Efficient utilisation of plant-based diets in the giant freshwater prawn, Marcrobrachium rosenbergii, varies according to individual, suggesting that it might be associated with differences in ...physiological and metabolic responses. Therefore, we aimed to investigate the individual differences in the growth response of shrimp fed to a soybean-based diet (SBM). Two hundred shrimp were fed SBM for 90 days, and specific growth rate (SGR) was determined individually. Fast- and slow-growing shrimp (F-shrimp vs. S-shrimp), with the highest and lowest 5% SGRs, respectively, were sampled to determine haemolymph chemistry and carcass composition. The hepatopancreas of these shrimps were used for transcriptome analysis through RNA sequencing (RNA-Seq). The results showed no significant differences in haemolymph chemistry parameters. In terms of carcass proximate composition, F-shrimp exhibited higher protein composition than did S-shrimp, suggesting that F-shrimp have higher protein anabolism. Using RNA-seq and real-time reverse transcription polymerase chain reaction (qRT-PCR), the expression levels of several genes encoding physiologic and metabolic enzymes were found to be upregulated in F-shrimp compared to in S-shrimp, suggesting that these enzymes/proteins mediated the efficient use of SBM-based diets for growth promotion in shrimp. Various DEGs associated with the immune system were observed, indicating a difference in immune processes between F- and S-shrimp. The expression of several housekeeping genes was found to be upregulated in S-shrimp. Collectively, the upregulated expression of several enzymes associated with physiological and/or metabolic processes and increased protein anabolism may be attributed to the efficient use of SBM for maximal growth in shrimp.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired ...phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.
The snakeskin gourami (Trichopodus pectoralis) exhibits sexual dimorphism, particularly in body size. Since the snakeskin gourami is usually marketed during sexual maturation, the sexual size ...dimorphism has become an economically important trait. Sex-biased gene expression plays a key role in phenotypic sexual dimorphism. Therefore, using high-throughput RNA sequencing (RNA-seq) technology, we aimed to explore the differentially expressed genes (DEGs) in ovary and testis during sex differentiation in juvenile snakeskin gourami. Our results revealed a number of DEGs were demonstrated to be overexpressed in ovary (11,625 unigenes) and testis (16,120 unigenes), and the top 10 female-biased (rdh7, dnajc25, ap1s3, zp4, polb, parp12, trim39, gucy2g, rtbs, and fdxr) and male-biased (vamp3, nbl1, dnah2, ccdc11, nr2e3, spats1, pih1d2, tekt3, fbxo36, and mybl2) DEGs were suggested to be mainly associated with ovary and testis differentiation, respectively. Additionally, using real-time reverse transcription polymerase chain reaction (qRT-PCR), validation of the differential expression of 21 genes that were previously shown to be related to gonad development was performed (ar, bHLH, cyp19a1, daz, dead-end, esrb, esrrg, gnrhr, gpa, gsg1l, hsd17B, mospd1, nanos-1, nanos-2, p53, piwi-1, piwi-2, rerg, rps6ka, tgf-beta, and VgR). The results showed a significantly positive correlation (0.84; P < 0.001) between the results of RNA-seq and qRT-PCR. Therefore, RNA-seq analysis in our study identified global genes that were associated with ovary and testis differentiation in the juvenile phase of the snakeskin gourami. Our findings provide valuable transcriptomic bioinformation for further investigation of reproductive biology and applications of sex manipulation.
The analysis of early gonadogenesis during larval development requires a molecular marker that is specifically expressed in the germ cell lineage, such as the vasa gene. In this study, we cloned and ...characterized vasa in the striped catfish (Pangasianodon hypophthalmus), and designated this as Phy-vasa. Phy-vasa contained all of the predicted consensus motifs that are shared among the vasa genes in other fish species, including RG and RGG repeats, ATPase motifs, and a DEAD-box, and phylogenetic analysis using various DEAD-box family proteins demonstrated that the Phy-vasa protein clustered within the Vasa family. Reverse transcription polymerase chain reaction (RT-PCR) indicated that Phy-vasa mRNA only occurred in the testis and ovary, and in situ hybridization showed that the gene was expressed only in the germ cells, with strong expression in the spermatogonia and oogonia. To investigate early gonadogenesis in catfish larvae, we undertook histological characterization and in situ hybridization using a Phy-vasa probe, which showed that migration of the primordial germ cells (PGCs) most commonly occurred in larvae at 2–10 days post-fertilization (dpf), the PGCs started to be surrounded by gonadal somatic cells at around 10–20 dpf, and rapid proliferation of the PGCs had begun by 30 dpf. These findings provide a valuable insight into early gonadal development in the striped catfish.
