Antinociceptive Analysis of Natural Monoterpenes Eugenol, Menthol, Carvacrol and Thymol in a Zebrafish Larval Model.

In the last decade, a considerable number of studies have broadened our knowledge of the nociceptive mechanisms of pain, a global health problem in both humans and animals. The use of herbal compounds such as eugenol, menthol, thymol, and carvacrol as analgesic agents has accompanied the growing interest in this area, offering a possible solution for this complex problem. Here, we aimed to explore how these natural substances-at three different concentrations (2, 5 and 10 mg/L)-affect the pain responses in zebrafish (Danio rerio) larvae exposed to 0.05% acetic acid (AA) for 1 min. By analysing the activity of acetylcholinesterase (AChE), 5'-ectonucleotidase and NTPDases, as well as aversion and exploratory behaviours, it was observed that that although all substances were effective in counteracting the pain stimulus, the concentration range within which they do so might be very limited. Eugenol, despite its acknowledged properties in fish anaesthesia, failed to alleviate the pain stimulus at low concentrations. Contrastingly, menthol exhibited the most promising results at the lowest concentrations tested. Overall, it is concluded that menthol might be a good analgesic for this species, qualifying it as a substance of interest for prospective studies.

Guidelines on Developmental Toxicity Tests: Brief Insights.

Developmental toxicology is a constantly evolving research field which needs to attend to a complex underlying regulatory network. In order to ensure human health and environmental safety, new substances have to be tested for toxic effects on reproduction and development, before being commercialized. Traditional in vivo mammalian models represent the intricacy of human development and provide more adequately an assessment of the interaction of chemical compounds with the reproductive system. However, in the last years, the directives are to reduce the use of vertebrate animals, promoting their use only as a last resort. Consequently, the interest on the development and validation of alternative tests, able to cover the various aspects of the reproductive cycle, has significantly increased. Reproductive toxicity is probably the most difficult endpoint to be replaced by alternative assays, since it should provide information on mechanism interactions essential for female and male fertility and also knowledge on the animal development during the first phases of its life cycle. This complexity explains the slow progress in implementing alternative models for reproductive toxicity safety assays. Alternative test models may be based on in vitro systems and nonmammalian animal models. Many biological processes have been successfully addressed using in vitro models, opening the possibility to study the interference of teratogenic compounds. Their validation and implementation have lagged behind, in part because of difficulties in establishing their predictability. Nevertheless, the advance toward the process of validation is crucial to replace and reduce the use of living animals. Based on the present state of the art, it is not probable that such testing strategies will completely replace the need to assess reproductive toxicity in vivo in the near future, but they will contribute to reduce animal tests and will provide important information. In this chapter, the approved guidelines for standard methods and alternative methods, according to their regulatory and scientific status, are enumerated and briefly described.

Development Features on the Selection of Animal Models for Teratogenic Testing.

Today, the use of animal models from different species continues to represent a fundamental step in teratogenic testing, despite the increase in alternative solutions that provide an important screening to the enormous quantity of new substances that aim to enter the market every year. The maintenance of these models is due to the sharing of similar development processes with humans, and in this way they represent an important contribution to the safety in the use of the compounds tested. Furthermore, the application of advances in embryology to teratology, although hampered by the complexity of reproductive processes, continues to prove the importance of sensitivity during embryonic and fetal development to detect potential toxicity, inducing mortality/abortion and malformations.In this chapter, essential periods of development in different models are outlined, highlighting the similarities and differences between species, the advantages and disadvantages of each group, and specific sensitivities for teratogenic testing. Models can be divided into invertebrate species such as earthworms of the species Eisenia fetida/Eisenia andrei, Caenorhabditis elegans, and Drosophila melanogaster, allowing for rapid results and minor ethical concerns. Vertebrate nonmammalian species Xenopus laevis and Danio rerio are important models to assess teratogenic potential later in development with fewer ethical requirements. Finally, the mammalian species Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus, phylogenetically closer to humans, are essential for the assessment of complex specialized processes, occurring later in development.Regulations for the development of toxicology tests require the use of mammalian species. Although ethical concerns and costs limit their use in large-scale screening. On the other hand, invertebrate and vertebrate nonmammalian species are increasing as alternative animal models, as these organisms combine low cost, less ethical requirements, and culture conditions compatible with large-scale screening. Their main advantage is to allow high-throughput screening in a whole-animal context, in contrast to the in vitro techniques, not dependent on the prior identification of a target. Better knowledge of the development pathways of animal models will allow to maximize human translation and reduce the number of animals used, leading to a selection of compounds with an improved safety profile and reduced time to market for new drugs.

