Karyology of pikeperch (Sander lucioperca)in the south Caspian Sea

Pikeperch (Sander lucioperca) is one of the economic fish species in the Caspian Sea, with no official report on its chromosome preparation and karyotype formula. For the metaphase preparation, 29 fry specimens of the fish weighting 10-25g each were used. Colchicine 0.01% at 0.7ml/100g of fish weight was injected in the muscular tissues. After 180 minutes, kidney and gill tissues were removed and hypotonized in KCL 0.075 M for 40 minutes, fixed with Carnoy mixture for 18-20 hours, homogenized and dropped on the slides, and stained with Giemsa 20% for 30 minutes. The prepared slides were swept by objective X100 light photomicroscope. The results showed that the chromosome number of this species is 2n = 48 and the chromosome arms is NF = 76. The karyotype formula of the species was found to be 2n = 1m + 13sm + 4st + 6a. Comparing the results of the study with those of other researchers showed that the pikeperch chromosome number in the South Caspian Sea is similar to the fish in other parts of the world but the type of chromosomes and karyotype formula is different

Mitogenic effects of phytohemagglutinin (PHA-P) and lipopolysacharide extracted from E.coli on cultured lymphocyte of the Caspian Sea ship sturgeon (Acipenser nudiventris)

The present study was designed to obtain the most effective dose for phtytohemagglutinin (PHA-P) and lipopolysaccharide (LPS) extracted from E. coli to optimize the lymphocyte culture method, the highest mitotic index (MI) and metaphase plates number of ship sturgeon, Acipenser nudiventris. Twenty specimens of two-year old A. nudiventris weighing on average 137g and with an average length of 32cm were used in this study. Different doses (0, 40, 60 and 80kgml ^(-1)) of PHA-P, LPS and a combination of the most effective dose of both mitogenic factors {(40 kgml super(-1)) PHA-P+ (80kgml super(-1)) LPS} were added to the culture media and mitotic indices were calculated for each treatment. Results indicated that a dose of 40kgml^(-1) PHA-P and 80kgml ^(-1) LPS produced the highest MI (6.26 and 3.02 respectively). Using higher concentrations of PHA-P and LPS resulted in decreased MI, whereas at lower doses of these mitogenic factors, mitotic arrest of cultured lymphocytes was observed. Using a combination of the most effective dose of the two mentioned mitogens {(40kgml ^(-1)) PHA-P + (80kgml super(-1)) LPS} yielded a MI of about 3.84. A dose of 40kgml ^(-1) PHA-P produced the highest MI (6.26), the highest number of lymphocytes was cultured and the largest number of metaphase plate was counted

Fertilizing ability of cryopreserved spermatozoa in the Persian sturgeon (Acipenser persicus) and stellate sturgeon (A. stellatus)

Motility of spermatozoa was studied on 12 and 7 specimens of Acipenser persicus and A. stellatus, respectively. The density measured to be 2.22±0.65x109ml-1 in A. persicus and 2.21±0.55x109ml-1 in A. stellatus. Semen samples were diluted with two extenders containing tris 118mM, sucrose 23.4mM, pH=8, egg yolk (20%), dimethyl sulfoxide (15%) and penicillin potassium (500IU/ml) and biociphus extender containing glycerol as a cryoprotectant at a ratio of 1:1 and then transferred to 0.5ml straws and frozen in a computer controlled low temperature apparatus and stored in liquid nitrogen for one week. To study fertilizing ability, the spermatozoa were then used to inseminate eggs after thawing. Mean sperm motility in fresh spermatozoa was 86.6% in A. persicus and 73.75% in A. stellatus which decreased to 32.2% (P<0.001) and 37.5% (P<0.001) in frozen spermatozoa, respectively. Also mean fertilization rate decreased from 90.4% to 30.7% in A. persicus and from 72% to 36.8% in A. stellatus

Primary culture of ovarian follicular cells of Sterlet, Acipenser ruthenus to develop an in vitro system

The aim of the present study was to develop an in vitro system for functional investigation of ovarian follicular cells in Sterlet, Acipenser ruthenus. Oocytes for the primary culture were obtained from the ovaries of a 6 years old Sterlet 729 g in weight and 47 cm in total length. The oocytes were in advanced vitellogenesis stage (PI >10). A part of the ovary (containing about 300 follicles) was removed, ovarian follicles isolated by manually removing those from the interstitial tissue and washed with sterile phosphate buffered saline (PBS) containing antibiotics and Amphotericin B. Follicular cells were separated by treating oocytes with 0.25% trypsin-EDTA in Ca2+ and Mg2+ free PBS and cultured in medium L-15 supplemented with 20% FBS, streptomycin sulphate (Gibco, 100 mg.ml-1), penicillin G potassium (Gibco, 100 IU.ml-1) and Amphotericin B (Gibco, 2.5 mg.ml-1) at 22 °C. The concentrations of Testosterone (T), Estradiol-17β (E2), Progesterone (P4) and 17α-hydroxyprogestron (17αOHP) in the medium were measured at days 3, 5 & 7 by the Enzyme-Linked Immunosorbent Assay. According to the results, the ovarian follicular cells of Sterlet proliferated in L-15 medium were steroidogenically active as expressed by the secretion of T, E2, P4 & 17αOHP. Testosterone was the dominant hormone secreted by cultivated follicular cells, which was correlated closely with the end of vitellogenesis in the isolated oocytes. Decrease in production of these hormones was greater at days 3 & 4 in comparison with those at days 5 & 6. By successfully culturing ovarian follicular cells of Sterlet in L-15 culture medium, an in vitro system was developed which enables functional studies to be carried out similar to the in vivo situation in the ovarian follicles

