Induction of triploidy in grass carp Ctenopharyngodon idella Valenciennes, 1844: comparison of cold & heat shocks

Triploidy in grass carp, Ctenopharyngodon idella Valenciennes, 1844, was induced on fertilized eggs to compare cold and heat shocks. Two simplified methods explained for verification of triploidy in grass carp. The cold shock (7 ˚C) was given in three treatments for 30 min starting 2.0, 2.5 and 4.0 min after fertilization. In cold shock, the start point (2.0 min after fertilization) showed the highest rate of triploidy (60.9%). Heat shocks were given at 38 ˚C, 40 ˚C and 42 ˚C, at 4.0 min after fertilization and lasted for 1.0 min. Produced larvae using heat shock 38 ˚C showed 10.8% triploidy, but no signs of triploidy were seen in other heat shock treatments. Verification of triploidy in grass carp was carried out using karyotyping and measurment of erythrocytes surface area and volume in fingerlings. Ratio of erythrocytes dimention and the size of their nuclei in triploids to diploids was 2.35 and 1.80, respectively. Comparison of results obtained from the application of cold and heat shocks indicated that cold shocks are more effective than heat shocks in the induction of triploidy in grass carp

Effects of starvation and re-feeding on some hematological and plasma biochemical parameters of juvenile Persian sturgeon, Acipenser persicus Borodin, 1897

The effect of starvation and re-feeding was investigated on growth, hematology and biochemical parameters in juvenile Persian sturgeon (Acipenser persicus). Three hundred and seventy five fish (108±0.63 g) were divided into five feeding groups. The control group (C) was fed to satiation three times a day during the experiment. The four groups were starved for 1 (W1), 2 (W2), 3 (W3), and 4 (W4) weeks respectively, and then fed to satiation during a 4 week re-feeding period. The results indicated that some parameters including final weight, specific growth rate ,body weight increase, plasma enzymes (ALT, Alanine aminotransferase, AST, Aspartat aminotransferase and ALP, Alkaline phosphatise, hematological parameters [Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH)]were significantly affected by feeding regimes. The plasma cortisol, hematocrit, lymphocytes, neutrophils, eosinophils, and monocytes were not affected by starvation and subsequent re-feeding. These findings showed that short term starvations had no significant negative effects on growth performance, most biochemical and hematological parameters in Persian sturgeon could recover when re-feeding resumed

Application of microsatellite markers to determine populations of the Persian sturgeon (Acipenser persicus) in the south of Caspian Sea

The objective of this study was to analyse the population genetic structure of the Persian sturgeon (Acipenser persicus) in Sefidrud and Gorganrud rivers watershed based on the characterization of microsatellite markers during 2006 - 2008. 100 samples of Persian sturgeon were collected from two regions. Four microsatellite loci (Ls68, Spl168, Spl173 and Afu68) were analyzed for the molecular characterization of this species which resulted in polymorphic patterns. DNA bands were analysed using Biocapt and GenAlex software package. A total of 109 alleles were observed of which the maximum number of alleles (17) were found in Spl168 locus which belonged to sturgeons from Sefidrud river's watershed and the minimum number of alleles (10) in Ls68 locus belonging to the sturgeons from Gorganrud river's watershed. Results of microsatellite analysis revealed that the differences between samples of two regions were not statistically significant (p>0.05), neither for the average number of alleles per locus nor for observed heterozygosities. The calculated Fst and Rst between two regions was 0.07 and 0.17 showing that the genetic difference was significant (p< 0.01). Samples from Sefidrud river's watershed in Spl173, Afu68 and Spl168 loci and samples of other regions in Afu68 and Spl168 loci were at Hardy-Weinberg equation. The genetic distance was calculated as 0.4 which represents a significant genetic difference between samples of two studied areas. In conclusion, this study suggests that the Persian sturgeons in two regions of the southern part of the Caspian Sea are genetically differentiated, therefore fisheries management of these unique stocks for restocking and conservation of gene pools is highly recommended

Identification of genetic marker for differentiation of Persian sturgeon (Acipenser persicus) from Russian sturgeon (A. gueldeustadtti)

