Influence of sampling conditions on blood chemistry values of Adriatic sturgeon Acipenser naccarii (Bonaparte, 1836)

Data on the blood chemistry of a chondrostean fish, the Adriatic sturgeon ( Acipenser naccarii ), are reported as measured with different sampling procedures, and as related to rearing conditions and age. Serum Cortisol, glucose, osmolality, Na⁺, Cl^-, Ca²⁺ and total protein concentrations were measured. Reference values for the blood chemistry of farmed sturgeon were measured on samples from resting undisturbed animals collected via a chronic indwelling catheter in the dorsal aorta that was implanted under anaesthesia. Following 24h recovery from catheterization, serum Cortisol, glucose and osmolality levels were 9.4 ng/ml, 58.8 mg/dl and 261.4 mOsm/kg, respectively. Furthermore, blood samples collected with the chronic indwelling catheters indicated that the surgical procedure of cannulation caused a stress response, with physiological changes that followed a pattern like that described in teleosts. Cortisol, glucose and osmolality were more sensitive to stress than the other variables measured. Sampling by cardiac puncture tended to be associated with elevated serum Cortisol levels in older, larger sturgeon, but not in young fish. Greater capture, confinement and handling stress in older, larger, sturgeon may have been responsible for this and other age-related differences in blood chemistry values measured following cardiac puncture. Within the same age class, both rearing conditions and temperature affected Cortisol, sodium and total protein concentrations significantly. Anaesthesia did not appear to reduce the degree of stress associated with cardiac puncture but altered serum ion concentrations.     

Endocrine changes during the previtellogenic stage of the great sturgeon,Huso huso (Linnaeus, 1758)

The study aimed to investigate changes in serum concentrations of sex steroids, thyroid hormones, stress indices, cholesterol, triacylglycerol, calcium and phosphorus, as well as the growth in the 3‐year old previtellogenic stage of great sturgeon, Huso huso over a 6‐month period. Forty individually marked fish (mean weight 6913 g) were divided among three circular tanks under a natural temperature and photoperiod. Every 1.5 months fish were weighed and blood samples taken for serum analysis. Fish showed significant changes in 17β estradiol, testosterone, triiodothyronine (T₃), thyroxine (T₄), adrenocorticotropic hormone, cortisol, cholesterol, and phosphorus, but not in triacylglycerol and calcium concentrations. From the January onset of the experiment to the end of the trial in July, there was a decrease in sex steroids and cortisol concentrations as well as a decline in T₃ concentrations from March and in T₄ from February to July. The mean weight of fish increased significantly from the beginning to the end of the experiment. Although the oocyte diameter increased from 197 to 202 μm, the difference was not significant. The histological data on oocytes suggest that the great sturgeon, like the other sturgeons, has a heterogeneous asynchronous ovarian development in the early stage of reproduction. This data might be useful to the further understanding of the physiology of Huso huso and/or to improve its culture. Moreover, it leads to a general increase in the understanding of the basic reproductive biology of this valuable chondrostean species.     

Morphometric study of Acipenser naccarii (Bonaparte, 1836) in fish farm individuals

The main objective of this study was to analyse differences and similarities in morphometric characteristics among specimens of the same year class of Acipenser naccarii (Bonaparte, 1836), comparing fish raised either in Italy or Spain. All specimens were Fl of captive broodstock in Italy.
Fifteen biometric variables were measured, and three derived indices were calculated, on 30 individuals of 3+ year class from Lombardy (Italy) and 90 individuals of 1+, 2+ and 3+ year class from Riofrio (Granada, Spain). A stepwise discriminate analysis (SDA) was carried out, and the results were contrasted using a non-hierarchical cluster analysis. The two groups proved to be similar, so a principal component analysis and a simple regression analysis were performed on all individuals, taking total length (Tl) as the independent variable and all the biometric variables as dependent.
The study revealed that although the Soljan index (i.e. ratio of mouth width to snout length) is related to Tl, and is a valid taxonomic characteristic, the CA index (i.e. relative position of the barbels) and FB index (i.e. ratio of snout length to width) are also valid characteristics for individuals longer than 57 cm, being independent of Tl.     