•Phy-vasa contained all of the predicted consensus motifs that are shared among the vasa genes in other fish species.•Phy-vasa mRNA only expressed in the testis and ovary.•Phy-vasa was strongly expressed only in the primordial germ cells (PGCs), spermatogonia and oogonia.•Migration of PGCs in the striped catfish most commonly occurred in larvae at 2–10 days post fertilization (dpf).•The PGCs started to be surrounded by gonadal somatic cells at around 10–20 dpf, and rapid proliferation of the PGCs had begun by 30 dpf.
Cryopreservation of testicular tissue would provide a useful tool to maintain spermatogonia, a precursor of gametes, for fish regeneration by germ cell transplantation. This study aimed to develop a ...cryopreservation method for spermatogonia by freezing the testicular tissue of striped catfish (Pangasianodon hypophthalmus) and testing whether cryopreserved spermatogonia exhibit transplantability. Freezing cryomedium containing L-15 medium and 1.3 M DMSO yielded the highest spermatogonia recovery rate after thawing. Supplementation with glucose and non-permeating cryoprotectants was not necessary to improve the recovery rate. We determined that an optimised equilibration time for cryopreserved testes in cryomedium included 60 min of equilibrium on ice before slow freezing (−1 °C/min), and subsequent thawing at 10 °C for 4–8 min. An Annexin V affinity assay and flow cytometry analysis were performed to identify apoptosis in cryopreserved testicular cells. Our results showed that cryoinjury of spermatogonia during cryopreservation could lead to apoptosis. Additionally, to determine the characteristics of cryopreserved spermatogonia at the molecular level, RT-qPCR was performed to quantify the expression of cxcr4b, dazl, dnd1, dnd2, nanos1, nanos2, and vasa. The results showed that compared to fresh testicular cells, frozen cells exhibited lower expression levels of cxcr4b and dazl. However, there were no significant differences in dnd1, dnd2, nanos1, and nanos2 expression levels. Although the expression level of vasa was lower in cryopreserved testes, both fresh and cryopreserved testicular cells showed positive vasa expression using in situ hybridisation. Furthermore, cryopreserved spermatogonia were transplanted into allogeniec recipient larvae and compared with fresh spermatogonia. Although the incorporation rate was lower than that of fresh spermatogonia, cryopreserved spermatogonia could be incorporated into the genital ridge of recipient larvae, demonstrating the transplantability of cryopreserved spermatogonia. Taken together, a method for cryopreservation of testicular tissue of striped catfish was optimised, and the cryopreserved testicular cells retained several spermatogenic stem cell characteristics, as well as transplantability.
•Cryomedium containing L-15 medium and 1.3 M DMSO was suitable for cryopreservation of testes in striped catfish.•Suitable thawing process included warming the cryopreserved testes at 10 °C for 4–8 min•Frozen cells exhibited lower expression levels of cxcr4b and dazl; however, there were no significant differences in dnd1, nanos1, and nanos2 expression levels.•Cryopreserved spermatogonia exhibit transplantability in allogenic recipient larvae.
