Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

A fluorescence‐based assay for in vitro screening of Saprolegnia inhibitors

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti‐Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence‐based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN‐1 viability dye combined with calcofluor white M2R, and is based on the formation of orange‐red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat‐killed and bronopol‐treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid‐treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN‐1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti‐Saprolegnia compounds.     

Nano-calcium carbonate reinforced polypropylene and propylene-ethylene copolymer nanocomposites: Tensile vs. impact behavior

Improvement of both the tensile and impact strength of the same polymeric material has always been a great challenge for the plastic industry. The study focuses on the effect of incorporation of calcium carbonate nanoparticles (0.3 wt% to 15 wt%) into three polypropylene (PP) based matrices viz. PP homopolymer, propylene-ethylene (PP-PE) copolymer and the blend of PP:PP-PE (30:70) to improve their impact behavior without hampering the tensile strength much. A loss in both the tensile and impact properties was observed in PP based nanocomposite. However, PP-PE based nanocomposites showed a significant improvement in impact strength (47 %) at 10 wt% loading with a loss of tensile strength by 22 %. To minimize this loss a blend of PP:PP-PE (30:70) was explored as a matrix. At 10 wt% loading, this matrix showed an improvement of 30 % in impact strength whereas the tensile loss was minimized to 10 %. Further, silane coupling agent which promoted good interfacial adhesion was used for best compositions. The variation of crystalline morphology of the nanocomposites with various formulations was analyzed using differential scanning calorimetry and X-ray diffraction.     

Diversity of root-knot nematodes in Moroccan olive nurseries and orchards: does <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> disperse according to invasion processes?

Background

Root-knot nematodes (RKN) are major pest of olive tree (Olea europaea ssp. europaea), especially in nurseries and high-density orchards. Soil samples were collected from main olive growing areas of Morocco, to characterize Meloidogyne species and to discuss the contribution of biotic and abiotic factors in their spatial distribution.

Results

RKN were found in 159 soil samples out of 305 from nurseries (52.1% occurrence) and in 11 out of 49 soil samples from orchards (23.2% occurrence). Biochemical and molecular characterisation (PAGE esterase and SCAR) revealed the dominance of M. javanica both in nurseries and orchards with minor presence of M. incognita only in nurseries, and M. arenaria in only one nursery. RKN were distributed on aggregated basis. Frequent presence of M. javanica in orchards might have come from nurseries. In contrast, the detection of M. incognita in nurseries alone suggests that this species could not reproduce in orchards because of either the competition with other plant-parasitic nematodes or unfit local habitats. The impact of environmental variables (climate, habitat origin and physicochemical characteristics of the substrates) on the distribution of Meloidogyne species is also discussed.

Conclusion

Olive nurseries in Morocco are not able to guarantee the safety of rooted plants. As a result, olive production systems are exposed to strong RKN invasion risks. Consequently, the use of healthy substrates in nurseries may prevent plant-parasitic nematode induction in orchards.

     

Reproductive resilience: a paradigm shift in understanding spawner‐recruit systems in exploited marine fish

A close relationship between adult abundance and stock productivity may not exist for many marine fish stocks, resulting in concern that the management goal of maximum sustainable yield is either inefficient or risky. Although reproductive success is tightly coupled with adult abundance and fecundity in many terrestrial animals, in exploited marine fish where and when fish spawn and consequent dispersal dynamics may have a greater impact. Here, we propose an eco‐evolutionary perspective, reproductive resilience, to understand connectivity and productivity in marine fish. Reproductive resilience is the capacity of a population to maintain the reproductive success needed to result in long‐term population stability despite disturbances. A stock's reproductive resilience is driven by the underlying traits in its spawner‐recruit system, selected for over evolutionary timescales, and the ecological context within which it is operating. Spawner‐recruit systems are species specific, have both density‐dependent and fitness feedback loops and are made up of fixed, behavioural and ecologically variable traits. They operate over multiple temporal, spatial and biological scales, with trait diversity affecting reproductive resilience at both the population and individual (i.e. portfolio) scales. Models of spawner‐recruit systems fall within three categories: (i) two‐dimensional models (i.e. spawner and recruit); (ii) process‐based biophysical dispersal models which integrate physical and environmental processes into understanding recruitment; and (iii) complex spatially explicit integrated life cycle models. We review these models and their underlying assumptions about reproductive success vs. our emerging mechanistic understanding. We conclude with practical guidelines for integrating reproductive resilience into assessments of population connectivity and stock productivity.     

