Effect of cryopreservation on post-thaw motility and viability of Grey mullet, Mugil cephalus sperm (Linnaeus, 1758).

In this study, we optimized a simple method of cryopreservation for Mugil cephalus sperm based on post-thaw motility and viability. A series of experiments were conducted by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN) surface. First, we carried out the cryopreservation using the extender V2E and cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (Me2SO) and dimethylacetamide (DMA) at a final concentration of 5% and 10%. We found that 10% of GLY, EG and Me2SO were more suitable compared to other CPAs. Then, different freezing heights (6, 8, 10 and 12 cm) above the LN surface were experimented with extender V2E and optimized CPAs. Then, 0.3 M of glucose, sucrose and trehalose were tested as extender along with optimized CPAs and freezing height. Additionally, the effect of fast-rate freezing and storage days (7, 30 and 180) on post-thaw sperm quality was documented using the factors optimized in earlier experiments. For all experiments, the fresh sperm was diluted at a ratio of 1:1 with cryomedium (CPA + extender), loaded into cryovials (2.0 mL) and frozen. The cryopreserved sperm was thawed at 30 °C for 90-120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium (0.3 M glucose + 10% EG) and frozen at 4 cm above the LN surface registered significantly (P < 0.05) highest post-thaw motility (73 ± 2%) and (71 ± 1%) viability. Fast-rate freezing has resulted in lower (about 30%) post-thaw motility and viability of sperm. The storage days (7, 30 and 180) did not have a significant effect on post-thaw sperm quality. Overall results show that using the factors optimized through this study, high-quality sperm can be obtained after cryopreservation.

Optimization and Physicochemical Characterization of Polysaccharide Purified from Sonneratia caseolaris Mangrove Leaves: a Potential Antioxidant and Antibiofilm Agent.

The Box-Behnken design was applied to determine the optimal parameters of the extraction condition by using the response surface methodology (RSM) from the leaves of Sonneratia caseolaris L. The result indicates the best-optimized conditions used for the extraction of polysaccharides at 84.02 °C temperature, 3.12 h time, and 27.31 mL/g for the water-to-material ratio. The maximum experimental yield of 8.81 ± 0.09% was obtained which is in agreement with the predicted value of 8.79%. Thereafter, low molecular weight polysaccharide (SCLP) was separated after sequentially being purified through column chromatography with a relative molecular weight of 3.74 kDa. The physicochemical properties were evaluated by characterization techniques such as FT-IR spectra, NMR spectrum, and SEM analysis. RP-HPLC analysis confirmed that SCLP was a heteropolysaccharide, majorly comprising rhamnose (28.25%), and xylose (27.17%) residues, followed by mannose (18.90%), and galactose (17.17%), respectively. Thermal analysis (TGA-DSC) results showed that SCLP is a highly thermostable polymer with a degradation temperature of 361.63 °C. X-ray diffraction patterns and tertiary structure analyses indicate that SCLP had a semi-crystalline polymer having a triple-helical configuration. Moreover, SCLP displayed potential antibiofilm ability for all the tested pathogens while stronger activity against Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, SCLP has potential in vitro antioxidant activity on DPPH, ABTS radical, superoxide, and Fe2+ chelating. These findings indicate that the polysaccharide has potentially been used in functional food, cosmetics, and pharmacological industries.

Effects of polysaccharide-based silver and selenium nanoparticles on growth performance, biochemical parameters, and immune response of Cyprinus carpio.

Avicennia marina mangrove leaves polysaccharide (AMLP) was used for the synthesis of polysaccharide-based selenium (AMLP-SeNPs) and silver nanoparticles (AMLP-AgNPs). The synthesized nanoparticles were further characterized by UV-Vis, DLS, FT-IR, X-ray diffraction, and HR-TEM analysis. A 60-day (8 weeks) feeding trial experiment was conducted to investigate the effects of AMLP, AMLP-SeNPs, and AMLP-AgNPs dietary supplementation on growth performance parameters, blood parameters, immunological and enzymatic profiles in Cyprinus carpio. The characterization results of AMLP-SeNPs and AMLP-AgNPs confirmed the formation of well-stabilized spherical nanoparticles with a mean particle size of 37.25 and 72.40 nm, respectively having a crystalline structure. The feeding experiment results demonstrated that 2 mg/kg of AMLP-SeNPs followed by 0.2 mg/kg of AMLP-AgNPs showed significantly (p Ë‚ 0.05) higher final weight, weight gain (WG), specific growth rate (SGR%), protein and lipid efficiency, and lower food conversion ratio as compared to other groups. The catalase, superoxidase dismutase, and glutathione peroxidase activity were significantly (p Ë‚ 0.05) higher in the group fed 2 mg/kg supplemented AMLP-SeNPs. Total protein and globulin contents were significantly (p Ë‚ 0.05) higher and albumin concentration was significantly lower in fish that received 2 mg/kg of AMLP- SeNPs as compared to control. A significant increase in serum HDL and decrease in LDL and MDA concentrations were observed in the group supplemented with 2 mg/kg of nano selenium. The body's crude lipid, protein, moisture, and ash were not significantly different from the control. The AMLP-SeNPs showed significantly (p Ë‚ 0.05) lower aspartate aminotransferase (AST), alanine aminotransferase (ALT), and higher alkaline phosphatase (ALP) activities compared to other test groups. The relative percentage survivability (RPS%) was higher in AMLP-SeNPs (84.6%) followed by AMLP-AgNPs (76.7%) after 8th weeks of supplementary diets as compared to control groups. Overall, the finding of these studies revealed that the inclusion of AMLP-SeNPs improved the growth performance and antioxidant defense system, enhance immune response, and provide resistance against Aeromonas hydrophila in Common carp.

