Ultrastructure and osmoregulatory function of the kidney in larvae of the Persian sturgeon Acipenser persicus

The localization of Na(+) , K(+) -ATPase (NKA) and the ultrastructural features of kidney were examined in larvae of the Persian sturgeon Acipenser persicus (L 31-41 mm total length and 182·3-417·3 mg). Investigations were conducted through light and electron microscopy and through immunofluorescence for NKA detection. The kidney nephrons consisted of a large glomerulus and tubules (neck, proximal, distal and collecting), which connected to the ureters. Posteriorly, ureters extended and joined together into a thin-walled ureter terminal sac. Ultrastructurally, the glomerular cells (podocytes) possessed distinctive pedicels that extended to the basal membrane. The proximal tubule (PT) showed two different cells. The cells lining the anterior part of PT possessed apical tall microvilli (c. 2·7 μm), a sub-apical tubular system, a basal nucleus and dense granules. Posteriorly in the cells, the sub-apical tubular system and granules were absent and round mitochondria associated with basolateral infoldings were found; the apical microvilli were reduced. Distal and collecting tubular cells showed the typical features of osmoregulatory cells, i.e. well-developed basolateral infoldings associated with numerous mitochondria. No immunofluorescence of NKA was detected in the glomeruli. A weak immunostaining was observed at the basolateral side of the cells lining the neck and PT. A strong immunostaining of NKA was observed in the entire cells of the distal tubules, collecting tubules and in some isolated cells of the ureters. In all immunostained cells, the basolateral region showed a much higher fluorescence and nuclei were immunonegative. In conclusion, the epithelial cells of kidney tubules had morphological and enzymatic features of ionocytes, particularly in the distal and collecting tubules. Thus, the kidney of A. persicus larvae possesses active ion exchange capabilities and, beside its implication in excretion, participates in osmoregulation.     

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997)

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative ¹H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 μM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 μM and 0 μM of AFP had the highest and the lowest motility percentages respectively and they were objected to ¹H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, β-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.     

Ontogeny and osmoregulatory function of the urinary system in the Persian sturgeon,Acipenser persicus (Borodin, 1897)

The structure of the kidney and the localization of Na⁺, K⁺-ATPase (NKA) immunopositive cells were examined throughout the postembryonic development of the Persian sturgeon, Acipenser persicus, from newly hatched prelarvae (10 mm) to 20 days post hatch (20 DPH) larvae (31 mm). Investigations were conducted through histology and immunohistochemistry by using the light and immunofluorescence microscopy. The pronephros was observed in newly hatched prelarvae. The cells lining the distal pronephric tubules and their collecting ducts showed laterally expressed NKA immunofluorescence that later extended throughout the whole cytoplasm. Mesonephrogenous placodes and pre-glomeruli were distinguished at 2 DPH along the collecting ducts posteriorly. Their tubules were formed and present in kidney mesenchyma, differentiated into neck, proximal, distal and collecting segments at 7 DPH when NKA immunopositive cells were observed. Their distal and collecting tubules showed an increasing immunofluorescence throughout their cytoplasm while the glomeruli remained unstained. From D 9 to D 17, the epithelial layer of pronephric collecting duct changed along the mesonephros to form ureters. Ureters, possessing isolated strong NKA immunopositive cells, appeared as two sac-like structures hanging under the trunk kidney. Since NKA immunopositive cells were not observed on the tegument or along the digestive tract of newly hatched prelarva, and also the gills are not formed yet, the pronephros is the only osmoregulatory organ until 4 DPH. At the larval stage, the pronephros and mesonephros are functional osmoregulatory organs and actively reabsorb necessary ions from the filtrate.     

Growth performance and body composition of sub-yearling Persian sturgeon, (Acipenser persicus, Borodin, 1897), fed different dietary protein and lipid levels

In order to evaluate the protein and energy requirement of Persian sturgeon (Acipenser persicus) sub‐yearlings, eight experimental diets containing two protein levels (40% and 45%) and four lipid levels (10%, 15%, 20% and 25%) were tested. Sturgeons (W₀ = 136.8 g) were fed the experimental diets to satiation four times daily for 150 days, resulting in a final mean weight of 375.8 g. Growth was significantly affected by lipid content of the diets. At 40% protein level, weight gain and specific growth rate (% per day) were significantly improved (P < 0.05) by increasing the dietary lipid (energy) content. Protein efficiency ratio (PER) was significantly affected by different dietary treatments for each dietary protein level tested, reaching a mean value of 3.58 in fish fed high lipid diets and a PER of 2.77 in low lipid diets. Results obtained in the present study suggest that the optimum dietary protein content for Persian sturgeon is 40%, with an estimated optimum protein‐to‐energy ratio of 18–20 mg kJ⁻¹.     