Effect of transferring lignocellulose-degrading bacteria from termite to rumen fluid of sheep on in vitro gas production,fermentation parameters,microbial populations and enzyme activity

The digestive tract of termite(Microcerotermes diversus) contains a variety of lignocellulose-degrading bacteria with exocellulases enzyme activity, not found in the rumen, which could potentially improve fiber degradation in the rumen. The objectives of the current study were to determine the effect of inoculation of rumen fluid(RF) with three species of bacteria isolated from termite digestive tract, Bacillus licheniformis, Ochrobactrum intermedium, and Microbacterium paludicola, on in vitro gas production(IVGP), fermentation parameters, nutrient disappearance, microbial populations, and hydrolytic enzyme activities with fibrous wheat straw(WS) and date leaf(DL) as incubation substrate. Inoculation of RF with either of three termite bacteria increased(P0.05) ammonia-N concentration compared with the control group(free of termite gut bacteria). Termite bacteria inoculation had no effect(P0.05) on gas production characteristics, dry matter, organic matter and neutral detergent fiber disappearance, pH, and concentration and composition of volatile fatty acids. Population of proteolytic bacteria and protozoa, but not cellulolytic bacteria, were increased(P0.05) when RF was inoculated with termite bacteria with both WS and DL substrates. Inoculation of RF with termite bacteria increased protease activity, while activities of carboxymethyl-cellulase, microcrystalline-cellulase, α-amylase and filter paper degrading activity remained unchanged(P0.05). Overall, the results of this study indicated that transferring lignocellulose-degrading bacteria, isolated from digestive tract of termite, to rumen liquid increased protozoa and proteolytic bacteria population and consequently increased protease activity and ammonia-N concentration in vitro, however, no effect on fermentation and fiber degradation parameters were detected. These results suggest that the termite bacteria might be rapidly lysed by the rumen microbes before beneficial effects on the rumen fermentation process could occur.     

Influence of nanochitosan/zeolite composite on growth performance,digestive enzymes and serum biochemical parameters in rainbow trout (Oncorhynchus mykiss)

Protective effects of nanochitosan/zeolite composite besides zeolite and chitosan/zeolite composite on rainbow trout growth, digestive enzyme activities and biochemical parameters were studied. Nanochitosan/zeolite hybrid composites with three different ratio of nanochitosan:zeolite (35:100, 3.5:100 and 0.35:100) were synthesized and analysed by X‐ray powder diffraction (XRD), thermal gravim (TG) analysis and scanning electron microscope (SEM) methods. Prepared composites as well as zeolite and control diets were fed to rainbow trout (50 g) for a period of 60 days. The addition of treated diets significantly improved growth performance compared to the control diet. Supplemental zeolite could only enhance the amylase activity in fish intestine, whereas other treatment groups could increase the pepsin activity besides intestinal alkaline phosphatase, trypsin and amylase activities. No differences were observed for intestinal acid phosphatase and lipase activities among the experimental diets. Meanwhile, serum total antioxidant activity and lipid peroxidation product, indicated by malondialdehyde (MDA), significantly increased and decreased, respectively, with some doses of administration, indicating the elevated antioxidant status in treatment groups. Serum‐specific marker enzymes, namely aspartate aminotransferase (AST) and alkaline phosphatase (ALP), were not affected in all groups. Meanwhile serum total protein in most treatment groups was significantly higher than the control group. Results showed that synthesized composites especially nanochitosan/zeolite composite at 5 g/kg had potential to enhance growth performance, digestive enzyme activities and some biochemical parameters in rainbow trout.     

Inhibition of postharvest senescence of green leafy vegetables by exogenous D-cysteine and L-cysteine as precursors of hydrogen sulphide

Hydrogen sulphide (H₂S) inhibits senescence in harvested fruit and vegetables but presents logistical, safety and regulatory issues to become a commercial treatment. D-cysteine and L-cysteine are semi-essential amino acids that are metabolised to hydrogen sulphide by plant tissues albeit by different pathways. This paper examines the effect of cysteine on postharvest senescence of three green leafy vegetables. Spraying pak choy leaves with 10 mmol D-cysteine, L-cysteine or DL-cysteine inhibited leaf senescence through a delayed loss of green colour expressed as market life, reduced respiration rate and reduced ethylene production. The beneficial effects of cysteine were similar to those achieved by fumigation with hydrogen sulphide. L-cysteine sprays on parsley and peppermint leaves also showed reduced leaf colour loss and respiration compared to untreated leaves. Cysteine, either as the racemate or individual enantiomers, is considered to have commercial potential for green leafy vegetables as it provides the beneficial effect of hydrogen sulphide but should be easier to register for commercial use due to the GRAS status of L-cysteine.     

