Optimization of protocols for microinjection-based delivery of cryoprotective agents into Japanese whiting Sillago japonica embryos.

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas » artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.

Effects of ultrasound on permeation of cryoprotectants into Japanese whiting Sillago japonica embryos.

Cryopreservation of fish embryos requires the swift uptake of considerable amounts of cryoprotectant (CPA) but this process is hampered by the low permeability of the egg chorion. This study examined the relative efficiency of ultrasound to promote the incorporation of CPAs in two different embryonic developmental stages (somites and tail elongation) of Japanese whiting Sillago japonica and performed a preliminary cryopreservation trial using the best conditions determined during the study. Embryos tolerated ultrasound densities up to 37.5 W/cm2 well for up to 3 min but had significant mortality at 50 W/cm2. Hatching rates of somites embryos sonicated at 37.5 W/cm2 for 1-3 min in 10 and 20% Me2SO solutions were comparable (61-72%) to that of sonication in artificial seawater (65-86%) but decreased sharply at the concentration of 30% (0-55%); at similar conditions, tail elongation embryos had comparatively lower survival. Me2SO content of sonicated embryos at the somites and tail elongation stages increased significantly by 58-191% and 27-123%, respectively, compared to controls exposed to Me2SO without ultrasound. Pre-exposure to Me2SO before sonication increased the CPA uptake further by 36% without impairing survival. A preliminary cryopreservation trial after ultrasound-mediated impregnation of somites embryos with a CPA solution containing 20% PG and 10% MeOH did not yield live embryos after freeze-thawing but resulted in a significant decrease of nucleation temperature and increase of the proportion of morphologically intact embryos after freeze-thawing. These results suggest that sonication might be useful for fish embryo cryopreservation although it may require combination with other techniques to enhance CPA permeation.

In vitro antioxidant and, α-glucosidase inhibitory activities and comprehensive metabolite profiling of methanol extract and its fractions from Clinacanthus nutans.

BACKGROUND: This study was aimed to evaluate antioxidant and α-glucosidase inhibitory activity, with a subsequent analysis of total phenolic and total flavonoid content of methanol extract and its derived fractions from Clinacanthus nutans accompanied by comprehensive phytochemical profiling. METHODS: Liquid-liquid partition chromatography was used to separate methanolic extract to get hexane, ethyl acetate, butanol and residual aqueous fractions. The total antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging and ferric reducing antioxidant power assay (FRAP). The antidiabetic activity of methanol extract and its consequent fractions were examined by α-glucosidase inhibitory bioassay. The chemical profiling was carried out by gas chromatography coupled with quadrupole time-of-flight mass spectrometry (GC Q-TOF MS). RESULTS: The total yield for methanol extraction was (12.63 ± 0.98) % (w/w) and highest fractionated value found for residual aqueous (52.25 ± 1.01) % (w/w) as compared to the other fractions. Significant DPPH free radical scavenging activity was found for methanolic extract (63.07 ± 0.11) % and (79.98 ± 0.31) % for ethyl acetate fraction among all the fractions evaluated. Methanol extract was the most prominent in case of FRAP (141.89 ± 0.87 µg AAE/g) whereas most effective reducing power observed in ethyl acetate fraction (133.6 ± 0.2987 µg AAE/g). The results also indicated a substantial α-glucosidase inhibitory activity for butanol fraction (72.16 ± 1.0) % and ethyl acetate fraction (70.76 ± 0.49) %. The statistical analysis revealed that total phenolic and total flavonoid content of the samples had the significant (p < 0.05) impact on DPPH free radical scavenging and α-glucosidase inhibitory activity. CONCLUSION: Current results proposed the therapeutic potential of Clinacanthus nutans, especially ethyl acetate and butanol fraction as chemotherapeutic agent against oxidative related cellular damages and control the postprandial hyperglycemia. The phytochemical investigation showed the existence of active constituents in Clinacanthus nutans extract and fractions.

Phenotypic divergence and biometric indices of silond catfish, Silonia silondia (Hamilton 1822) populations inhabiting the coastal rivers of Bangladesh.

To observe phenotypic differentiation among populations of Silond catfish, Silonia silondia (Hamilton 1822), a multilinear approach was used. A sum of 180 samples from three coastal rivers (Meghna, Payra, and Kirtankhola) of the Bangladesh coast were scooped up to comprehend whether distinct populations of S. silondia could be separated from one another due to adaptive divergence. The findings of this study reflect the first attempt to determine the stock structure, morphological characters, length-frequency distributions, length-length relationships, length-weight relationships, condition factors, relative weight, and form factor of S. silondia in the coastal rivers of Bangladesh. Important discrepancies between the means of the three populations were revealed using univariate and multivariate analysis of variance (p < 0.01). In principal component analysis, the first and second principal components described 83.546% and 4.302% of the total variation, respectively. The step-wise discriminant function analysis revealed two variables that separated the populations significantly. Besides, a dendrogram based on Euclidean distances accurately separated the populations. In a one-way analysis of variance study, nineteen out of twenty-one morphometric characters showed significant variation (p < 0.01)among three populations. The length parameters based on the length-length relationships of each sample were found to be highly significant (p < 0.01). The length-weight relationships exhibited that the b value fluctuated from 2.796 (Kirtankhola) to 3.498 (Meghna). The Fulton's condition factor was estimated in the current study for this species with an average value ranging from 1.12 to 1.35. The calculated form factor values of this species were 0.0016, 0.0054, and 0.0110 for Meghna, Payra, and Kirtankhola river, respectively. Therefore, this study will expectantly inform fisheries taxonomists about the species' current stock structure, intraspecific phenotypic divergences, and aid in its management and conservation in similar ecosystems in Bangladesh and around the world.

Nutritional profiling of some selected commercially important freshwater and marine water fishes of Bangladesh.

The purpose of the study was to investigate the proximate composition and mineral content of Bangladesh's economically important freshwater and marine water fish. Proximate composition and mineral content was determined according to the Association of Official Agricultural Chemists (AOAC) standard method. All of the factors had a substantial variation (p < 0.05), according to the findings. The maximum protein content was observed in Lates calcarifer (18.673%) and minimum in Pangasius pangasius (15.616%). The content of lipid among the species varied from 0.316% to 13.396%, with Mugil cephalus having the highest lipid content and Channa striata having the lowest. The moisture content ranges from 68.343% to 81.160%. All the fishes have an average ash content of 0.850%-4.350%. The energy content is also significantly higher in marine water fishes. The mineral content was highly variable. Calcium content was lowest in Pangasius pangasius (0.555%) and highest in Setipinna phasa (3.495%). The magnesium content ranged between 0.281% and 1.885%. Phosphorus was lowest in Lepturacanthus savala (0.826%) and highest in Setipinna phasa (2.114%). The amount of sodium, potassium, and sulfur was relatively less for all fish species but there were substantial differences across the twelve samples. The PCA biplot's for proximate composition analyses has demonstrated positive affinity only between Lates calcarifer and Mugil cephalus in case of ash, lipid, and carbohydrate whereas Setipina phasa, Mugil cephalus, Lutjanus lutjanus, and Oreochromis mossambicus were grouped together with magnesium, phosphorus, calcium, and sulfur in the case of mineral content. Overall, the marine water fishes can be a good food item in terms of nutrition which could provide better health benefits for human.