Embryonic development of adriatic sturgeon,Acipenser naccarii (Bonaparte 1836), in farming conditions: a guide

The sequence and timing of the embryonic development of Adriatic sturgeon (Acipenser naccarii) are described at a constant temperature (17 ± 1 °C), from fertilisation to hatch in a hatchery. Fertilised eggs, obtained from one female and two males, were held in a flow‐through system. On the first day, embryos were sampled every 20 min during the first 12 h, and every 40 min in the following 12 h. Embryos were sampled hourly on the second day. On the third and fourth days, embryos were sampled every 2 h, and every 3 h on the fifth and sixth days. The first cleavage furrow appeared at 1 h 40 min postfertilisation. Blastulation concluded at 12 HPF (hours postfertilisation), when the dorsal blastopore lip formed. The completion of gastrulation was at 18 HPF, closing of the neural tube at 23 HPF and formation of the s‐shape heart at 31 HPF. The mass hatch occurred at 120 HPF. A. naccarii is a relatively common sturgeon species in aquaculture and possesses similar developmental patterns to those of other sturgeons; hence, we can use this species as a model for further studies about sturgeon species which are more difficult to breed artificially. Information about the embryonic development of the Adriatic sturgeon could assist restocking programmes.     

Modifying the physiological responses to handling stress in beluga sturgeon Huso huso (Linnaeus, 1758); Interactive effects of feeding time and dietary fat

The present study investigated the interaction of feeding times with two dietary fat levels on physiological responses to handling stress in juvenile Beluga sturgeon. Fish were fed with two different diets (high energy; HE: 24% fat and low energy; LE: 12% fat) for 8 weeks at two feeding times; 09:00 and 16:00 (during the day) or 21:00 and 04:00 (during the night). At the end of the trial, blood samples were taken and the resting fish were held in a net out of water for 90 s as the handling stressor. Three hours after application of stress, post‐stress blood was taken. Cortisol, glucose, and lactate concentrations were considered as stress indicators. The mean values of cortisol and lactate levels did not reveal a significant difference between pre‐ and 3‐hr post‐stress samples, but the average concentration of glucose showed a significant difference. Cortisol, lactate and glucose concentrations were not influenced by the difference in the diets. Moreover, the cortisol and lactate concentrations were not affected by the different feeding times, while glucose levels were significantly affected by the feeding times with the lowest level in fish fed during the day. With respect to stress indicators, the results revealed that feeding times affected pre‐ and post‐stress secondary response to handling stress, but the rate and magnitude of metabolites (cortisol, glucose and lactate) were not affected by dietary fat levels. Therefore, it is necessary to examine the best feeding times with the interaction of feed ingredients in sturgeon farming.     

Cardiovascular responses to hypoxia in the Adriatic sturgeon (Acipenser naccarii)

The in vivo cardiovascular responses to hypoxia, and the intrinsic functional characteristics of the heart in vitro , were determined, and compared, in the Adriatic sturgeon (Acipenser naccarii). During exposure to hypoxia in vivo , blood oxygen content (Cao₂) declined as water 0₂ partial pressure (Pwo₂) was reduced, despite an increase in haematocrit. The main cardiovascular response was a reduction in dorsal aortic blood pressure, with a slight bradycardia, while cardiac output remained constant. Reduced oxygen content of the perfusate had significant inhibitory effects on the intrinsic performance of the heart in vitro , causing a reduction in the heart rate; a reduction in the sensitivity of responses to increased preload (Frank-Starling response), and a more rapid decline in power output and stroke volume when afterload was increased. Overall, the in vitro results suggest that hypoxia depresses the contractility of the heart (i.e. its inotropic responses). The reduction in dorsal aortic pressure in vivo may, therefore, counteract the depressive effects of hypoxia on heart contractility, and thereby avoid a hypoxic depression of cardiac output.     

