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We evaluated the cryoprotective effects of methanol on zebrafish sperm at different concentrations, exposure times, and stages during cryopreservation. Samples were collected by crushing of dissect...

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA and methanol, membrane integrity was significantly decreased (70-75%) compared to controls. With catalase, sperm cells were maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

A critical challenge for the practical use of the layered O3-type binary nickel manganese oxides for sodium-ion batteries is the poor structural stability during extended cycling. The approaches of constructing O3/P2 hybrid composites can partially improve the cycling stability, but general approaches sacrifice the advantages of high capacity and low cost of the O3-type cathodes due to excessive sodium deficiency and lithium substitution. Here, we rationally design a serial of novel O3-majority hybrid Na0.9-xNi0.45Mn0.55O2 (x = 0.02, 0.04 and 0.08) cathodes, which exhibit high capacities while maintaining exceptional long-term stability. Particularly, the optimized O3/P2 Na0.88Ni0.45Mn0.55O2 composite delivers 106.7 mA h·g−1 with 71.1% capacity retention after 250 cycles at 1 C (1C = 150 mA g−1), the cyclability is 32% higher than that of the O3 Na0.9Ni0.45Mn0.55O2 cathode; and it also delivers a initial discharge capacity of 75.9 mA h·g−1, maintaining 72.4% capacity retention after 1000 cycles at 10 C. More importantly, the post-cycling analyses demonstrate O3/P2 hybrid phases successfully suppress the structural degradation of Na0.9Ni0.45Mn0.55O2 during battery operation. This study provides new perspectives in designing high performance cathodes for sodium-ion batteries.

This review is focused on the status and summary of larval cryopreservation in aquaculture mollusks. A total of 26 publications were identified addressing 14 molluscan bivalve species. Most studies were conducted on trochophores and D-larvae, a few studies were on umbo larvae, and no study was found on pediveligers. Based on the post-thaw viability, there is no general conclusion about the best larval stage for cryopreservation. The research topics of most publications focused on developing or improving cryopreservation protocols through exploring one or more vital factors of the cryopreservation procedure. The most dominant cryoprotectant agents (CPAs) used for molluscan larval cryopreservation were dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG) at final concentrations of 5–15%. Non-permeable CPAs, such as glucose, fructose, sucrose, trehalose, and polyvinylpyrrolidone (PVP), were dominantly used as additives. Most cooling processes started from 0 °C, 4 °C, or larval culture temperature to −10 °C or − 12 °C at −1 °C/ min and holding at this temperature for 5–15 min with or without “seeding”, and then continued to cool to around −35 °C at −0.3 to −2.5 °C/ min (mostly at −0.5 and − 1 °C/ min) followed by a 5–10 min holding period. All frozen samples were then transferred into liquid nitrogen (−196 °C) for long-term storage. Evaluation of post-thaw viability was reported over a wide range of parameters with no standard criteria, for example, motile post-thaw larvae were considered alive but no definition of swimming velocity. A sharp decrease of survival was often reported after a period of culture although immediate post-thaw survivals were reported high in several studies. Overall, no general optimal protocol could be concluded. Post-thaw larvae were reported to survive beyond metamorphosis in Pacific oysters and even to the adult stage and reproduced successfully after 3 years of culture, indicating the promising future of this technology. Till now, no study has been reported on germplasm cryobanking by use of molluscan larval cryopreservation. Pathways and aspects for promoting molluscan larval cryopreservation technology for germplasm repository establishment and commercial seed production were discussed.

COVID-19 is a highly contagious disease caused by a novel coronavirus SARS-CoV-2. The interaction between SARS-CoV-2 spike protein and the host cell surface receptor ACE2 is responsible for mediating SARS-CoV-2 infection. By analyzing the spike-hACE2 interacting surface, we predicted many hot spot residues that make major contributions to the binding affinity. Mutations on most of these residues are likely to be deleterious, leading to less infectious virus strains that may suffer from negative selection. Meanwhile, several residues with mostly advantageous mutations have been predicted. It is more probable that mutations on these residues increase the transmission ability of the virus by enhancing spike-hACE2 interaction. So far, only a limited number of mutations has been reported in this region. However, the list of hot spot residues with predicted downstream effects from this study can still serve as a tracking list for SARS-CoV-2 evolution studies. Coincidentally, one advantageous mutation, p.476G>S, started to surge in the last couple of weeks based on the data submitted to the public domain, indicating that virus strains with increased transmission ability may have already spread.