Enhancement of growth, innate immunity, and disease resistance by probiotic Enterococcus faecium MC-5 against Aeromonas hydrophila in Indian major carp Cirrhinus mrigala.

E. faecium MC-5 was isolated and characterized from the gut content of C. carpio and supplemented with a pellet diet at the ratio of 1.0 × 106, 1.0 × 109, and 1.0 × 1012 cfu g-1. Then it was fed to Cirrhinus mrigala for 42 days and the growth performances were evaluated. The growth performance of experimental groups was significantly (P < 0.05) increased than the control group. During 21 and 42 days of the feeding experiment, all the tested immunological parameters such as phagocytic, respiratory burst, lysozyme, acid phosphatase (ACP), superoxide dismutase (SOD), myeloperoxidase activities, and IgM level of experimental groups were significantly (P < 0.05) increased than a control group. After 21 and 42 days of feeding experiment, the fishes were challenged with A. hydrophila, and the relative percentage of survival of experimental groups (PT-I to PT-III) over the control group was recorded from 30.94 to 61.53 and 38.04-77.52 %, respectively. During the challenge experiment, A. hydrophila load was enumerated from dead fish at every 7-day interval. The A. hydrophila load in both the tissues of experimental groups decreased positively from the 7th to 14th day of challenge duration. Overall, the results suggested that the addition of E. faecium MC-5 enhanced the growth performance, immunological response, disease resistance to A. hydrophila, and antioxidant defense system in C. mrigala. Hence these results suggest that E. faecium MC-5 could be used as an advantageous probiotic supplement in aquaculture production.

Characterization of potential probiotic bacteria Enterococcus faecium MC-5 isolated from the gut content of Cyprinus carpio specularis.

The goal of this study was to determine the unique characteristics of Enterococcus faecium MC-5, a probiotic bacteria isolated from the intestine of a fish, Cyprinus carpio specularis, collected from Dal Lake in Srinagar, Kashmir, India. For this, the important valuable probiotic attributes, some functional properties, and safety assessments were analyzed in-vitro for the strain MC-5. The strain E. faecium MC-5 exhibited high resistance to low pH, high bile salt, lysozyme, and phenol. The strain MC-5 showed excellent auto- and co-aggregation properties and displayed remarkable hydrophobicity towards various tested hydrocarbons which suggested that the strain possesses venerable adhesion properties. Apart from these, the cell-free supernatant (CFS) of strain MC-5 exhibited phenomenal antimicrobial activity against the tested pathogens. A scanning electron microscope (SEM) image revealed strain MC-5 finely adhered to human colon adenocarcinoma cells (HCT-15 cells). The strain MC-5 showed high bile salt hydrolase activity and excellent cholesterol removal ability of 70.27%. The intact cells of strain MC-5 also showed strong DPPH scavenging activity. The EPS produced by E. faecium MC-5 inhibited the adhesion of Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica on HCT-15 cells with maximum inhibition rates of 41.82, 40.34, and 55.51%, respectively for displacement assay, which was higher as compared to exclusion (26.06, 26.11, and 39.23%) and competition assays (30.06, 26.7, and 41.20%). Strain MC-5 did not exhibit hemolysis and was also found susceptible to vancomycin and other clinically important antibiotics. When evaluating all the results from the present study, it is propounded that strain MC-5 has enviable probiotic characteristics and thus can be used as bio-protective cultures and/or bio-shield in food and pharmaceutical industries.

Functional and molecular characterization of a cold-active lipase from Psychrobacter celer PU3 with potential antibiofilm property.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129-135 AA) and R2 (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Haloalkaline Lipase from Bacillus flexus PU2 Efficiently Inhibits Biofilm Formation of Aquatic Pathogen Vibrio parahaemolyticus.

A gene encoding lipase enzyme from Bacillus flexus PU2 was cloned and expressed in E. coli BL21 (DE3) pLysS and purified protein having the molecular weight of 34 kDa. This lipase was found to be alkaline (pH 9) and slightly thermophilic. This lipase was observed to retain its activity in the presence of methanol, ethanol, DMSO, and acetone. Ferrous sulfate, copper sulfate, and manganese sulfate highly enhanced the lipase activity. All the surfactants and detergents were found to inhibit the enzyme activity, whereas the bleaching agent hydrogen peroxide was found to increase the activity. This lipase was observed as a metalloenzyme, and its activity was highly inhibited by EDTA. Also, it is moderately halophilic and can retain the activity between 0.2 and 0.8 M NaCl. Biofilm inhibitory potential of purified lipase was tested against pathogenic Vibrio parahaemolyticus, and the minimal inhibitory concentration observed was 350 U. Different concentration of this enzyme significantly changed the morphology and biofilm density of V. parahaemolyticus and was evinced by SEM and CLSM imaging. The transcriptome levels of genes responsible for biofilm formation, motility, and virulence such as, motX, fliG, and trh were significantly downregulated with lipase treatment.