فصلنامه محیط زیست جانوری
سال پنجم شماره 2 (تابستان 1392)

Alcian blue-Alizarin red staining protocol during different growth stages of Beluga (Huso huso), as a model of sturgeon fishes, for skeletal ontogeny studies was prepared successfully. For an optimal staining protocol design both fish size and their morphological characteristics at each developmental stage were considered, since such parameters notably influence the staining of tissues. Three developmental windows were determined for an optimal staining procedure: (i) 11-20 mm, (ii) 29-30 mm, and (iii) more than 30 mm total length (TL). Generally, for skeletal staining, alcian blue and alizarin Red are used for stain cartilage and bone respectively. The incubation times of the different solutions were adjusted to allow the stain penetration for revealing the integrity of cartilaginous and bony tissues without significant tissue degradation. Since it was possible to detect the first cartilaginous and mineralized structures in specimens as small as 11 and 20 mm TL, respectively, this procedure is a useful tool to study the sturgeon fishes skeletal ontogenesis, to precociously diagnose skeletal malformations in small larvae and eventually to better characterize the effect of different environmental and ⁄ or nutritional factors on the ossification status of specific skeletal components.

The effects of Highly Unsaturated Fatty Acid (HUFA) and different vitamin C levels were investigated on salinity tolerance in Persian Sturgeon larvae. Artemia urmiana cysts were hatched with standard method and the nauplii were enriched with a imported emulsion (ICES30/4), homemade sturgeon ovary oil both supplemented with three vitamin C levels and fed to the fish larvae as live food for 20 days. At the end of experiments, fatty acids profile in Artemia nauplii and fish larvae, salinity tolerance in fish larvae exposed in different water salinities during different times were determined. Results showed that, the survival rate and salinity tolerance of fish in high water salinity were increased significantly (P<0.05) in all treatments compared to the control fish larvae which were fed Artemia nauplii unenriced. All the main factors have significantly effects (P<0.05) on salinity tolerance of sturgeon fish larvae when exposed in 12 ppt salinity after 120 h but there are not any differences in fishes salinity tolerance between the HUFA and vitamin C combination treatments (P>0.05). Survival rate in the main treatments IES30/4, vitamin C (20%) and 24 h enrichment period were 100%±1.00, 99%±1.00, 99%±1.00 respectively which caused the highest salinity tolerance in fish larvae. Also, omega 3 and omega 6 fatty acids in all Artemia nauplii and sturgeon fish larvae treatment especially those enriched with the commercial emulsion increased significantly compared to control groups (P<0.05).

As important of cartilage tissue in sturgeons, for this study cartilage tissue of vertebral column of 40 days, one and 4 years old of Persian sturgeon, after preparation were fixed in buen fixative and 4% Buffered Glutaraldehyde and then they have been provided to pass the other histological procedure for study under light and electrom microscopic study. Study of cartilage tissue of vertebral column of Persian sturgeon showed prichondrium, chondroblast and chondrocyte are the structure of it and a large number of mature cells have origin and the same lacunae. In ultra structural study of cartilage cells chondroblasts have more aggregation inter cellular organell than to the chondrocytes. This finding in 40 days, than to the one and 4 years old of Persian sturgeon was clear but all of them have no smooth membrane. Because there is not any study on cartilage tissue in Persian sturgeon this study was done.

The aim of the study was to investigate the replacement of fish oil by soybean oil and the effects on the growth performance, survival and fatty acid metabolism in Persian sturgeon fingerlings. Three iso-nitrogenous and iso-lipidic experimental diets containing 100% fish oil, 50 and 100% soybean oil were fed to triplicate group of fingerlings (initial weight 210 g). No significant difference was observed in the case of weight gain, specific growth rate, feed conversion ratio and survival (P > 0.05). Fillet fatty acid composition was analyzed and the results showed that fillet fatty acid composition was highly affected by dietary fatty acid profile and the highest accumulation rate of linoleic acid (C18:2n6) and linolenic acid (C18:3n3) was observed in 100% soybean and fish oil respectively. Also feeding 50% soybean oil stimulated elongation and desaturation pathway of C18 PUFA in fingerlings and the highest amount of EPA, DHA arachidonic acid (AA) was observed in this treatment. Data obtained show that replacing fish oil with 100% soybean oil had inhibitory effect on the elongation and desaturation of C18 PUFA and the lowest amount of DHA and AA obtained in fingerlings fillet.