This study aimed to investigate the potential probiotic
spp. from the intestine of Nile tilapia in order to construct a recombinant probiotic for the enhancement of the Nile tilapia immune response. ...One hundred bacterial isolates from the intestine of Nile tilapia were characterized for species identification using the 16s ribosomal RNA (rRNA). Only
isolates with exhibited antagonistic activity were investigated for their biological functions, which included protease-producing capacity, bile salts and pH tolerance, antibiotic susceptibility, and pathogenicity tests. According to the best results,
isolate B29, as closely related to
, was selected to construct a recombinant probiotic for the delivery of CC chemokine protein (pBES
-CC). The existence of recombinant probiotics was confirmed by Western blotting before the feeding trial. In addition, the CC chemokine mRNA level was quantified in the intestine of fish fed probiotics after 30 days of feeding. Total immunoglobulin, lysozyme activity, alternative complement 50 activity (ACH50), and phagocytic activity of fish fed either wild-type or recombinant probiotics were significantly increased, indicating that probiotics could stimulate the Nile tilapia immune system through different processes. Interestingly, the dietary supplementation of recombinant probiotics has a stronger immune response enhancement than the wild-type strain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
This study aims to shed light on the association between non-volatile and volatile compounds related to flavor/taste characteristics as well as sensory acceptability of Nile tilapia fillet ...(Oreochromis niloticus) cooked by various sous-vide (SV) conditions (50−60 ℃, 30−60 min), with fish cooked with boiling water used as control. Higher temperatures and longer processing times of SV cooking led to greater protein and lipid oxidation as indicated by the increase in total sulfhydryl (-SH), carbonyl, free fatty acid (FFA) contents as well as peroxide values (PV) and thiobarbituric acid reactive substance (TBARS) values. The differences in flavor/taste components including adenosine triphosphate (ATP)-related compounds, free amino acids (FAAs) and volatiles were also obtained, which directly affect sensory acceptability as evaluated by using the hedonic scale. Based on principal component analysis (PCA) results, the acceptability score was strongly correlated with inosine monophosphate (IMP) and acetoin, which seem to be the most crucial flavor enhancers for cooked tilapia. Among all samples, tilapia processed at 60 °C for 45 and 60 min, which contained significantly higher IMP and acetoin (p < 0.05) than others, had significantly higher flavor-liking and overall-liking scores, with a more than 7.5 meaning for high acceptability (p < 0.05), indicating the optimal SV conditions for tilapia fillet. Overall, the present finding indicated that the SV-cooking technique, at the optimal conditions, can improve the meat quality of cooked fish, in terms of flavor/taste characteristics, compared with traditional cooking (control).
Combining germ cell cryopreservation with transplantation is a powerful tool for preserving the genetic resources of various fish strains. In this germ cell transplantation system, since only type A ...spermatogonia (ASGs) among the various testicular cells have the potency to colonize recipient gonads and resume gametogenesis, techniques for visualization, isolation, and tracing of ASGs facilitate ease of manipulation. Here we established an ASG visualization technique in two Salmo species: brown trout and Atlantic salmon. We applied a monoclonal antibody (antibody No. 95), which specifically recognizes a cell surface antigen of ASGs obtained from rainbow trout, against Salmo species. Immunohistochemistry studies showed that antibody No. 95 also specifically detected the ASGs of brown trout and Atlantic salmon. Furthermore, marker-gene analyses of brown trout testicular cells sorted by flow cytometry using Alexa flour 488-conjugated antibody No. 95 revealed that fluorescent-positive cells possessed molecular characteristics of ASGs. Finally, to investigate the transplantability of antibody-positive cells, ASGs pre-stained with antibody No. 95 were transplanted into the peritoneal cavity of rainbow trout larvae. As a result, fluorescent cells incorporated into the recipient gonads, suggesting that the visualized ASGs possess transplantability. Thus, it is expected that the ASG visualization methods developed in this study will facilitate the development of more efficiency of germ cell transplantation techniques in Salmo species.
•Monoclonal Antibody recognizing type A spermatogonia (ASGs) of Salmo was selected.•Live ASGs could be visualized and isolated by the antibody labeled with Alexa 488.•ASGs visualized by the antibody could be used for germ cell transplantation.•These techniques facilitate ease of germ cell manipulation in Salmo species.
In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, ...high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.
We succeeded to enrich transplantable germ cells in rainbow trout using novel monoclonal antibodies by magnetic‐activated cell sorting. This method could be successfully applied to various Pacific salmonids.
The effects of various sous-vide (SV) cooking conditions (50-60℃, 30-60 min) on physicochemical properties related to the texture characteristics, protein structure/degradation, and sensory ...acceptability of tilapia fillet (
) were investigated. With an increasing temperature and processing time of SV cooking, protein degradation (of both myofibrils and connective tissue) was more pronounced, as evaluated by the decrease in water- and salt-soluble proteins, total collagen, as well as the changes in the ratio of secondary protein structures (α-helix, β-sheet, β-turn, etc.), which were determined by synchrotron-FTIR (SR-FTIR). These degradations were associated with the improvement of meat tenderness, as estimated by shear force and texture profile analyzer (TPA) results. Among all SV conditions, using 60 ℃ for 45 min seems to be the optimal condition for tilapia meat, since it delivered the best results for texture characteristics and acceptability (
< 0.05). Moreover, principal component analysis (PCA) results clearly demonstrated that the highest texture-liking score of this condition was well associated with the intensity of β-sheets, which seem to be the crucial component that affected the texture of SV-cooked tilapia more so than other parameters. The findings demonstrated the potential of SR-FTIR to decipher the biomolecular structure, particularly the secondary protein structure, of SV-cooked tilapia. This technique provided essential information for a better understanding of the changes in biomolecules related to the textural characteristics of this product.
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