Laser irradiation as a novel alternative to detach intact particulate matter collected on air filters.

Airborne particulate matter (PM) is collected on specific filters. For subsequent testing, the PM should be detached intact from the filter. Liquid extraction (LE), the standard method to detach PM from air filter surfaces, is challenging and can be tedious. Laser irradiation has been used to characterize PM on filters, but not to detach PM from filters for subsequent testing. A feasibility study was conducted to assess the potential of laser irradiation to detach PM from air filters. Laser-detached PM was deposited on a pre-weighed glass plate. PM detachment and collection were conducted in a single step. PM-coated air filters were subjected to visual inspection, gravimetric assessment of captured PM, and spectroscopic scanning (ATR-FTIR, SEM-EDS, and XRD) before and after laser irradiation. Laser irradiation PM detachment efficiency was up to 78 %. Functional groups, elements, and minerals of PM collected on filter surfaces disappeared or significantly decreased after irradiation, demonstrating detachment, without suffering a change in their nature. No evidence of filter fragments was found in the detached PM. Laser irradiation was i) an easy, ii) rapid, and iii) single step procedure that iv) detached PM, v) didn't detach filter fragments, vi) didn't change PM composition, and vii) is amenable to automation and high throughput. Laser irradiation to detach PM from air filters as an alternative to LE is worthy of further study and development.

An Endeavor to Find Starter Feed Alternatives and Techniques for Zebrafish First-Feeding Larvae: The Effects on Viability, Morphometric Traits, Digestive Enzymes, and Expression of Growth-Related Genes.

Low and variable growth and survival rates (SR) of 6-10 days postfertilization zebrafish larvae are a problem. This problem seems to be linked to starter feed characteristics. This study is an attempt to find alternatives to address these requests. For this, larvae were fed fresh and lyophilized microalgae (Chlorella, Scenedesmus, and Haematococcus), egg yolk (YOLK), lyophilized Artemia nauplii (LAN), and a combination of them. The lowest SR was observed in algae-fed larvae. All died on day 11 showing an emaciated appearance, similar to starved larvae. The highest SR was observed in YOLK- and LAN-fed larvae, which also showed an elongated anterior part of the body. Negative correlations of SR with vegfaa (vascular endothelial growth factor) and morphometric traits with igf2a (insulin-like growth factor) were also found and supported by changes at the molecular level. The presence of algae in the digestive tract of the larvae and the observation of fecal droppings indicate that the algae have an appropriate size and are palatable. The increase in the digestive enzyme activity shows the larval effort to digest the algae. The fact that the algae-fed larvae died even before the larvae were kept in starvation indicates the dramatic amount of energy that the larvae spent in microalgae digestion. Although both YOLK- and LAN-fed larvae had the highest SR, LAN group started to feed on Artemia nauplii sooner. This can be linked to the delayed growth in YOLK-fed larvae and an accelerated growth in the case of LAN-fed group. LAN is an expensive feed with negative effects on water quality, whereas YOLK is a cheap and nutritionally balanced feed with fine granular texture that contributes to a larval SR similar to LAN without affecting water quality. In conclusion, microalgae cannot be considered a suitable starter food for zebrafish, whereas LAN and YOLK can be considered good starter feeds.

Imatinib mesylate effects on zebrafish reproductive success: Gonadal development, gamete quality, fertility, embryo-larvae viability and development, and related genes.

Imatinib (IM) is a tyrosine kinase (TK) inhibitor (TKI) used to treat chronic myeloid leukemia. Clinical case reports and a few laboratory mammal studies provide inconclusive evidence about its deleterious effects on reproduction. The aim of the current study was to evaluate the potential of zebrafish to characterize IM-induced effects on reproduction and clarify IM effects on reproductive success. To this end, we exposed adult zebrafish to four concentrations of IM for 30 days followed by a 30-day depuration period. IM exposure caused a concentration-dependent, irreversible, suppression of folliculogenesis, reversible decrease in sperm density and motility, decreased fecundity and fertility, but no significant change in atretic follicle abundance. We also observed IM-induced premature hatching, but no significant change in embryo-larvae survivability. However, we found significant IM-induced morphometric malformations. IM decreased expression of vegfaa and igf2a (two reproductive-, angiogenic-, and growth-related genes) in testes and ovaries. The results demonstrate IM can induce significant changes in critical reproductive endpoints and zebrafish as a suitable model organism to show effects of IM on reproduction. The findings suggest that TKI effects on reproductive success should be considered.