Protocol of Geometric Morphometrics for Teratogenicity Testing.

Geometric morphometrics (GM) enables a quantitative study of shapes and forms allowing the identification and characterization of teratogenic malformations. The GM methodology offers several advantages in comparison to traditional biometric methods, such as higher detail and precision analysis. In this chapter, we describe the recent application of the Procrustes method with ImageJ and MorphoJ programs in the characterization of developmental malformations. With this methodology, we are a step closer to being able to assign molecular pathways or unique signatures to a specific teratogen according to the produced phenotypes or to cluster unknown compounds.

Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

Non-Invasive Biomarkers in Saliva and Eye Infrared Thermography to Assess the Stress Response of Calves during Transport.

Animal transport is currently a stressful procedure. Therefore, animal-based indicators are needed for reliable and non-invasive welfare assessment. Saliva is a biospecimen with potential validity for the determination of cortisol and oxidative stress, although its use to assess calf welfare during transport has never been tested. Similarly, the applicability and reliability of infrared thermography to assess temperature change during calves' transport have never been evaluated. These objectives were outlined following the known and growing need to identify non-invasive methodologies for stress assessment in bovines. This study was conducted on 20 calves of the Arouquesa autochthone breed, at about nine months of age, during their transport to slaughter. For each animal, saliva samples and thermographic images of the eye were collected at three time points: before transport, after transport, and at slaughter. The saliva was then processed to measure cortisol levels and oxidative stress parameters (reactive oxygen species, thiobarbituric acid reactive substance, carbonyls, and advanced oxidation protein products), and the images were analyzed using FLIR Tools+ software. There was an increase in cortisol concentration and oxidative stress parameters (reactive oxygen species, thiobarbituric acid reactive substance, carbonyls, and advanced oxidation protein products) in saliva after transport. An increase in eye temperature triggered by transport was also observed. The cortisol and eye temperature results at slaughter were returned to values similar to those before transport; however, the values of oxidative stress remained increased (mainly TBARS values). These non-invasive techniques seem to be reliable indicators of stress in bovine transport, and oxidative stress parameters in saliva may be a persistent marker for welfare assessment.

Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects of Thymol and 24-Epibrassinolide in Zebrafish Larvae.

Thymol (THY) and 24-epibrassinolide (24-EPI) are two examples of plant-based products with promising therapeutic effects. In this study, we investigated the anti-inflammatory, antioxidant and anti-apoptotic effects of the THY and 24-EPI. We used zebrafish (Danio rerio) larvae transgenic line (Tg(mpxGFP)i114) to evaluate the recruitment of neutrophils as an inflammatory marker to the site of injury after tail fin amputation. In another experiment, wild-type AB larvae were exposed to a well known pro-inflammatory substance, copper (CuSO4), and then exposed for 4 h to THY, 24-EPI or diclofenac (DIC), a known anti-inflammatory drug. In this model, the antioxidant (levels of reactive oxygen species-ROS) and anti-apoptotic (cell death) effects were evaluated in vivo, as well as biochemical parameters such as the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), the biotransformation activity of glutathione-S-transferase, the levels of glutathione reduced and oxidated, lipid peroxidation, acetylcholinesterase activity, lactate dehydrogenase activity, and levels of nitric acid (NO). Both compounds decreased the recruitment of neutrophils in Tg(mpxGFP)i114, as well as showed in vivo antioxidant effects by reducing ROS production and anti-apoptotic effects in addition to a decrease in NO compared to CuSO4. The observed data substantiate the potential of the natural compounds THY and 24-EPI as anti-inflammatory and antioxidant agents in this species. These results support the need for further research to understand the molecular pathways involved, particularly their effect on NO.

