Antonio Sergio Varela Junior

Toxic effects of the herbicide Roundup in the guppy Poecilia vivipara acclimated to fresh water

Although it is believed that glyphosate-based herbicides are relatively nontoxic to humans, its broad use in agriculture and consequent contamination of aquatic systems is a concern. In the present study, reproductive (sperm quality) and biochemical parameters (acetylcholinesterase and glutathione S-transferase activity, lipoperoxidation, and antioxidant capacity against peroxyl radicals) were evaluated in adult guppies (Poecilia vivipara) acclimated to fresh water and exposed (96 h) to environmentally realistic concentrations of glyphosate (130 and 700 μg L(-1)) as the commercial formulation Roundup. Male guppies exposed to Roundup showed a poorer sperm quality, measured as reduced plasmatic membrane integrity, mitochondrial functionality, DNA integrity, motility, motility period and concentration of spermatic cells, than those kept under control condition (no Roundup addition to the water). Most of the spermatic parameters analyzed showed strong association to each other, which may help to understand the mechanisms underlying the observed reduction in sperm quality. Exposure to Roundup did not alter the biochemical parameters analyzed, though differences between genders were observed and deserve further investigations. Findings from the present study suggest that exposure to environmentally relevant concentrations of Roundup may negatively affect at long-term the reproduction of P. vivipara, with consequent changes in fish populations inhabiting environments contaminated with the herbicide.

Effect of glyphosate on the sperm quality of zebrafish Danio rerio.

Glyphosate is a systemic, non-selective herbicide widely used in agriculture worldwide. It acts as an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase by interrupting the synthesis of essential aromatic amino acids. This pathway is not present in animals, although some studies have shown that the herbicide glyphosate can affect fish reproduction. In this study, the effect of glyphosate on sperm quality of the fish Danio rerio was investigated after 24 and 96 h of exposure at concentrations of 5mg/L and 10mg/L. The spermatic cell concentration, sperm motility and motility period were measured employing conventional microscopy. The mitochondrial functionality, membrane integrity and DNA integrity were measured by fluorescence microscopy using specific probes. No significant differences in sperm concentration were observed; however, sperm motility and the motility period were reduced after exposure to both glyphosate concentrations during both exposure periods. The mitochondrial functionality and membrane and DNA integrity were also reduced at the highest concentration during both exposure periods. The results showed that glyphosate can induce harmful effects on reproductive parameters in D. rerio and that this change would reduce the fertility rate of these animals.

Effects of exposure to cadmium in sperm cells of zebrafish, Danio rerio

Cadmium is a natural element found in the earth’s crust; it is usually associated with other metals, but due to the impacts caused by human activity, its concentration has increased in the aquatic environment. This metal may damage aquatic animal reproduction, decreasing the rate of fertilization of organisms such as fish. Thus, this study aimed to evaluate the in vitro toxicity of different concentrations of cadmium (0 (control), 0.5, 5, and 10 μg/L) using sperm cells of model organism zebrafish, Danio rerio. Structural parameters, including integrity and fluidity of the plasma membrane, concentration of oxygen species, mitochondrial function and DNA fragmentation were measured by flow cytometry. The following sperm movement parameters were also measured using the computer assisted sperm analysis (CASA) system: motility, time of motility, curvilinear velocity, average path velocity and straight line velocity in μm/s. Significant effects were observed on path speed, straight speed, curvilinear velocity, motility time, progressive and total motility, and plasma and DNA integrity. The results showed that cadmium can negatively affect some reproductive parameters in D. rerio, which may reduce the fertility rate of these animals.

GH overexpression decreases spermatic parameters and reproductive success in two-years-old transgenic zebrafish males

Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis.

Melatonin delivery by nanocapsules during in vitro bovine oocyte maturation decreased the reactive oxygen species of oocytes and embryos

In this work, a promising approach to increase the advantageous properties of melatonin through its encapsulation into lipid-core nanocapsules (LNC) was examined. Oocytes were treated during in vitro maturation with non-encapsulated melatonin (Mel), melatonin-loaded lipid-core nanocapsules (Mel-LNC), and unloaded LNC. Cytotoxicity, meiotic maturation rate, development to the blastocyst stage, reactive oxygen species (ROS) and glutathione levels, mean cell number and apoptotic cell/blastocyst, and mRNA quantification were evaluated. Both Mel and Mel-LNC enhanced in vitro embryo production, however, Mel-LNC proved to be more effective at decreasing ROS levels and the apoptotic cell number/blastocyst, increasing the cleavage and blastocyst rates, up-regulating the GPX1 and SOD2 genes, and down-regulating the CASP3 and BAX genes. Mel-LNC could penetrate into oocytes and remain inside the cells until they reach the blastocyst stage. In conclusion, when melatonin was encapsulated in LNC and applied during in vitro oocyte maturation, some quality aspects of the blastocysts were improved.

The reproductive biology of the plata pompano, Trachinotus marginatus (Teleostei: Carangidae), in southern Brazil

The Plata pompano, Trachinotus marginatus (Cuvier, 1832), is an endemic carangid of the Southwestern Atlantic Ocean, occurring from Rio de Janeiro to Uruguay. This study describes the reproductive period, spawning type, the size at first gonadal maturation and the length-weight relationship of individuals sampled from landings of the artisanal and commercial fishing fleets in Rio Grande that operate along the coast of Rio Grande do Sul state, southern Brazil (~ 32oS) to the Uruguayan border (~ 34oS). Monthly collections from September 2008 through January 2010 yielded 274 individuals ranging from 142 to 444 mm in total length (TL). The gonadal development stages were defined according to the histological examination of the ovaries and the testes. The relationship between TL (mm) and total weight TW (g) was statistically different between males (TW = 0.000463*TL2.7655) and females (TW = 0.000361*TL2.8131), showing negative allometric growth for both sexes. The sizes at first maturity were 187.2 mm and 254.9 mm for females and males, respectively. The presence of two modal groups of oocyte diameters suggested that total spawning occurred. The interpretation of the monthly variations of the condition factor and gonadosomatic index, which are associated with higher frequencies of the more advanced stages of gonadal development, identified the spring and summer months as the reproductive period, with a peak in the reproductive activity during November and January. These results suggested that the species has an opportunistic reproductive strategy.