Zebrafish as a Model to Screen the Potential of Fatty Acids in Reproduction.

Sperm quality is an important topic in general health, chemotherapy, and gamete preservation technology. Fatty acid (FA) composition of membranes, which is influenced by the diet, plays key roles in sperm biology and quality. Dietary supplementation with natural products can be used as a technique to screen potential agents to protect, modify, and recover sperm quality. In this study, zebrafish (male [♂-ZF] and female [♀-ZF]) were fed a single cultivar olive oil (OO) bioencapsulated in Artemia. OO-treated ♂-ZF had higher (p < 0.05) sperm density and motility compared to the Artemia nauplii (AN). A significant difference was also observed in follicle abundance at different stages of gametogenesis, and a nonsignificant increase in total fecundity between OO-treated ♀-ZF and the AN, although in OO-treated ♀-ZF, mature follicles had a smaller diameter. A higher fertility rate (FR) was observed in OO-treated pairs compared to the other groups. Hatching in the OO-treated fish was accelerated, although no significant differences could be found in terms of hatching rate (HR) and embryo/larval survival rate (SR). These findings in FR, HR, and SR were also confirmed in male and female replacement mating trials. Taken together, this study shows that altering the FA ratios in the diet has a clear impact on several reproductive parameters in the zebrafish, adding new information about the nutritional requirement of this model species.

Morphometric characteristics and time to hatch as efficacious indicators for potential nanotoxicity assay in zebrafish.

Although the effects of nano-sized titania (nTiO2 ) on hatching events (change in hatching time and total hatching) in zebrafish have been reported, additional consequences of nTiO2 exposure (i.e., the effects of nTiO2 -induced changes in hatching events and morphometric parameters on embryo-larvae development and survivability) have not been reported. To address this knowledge gap, embryos 4 h postfertilization were exposed to nTiO2 (0, 0.01, 10, and 1000 µg/mL) for 220 h. Hatching rate (58, 82, and 106 h postexposure [hpe]), survival rate (8 times from 34 to 202 hpe), and 21 morphometric characteristics (8 times from 34 to 202 hpe) were recorded. Total hatching (rate at 106 hpe) was significantly and positively correlated to survival rate, but there was no direct association between nTiO2 -induced change in hatching time (hatching rate at 58 and 82 hpe) and survival rate. At 58, 82, and 106 hpe, morphometric characteristics were significantly correlated to hatching rate, suggesting that the nTiO2 -induced change in hatching time can affect larval development. The morphometric characteristics that were associated with change in hatching time were also significantly correlated to survival rate, suggesting an indirect significant influence of the nTiO2 -induced change in hatching time on survivability. These results show a significant influence of nTiO2 -induced change in hatching events on zebrafish embryo-larvae development and survivability. They also show that morphometric maldevelopments can predict later-in-life consequences (survivability) of an embryonic exposure to nTiO2 . This suggests that zebrafish can be sensitive biological predictors of nTiO2 acute toxicity. Environ Toxicol Chem 2018;37:3063-3076. © 2018 SETAC.

A Simple and Inexpensive Microinjection System for Zebrafish Embryos and Larvae.

Microinjection is a widely used technique to inject defined volumes and concentrations of substances and explore their physiological function in vivo. The technique has been particularly successful with zebrafish embryos; however, the injection equipment can be relatively expensive and therefore available only to well-funded laboratories. In this study, a simple, cheap, easy-to-assemble, and easy-to-use setup with a straightforward, accurate, and efficacious calibration method is introduced. The accuracy of this calibration method was tested by comparing with the results of calibration methods that are currently used in high-cost systems. Injection success with this low-cost system was verified based on the presence of injected dyes in zebrafish embryos, the absence of any significant morphological and behavioral differences between 3,4,-dichloroaniline-treated and untreated embryos, and larval viability.