Comparative morphology of the pectoral fin spine of the Persian sturgeon Acipenser persicus,the Russian sturgeon Acipenser gueldenstaedtii,and the Starry sturgeon Acipenser stellatus in Iranian waters of the Caspian Sea

The morphological characteristics of the pectoral fin spine were compared in three species of sturgeon, the Persian sturgeon (Acipenser persicus), the Russian sturgeon (Acipenser gueldenstaedtii), and the Starry sturgeon (Acipenser stellatus), all sampled from the Caspian Sea. On the basis of morphological characters of the pectoral fin spine, 62.2% of the individuals were correctly classified into separate groups. The cluster analysis also divided the three species into two major subgroups. Acipenser persicus and A. gueldenstaedtii were grouped together, suggesting a similar evolutionary basis. Significant morphological heterogeneity in pectoral fin spine characteristics was observed among the three sturgeon species. Principal component analysis identified the largest differences were in the pectoral fin spine size and the angle between distal pectoral fin spine and the horizontal line (A°). The first and second principal components (PC1 and PC2) of all observations accounted for 64.19% and 14.33% of the total variation, respectively. The combination of all analyses showed the relevance of applying pectoral fin spine shape for interspecific distinction of the three species of sturgeons.     

The transcription of l-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbate, during development of Persian sturgeon Acipenser persicus

l-Gulono-gamma-lactone oxidase (GULO) is a key enzyme for the biosynthesis of ascorbate, which is essential for several cellular functions. In the present study, mRNA expression of GULO gene was evaluated during the early development of Persian sturgeon. First, because there are no comparative studies that have established suitable quantitative real-time PCR reference genes in sturgeons for any physiological conditions, we evaluated six candidate reference genes (ACTB, RPL13, UBQ, RPL6, GAPDH and EF1A) during the early development of Persian sturgeon. The most stable mRNA expression was obtained with RPL6 and ACTB, whereas the least stable was RPL13. After normalization using RPL6, ACTB and RPL6/ACTB combination, the mRNA expression of GULO was highest at the embryonic stage (2days before hatching; P<0.05) and started to decline from hatching of larvae to the rest of the developmental time-points. This suggests that the vitamin C requirements are highest during early life stages, and it is likely that the changes in GULO mRNA expression are associated with changes in GULO enzyme activity.     

Anesthesia of wild female Persian sturgeon,Acipenser persicus (Borodin, 1897) breeders during controlled propagation: effects on hematological parameters,stress response and reproductive performance

This study examined the effects of anesthesia on the hematological and biochemical parameters as well as the reproductive performance of wild female Persian sturgeon, Acipenser persicus, during controlled spawning. Fourteen mature females were divided into two groups: ‘anesthetized’ and ‘non‐anesthetized’. All activities including transportation, catheterizing and handling were performed with both groups: (i) under anesthesia (150 ppm clove oil), and (ii) without anesthesia. After 10 days storage and handling, blood samples were taken from all fish after anesthesia. No significant differences were found in the reproductive performance of either group. However, differences were found in the hematological parameters, with values being significantly higher in the non‐anesthetized group, including neutrophils (34.36 ± 6.33% vs 23.63 ± 5.22%), monocytes (2.84 ± 1.70% vs 1.27 ± 0.64%), mean corpuscular volume (321.3 ± 39.40 pg vs 228.0 ± 24.46 pg) and mean corpuscular hemoglobin (106.9 ± 15.70 fl vs 76.50 ± 7.50 fl). Significantly lower values were found in the non‐anesthetized group for lymphocytes (60.68 ± 7.25% vs 73.54 ± 4.80%), Hb (4.62 ± 0.74 mg dl^?1 vs 6.28 ± 1.21 mg dl^?1), Hct (13.86 ± 1.76% vs 18.84 ± 3.85%), red blood cell (0.43 ± 0.05 cell mm^?3 vs 0.85 ± 0.13 × 10⁶ cell mm^?3) and white blood cell (22 403 ± 2240 cell mm^?3 vs 35 318 ± 3084 cell mm^?3). The non‐anesthetized fish had significantly higher cortisol levels compared to the anesthetized group (62.33 ± 8.85 ng ml^?1 vs 46.12 ± 8.07 ng ml^?1). There was no difference in plasma glucose levels between groups. It is concluded that the use of clove oil as an anesthetic is suitable for handling of wild female Persian sturgeon in controlled propagation programmes. However, further research is needed to determine a standardized protocol for the safe application of anesthesia for use in sturgeons in general.     