Optimal stocking density for beluga,Huso huso,and ship sturgeon,Acipenser nudiventris during the grow‐out phase

The influence of three different initial stocking densities (SD) in flowthrough systems was evaluated on growth performance and feed utilizaition in beluga, Huso huso, and ship sturgeon, Acipenser nudiventris, juveniles in three different grow‐out phases for 228 days. In each grow‐out phase, fish were randomly distributed in 18 concrete square tanks (2.0 × 1.0 × 1.2 m³; 2 m³ of volume) according to the experimental SD. At the first phase, both fish species were subjected to three SD including 1.5, 3 and 6 kg/m² for 60 days. In the second phase, fish were submitted to three SD including 3, 6 and 9 kg/m², whereas in the third phase, beluga were stocked at 6, 9 and 12 kg/m² and ship sturgeon were stocked at 6, 8 and 10 kg/m² and each phase lasted for 12 weeks. In both species, the growth performance and feed intake (FI) significantly decreased with increasing SD at the end of the first phase (p < 0.05). In the second phase, growth performance and FI in beluga decreased with increasing SD, whereas in ship sturgeon neither growth nor FI were affected by SD. Interestingly, the growth performance and feed utilization in both species were not decreased with increasing SD in the third phase indicating adaptability of these species to high stocking density during grow‐out period. According to the result of this study, the appropriate initial SD for beluga within the range of 100–500, 500–2,000 and 1,500–3,000 g were at 1.5, 6 and 12 kg/m², respectively in an open flow‐throw system. Regarding to ship sturgeon, initial SD at 1.5 and 10 kg/m²recommended for fish within the range of 100–300 and 300–1,200 g, respectively in an open flow‐throw system.     

Isolation and characterization of microsatellites in the Adriatic sturgeon (Acipenser naccarii)

We isolated and characterized seven polymorphic microsatellite loci from a partial genomic library of the Adriatic sturgeon (Acipenser naccarii) enriched for GATA repeats. Variability was tested in 20 specimens of this endangered species. Our data support the tetraploid condition for A. naccarii and will be useful for the investigation of the remnant genetic variability of this species as well as for genetic tagging studies.     

Behavioural responses to various chemical incentives in hybrid beluga x Russian sturgeon (Huso huso x Acipenser gueldenstaedtii) fry

Behavioural contact feeding responses to various chemical incentives were studied in hybrid beluga x Russian sturgeon ( Huso huso x Acipenser gueldenstaedtii ) fry. Solutions of 29 amino-acids (10–2 M); aqueous extracts of various vegetable, animal and microbiological components of commercial fish diets (30 min infusion of 100 mg in 100 ml), and various artificial and natural substances were tested. The most attractive amino-acids were lysine, methionine and alanine. Most of the dietary extracts tested did not stimulate feeding behaviour, indicating that widespread commercial sturgeon diets (such as ST-07 or ST-04Az) consist of components with little attractiveness for sturgeon fry. In most cases extruded diet components stimulated more bites than granulated ones. Among the diet components, only fish protein concentrate (FPC), dry milk and krill meal stimulated feeding behaviour. Flavour enhancers such as concentrated low molecular weight nitrogenous substancs (CLMNS), sodium glutamate, and "Finnstim" all stimulated feeding behaviour of fry. Some of these substances are recommended as flavour enhancers in sturgeon diets.     

Production of recombinant great sturgeon (Huso huso,Linnaeus, 1758) growth hormone (GH) by Pichia pastoris (Guillierm, 1956)

In this research, the encoding cDNA of growth hormone (GH) was cloned from the pituitary gland of great sturgeon Huso huso (three adults: two females and one male, 7–9 years old, 70–90 kg, reared in concrete ponds). In order to obtain the great sturgeon recombinant GH expression in Pichia pastoris, the mature encoding cDNA was first cloned in TA vector PTZ57R and then sequenced. After confirmation of the correct GH sequence, the GH coding sequence was subcloned into pHILS1 expression vector. The yeast Pichia pastoris GS115 strain was transformed with the expression plasmid. Results obtained from this study showed that great sturgeon GH recombinants were expressed upon induction with methanol and exported into the medium. The level of expression was examined using RNA analysis, SDS‐PAGE, and western blot analysis. RNA analysis of the recombinant strains showed a sharp, specific band in 800 bp. The specific band in transformants indicated the presence of GH RNA in the yeast. SDS‐PAGE and western blot analysis showed a specific 21 kDa band for the growth hormone. Culture conditions were optimized for pH = 6 and incubation time (after 24 h induction, peaking at 72 h) for maximal protein production. The results provide useful information for the future production of recombinant growth hormones in other sturgeon species.     