Searching low-cost porous carbon is crucial for the future development of supercapacitors in a wide application range. In this paper, rice husk derived carbon (RHC) was prepared through direct carbonization of rice husk in a tube furnace without inlet of N2. RHC possesses relatively high pore volume, specific surface area and specific capacitance. The high specific surface area of RHC is attributed to the self-activation of rice husk by H2O, CO2 produced in the carbonization process. RHC was further treated by alkali etching to prepare a porous carbon (referred as RHPC). Owing to the removing of SiO2 in RHC, RHPC exhibits higher specific surface area and specific capacitance than RHC.

The development of the layered cathode material for sodium ion batteries are hindered by synthesis approaches. The sol-gel method is a promising way to prepare the cathodes due to the advantage of mixing the raw product at atomic or molecular level, while the selection criteria for complexing agents are still unclear. Herein, the Na0.9Ni0.45Mn0.55O2 cathode is successfully prepared via sol-gel method, by using sucrose, glucose, citric acid, and ethylene diamine tetra-acetic acid as the chelating agent, respectively. The effects of different chelating agents on the morphologies, structural and electrochemical properties are studied in detail. Electrochemical properties prove that the sample using citric acid shows the best electrochemical performance, delivering a capacity of 96 mAh g−1 and 48% retention after 300 cycles at 1C, and 59.6 mAh g−1 of capacity at 10 C rate, which are significantly higher than those of the other samples. XRD and HRTEM are conducted for the cycled electrodes, which demonstrate that the structure of the optimized sample maintains better phase stability and interfacial stability. This work is of considerable significance in understanding the selection criteria for the synthesis of layered cathode chelators for sodium-ion batteries by the sol-gel method.

Ni-rich cathode materials, one of the most promising cathodes for high-energy lithium-ion batteries, are still suffered from interfacial instability and bulk degradation. Herein, Zr-doped and Li6Zr2O7-coated LiNi0.8Co0.1Mn0.1O2 cathode, and Zr-doped Li6Zr2O7–LiNi0.8Co0.1Mn0.1O2 composite are successfully prepared via a smart one-step calcination process. The attained dual-modified architecture allows the optimized sample exhibiting enhanced rate performance while maintaining long-term stability at room temperature (82.13% after 200 cycles at 1 C rate) and even at elevated temperature. Further studies reveal that the delayed temperature-driven phase transition and the suppressed interfacial degradation can be addressed with the synergetic effects provided by the Zr-doping and Li6Zr2O7-coating. The Zr doping could improve bulk stability by reducing cation disorder. The conductive Li6Zr2O7 surface coating enhances the interfacial stability of the cathode materials while improving the electrochemical kinetics. This smart modification strategy renders Zr modification a viable modification method to enhance the electrochemical performance and structural properties of Ni-rich cathode materials.

Control of sperm concentration is required to ensure consistent and reproducible results for cryopreservation and in vitro fertilization protocols. Determination of sperm concentration is traditionally performed with a counting chamber (e.g., hemocytometer), or more recently with a spectrophotometer. For small-sized biomedical model fishes, the availability of sperm sample is limited to microliters, so it is desirable to develop fast and accurate approaches for concentration determination that also minimize sample use. In this study, a new approach was developed for sperm concentration determination using a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) with simultaneous measurement of sperm membrane integrity after fluorescent staining with SYBR(®) -14 and propidium iodide (PI) in sperm from Zebrafish Danio rerio. The goal was to develop a protocol for simultaneous determination of sperm quality and quantity by flow cytometry. The objectives were to (1) determine the effects of sample volume (250 and 500 µl) and analysis volume (10 and 50 µl) on the accuracy of particle counting using standard volumetric validation beads; (2) identify the effective range of sperm concentrations that flow cytometry can measure; (3) test the precision and reproducibility of the sperm concentration measurements; and (4) verify the flow cytometry approach by comparison with measurement with a hemocytometer and a microspectrophotometer. Sample volumes of 250 and 500 µl and analysis volumes of 10 and 50 µl did not affect bead count with the factory-set flow rates of "medium" or "fast," and the precision and accuracy was retained across a concentration range of 1 × 10(3) -1 × 10(7) cells/ml. The approach developed in this study was comparable to traditional methodologies such as hemocytometer or microspectrophotometer. This study provides an efficient, accurate, and rapid method for determination of sperm concentration using flow cytometry while providing simultaneous assessment of sperm membrane integrity. Such approaches can reduce the time needed for quantity assessment and maximize the use of valuable sperm samples. © 2015 International Society for Advancement of Cytometry.