This study aimed to survey development of eye Beluga (Huso huso) from hatching time to 32 days after hatching and also determine the structure and organization of the various layers of development of eye and it was at this stage. At hatching, Great sturgeon larvae possess an undifferentiated lens and retina and the eye begins to differentiate at 2 days post-hatch. The most obvious changes of eye structural Beluga occurred between hatching time and 4 days after hatching when At this age, the eye wall has a developed and organized location, lens, iris, choroid, sclera, cornea and the vitreous cavity and at 8 days after hatching, all retinal layers observed quite distinct.

The present study was carried out with aims at dettecting the effects of different concentrations of soy - phytoestrogens Genistein (GE) and equol (EQ) on farmed female beluga 5 - year - old during a year. Testosterone (T) level was 0.3 ± 0.06 ng/ml at the start and reached to a maximum (21.04 ± 1.91 ng/ml) at EQ 0.4 g/kg at the end of experiment (P<0.05). 17β - estradiol (E2) levels increased significantly at some concentrations of GE and EQ at the end than start and reached to a maximum (12.6 ± 1.04 ng/ml) at EQ 0.4 g/kg at the end of experiment (P<0.05). 17α- hydroxy progesterone (17α - OHP) showed no significant difference. In conclusion, equol at concentration 0.4 g/kg showed more powerful positive effects on testosterone (T) and estradial (E2) than other concentrations of equol and genistein in farmed female Huso huso. Comparatively, EQ showed more estrogenic effects on ovary development in comparison to GE concentrations.

In this study, the effects of the Atrazine were studied on some blood biochemistry parameters in 135Acipenser nudiventris during a 96 hours period. These examinations were made by 4 treatments and each treatment repeated these exams three times with a blank. The amount of LC50-96h of Atrazine was estimated 32.01 mg per liter. Albumin showed significant difference at some concentrations (P<0.05). The Cholesterol level in all concentrations showed significant decrease in comparison with the blank in time. The Triglyceride had its minimum amount in the concentration of 37 mg per liter, which had a significant difference with respect to the blank and some other treatments (P<0.05). Hematocrit percent increased meaningfully with the increase of the Atrazine concentration, and the red blood cells (RBC) decreased meaningfully in different treatments. Hemoglobin showed meaningful tolerance in different times and concentrations. Changes in the average cellular volume had a meaningful difference with the increase of the Atrazine dose and in different treatments and times. The average changes in the hemoglobin corpuscular showed a meaningful difference in different treatments and different times. The average of corpuscular hemoglobin in 24 hours had a meaningful increase in the most of the treatment in comparison with the blank. Neutrophils have increased meaningfully with the increase of concentration and with passing time. Lymphocytes have increased meaningfully with the increase of the Atrazine concentration in comparison to the blank. The monocytes have increased meaningfully in time and with the increase of the Atrazine concentration. According to the table of the toxicity levels, Atrazine is considered as ‘toxic’ for Acipenser nudiventris and we can use the changes in the desired parameters as tools in analyzing the status in the pathobiology of fishes.

Allometric growth pattern of Sterlet (Acipenser ruthenus) was studied during larval development from hatching up to 50 days post hatching. For this experiment, in total 180 specimens were sampled and their left side photographed using a digital camera. Morphometric parameters of this study obtained from 2D pictures of specimens using the software ImageJ and allometric growth pattern was calculated as a power function of total length using non-transformed data: Y=axb. Results revealed that morphogenesis and differentiation during early ontogeny i.e. prelarval stages is more intense than larval stage. The allometric growth pattern of the tail length, head length and head width were positive allometric up to 14, 17 and 20 dph, respectively. This can be as result of vital functions importance including swimming, respiration and sensory systems during this period. The timing of finfold differentiation into unpaired fins (11-14 dph) is similar to that of tail growth pattern. Differentiation into unpaired along with growth of the tail and negative allometry of caudal peduncle in day 7 dph can be related to improve of fish swimming ability.