Protective effects of 24-epibrassinolide against the 6-OHDA zebrafish model of Parkinson's disease.

The molecular processes behind Parkinson's disease (PD) remain under debate although mitochondrial oxidative stress generation has been proposed as a fundamental contributor. In this context, different brassinosteroids have shown neuroprotective action hampering oxidative stress. This study determined the effects of 24-Epibrassinolide (24-EPI) against 6-hydroxydopamine- (6-OHDA-) induced toxicity using the zebrafish embryonic model. Embryos were exposed to 250 µM 6-OHDA or co-exposed to 24-EPI (0.01, 0.1, and 1 µM) for 3 days, starting at 48 h post-fertilization (hpf). During the experimental period, developmental parameters were assessed. At 120 hpf, larvae were tested for behavioural phenotypes with different biochemical biomarkers and tyrosine hydroxylase- (TH-) reactive neurons being also assessed. Exposure to 6-OHDA induced a decrease in body length while no other morphological phenotypes were noticed. A significant decrease in TH-neurons immunofluorescence, a decreased locomotion (speed and distance moved), and an increased absolute angle were found in 6-OHDA-exposed embryos. These outcomes were rescuable by the co-exposure with 24-EPI. Surprisingly, the direct effects of 6-OHDA on reactive oxygen species (ROS) were not observed in the present study supporting the involvement of other molecular pathways in the 6-OHDA-induced effects during embryonic development. Overall, the results obtained confirm PD-like symptoms induced by 6-OHDA during embryonic development which were reverted by 24-EPI. Although antioxidative signalling pathways deserve further scrutiny, the findings support the further investigation of 24-EPI neuroprotective effects.

Study on the antineoplastic and toxicological effects of pomegranate (Punica granatum L.) leaf infusion using the K14-HPV16 transgenic mouse model.

Punica granatum L. (pomegranate) has been used in functional foods due to its various health benefits. However, the in vivo biological potential of its leaf remains little known. This study has aimed to characterize the antineoplastic and toxicological properties of using pomegranate leaf infusion (PLI) on transgenic mice carrying human papillomavirus (HPV) type 16 oncogenes. Thirty-eight mice were divided into 3 wild-type (WT) and 3 transgenic (HPV) groups, with exposure to 0.5% PLI, 1.0% PLI, and water. The animals' body weight, drink and food consumption were recorded. Internal organs, skin samples and intracardiac blood were collected to evaluate toxicological parameters, neoplastic lesions and oxidative stress. The results indicated that PLI was safe as no mortality, no behavioural disorders and no significant differences in the levels of microhematocrit, serum biochemical markers, internal organ histology, and oxidative stress was found among the WT groups. Histological analysis revealed that HPV animals that consumed PLI exhibited reduced hepatic, renal and cutaneous lesions compared with the HPV control group. Low-dose PLI consumption significantly diminished renal hydronephrosis lesions and relieved dysplasia and carcinoma lesions in the chest skin. Oxidative stress analysis showed that low-dose PLI consumption may have more benefits than high-dose PLI. These results suggest that oral administration of PLI has the potential to alleviate non-neoplastic and neoplastic lesions against HPV16-induced organ and skin injuries, though this requires further scientific research studies.

Brassica oleracea Var italica by-Products Prevent Lipid Accumulation and Cell Death in a Liver Cell Model of Lipid Toxicity.