Agronomic traits at the seedling stage,yield, and fiber quality in two cotton (Gossypium hirsutum L.) cultivars in response to phosphorus deficiency

ABSTRACT

Cotton is critical for phosphorus demands and very sensitive for its deficiency. However, identifying the effect of low-phosphorus tolerance on cotton growth, yield, and fiber quality by reducing phosphorus consumption. This may help to develop phosphorus-tolerant high-yielding cotton cultivars. In a two-year repeated (2015 and 2016) hydroponic experiment (using 0.01 and 1 mM KH₂PO₄), two cotton cultivars with phosphorus sensitivity (Lu 54; a low-phosphorus sensitive and Yuzaomian 9110; a low-phosphorus tolerant) were screened on the base of agronomic traits and physiological indices through correlation analysis, cluster analysis and principal component analysis from 16 cotton cultivars. Low phosphorus nutrition reduced the plant height, leaf number, leaf area, phosphorus accumulation and biomass in various organs of seedlings. The deficiency negatively affected the morphogenesis of seedlings, as well as yield and fiber quality. Further, these screened cultivars were tested in a pot experiment with 0, 50, 100, 150, 200 kg P₂O₅ ha^?1 during 2016 and 2017. It was found to have a significant (P< 0.05) difference in boll number, lint yield, fiber strength, and micronaire at the harvest. Furthermore, after collectively analyzed the characteristics of Lu 54 and Yuzaomian 9110, there were six key indices that could improve the low phosphorus tolerance of cotton cultivars. These were root phosphorus accumulation, stem phosphorus accumulation percentage, leaf and total biomass of seedlings, seed cotton weight per boll and fiber length.     

Marine-Derived Chitosan Nanoparticles Improved the Intestinal Histo-Morphometrical Features in Association with the Health and Immune Response of Grey Mullet (Liza ramada)

Marine-derived substances are known for their beneficial influences on aquatic animals’ performances and are recommended to improve intestinal health, immunity, and anti-oxidative status. The present study investigates the role of chitosan nanoparticles on the intestinal histo-morphometrical features in association with the health and immune response of Grey Mullet (Liza ramada). Chitosan nanoparticles are included in the diets at 0, 0.5, 1, and 2 g/kg and introduced to fish in a successive feeding trial for eight weeks. The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) parameters are significantly increased while feed conversion ratio (FCR) decreases by chitosan nanoparticles compared to the control (p < 0.05). The morphometric analysis of the intestines reveals a significant improvement in villus height, villus width, and the number of goblet cells in chitosan-treated groups in a dose-dependent manner. Additionally, there is a positive correlation between the thickness of the enterocyte brush border and the chitosan dose, referring to an increasing absorptive activity. Histologically, the intestinal wall of Grey Mullet consists of four layers; mucosa, sub-mucosa, tunica muscularis (muscular layers), and serosa. The histological examination of the L. ramada intestine shows a normal histo-morphology. The epithelial layer of intestinal mucosa is thrown into elongated finger-like projections, the intestinal villi. The values of hemoglobin, hematocrit, red blood cells (RBCs), total protein (TP), albumin, and globulin are significantly increased in fish fed 1, and 2 g/kg of chitosan nanoparticles compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest levels of TP and albumin are observed in fish fed 1 g/kg diet (p < 0.05). The lysozyme activity and phagocytic index are significantly enhanced by feeding chitosan nanoparticles at 0.5, 1, and 2 g/kg, whereas the phagocytic activity is improved in fish fed 1 and 2 g/kg (p < 0.05). The highest lysozyme activity and phagocytic index are observed in fish fed 1 g/kg. SOD is significantly activated by feeding chitosan nanoparticles at 1 g/kg. Simultaneously, glutathione peroxidase (GPx) and catalase (CAT) activities also are enhanced by feeding chitosan at 1 and 2 g/kg, compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest GPx and CAT activities are observed in fish fed 1 g/kg (p < 0.05). Conversely, the malondialdehyde (MDA) levels are decreased by feeding chitosan at 1 and 2 g/kg, with the lowest being in fish fed 1 g/kg (p < 0.05). To summarize, the results elucidate that L. ramada fed dietary chitosan nanoparticles have a marked growth rate, immune response, and anti-oxidative response. These improvements are attributed to the potential role of chitosan nanoparticles in enhancing intestinal histo-morphometry and intestinal health. These results soundly support the possibility of using chitosan nanoparticles at 1–2 g/kg as a feasible functional supplement for aquatic animals.