In this study electrolytes and sex steroids levels of beluga (Huso huso) were determined. Forty-one fish selected randomly from different age groups (4, 6, 7 and 8 year-old) including 23 females and 18 males in Shahid Marjani Sturgeon Culture Center located in the Golestan Province (Gorgan, Iran). Blood sampling was taken from the caudal vein of fish. After serum separation, electrolytes (calcium, sodium and potasium) and sex steroids were determined. No significant differences were seen between male and female for blood serum electrolytes. There is not any difference in electrolytes fluctuation of Beluga between males and females when evaluated with sex maturation. The results of linear regression analysis in male and female showed that with the increase in gonad maturation the level of calcium was increased. Testosterone and 17α-hydroxyprogestrone levels showed significant difference in males at the stage V. The level of estradiol-17β (E2) in males showed chaotic fluctuations in different stages. By six years, all males could be separated from females based on T content (>5 ng/ml). In females, T, E2 and 17-OHP levels were relatively low before vitellogenesis, but during the vitellogenesis (stage IV) the level of T and E2 dramatically increased. The level of 17α-hydroxyprogestrone showed chaotic fluctuations in different stages. In conclusion, T, E2 and 17-OHP hormones are most important indices of sexual maturation.

In this study, taste preference of juveniles of Persian sturgeon were identified to 4 concentrations of sucrose (10, 1, 0.8 and 0.5) and 4 concentrations of calcium chloride (10, 1, 0.5 and 0.1) by using behavioral assay method. The fish used were divided into 3 weight groups: small group (average weight was 7-10 g), medium group (15-20 g) and large group (23-28 g). The results of index of palatability, for both intra-oral and extra-oral receptions, revealed that the differences among 3 weight groups were not significant generally (p> 0.05). It is also found that calcium chloride had positive effect on extra-oral taste reception in all 3 weight groups. In other words, the index of extra-oral palatability increased by increasing concentration. The maximum levels of index of intra-oral palatability for calcium chloride were observed at concentrations of 1% (for small group) and 10% (for medium and large group). But sucrose had negative effect on number of catching acts, just for large group, and it had also negative effect on consumption of granules for all 3 weight groups. Based on these results, we can say addition of 0.5-1 % calcium chloride in juveniles of Persian sturgeon diet can improve feeding status in this species.

Blood indexes are very important in fish physiology, the more knowledge of blood indexes of fishes, the more ability of breeding and culturing of them. In the present study, The correlation between biochemical factors of blood (calcium, magnesium, total protein, cholesterol and glucose) with weight of female fishes and some biological characteristics of gonad (e.g. fertilization rate, fluid egg rate, gonadosomatic index, polarization index and oocyte diameter) in 9 female of the migratory population of Sterlet (Acipenser ruthenus) and 3 female of the migratory population of Siberian sturgeon (Acipenser baeri) in Spring of the 2012 were determined. In Sterlet, significant negative correlations were observed between blood Ca+2 with oocye diameter and fertilization rate (P<0.05). Also there was significant negative correlations between blood Ca+2 with fluid egg rate and gonadosomatic index (P<0.01). There was an invert correlation between total protein with oocyte diameter and fluid egg rate (P<0.05). Relationship between blood cholesterol with oocyte diameter, fluid egg rate, and fertilization rate and gonadosomatic index was direct, but this correlation was not significant. In Siberian sturgeon, there was an invert correlation between blood glucose and body weight (P<0.05). Also there was significant positive correlation between total protein and gonadosomatic index (P<0.05). Blood calcium was positively correlated with body weight, polarization index, fluid egg rate and fertilization rate but these correlations were not significant. Relationship between total protein and blood cholesterol with body weight, fluid egg rate, fertilization rate was negative that was not significant.