Obesity, a rising concern in the Eastern world, encompasses several co-morbidities, namely non-alcoholic fatty liver disease (NAFLD). Potential natural-based interventions to decrease the burden of obesity complications are being investigated. Many of the edible parts of plants are not sold for consumption and end up as massive waste, losing nutritional potential. In fact, a sizeable amount of waste is generated within the different steps of the food supply chain, representing a massive loss of both plant material and natural resources. A good example is Brassica by-products (BBPs). The objective of this work was to investigate the effect of three different extracts from broccoli (Brassica oleracea var italica) by-products in an in vitro model of free fatty acid (FFA)-induced lipotoxicity using human hepatoma HepG2 cells. Broccoli leaf, stalk, and inflorescence extracts induced a dose-dependent decrease in the cell viability of HepG2 cells. However, the maximal non-lethal concentrations of leaves, stalks, and inflorescences (10 µg/mL) did not compromise mitochondrial function or neutral lipid accumulation in HepG2 cells. The extracts significantly decreased FFA-induced lipid accumulation in HepG2 cells either in a co-incubation or pre-incubation strategy. The broccoli extracts' capacity to prevent the FFA-induced decrease in catalase activity in HepG2 may explain the observed effects.

Mitigation of nicotine-induced developmental effects by 24-epibrassinolide in zebrafish.

Nicotine is a highly addictive substance that can cause teratogenic impacts in the embryo through redox-dependent pathways. As antioxidants, naturally occurring chemicals can protect cells from redox imbalance. The purpose of this study was to evaluate the effectiveness of 24-epibrassinolide (24-EPI), a natural brassinosteroid with well-known antioxidant properties, in protecting zebrafish embryos against nicotine's teratogenic effects. For 96 h, embryos (2 h post-fertilization - hpf) were exposed to 100 µM nicotine, co-exposed with 24-EPI (0.01, 0.1, and 1 µM), and 24-EPI alone (1 µM). Lethal and sublethal developmental characteristics were evaluated during exposure. Biochemical tests were performed at the conclusion of the exposure, and distinct behavioural paradigms were analysed 24 h later. Nicotine exposure resulted in a higher proportion of larvae with deformities, which were decreased following co-exposure to 24-EPI. Nicotine exposure also caused an increase in oxidative stress as observed by the increased activity of superoxide dismutase and catalase accompanied by an increase in the malondialdehyde levels. Besides, metabolic changes were noticed as observed by the increased lactate dehydrogenase activity that were hypothesised to be associated to nicotine-induced hypoxia which may be responsible for the increased oxidative damage. In addition, locomotor deficits were observed as well as a decrease in the acetylcholinesterase activity denoting nicotine-induced cognitive dysfunction. However, co-exposure to 24-EPI alleviated behavioural deficits and improved nicotine-induced emotional states. Overall, and although further studies are required to clarify these effects, 24-EPI showed promising ameliorative properties against the teratogenic effects induced by nicotine.

Growth Performance, Carcass and Meat Traits of Autochthonous Arouquesa Weaners Raised on Traditional and Improved Feeding Systems.

Arouquesa is an autochthonous bovine breed known for its Arouquesa PDO beef labeling. There are several production systems under the definition of PDO labeling. This study aimed to compare the effect of different production systems on carcass and meat traits for the Arouquesa breed. Two trials differing in diet and weaning age were conducted. The first trial included a TF group fed the traditional way and weaned at 9 months; a TF + S1 group, equal to TF, but with a starter supplement; and finally, a S1 + S2 group that was fed with a starter and a growth supplement and weaned at 5 months. The second trial was composed of a TF + S3 group fed like the TF + S1 group but reared until 12 months with a finishing supplement, and finally, the S3 group fed like the S1 + S2 group but reared until 12 months. In the first trial, the TF + S1 and S1 + S2 groups showed higher final live weight and average daily gain. In the second trial, we observed differences in the subcutaneous fat that was higher in the S3 group. Regarding meat traits, we observed differences in exudative and cooking losses in the first trial. In general, supplementation improved meat production without affecting meat quality parameters.