The high voltage application of the LiNi0.5Co0.2Mn0.3O2 cathode is still hindered by its unstable surficial structure and poor capacity retention. Surface coating has been proved to be an effective strategy to deal with these problems. However, previous multistep surface coating approaches greatly complicate the synthesis procedure. In this work, a simple and large-scale solid-state approach is developed to prepare lanthanum-based oxides co-coated LiNi0.5Co0.2Mn0.3O2 cathode materials. Electrochemical tests indicate that the co-coated samples exhibit superior cycle life and higher rate performance than those of the pristine sample. Further studies reveal that the enhanced interface stability and facilitated electron transportation can be attributed to the synergetic contribution provided by the lanthanum-based oxides co-coating layer. The present work demonstrates that our approach can achieve cathode synthesis and surface coating synchronously, and shows great potential to apply on a large scale and extent to all Ni-rich cathodes.

Knowledge of sperm motility activation for viviparous fishes has been limited to study of several species in Poeciliidae, and the dissociation of sperm bundles is poorly understood. The goal of this study was to use the endangered Redtail Splitfin (Xenotoca eiseni) as a model to investigate the activation of sperm from viviparous fish by study of free sperm and spermatozeugmata (unencapsulated sperm bundles). The specific objectives were to evaluate the effects of: (1) osmotic pressure and refrigerated storage (4 ºC) on activation of free sperm, (2) osmotic pressure, ions, and pH on dissociation of spermatozeugmata, and (3) CaCl(2) concentration and pH on sperm membrane integrity. Free sperm were activated in Ca(2+)-free Hanks’ balanced salt solution at 81–516 mOsmol/kg. The highest motility (19 ± 6%) was at 305 mOsmol/kg and swim remained for 84 h. Glucose (300–700 mOsmol/kg), NaCl (50–600 mOsmol/kg), and KCl, MgCl(2), and MnCl(2) at 5–160 mM activated sperm within spermatozeugmata, but did not dissociate spermatozeugmata. CaCl(2) at 5–160 mM dissociated spermatozeugmata within 10 min. Solutions of NaCl-NaOH at pH 11.6 to 12.4 dissociated spermatozeugmata within 1 min. The percentage of viable cells had no significant differences (P = 0.2033) among different concentrations of CaCl(2), but it was lower (P < 0.0001) at pH 12.5 than at pH between 7.0 to 12.0. Overall, this study provided a foundation for quality evaluation of sperm and spermatozeugmata from livebearing fishes, and for development of germplasm repositories for imperiled goodeids.