Effect of starvation and re‐feeding on growth performance and content of plasma lipids,glucose and insulin in cultured juvenile Persian sturgeon (Acipenser persicus Borodin, 1897)

The effect of starvation and subsequent re‐feeding to satiation on compensatory growth performance, insulin and blood serum values were investigated in juvenile Persian sturgeon (Acipencer persicus) with an average weight 108.04 ± 0.28 g (mean ± SEM) and in the same rearing condition over an 8‐week period. Sturgeons were allocated to one of five feeding treatments: controls (C, continuous feeding), W1 (1 week starvation), W2 (2 weeks starvation), W3 (3 weeks starvation) and W4 (4 weeks starvation), followed by a single 4 weeks of re‐feeding to satiation. Changes in growth performance and blood serum indices were examined at the end of weeks 4 and 8. Body weight, specific growth rate (SGR), condition factor (CF) and weight gain were determined to have significantly decreased during starvation. Fish starved for 1 week reached the same weight as the control fish after re‐feeding for 4 weeks, indicating that complete compensatory growth occurred. Although the specific growth rate in W2, W3 and W4 fish was greater than that in the control fish after re‐feeding, W2, W3 and W4 fish did not reach the same body weight as control fish at the end of re‐feeding period, and showed partial compensation only. Blood plasma, glucose and insulin concentrations did not change significantly during starvation and re‐feeding (P > 0.05). This suggests that sturgeon are able to maintain glycaemia during starvation, probably due to their non‐carbohydrate dietary source. Plasma total lipid and triglyceride levels increased in starvation treatments, whereas the increases were significant only in W3 treatment (P < 0.05). After a 4‐week re‐feeding period, their levels decreased in comparison to the starvation periods. Increases in plasma total lipid and triglyceride levels appear to be due to their roles as preferred nutrients for mobilization in Persian sturgeon and the magnitude and duration of compensatory growth depended on the length of food deprivation.     

Application of microsatellite markers for genetic conservation and management of Persian sturgeon (Acipenser persicus,Borodin, 1897) in the Caspian Sea

A study was conducted to ascertain the genetic structure and the level of heterozygosity of Acipenser persicus in the Caspian Sea. A total of 167 fish were randomly collected from Turkmenistan, Russia and two regions of Iran. The number of alleles of eleven microsatellite markers ranged from 3 to 21 and the mean observed values of heterozygosity were 0.56 ± 0.20, 0.64 ± 0.14, 0.67 ± 0.16, and 0.64 ± 0.11. The observed heterozygosity was lower than the expected levels. The observed low genetic differentiation indicates that all populations are closely related. Hence, inbreeding is a potential problem, which should be taken into consideration in future breeding programs to avoid a further decline in genetic diversity.     

Molecular basis of the digestive functionality in developing Persian sturgeon (Acipenser persicus) larvae: additional clues for its phylogenetic status

Persian sturgeon (Acipenser persicus) is a critically endangered species, mainly due to overexploitation for its caviar. The permanence of populations of this species in the Caspian Sea is fully dependent on restocking programs. Accordingly, it is considered as an interesting target for aquaculture for both restocking and commercial purposes. In addition, as a Chondrostei, it exhibits one of the slowest rates of molecular evolution among vertebrates and is propounded as an excellent candidate for phylogenetic analysis and evolutionary biology. In this study, the early ontogeny of some key digestive enzymes precursors was determined at molecular level, aiming to obtain basic knowledge on the acquisition of digestive capacity of this species, and at the same time, to advance in its phylogenetic status from the point of view of digestion. For this purpose, A. persicus cDNAs for β-actin (actb; used as an internal reference gene), bile-salt activated lipase (cel), trypsinogen 1 (try1), pepsinogen (pga), and gastric proton pump (atp4a) were amplified and cloned, and their subsequent expressions were measured by quantitative real-time PCR during the first 34 days post hatch (dph). Two isoforms for pga and at least six for try1 were obtained in this study, probably due to the additional genome duplication which sturgeons suffered along evolution. Phylogenetic analysis of the deduced amino acids sequences from the studied genes demonstrated that this species has a close evolutionary distance to Holostei, coelacanths, and tetrapods, including amphibians, reptiles, birds and mammals. According to our results, expression of all the genes increased gradually over time and reached maximum levels around 18 dph. This pattern, which was comparable to length and weight data, could indicate that around 3 weeks after hatching, the digestive capacity of the Persian sturgeon changes from larval to juvenile mode.