Evaluation of the developmental effects of a glyphosate-based herbicide complexed with copper, zinc, and manganese metals in zebrafish.

The use of glyphosate-based herbicides (GBH) has increased dramatically, being currently the most used herbicides worldwide. Glyphosate acts as a chelating agent, capable of chelate metals. The synergistic effects of metals and agrochemicals may pose an environmental problem as they have been shown to induce neurological abnormalities and behavioural changes in aquatic species. However, as their ecotoxicity effects are poorly understood, evaluating the impacts of GBH complexed with metals is an ecological priority. The main objective of the study was to evaluate the potentially toxic effects caused by exposure to a GBH (1 µg a.i. mL-1), alone or complexed with metals (Copper, Manganese, and Zinc (100 µg L-1)), at environmentally relevant concentrations, during the early period of zebrafish (Danio rerio) embryo development (96 h post-fertilization), a promising model for in vivo developmental studies. To clarify the mechanisms of toxicity involved, lethal and sublethal development endpoints were assessed. At the end of the exposure, biochemical and cell death parameters were evaluated and, 24 h later, different behavioural responses were assessed. The results showed that metals induced higher levels of toxicity. Copper caused high mortality, low hatching, malformations, and changes in biochemical parameters, such as decreased Catalase (CAT) activity, increased Glutathione Peroxidase (GPx), Glutathione S-Transferase (GST), reduced Glutathione (GSH) and decreased Acetylcholinesterase (AChE) activity, also inducing apoptosis and changes in larval behaviour. Manganese increased the activity of SODs enzymes. Zinc increased mortality, reactive oxygen species (ROS) levels, superoxide dismutase activity (SODs) and caused a decrease in AChE activity. Embryos/larvae exposed to the combination of GBH/Metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with the mechanisms of metal toxicity at the biochemical, physiological, and behavioural levels of zebrafish.

Beneficial Effects of Broccoli (Brassica oleracea var italica) By-products in Diet-induced Obese Mice.

BACKGROUND/AIM: Obesity currently affects the whole world, with greater incidence in high-income countries, with vast economic and social costs. Broccoli harvest generates many by-products equally rich in bioactive compounds with potential anti-obesity effects. This study aimed to evaluate the anti-obesity effects of broccoli by-products flour (BF) in obese mice. MATERIALS AND METHODS: A commercial high-fat diet formulation (representing a Western diet) was used to induce obesity in mice. BF (0.67% or 1.34% weight/weight) was incorporated as a chemoprevention compound into a control and a hypercholesterolemic diet, at two different concentrations, and fed for 14 weeks to C57BL/6J mice. For a therapeutic approach, two groups were fed with the hypercholesterolemic diet for 10 weeks, and then fed with BF-supplemented diets in the last 4 weeks of the study. RESULTS: BF supplementation helped to maintain a lower body weight, reduced adipose tissue accumulation, and enhanced the basal activity of superoxide dismutase and glutathione S-transferase. Although BF supplementation tended to reduce the relative liver weight increased by the Western diet, the differences were not significant. CONCLUSION: BF appears to have a beneficial effect in preventing weight gain and fat accumulation induced by hypercholesterolemic diets.

The Effect of Weaning and Slaughter Age on the Physicochemical and Sensory Characteristics of Arouquesa Beef-A PDO Portuguese Meat.

(1) Background: Autochthonous breeds meat is well accepted due to its sensory characteristics, perceived low environmental impact, and animal welfare. We aimed to evaluate the effect of weaning and slaughter age on the physicochemical and sensory characteristics of Arouquesa, a Portuguese Protected Designation of Origin (PDO) meat and to evaluate the psychological effect of knowing the weaning age on the consumer's hedonic evaluation. (2) Methods: Meat from 26 animals was assigned to 4 groups, with combinations of weaning (W) at 9 or 5 months and slaughter (S) at 9 or 12 months: W9-S9, W9-S12, W5-S9, and W5-S12. The meat was analysed for pH24h, colour (L*a*b*), cooking losses and shear force. A Check All that Apply test was made with 70 consumers; they were also asked to punctuate the hedonic appreciation of anonymous and weaning age-identified meat. (3) Results: W9-S9 were more tender, had lower shear force, and was juicier than W5-S9. When animals were slaughtered at 12 months, there were no differences in the physicochemical and sensory characteristics between the weaning ages. The effect of information about the weaning age influences the consumer's hedonic evaluation, as revealed by the comparison between the anonymous and identified samples. (4) Later weaning resulted in more tender meat when the slaughter was at 9 months and positively impacted consumer perception.