Sperm cryopreservation is a valuable tool in germplasm preservation and breeding. Despite many studies, reliable and routine cryopreservation of oyster sperm remains a challenge. The goal of this study was to develop a reliable protocol for sperm cryopreservation of the eastern oyster Crassostrea virginica with high-throughput processing by using two types of 0.5-ml straws (French and CBS™ high security straws). The objectives were to: 1) evaluate the effect of 10% methanol, dimethyl sulfoxide (DMSO), and propylene glycol as cryoprotectants at cooling rates of 5, 20 and 40 °C/min from 5 to − 80 °C and thawing at 30, 40 and 50 °C; 2) evaluate the effect of cooling rates of 10, 15, 20, 25 and 30 °C/min with 10% DMSO as cryoprotectant and thawing at 40 °C; 3) evaluate the effect of equilibration time (10–60 min) before freezing; 4) evaluate the effect of sperm concentrations from 1 × 10 8 to 1 × 10 9 for freezing, and 5) verify the established protocol by freezing sperm from 16 individual males. Among the three cryoprotectants, DMSO yielded the highest post-thaw motility at a cooling rate of 20 °C/min when thawed at 30 or 40 °C. Further evaluation of cooling rates of 10, 15, 20, 25 and 30 °C/min showed that 20 or 25 °C/min yielded the highest post-thaw motility (34 ± 5%) and fertility (77 ± 12%) for French straws and CBS straws (28 ± 3% and 69 ± 14%). Equilibration times of 10 to 60 min did not cause significant differences in post-thaw motility when freezing with 10% DMSO at a cooling rate of 25 °C/min. Also, sperm concentrations ranging from 1 × 10 8 to 1 × 10 9 at freezing did not cause significant differences in post-thaw motility. Sperm concentration after thawing was not different compared to that before freezing, and no agglutination was observed in the post-thaw samples. Finally, after thawing, sperm cryopreserved from 16 males with this protocol showed 58 ± 24% fertility (from 18 to 86%) for French straws, and 54 ± 21% fertility for CBS straws (from 18 to 95%). Overall, this study provided a reliable protocol for sperm cryopreservation in the eastern oyster with potential for high-throughput processing which can produce thousands of straws per day with homogenous and reliable quality.

Diierent durations of a cytochalasin B (CB) treatment were tested for tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis Say. Cytochalasin B, 0.67 mg L 1 , was applied to newly fertilized eggs at 8^10-min post fertilization and removed when in the untreated eggs: (1) Polar body 1 (PB1) was released in 90% of the eggs and polar body 2 (PB2) began to form (T1); (2) Polar body 2 was released in about 25^30% of the eggs (T2); (3) Polar body 2 was released in about 70^75% of the eggs (T3); or (4) eggs began to enter mitosis I or the polar lobe began to form (T4). Three replicates were produced using diierent sets of parents. The ploidy of resultant larvae and juveniles was determined by £ow cytometry. Blocking PB1 alone in T1 groups produced mostly tetraploids, and longer CB treatments in T2 and T3 resulted in increasing numbers of pentaploids. In T4 groups where both PB1 and PB2 were inhibited, larvae were predominantly pentaploids. Pentaploid larvae were arrested at the trochophore stage. The majority of tetraploid larvae died as trochophores, although a small fraction reached D-stage. Among 478 juvenile clams sampled from a T1 group, three (0.6%) were con¢rmed as tetraploids. This study shows that tetraploid embryos can be produced at high e⁄ciencies (40^90%) by blocking meiosis I. Tetraploids produced by meiosis inhibition in normal eggs are viable in M. lateralis, but their survival beyond metamorphosis is extremely low.

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: 1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me2SO), N, N- dimethylacetamide (DMA), N, N,-dimethyl formamide (DMF), and glycerol with concentrations of 5, 10, and 15% for 60 min of incubation at 4 °C; 2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and 3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5 and 10%) did not change the sperm motility after 30 min; Me2SO, DMA, and DMF (10 and 15%) and glycerol (5, 10 and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 °C/min to 25 °C/min) and were compared to Me2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ≤ 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ≤ 10% which was significantly lower than that of methanol and ME. With Me2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ≤ 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.

Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (

Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38 degrees C), cold shock (1, 4, and 7 degrees C), and nocodazole (0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23 degrees C, and lasted for 10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae and juveniles was determined with flow cytometry. Heat shock of 35 and 38 degrees C was effective in inhibiting mitosis I, producing 54% to 89% tetraploid larvae. Heat shock of 32 degrees C accelerated embryonic development without inhibiting mitosis or producing tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids in the 4 degrees C and 7 degrees C treatment groups. Cold shock of 1 degrees C produced 31% tetraploid larvae in one replicate, with none surviving to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids. This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none of the induced tetraploids survived to juvenile stage.