The use of Probiotics for increasing the growth and resistance against disease are well documented in aquaculture. We conducted a study to determine the effects of probiotic (Primalac) on the growth, survival and body composition in Acipenser persicus fingerlings. The commercial Probiotic, Primalac, were mixed thoroughly with the artificial feeds at concentration of 0.0 (control), 0.05, 0.1 and 0.15 % of dry diet and fed to healthy fish (14.676±0.813 g) at 2 – 3.5 % of their body weight daily in two split doses for 15 weeks. The trail was carried out in 12 PVC tanks 500 Liter which about 200 liter of water. That all tanks 10 juvenile Persian sturgeons were stocked. During the experimental period, weight and length of fish were recorded at an interval of 14 days. Results showed that specific growth rate (SGR), body weight gain (BW %), net protein utilization (PER) and feed efficiency (FE) showed significantly (P<0.05) different among the experimental groups. The present study results showed that probiotic treatments indicated no significant difference were observed in body chemichal compositions (P>0.05).

In the present study, we tried to cultivate neural stem cells from different regions of Persian sturgeon (Acipenser persicus) brain, as a chondrostei fish. For this, telencephalon, mesencephalon and metencephalon of the brain were separated, cultivated in DMEM/F12 medium supplemented with fetal bovine serum (20% FBS) and maintained at 250C with 5%CO2. Medium was changed every 3s days and replaced with fresh medium. After 2-3 weeks, cells were confluent and passaged. Cells of metencephalon (cerebellum) culture from Persian sturgeon brain (CPS) are still alive after about 7 months and 9 passages and their stocks are available in the laboratory. The cell cultures have been cryopreserved using liquid nitrogen. The survival rate of cells was 70% after thawing. In immonocytochemistry survey, cells were immonopositive for anti-nestin antibody, as a neural stem cell marker. The obtained cultures could be considered as a constant long term culture because of their stability, and could be used in different studies.

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis,and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDSPAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. The thermal denaturation properties of the Hbs were investigated by differential scanning calorimetry (DSC). UV–vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb–oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity, hydrophobicity, themostability and alpha-helix secondary substructures of fish hemoglobins and environmental partial pressure of oxygen.

In present study, we investigated and compared serum biochemical (Calcium, Magnesium, Total Protein and Glucose) levels in three groups of female Persian sturgeon (A. persicus). The gonadal maturation was determined by gonadosomatic index (GSI). There was significant difference among GSI in three groups of female Persian sturgeon (P<0.05). The lowest GSI belonged in immature female and the highest GSI belonged in mature females. The result showed that, there was significant difference among calcium, total protein and glucose levels in three groups of female Persian sturgeon (P<0.05). Calcium level in maturing females (2.86±0.78 mg/dl) was higher than in immature (0.29±0.12 mg/dl) and mature females (0.07±0.05 mg/dl). While, total protein level in mature female (6.71±0.87 g/dl) was higher than in immature (2.37±0.28 g/dl) and maturing females (2.81±1.13g/dl). The level of glucose in maturing, immature and maturing females was measured 132.63±12.98 mg/dl, 118±3.7 mg/dl and 78±0.29 mg/dl, respectively. No significant difference observed in magnesium concentration of three groups of female Persian sturgeon (P>0.05).

In the present study, the effect of different levels of dietary nucleotides (Vanazhen) as a food attractant on the growth rate and feeding rate of Persian sturgeon (Acipenser persicus) were studied. Fish with an average weight of 42/37 ± 2/12 g and mean length of 23/67 ± 0/61 cm were divided to 4 treatments with different levels of nucleotides in the diet (0, 0/25, 0/35 and 0/50%) with three replications containing 12 fish per tank. After 10 weeks feeding, survival, growth and feeding indices such as weight gain, specific growth rate, condition index, feed conversion and feed efficiency ratio were examined and the significance of them were analyzed using One-way ANOVA analysis software Spss 18. The best results in terms of weight and length compared to the weight gain, feed conversion ratio, specific growth rate and feed efficiency ratio from treatment 2 (0/35% of the nucleotides). This study showed that adding nucleotides to increase food intake, reduce food losses and thus increase the growth rate of sturgeon.