First Study of Pharingomyia picta and Cephanemyia auribarbis in Wild Populations of Red Deer (Cervus elaphus) in Portugal.

Nasopharyngeal myiasis in red deer has never been studied in Portugal. For this reason, from December 2015 to February 2016 a study was derived on hunted red deer in Idanha-a-Nova county to evaluate the Diptera larvae presence. During the initial examination on the spot, the larvae was analysed at the nasopharynx. When present, larvae were collected for further species identification. The infestation prevalence was 50.6 ± 7.61% (CI 95%), and the mean parasitism intensity was 11.38 larvae per affected host. Two larvae species were found, Pharyngomyia picta and Cephenemyia auribarbis, both on single or concomitant infection, with the first species having a higher prevalence and mean infestation intensity than the second. The larvae's prevalence was significantly higher (X2 = 4.35; p = 0.0370) in males (62.16 ± 11.05%) than in females (41.30 ± 10.06%). Within age groups, younger animals showed a higher prevalence. This study shows the presence of P. picta and C. auribarbis larvae in the wild red deer population in Portugal for the first time. The high parasitic prevalence and mean intensity highlight the importance of this parasitosis in this wild species. For this reason, more scientific research is required to accurately assess its prevalence in other geographic areas, and evaluate the risk factors as well effects of this parasitosis on the deer population.

Behavioural impairment and oxidative stress by acute exposure of zebrafish to a commercial formulation of tebuconazole.

Tebuconazole is a systemic follicular fungicide known to cause diverse problems in non-target organisms namely associated to the pure active ingredient. As such, the objective of this work was to evaluate developmental changes induced by a tebuconazole commercial formulation to a non-target animal model. Zebrafish embryos at ± 2 h post-fertilization were exposed to tebuconazole wettable powder concentrations (0.05, 0.5 and 5 mg L-1) for 96 h with developmental toxicity assessed throughout the exposure period and biochemical parameters evaluated at the end of the exposure. Behavioural assessment (spatial exploration and response to stimuli) was conducted 24 h after the end of the exposure. While no developmental and physiological alterations were observed, exposure to tebuconazole resulted in an increased generation of reactive oxidative species at the 0.05 and 0.5 mg L-1 concentrations and a decreased GPx activity at the 0.5 mg L-1 concentration suggesting a potential protection mechanism. There was also a change in the avoidance-escape behaviour supporting an anxiolytic effect suggesting possible alterations in the central nervous system development demanding further studies.

Effect of a Sub-Chronic Oral Exposure of Broccoli (Brassica oleracea L. Var. Italica) By-Products Flour on the Physiological Parameters of FVB/N Mice: A Pilot Study.

Brassica by-products are a source of natural bioactive molecules such as glucosinolates and isothiocyanates, with potential applications in the nutraceutical and functional food industries. However, the effects of oral sub-chronic exposure to broccoli by-product flour (BF) have not yet been evaluated. The objective of this pilot study was to analyse the effects of BF intake in the physiological parameters of FVB/N mice fed a 6.7% BF-supplemented diet for 21 days. Glucosinolates and their derivatives were also quantified in plasma and urine. BF supplementation significantly decreased (p < 0.05) the accumulation of perirenal adipose tissue. Furthermore, mice supplemented with BF showed significantly lower (p < 0.01) microhematocrit values than control animals, but no impact on the general genotoxicological status nor relevant toxic effects on the liver and kidney were observed. Concerning hepatic and renal antioxidant response, BF supplementation induced a significant increase (p < 0.05) in the liver glutathione S-transferase (GST) levels. In BF-supplemented mice, plasma analysis revealed the presence of the glucosinolates glucobrassicin and glucoerucin, and the isothiocyanates sulforaphane and indole-3-carbinol. Overall, these results show that daily intake of a high dose of BF during three weeks is safe, and enables the bioavailability of beneficial glucosinolates and isothiocyanates. These results allow further testing of the benefits of this BF in animal models of disease, knowing that exposure of up to 6.7% BF does not present relevant toxicity.

