maintained by artificial reproduction allowing for the harvest of sturgeons in this basin. Shortened spawning migrations in regulated rivers and long-term artificial selection of the ripest fish at the hatcheries have resulted in high functional maturity of sturgeon spawners in the sea near the river mouth. The traditional technology of artificial sturgeon reproduction is non-effective under modern ecological conditions. The use of ecological and hormonal methods to control seasonal propagation of stellate sturgeon, A.stellatus , Russian sturgeon, A.gueldenstaedti , beluga, Huso huso , and sterlet, A.ruthenus , make it possible to stagger the sexual cycle of breeders from the various biological groups for early (5 months) or later (6 months) spawning. Use of these methods preserves the natural heterogeneity of sturgeon populations maintained through stocking, and also allows for a considerable increase in the quantity of fry reared at the same production base.
The use of the synthetic analogue of mammalian gonadotropin-releasing hormone (Gn-RHa) is better for inducing maturity of the ripest brood fishes, especially for stellate sturgeon, in comparison with the traditional pituitary injections. The schedule of injections was determined for the various species and seasons of spawning.
Short-term rearing of larvae in ponds (no more than 15 days) and subsequent adaptation to sea water in brackish lagoons (1–5 %o) allows for repeated use of nursery ponds in sturgeon hatcheries and a considerable increase in the quantity and survival of sturgeon juveniles in the sea.
The restoration of the sturgeon population structure in the Sea of Azov is not possible without developing a genetic collection of the fish under a controlled culture regime.     

Comparative coelomic fluid composition of sterlet sturgeon Acipenser ruthenus Linnaeus, 1758, Siberian sturgeon Acipenser baerii Brandt, 1869, and Russian sturgeon Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833

The objectives of this study were to compare the physicochemical properties of coelomic fluid (CF) in three sturgeon species, sterlet sturgeon Acipenser ruthenus (age 5–8 years), Siberian sturgeon Acipenser baerii (age 15–20 years), and Russian sturgeon Acipenser gueldenstaedtii (age 13–18 years). For the study, CF was collected by plastic pipette from the eggs of mature female sterlet sturgeon (N = 10), Siberian sturgeon (N = 7) and Russian sturgeon (N = 4); osmolality, pH, ionic composition (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?), glucose, cholesterol and total protein of the CF were then analyzed. Mean concentration of K⁺ (4.4–6.11 mmol/L), Cl^? (89.8–98.0 mmol/L), Ca²⁺ (0.87–0.96 mmol/L), Mg²⁺ (0.57–0.67 mmol/L), cholesterol (0.13–0.16 mmol/L), total protein (2.41–3.57 g/L), pH (7.92–7.98), and osmolality (190–213 mOsm/kg) of CF were not significantly different among these three species. However, the mean concentration of Na⁺ was significantly lower in sterlet sturgeon (104.6 ± 7.7 mmol/L; p < .05) than in the Siberian (126.4 ± 6.2) and Russian (123.0 ± 5.9) sturgeon. For these three species, Na⁺, K⁺, Cl^?, and Mg²⁺ were the dominating ions; several significant correlations were observed among these ions and other physiochemical properties of CF. This is the first report on the ionic and biochemical composition of the CF of sterlet, Siberian and Russian sturgeon, which can be used as a reference point for further development of artificial media for the short‐term storage of unfertilized sturgeon eggs as well as for the standardization of the fertilization protocol for these species in controlled reproduction.     

Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

Fertility of females of sturgeon hybrids obtained from species with different levels of ploidy (Acipenser ruthenus and A. dauricus) and their cloning

Main purpose of this work is the identification of females of artificial sturgeon hybrids capable to produce unreduced oocytes. The importance of this task is due to the ability to receive clonal all-female lines. Experiments were performed on the previously obtained reciprocal hybrids of sterlet, Acipenser ruthenus (S) with ~120 chromosomes and kaluga, Acipenser dauricus (K) with ~260 chromosomes. Karyotypes of backcross hybrids of (S × K) female (obtained by crossing sterlet female with kaluga male) and sterlet male included 180 – 190 chromosomes. This means that (S × K) female produced eggs with ~125 chromosomes and its karyotype consisted of ~250 chromosomes. This number was confirmed by a comparative analysis of erythrocyte size in this female and species with different ploidy. Karyotype with ~250 chromosomes can occur in (S x K) female only as a result of fertilization of a diploid sterlet egg (120 chromosomes) with kaluga haploid sperm (~130 chromosomes). Eggs of hybrid fertile (S × K) female, inseminated with inactivated sperm of Amur sturgeon and sterlet, developed into viable gynogenetic offspring, confirmed by the analysis of five microsatellite loci in this progeny, (S x K) female, and males used for UV-inactivated sperm. These data allow us to propose a method for obtaining fertile females of sturgeon hybrids from species with different ploidy. For this, experimentally obtained diploidized eggs from diploid 120-chromosome species must be fertilized by 250–270-chromosome male. Karyotypes of backcross hybrids of (K × S) female (obtained by crossing kaluga female with sterlet male) and sterlet male included ~250 chromosomes and hybrids of this female with kaluga male had ~320 chromosomes. These results proved an ability of hybrid (K × S) female to produce unreduced eggs, resulting in triploid backcrosses. The absence of reduction during egg development is well known in clonal forms (species) of vertebrates, which are of hybrid origin, and in artificially created fish hybrids. However, this has not been reported previously for sturgeons. Insemination of eggs of (K × S) female with UV-inactivated sperm of sterlet and Amur sturgeon led to offspring generation for which the genetic identity to their mother was proved using microsatellite analysis. That is, clonal inheritance was observed. These results suggest the possibility of developing a technology to produce all-female offspring. Artificial production of clonal lines in hybrid vertebrates can be also considered as experimental reproduction of the first stages of reticular speciation in nature.     

Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

High swimming and metabolic activity in the deep-sea eel Synaphobranchus kaupii revealed by integrated in situ and in vitro measurements

Several complementary studies were undertaken on a single species of deep-sea fish (the eel Synaphobranchus kaupii) within a small temporal and spatial range. In situ experiments on swimming and foraging behaviour, muscle performance, and metabolic rate were performed in the Porcupine Seabight, northeast Atlantic, alongside measurements of temperature and current regime. Deep-water trawling was used to collect eels for studies of animal distribution and for anatomical and biochemical analyses, including white muscle citrate synthase (CS), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and pyruvate kinase (PK) activities. Synaphobranchus kaupii demonstrated whole-animal swimming speeds similar to those of other active deep-sea fish such as Antimora rostrata. Metabolic rates were an order of magnitude higher (31.6 mL kg(-1) h(-1)) than those recorded in other deep-sea scavenging fish. Activities of CS, LDH, MDH, and PK were higher than expected, and all scaled negatively with body mass, indicating a general decrease in muscle energy supply with fish growth. Despite this apparent constraint, observed in situ burst or routine swimming performances scaled in a similar fashion to other studied species. The higher-than-expected metabolic rates and activity levels, and the unusual scaling relationships of both aerobic and anaerobic metabolism enzymes in white muscle, probably reflect the changes in habitat and feeding ecology experienced during ontogeny in this bathyal species.     

The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms

The genetic polymorphism of ten sturgeon species that inhabit the territory of the Russian Federation (Russian sturgeon, Siberian sturgeon, Amur sturgeon, Sakhalin sturgeon, Persian sturgeon, ship sturgeon, sterlet, stellate sturgeon, beluga, and kaluga) was examined at five microsatellite loci (Afug41, Afug51, An20, AoxD161, AoxD165) (in total, 3821 individuals). The examined loci were successfully amplified with the same primer set in all species tested and demonstrated a high level of variation. Alleles specific to different species have been identified, which allows them to be used to identify species of sturgeon and their food products. In addition, the possibility of identifying hybrid forms was demonstrated. The assignment test performed in the STRUCTURE software program showed a high probability of correctly assigning each individual to its species based on genotyping with five microsatellite loci examined (96–98%, on average). However, for Russian and Persian sturgeon, the rate of proper species assignment was considerably lower (75 and 84%, respectively).

     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

Serum levels of testosterone, 11‐ketotestosterone and oestradiol‐17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment

Changes in serum concentrations of two androgens, testosterone (T) and 11‐ketotestosterone (11KT), and oestradiol‐17β(E2) in male and female giant sturgeon Huso huso , Russian sturgeon Acipenser gueldenstaedtii and stellate sturgeon Acipenser stellatus were studied at different stages of gonadal maturity and after final maturation induced by hormonal treatment. Both male and female fish displayed a distinct increase in serum steroid concentrations during gonadal development. 11KT levels were significantly higher in males than females, with a positive correlation detected between 11KT and T concentrations. In maturing males and females, higher values of both 11KT and T were observed in stellate sturgeon compared to giant and Russian sturgeons. Vitellogenesis and high E2 levels were correlated in maturing sturgeon females.     

Assessment of Extinction Risk and Reasons for Decline in Sturgeon

Sturgeon populations in the Danube River have been affected by a combination of hydropower development, over-harvesting, habitat degradation from agricultural and industrial practices and from urbanization. The effects of these changes have been monitored on six sturgeon species inhabiting the Danube River. Two of them are resident species, while the other four migrate to the river for spawning. Atlantic sturgeon (Acipenser sturio) has completely disappeared from this region. Ship sturgeon (Acipenser nudiventris) is very rare in professional fishing catches. Beluga (Huso huso), Russian sturgeon (Acipenser gueldenstaedtii), stellate sturgeon (Acipenser stellatus) and sterlet (Acipenser ruthenus) are endangered with different levels of extinction risk. Here, we model the time dependence of the beluga and Russian sturgeon catch in the Serbian part of the Danube River. Predicted extinction of Russian sturgeon was estimated to fall around the middle of the century, and for beluga approximately at middle of the millennium. Suggestions for sturgeon conservation measures on a national level and coordination of all relevant institutions in Serbia are also presented.