Fluorescent transgenic zebrafish as a biosensor for growth-related effects of methyl parathion

Transgenic fish models are potential alternative subjects in toxicological studies, since they can provide in vivo information on the deleterious effects of different substances. Here, we used a transgenic zebrafish (Danio rerio) lineage, which expresses a destabilized fluorescent protein (DsRED) driven by the myosin light chain promoter (Mylz2), in order to propose a new research tool for environmental biomonitoring. For validating the MYO-RED lineage, we exposed fish to the organophosphorated pesticide methyl parathion (MP). The effect of MP on fish growth was assessed by evaluating weight, length, condition factor and muscle fiber diameter. All factors suffered reduction at both tested concentrations (0.13μM and 13μM of MP). Similarly, fluorescence intensity decreased in a concentration-dependent manner, suggesting muscle protein catabolism. However, DsRED gene expression lowered only at the higher MP concentration. Results indicate that the MYO-RED transgenic zebrafish is an interesting model for detecting the growth-related effects of pollutants. Destabilized proteins such as reporter genes are apparently sensitive biomarkers, since effects were observed even at the lower, environmentally acceptable concentration. Therefore, this transgenic fish is a promising candidate model for sensitive, fast, and easy environmental monitoring.

Concentração de lactato de cálcio e tempo de incubação sobre a capacidade de adesão e penetração de espermatozoides suínos na membrana perivitelina do ovo da galinha

This study tested the effect of different calcium lactate concentrations and incubation periods on the binding and penetration capacity of swine sperm to the perivitelline layers (PL) of chicken eggs, with the expectation of developing a test to estimate the potential fertility of boars used as sperm donors. In the first experiment, semen samples (n=12) were incubated with TCM medium ant two calcium lactate levels: 1.1 and 2.2µg mL-1. After the set up of fixed calcium lactate level (1.1µg mL-1), the second experiment compared three incubation periods: 10, 15 and 20min. The observed variables were binding rate (BR) and number of bound sperm (NB) in the outer PL, and penetration rate (PR) and number of holes (NH) in the inner PL. The BR to the outer MP was 100%, regardless of the tested concentrations and incubation periods. The NB to the outer perivitelline layer and the PR and the NH in the inner perivitelline layer were greater with 1.1µg mL-1 calcium lactate than 2.2µg mL-1. The NB in the outer PL was greater with 20min incubation than with shorter period (P<0.05). Using 10min incubation, there was no penetration in the inner PL, but, with incubation for 20min, the PR and the NH in the inner PL were greater than those with incubation for 15 min (P<0.05). Using 1.1µg mL-1 calcium lactate and incubation for 20min, the in vitro test using PL can be efficiently conducted to assess important steps of the fertilization process.

Effects of chitosan-coated lipid-core nanocapsules on bovine sperm cells

Toxicology studies have a pivotal role for selection of new nanosystems. As lipid-core nanocapsules (LNC) rise as a potential system not only for drug delivery but also for immunotherapy and gene therapy, the demand for models of toxic screening increases, and sperm arises as a promising model due to the easiness to evaluate its viability parameters. LNCs were coated with chitosan, chitosan-coated lipid-core nanocapsules (LNC-CS), in order to modify the nanocapsule surface. We evaluated the toxicity of LNC and LNC-CS after incubation with bovine sperm in different concentrations (2.5%, 5%, 10%, 20%, 40% and 80%) (v/v) and periods of exposure (0h and 1h). CASA parameters and flow cytometry assays were performed to assess LNC and LNC-CS effects. The results corroborated with previous studies indicating that there is no toxicity from LNCs and LNC-CS below 40% (v/v) concentration.

Histopathological Analysis of UVB and IR Interaction in Rat Skin

To determine the chronic skin effects caused by the interaction of infrared and ultraviolet B radiations, male Rattus norvegicus (Wistar) (2 months old) were exposed for 15 days to infrared radiation (600–1500 nm, with a peak at 1000 nm, n = 12) for 30 min (1080 J cm−2) (IRo); to ultraviolet B radiation (peak emission at 313 nm, n = 9) for 90 min (55.08 J cm−2) (UVB); to infrared radiation followed after 90 min by ultraviolet B (n = 6) (IRUVB) and to ultraviolet B followed after 90 min by infrared radiation (n = 9) (UVBIR). Skin samples were collected and histopathological analysis showed the presence of acanthosis, parakeratotic and orthokeratotic hyperkeratosis, intraepidermal pustules, keratin pearls, detachment of epidermis, collagen necrosis, inflammatory infiltrate, vasodilation, basal cell vacuolization and superficial dermis degeneration both in UVB and UVBIR treatments. IRUVB animals showed the same characteristics as above except for parakeratotic hyperkeratosis, keratin pearls and superficial dermis degeneration. To conclude, infrared radiation exposure after ultraviolet B irradiation increases skin damage without protecting the tissue, while infrared radiation exposure before ultraviolet B irradiation showed a protective effect against ultraviolet skin damage.