MS-222 and Propofol Sedation during and after the Simulated Transport of Nile tilapia (Oreochromis niloticus).

The use of anesthetics has been suggested as a strategy to hamper live fish transport-induced stress. Still, there is insufficient data available on the use of alternative anesthetics to MS-222. This study investigated the use of propofol to mitigate stress in Nile tilapia (Oreochromis niloticus, 143.8 ± 20.9 g and 20.4 ± 0.9 cm) during a 6 h simulated transport. Individuals (n = 7) were divided into three groups: control, 40 mg L-1 MS-222, and 0.8 mg L-1 propofol. A naïve group non-transported was also considered. During the 6 h transport and 24 h after, the response to external stimuli, opercular movements, water quality parameters, behavior, blood hematology and other physiological values, the histopathology of the gills, the quality of the fillet, and oxidative-stress changes in gills, muscle, brain, and liver were evaluated. Propofol increased swimming activity of fish but decreased opercular movements and responses to external stimuli, indicating oscillations of the sedation depth. Water pH and glucose levels increased, while hematocrit (HCT) and lactate decreased in propofol groups at 6 h. At this time-point, MS-222 also induced a decrease in the HCT and lactate levels while increasing cortisol levels. Despite these effects, the stress-related behaviors lessened with anesthetics compared to the control group. After the recovery period, physiological responses normalized in animals from both anesthetic groups, but the control still had high cortisol levels. Overall, propofol is a good alternative for the transportation of this species, showing efficient sedation without compromising health or fillet quality. However, further pharmacodynamics and pharmacokinetics knowledge is required to support its use in aquaculture settings.

Inflammatory, Oxidative Stress, and Apoptosis Effects in Zebrafish Larvae after Rapid Exposure to a Commercial Glyphosate Formulation.

Glyphosate-based herbicides (GBH) are the most used herbicides in the world, carrying potentially adverse consequences to the environment and non-target species due to their massive and inadequate use. This study aimed to evaluate the effects of acute exposure to a commercial formulation of glyphosate, Roundup® Flex (RF), at environmentally relevant and higher concentrations in zebrafish larvae through the assessment of the inflammatory, oxidative stress and cell death response. Transgenic Tg(mpxGFP)i114 and wild-type (WT) zebrafish larvae (72 h post-fertilisation) were exposed to 1, 5, and 10 µg mL-1 of RF (based on the active ingredient concentration) for 4 h 30 min. A concentration of 2.5 µg mL-1 CuSO4 was used as a positive control. Copper sulphate exposure showed effectiveness in enhancing the inflammatory profile by increasing the number of neutrophils, nitric oxide (NO) levels, reactive oxygen species (ROS), and cell death. None of the RF concentrations tested showed changes in the number of neutrophils and NO. However, the concentration of 10 µg a.i. mL-1 was able to induce an increase in ROS levels and cell death. The activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), the biotransformation activity, the levels of reduced (GSH) and oxidised (GSSG) glutathione, lipid peroxidation (LPO), lactate dehydrogenase (LDH), and acetylcholinesterase (AChE) were similar among groups. Overall, the evidence may suggest toxicological effects are dependent on the concentration of RF, although at concentrations that are not routinely detected in the environment. Additional studies are needed to better understand the underlying molecular mechanisms of this formulation.