An Affordable Intraperitoneal Injection Setup for Juvenile and Adult Zebrafish.

Intraperitoneal (IP) injections are an effective and reproducible route of drug administration. However, current IP equipment can be either costly, inaccurate, or unsafe for zebrafish. We describe a simple, low-cost IP setup, which can be easily assembled from inexpensive and readily available parts, and which provides a safe, reproducible, and accurate IP-injection method for experimenters.

Experimental Assessment of the Efficacy of Five Veterinary Broad-Spectrum Anthelmintics to Control the Intestinal Capillariasis in Zebrafish (Danio rerio).

Zebrafish in research facilities are frequently infected with capillarids. Since the health status (as a nonprotocol source of variation) of zebrafish can affect the validity of experiments, it is important to develop therapies for common zebrafish diseases. Regarding the likelihood of (1) the development of drug resistance and (2) the loss of the efficacy of a drug for laboratory zebrafish, the availability of alternatives for treatment is of direct importance. The efficacy of five dewormers from the same or different therapeutic groups was assessed in the current study. The exposure to each drug was repeated in triplicate (i.e., 3×100 fish in each treatment). The (1) elimination of parasite eggs, larvae, or adults from fresh fecal droppings (as the first main criterion) and (2) dissection of one-third of treated fish (i.e., 100 fish per drug) and examination of their gut contents (as the second major criterion) were considered to verify the efficacy of the drugs to eradicate the infection. Mebendazole (meb), praziquantel (pra; after the first round of treatment, i.e., six-fold administration, twice a day, for 3 days), fenbendazole (fen; after the second round of treatment), and ivermectin (ive; just after two administrations: twice during a day, i.e., a successful treatment with the smallest therapeutic effort) eradicated the infection, while albendazol (alb) was ineffective, although alb in a combined therapy with fen was successful. No age-, sex-, or disease severity-dependent responses to drugs were observed. The meb, pra, and ive were eliminating parasite eggs effectively in contrast with fen (that just was effective on adults). The drugs produced no observable side effects in zebrafish.

Efficacy of the hatching event in assessing the embryo toxicity of the nano-sized TiO2 particles in zebrafish: a comparison between two different classes of hatching-derived variables.

The aim of the present study was to evaluate the nano-TiO2 toxicity to zebrafish embryos through evaluating the success in hatching in relationship with hours post-exposure instead of considering just the total hatching rate. Zebrafish embryos 4h post-fertilization were exposed to nTiO2 (0, 0.01, 10, and 1000 µg mL(-1)) for 130 h. The hatching rate (HR) was calculated for each concentration (treatment). The HR magnitude was significantly (p<0.001) correlated (using simple regression) to hours post-exposure time interval (hpe; 34, 58, 82, 106, and 130), noted as HR.hpe. The HR descriptive statistics (HRds) and the parameters of the regression models (i.e., constant, x, F, and r(2)) were recruited to define 15 HRds- and 4 h.hpe-derived variables, respectively. The efficacy of the variables was evaluated. Exposure to nTiO2 led to a significant: premature hatching and general decrease in time required for normal hatching; and change in HR and hpe interrelations in a dose-dependent manner. The major change in hatchability between the treatment and control occurred at 58 hpe (62 hpf), when the treatment with nTiO2 induced significant premature hatching compared to only 6% of the hatched embryos in the control at the same time point. EC10 and EC50 values that cause premature hatching at 58 hpe for nTiO2 are 0.073 µg mL(-1) and 107.2 µg mL(-1) respectively. In general(,) this study shows multivariate differences among exposure concentrations of nTiO2 recruiting hatching-derived endpoints.

Embryo and larva development in Dentex dentex, a marine pelagophil teleost: an endeavor to find a series of new fatty acid interrelations.

Although the fatty acid (FA) requirements of marine teleosts not only are a function of the amount of each FA individually, but also of the relative proportions of the FAs; mostly, the interactions have been ignored and merely limited to a few interrelations of 20:4[n-6], 20:5[n-3], and 22:6[n-3]. To address this shortcoming, viable eggs of Dentex dentex were obtained from broodfish in captivity. Nine viability parameters (VP) (i.e., floating rate (FR), hatching rate (HR), and survival rate (SR) from day 0 to 5 posthatch (dph)) that are currently used in mariculture systems were determined. Egg FAs were characterized and quantified. One hundred and twenty ratios were made based on the FA contents estimated. Sixty-four ratios were significantly and strongly correlated to embryo/larva success through 201 simple regression models (r(2) = 0.640-0.948; P = 0.006-P < 0.001). Of the 201 significant relationships found, -12, -5, -2, -3, -22, -23, ∼21, and -13% show the relations of the egg FA ratios with FR, HR, and SR at 0, 1, 2, 3, 4, and 5 dph, respectively. All the FAs characterized in this study were significantly correlated to VPs through either their individual relative proportions or relative proportion of their combinations. This study, for the first time, presents a series of new FA interrelations and uncovers their biological meanings under both basic and applied aspects through correlating them to embryo/larva success.

Evaluation of quantitative importance of egg lipids and fatty acids during embryos and larvae development in marine pelagophil teleosts: with an emphasis on Dentex dentex.

Quantitative compositin of Dentex dentex eggs lipid classes (LCs) and fatty acid (FA)s were measured and correlated to embryo and larva viability parameters (egg buoyancy, floating rate (FR), hatching rate, HR and survival rate, SR, at 0-5 days post hatch, dph) through 43 formulated models (38 using FAs, as independent variable, [R(2) = 0.641-0.859] and 5 using LC [R(2) = 0.459-0.739]). The sphingomyelin, phosphatidylcholine and 20:2(n-6) were significantly correlated with FR; phosphatidylserine + phosphatidylinositol with SR at 1 dph; steryl ester + waxes/triacylglycerol with HR and SR at 0 dph; 14:0 with SR at 1-5 dph; 18:1(n-9), summation operator18:1, 20:5(n-3)/20:4(n-6), summation operatormonounsaturated FAs (MUFAs), 20:5(n-3), 22:6(n-3)/20:5(n-3) and 18:3(n-3) with SR at 2-5 dph; 20:5(n-3) + 20:4(n-6) with FR and SR at 2-5 dph. It was also found that a 14:0, 18:1(n-9), summation operatorMUFA, 18:2(n-6), 18:3(n-3), 20:5(n-3), 22:6(n-3)/20:5(n-3) and 20:5(n-3)/20:4(n-6) equal to 3.3, 16.5, 32.5, 9, 1.3, 8, 1.5 and 7, respectively, perhaps can provide a SR at 3 and 5 dph of >70%. The results indicated the importance of the egg LCs and FAs quantitative composition for common dentex embryonic and larval development and a potential use of these factors to predict egg quality in teleosts.

Quantitative composition of vitellogenin-derived yolk proteins and their effects on viability of embryos and larvae of common dentex (Dentex dentex), a marine pelagophil teleost.

To evaluate the importance of the quantitative composition of vitellogenin (Vtg)-derived egg yolk proteins during initial events of Dentex dentex life, viable eggs were obtained from captive broodstocks. Viability parameters (Vps; egg buoyancy (FR), hatching rate (HR), and larval survival rate (SR) at 0, 3, and 5 days posthatch (dph)) were determined for 70 egg batches. Vtg-derived yolk proteins were purified from the eggs using cation-exchange chromatography, separated and detected by SDS-PAGE, identified and characterized using mass spectrometry, quantified using image densitometry, and finally correlated to VPs. The 77.97, 57.22, 22.22, 17.69, and 16.95 kDa proteins were identified as proteolytic products resulted from both VtgA and B. All the Vtg-derived yolk proteins were significantly correlated to VPs with a maximum explanatory effect (R(2)) of 0.634. The concentration of the 57.22 kDa protein was significantly correlated with PR, the 77.97, 57.22, 22.22, 17.69, and 16.95 kDa proteins with HR and SR at 0 dph, the 22.22, 17.69, and 16.95 kDa proteins with SR at 3 and 5 dph. The results showed the importance of yolk protein content in the egg for embryogenesis and early larval development and indicated its potential use to predict egg quality in teleosts.