The Persian sturgeon (Acipenser persicus) is more abundant sturgeon species in the South Caspian Sea and consist the highest proportion of Iranian Caviar, meat as well as bringing maximum foreign currency income, however from systematic point of view and differentiation of this species from Russian sturgeon (Acipenser gueldenstadttii) a serious challenging issues remain, where some Russian scientist are believe that the Persian sturgeon is not as an valid species and consider it as a subspecies of Russian sturgeon. This research conducted with the objective of identification and introducing a molecular marker based on specific DNA for differentiation of two species of Persian sturgeon and Russian sturgeon via a proved molecular marker method. For this purposes 8 different molecular approaches such: Microsatellite, AFLP, RAPD, sequencing of Cytb, 16sDNA, ND5, Growth Hormone gene and finally Single Nucleotide Polymorphism (SNP) were investigated. Based on applied methodology, between 5 to 16 caudal fin tissues were sampled for each species from different region of the Caspian Sea, Sefiedrud River, Ural and Volga rivers. Following DNA extraction, its quality and quantity were determined and the PCR experiment has been conducted using 5-110 primers according to various methods and type of gene. The PCR products were electrophoresed on Polyacrilamid or agarose gels and followed by silver and Ethidium Bromide staining. In RAPD method, polymorphic DNA band was cut on the gel followed by purification and then the segments were cloned in vector in Top10 strain of E.coli, and then sequenced. Meanwhile for Growth Hormone gene in Persian and Russian sturgeon the MEGA 4, Gene runner software were used to design the appropriate primers for PCR amplification. The PCR products were cloned in PTZ57R/T vector and transformed in Top10 E.coli strain and sequenced finally. For all other genes, similar methods were applied for PCR amplification and its products were sequenced and statistical analysis as well as phylogenetical tree was performed. In Single Nucleotide Polymorphism (SNP) method, after genomic library construction, in total 14.4 billion nucleotides were sequenced and similarity/ differentiation analysis of two species were investigated using specific bioinformatic software. Results indicated that Microsatellite and AFLP methods showed high level of genetic variation both within and between species. The Cytb gene, when 4 sample sequences from each species were compared two species were differentiated, however when analysis repeated over 15 samples, the sequence comparison couldn't differentiate two above mentioned species. Full sequence comparison of 16sDNA and mtDNA-ND5 gene showed variation in some nucleotide in both species of Persian and Russian sturgeon but no significant. Results of sequences obtained from cloned segment with RAPD method and also specific primer design based on produced sequences could succeed to discover a variable DNA band that able to differentiate two species from each other. Results of the present study also showed that the growth hormone gene (GH) of Persian and Russian sturgeon consists of 645 nucleotide that translate to 214 Amino Acids. The sequence comparison indicated that the gene coding growth hormone in Persian and Russian sturgeon had the highest similarity with GH of Mammals (71%), Anguilaformes (63%) and less similarity with bony fish (37%). Phylogenetic analysis indicates that Persian and Russian sturgeon in compare to other organism are ancient species and this gene is originated from a common ancestor. At present study the most appropriate results obtained from Single Nucleotide Polymorphism (SNP) method by sequencing 14.4 billion nucleotide from genome of two species of Persian and Russian sturgeon from North and the South Caspian Sea could prove that the Persian sturgeon is a valid and independent specie. This excellent results is the biggest scientific achievement for differentiation of two highly commercial important sturgeon species in the Caspian Sea in last two decades

Investigation on possibility of separation of Acipenser persicus and Acipenser gueldenstaedtii using molecular cytogenetics techniques

Location of family Satellite DNA (HindIII SatDNA) on chromosomes of Acipenser persicus and Acipenser gueldenstaedtii was analyzed using Fluorescent in Situ Hybridization (FISH) technique to determine the genetic differences between these two species. After obtaining suitable metaphase plates using leukocyte culture and HindIII SatDNA extraction from genome of the fish under study, extracted SatDNA from genome of A. gueldenstaedtii was used as a FISH probe. The probe was labeled with spectrum orange (Orange-dUTP) and hybridized with chromosomes of the fish under study. Analysis of SatDNA sequences isolated from A. persicus and A. gueldenstaedtii showed the presence of 163bp for A. persicus and 168bp for A. gueldenstaedtii. In the study of metaphase plates 66±4 signals were detected from the hybridization of the probe used with chromosomes of A. persicus and 68±3 signals were detected from the hybridization of the probe with chromosomes of A. gueldenstaedtii. Due to presence of numerous microchromosomes and heterogenous and large hybridization signals, it was impossible to identify the precise position of signals on chromosomes. The following results are worth mentioning: 1. developing leukocyte culture method for A. gueldenstaedtii and completing it for all of the Caspian Sea sturgeons. 2. Developing a suitable culture medium for the Caspian Sea sturgeons. 3. Isolating sturgeons HindIII Satellite DNA family from A. persicus and A. gueldenstaedtii, determining sequences and registering the sequences in the NCBI gene bank (FJ429174; FJ 94465). 4. Developing a protocol for FISH technique in A. persicus and A. gueldenstaedtii which has opened a new era in aquaculture genetic studies entitled Molecular cytogenetics in the country. Considering its potential applications, this technique can be applied to other aquatic species