Purification and characterization of lysozyme in Persian sturgeon, Acipenser persicus (Borodin, 1897) from the Southwest Caspian Sea

Lysozyme (N-acetylmuramide glyconohydrolase, (EC 3.2.1.17)) is a unique enzyme which cleaves the β-1,4 linkages of N-acetylmuramic and N-acetylglucosamine of the peptidoglycan, which leads to the lysis of the bacterial cell wall. Lysozyme, as a self-defense enzyme, is produced in many organs of vertebrates. The present study describes purification and characterization of lysozyme from Acipenser persicus (Borodin, 1897). After the extraction process, ion exchange chromatography was utilized to purify the enzyme. The SDS-PAGE analysis confirmed that the molecular weight was about 14 kDa. Moreover, some of the biochemical properties such as optimum temperature, pH and the effect of metal ions on the activity of purified enzyme were investigated. Based on the results the optimum activity and pH were obtained at 50 °C and 6.5 respectively. The purified lysozyme was active in the presence of different salts including NaCl (0–0.125 M), KCl (0.075–0.125 M), MgCl2, and CaCl2 (0.005 M). Kinetic parameters were also calculated

Gynogenesis in Persian sturgeon (Acipenser persicus) and beluga (Huso huso)

The objective of the present study was to determine the possible production of Persian sturgeon (Acipenser persicus) and Beluga (Huso huso) gynogen/triploids and also to determine the most appropriate type of thermal shock and the duration of induced shock after fertilization. Persian sturgeon and Beluga spawners were collected from Guilan's sturgeon catch stations and transported to the Shahid Beheshti sturgeon hatchery for artificial breeding and restocking programs. Ovulated eggs and sperms were collected based on common procedures in hatcheries. In order to separate the seminal fluids and dilute the milts, sperms were centrifuged at 6000 rpm for 20 min. and seminal fluids stored in refrigerator for further use. Sperm motility was investigated. In order to determine the best duration for radiation, the milt was diluted (1:9) with immobilizing solution. Samples of diluted milt were placed for UV irradiation (UV lamp model UVG-54, 254 nm, made by UVP America) for 0.5, 1, 1.5, 1.45, 2, to 5 min. The motility of radiated sperms and controls were examined under the light microscope and the motility curve was drawn. For application of thermal shock two types of heat shock (32, 34 and 37°C) and cold shock (0±1°C) were used for duration of 2.5 and 60 min respectively. Both thermal shock were applied at 12, 15, 18 min after fertilization. Four experimental groups were designed including; normal eggs as control group and sperms without UV thermal shock), gynogenesis (Sperm irradiated with UV and thermal shock were applied), triploid (thermal shock without radiation by UV on sperm) and haploid group (without thermal shock but using irradiated sperm for fertilization). Verification of the success of treatments was assessed using genetic analysis on sturgeon larvae and fingerlings. In triploids the total surface area, volume of cells and nucleus as well as chromosome number were determined. To identify a gynogenetic larva, microsatellite markers were used to analysis specific loci by using primers designed for lake sturgeon. The results were analyzed using SPSS, Excell software. To determine the significant levels between various parameters and comparison between controls and various treatments, one way of Analysis of Variance (ANOVA) was used. Whenever the significant level was observed to determine its level a Duncan test were examined. Results of present study showed that the best duration for UV radiation on sperms of Beluga was 105-110 seconds. Average fertilization rate for control Beluga was 51%, while in heat shock group it was 2-5 % and in cold shock it was 44.6%. There was a significant difference in fertilization rate in cold shock group compared to heat shock group (P<0.05), however no difference was observed between 32 and 34°C treatments. The average survival rate of larvae in control group was 51%, while in heat shock treatment (32 and 34°C) it was very low close to zero. However in cold shock treatment the results was better and hatching percentage of larvae was between 30 -35%. Triploid treatment showed better results than gynogenesis group. A minimum triploid larvae obtained from heat shock was zero but using cold shock, the maximum number of 170 specimen was harvested. There was no significant difference in the number of larvae obtained between 32 and 34° C treatments (P<0.05). Although some difference was observed on large and small axes, surface areas and volume of red blood cells but no significant differences were observed between control and triploid groups (P 0.05). In the meantime, the chromosome number in triploid beluga was (3N=177±3) as compared to diploid 2N= 118±3, which indicated an extra set of chromosome (n=60) in triploid fish. Totally 26.6% of investigated fish was triploids. Microsatellite molecular markers clearly differentiate gynogenetic fish on the bases of allele inheritance of male and female parents, and were proven that this technique can clearly identify allelic inheritance of parents to offspring. In Persian sturgeon in compare to beluga a different results were observed. Heat shock (37°C) not present any positive results therefore has no application in induce gynogenesis on this species, also no significant difference was observed between 32 and 34°C treatment. Cold shock showed better results, especially when duration of UV radiation was adjusted to 105 seconds. Molecular analysis using microsatellite marker positively proved the gynogenetic offspring by counting the allelic inheritance. However Persian sturgeon as a tetraploid species (2N=240) has its difficulty on scoring the banding patterns. We highly recommend disomic primers application for allelic inheritance on gynogene Persian sturgeon