Purification and characterization of the hemoglobin components of Adriatic sturgeon (Acipenser naccarii) blood

The oxygen binding properties of Acipenser naccarii hemoglobins have been investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes and hemolysate in the presence of the physiological cofactors GTP and chloride ions. In particular, the oxygen equilibrium reveal a very low sensitivity of the hemolysate to the chloride ions and temperature while in the presence of organic phosphates their oxygen affinity decreased strongly. The electrophoretic analysis of the hemolysate of the sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. The partial amino acid sequence of the a and P chain of the single hemoglobins was also established.     

The effect of feeding rate on growth of pectoral fin spine of juvenile great sturgeon Huso huso (Linnaeus, 1758)

The effect of two feeding rates (0.5 and 1.5% of total body weight) was assessed on the growth of pectoral fin spines of captive juvenile great sturgeon, Huso huso, after second year of life. The fish received Oxytetracycline (OTC) twice in the first and second years of their lives under basic diet. During the 5‐month experimental period, juveniles (mean 1,187.4 g, 0.1 standard deviation [SD]), n = 50) were reared with two feeding rates under similar conditions in 10 fiberglass tanks (1.5 m³). The fish were fed manually with a commercial diet twice a day (35% Biomar, Nersac, France) throughout the experiment. The OTC marks were distinguished in all pectoral fin spine sections under ultraviolet light. The means of the first and second annular radii were 806.6 µm (27.2 SD) and 2,246.5 µm (50.2 SD), respectively. The marginal increment analysis beyond the second OTC mark revealed a significantly smaller marginal increment for low feeding rate treatment (143.9, 11.2 SD) as compared to the high feeding rate one (269.0, 14.6 SD). The results indicate the slower growth rate in the fish fed the low feeding treatments seen in the pectoral fin spine formation, which can be used as an indicator of recent feeding history in sturgeon juveniles. The best daily feeding rate for great sturgeon of 2,460 g was determined to be 1.5% body weight/day in this study.     

Morphometric comparison of the cephalic region of cultured Acipenser baerii (Brandt, 1869), Acipenser naccarii (Bonaparte, 1836) and their hybrid*

Geometric morphometric techniques were used in the characterization of morphological differences of the cranial region in cultured Acipenser baerii and A. naccarii and their hybrid. Species identification in sturgeons sometimes is difficult, because of the morphological plasticity of individual variability and the small differences between taxa. Identification is also complicated by human intervention and manipulation (e.g. aquaculture, hybridization, pollution). This study indicates how the differences between two species and their hybrids can be quantified and accurately visualized via geometric morphometrics. This technique may serve as a useful methodology to supplement analytical studies on morphometrics and systematics in Acipenseridae.     

Factors influencing the natural reproduction decline in the beluga (Huso huso,Linnaeus, 1758), Russian sturgeon (Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833), and stellate sturgeon (A. stellatus,Pallas, 1771) of the Volga–Caspian basin: A review

The review describes the changes in natural reproduction of three important sturgeon species in the Volga–Caspian basin: (a) the beluga (Huso huso Linneaus, 1758), (b) the Russian sturgeon (Acipenser gueldenstaedtii Brandt &Ratzeburg, 1833), and (c) the stellate sturgeon (Acipenser stellatus Pallas, 1771). Since the past 60 years, these species responded to severe influences of natural and anthropogenic factors. On the basis of original and published data, an analysis has been made of (a) the numbers of larvae migrating from spawning sites (according to plankton net survey), (b) fecundity and histological anomalies in gonad development, (c) the numbers of adult sturgeons in the Caspian Sea and of spawners migrating to the Volga River (according to trawl and beach seine survey), and (d) foraging resources for the sturgeons. The results show that their natural reproduction in the Volga–Caspian basin has declined drastically during the past decades under the impact of (a) fluctuations of the Caspian Sea level and flow discharge from the Volga River, (b) blockage of sturgeon migration routes and loss of spawning sites because of dam construction, (c) water pollution in the lower reaches of the Volga River and in the Caspian Sea, and (d) intensive and selective illegal and unreported fishing. The relative significance of these factors has been changing during the study period.