Study of kutum (Rutilus frisii kutum) population using mtDNA with PCR

Study of Mahisefid population using mtDNA with PCR Mahisefid diversity was studied in four rivers including Lamir, Sefidrood, Shirrood and Tajan. Total sample was collected from four rivers when fish migrat to river for spawning, 100 samples from Sefidrood, 98 samples from Lamir, 48 samples from Shirrood and 48 samples from Tajan. DNA was extracted with phenolcholorophorm. Samples were used for RFLP, the PCR product were digested by 20 restriction enzymes as follow: TasI, HaeIII, HinfI, HincII, SalI, DraI, AccI, AvaII, XhaI, BshNI, AvaI, BclI, BshII, MspI, PstI, RsaI, SdnI, TaqI, TruI, VspI. The four restriction enzymes including: TasI, HaeIII, HinfI, HincII showed diversity, 6 enzyme didn t have any restriction and 14 enzyme showed monomorphic. Total 20 haplotype studied that haplotype AAAA and BAAA had most frequency. The average haplotype frequency of AAAA was 29.93% and the average haplotype frequency of BAAA was 27.55%. 2- Study of population of Mahisefid with microsatellite markers 120 specimens of R.frissi kutum were used from four rivers to test thirty primers (30 samples from each river) of which 8 primers showed polymorphism. A large variation in heterozygosity average over all samples was observed among loci, that ranged from 0.13 to 0.91. For a given locus, observed heterozygosity varied greatly among the samples. For example, in Lamir 0.07 at SPY5 and 1 in Sefid Rud at CA1. Tajan had an observed heterozygosity of only 0.17, whilst the Lamir had an observed heterozygosity of 0.07 at locus SPY5. At locus SPY4, Tajan and Sefid Rud are the same and (0.53) and Shir Rud is 0.83 and Lamir is 0.47. However, despite these differences, there was clear difference in average heterozygosity observed between the samples. To investigated of Hardy-Weinberg Equilibrium in all locus and all rivers deviate showed from Hardy-Weinberg Equilibrium was significantly different ( P 0.05) between all samples except Lamir River and Sefid Rud River. The highest Fis was in Lamir River (0.84) in locus SPY5 and in Sefid Rud River in locus SPY5 (0.71), in locus CA12 (0.67) in Shir Rud River and SPY5 (0.63) in Tajan River. The lowest Fis is in locus CA1 (-0.55) in Sefid Rud River, in locus CA1 (-0.47) in Tajan River, in locus CA1 (-0.28) in Lamir River and in locus SPY6 (0.16) in Shir Rud River

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

Investigation of possibility of identification sex marker in beluga (Huso huso) and Persian sturgeon (Acipenser persicus) by using of molecular techniques

Due to high maturation age in sturgeons and lack of morphologic differences between male and female even in brood stocks, sex determination is difficult in these species. In this research with using of AFLP approach and 100 primer combinations, male and female genomic DNA of 20 individuals in Persian sturgeon (Acipenser persicus) and beluga (Huso huso) were investigated. Ligation was carried out with using of MseI and EcoRI, and then adapters were ligated with using of T4 DNA Ligase. Fragments amplification was done through two steps PCR and electrophoresis on denature poly acrylamid and stained by silver staining. Data derived from banding patterns were scored as o (absence) and 1 (Presence). A set of 100 (Eco+3 and Mse+4) primer combinations in A. persicus and H. huso yielded approximately a total of 3771 and 3779 scorable bands, respectively of which 30% in A. persicus and 29.6% in H. huso were polymorphic. The fragments ranged from 50 to 600 bp without revealing any sex specific markers. So we used cDNA-AFLP approach in order to analysis of gene expression in 8 female and 8 male Persian sturgeon gonads. Results revealed two cDNA markers in female gonad (TDF1, TDF2) and they verified with RT-PCR in male and female gonads cDNA. But unfortunately they didn’t verify in genomic DNA. According to this research results and previous researches, it seems that sturgeons may have not sex chromosomes or the methods were used couldn’t determine them

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic

Gene selection for optimal prediction of cell position in tissues from single-cell transcriptomics data

Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pairrule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes than in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues