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Edwardsiella tarda is a crucial pathogenic bacterium in tropical aquaculture. This bacterium was recently isolated from tambaqui (Colossoma macropomum), a commercially important fish species in Brazil. This study assessed the antimicrobial susceptibility, pathogenicity, and genetic diversity of the tambaqui-derived E. tarda isolates. Fourteen bacterial isolates isolated from tambaqui were identified as E. tarda by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and dnaJ gene sequencing. Antimicrobial susceptibility tests were conducted against seven drugs using the disc diffusion assay. The pathogenicity test conducted by intraperitoneal injection of 2.4 × 107 colony-forming units (CFU) fish-1 of E. tarda (ED38-17) into tambaqui juveniles eventually revealed that neither clinical signs nor death were present. However, splenomegaly and whitish areas in the spleen and kidneys were observed. The histological investigation also revealed granulomatous splenitis, nephritis, and hepatitis occurring internally. Repetitive extragenic palindromic-PCR fingerprinting separated the 14 isolates into three genetic groups. The antibiogram revealed that all E. tarda isolates were wild-type (WT) to florfenicol (FLO), norfloxacin (NOR), neomycin (NEO), erythromycin (ERY), and oxytetracycline (OXY); however, some were non-wild-type to sulfamethoxazole/trimethoprim (7.1%) and amoxicillin (21.4%). Therefore, through experimental infection, E. tarda ED38-17 could induce pathogenic effects in C. macropomum. Additionally, three distinct genetic types were found, and the E. tarda isolates were WT to FLO, NOR, NEO, ERY, and OXY. These findings raise awareness of a bacteria causing unseen lesions, a pathogen that will potentially impact tambaqui aquaculture in the future.

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A preservation protocol has not been established for Colossoma macropomum oocytes, and its development may improve the production and breeding programs of this South American fish species. Thus, this study aimed to determine the effect of different methods and protocols for the preservation of C. macropomum oocytes. Seven experiments were conducted throughout the breeding season of this species. The oocytes were collected and stored in sterile conditions. Preserved oocytes were subjected to storage in the following treatments: room temperature (RT, 27 °C), centrifugation followed by ovarian fluid removal (Cen), vacuum (Vac), chilled temperature (ChT), centrifugation and vacuum (CV), vacuum and chilled temperature (VChT), and centrifugation, vacuum, and chilled temperature (CVChT) in dry sterilized plastic containers and plastic bags. Chilled storage was tested at 4 and 13 °C. Fertilization and hatching rates were assessed at 0, 30, 60, 90, and 120 min after stripping (MAS) for preservation protocols. The larval malformation rate was analyzed at 0 and 30 MAS for RT and ChT. Quantitation and identification (by mean of MALDI-TOF MS) of bacteria were performed at 0, 60, 90, and 120 MAS, and scanning electron microscopy (SEM) analyses were carried out at 0, 60, and 90 MAS. The fertilization and hatching rates decreased over preservation time and breeding season. RT samples fertilized at 0, 30, and 60 MAS yielded similar fertilization rates at both the beginning and end of the season. By the end of the season, oocytes from treatment ChT at 13 °C 30 MAS yielded higher fertilization and hatching rates, and a lower percentage of larvae malformation than RT 30 MAS. The treatment ChT at 4 °C triggered low a fertilization rate. The treatments ChT (13 °C) and Cen provided good fertilization rate when used alone and with other approaches, i.e., treatments VChT, CV, and CVChT. The treatment Vac presented inconsistent results, so no conclusion could be made. Bacterial colony counts were low (10-1.6 × 105 CFU-mL-1), and a total of 18 bacteria species were identified in all batches analyzed; however, the treatments did not influence the number of bacteria. C. macropomum female breeders presented distinct bacteria species in their oocytes and the presence of bacteria did not impair oocyte quality until 120 MAS. Moreover, SEM analyses showed that the micropyle was not occluded during oocyte storage, and ovarian fluid was observed on the surface of chilled oocytes. Therefore, Colossoma macropomum oocytes could be preserved under chilled storage at 13 °C for 30 min throughout its breeding season.

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The cryopreservation process causes damage to sperm structures and supplementation of the cryoprotective medium is an alternative to reduce these damages. The aim of this study was to determine the effect of melatonin supplementation on post-thaw sperm quality in Prochilodus lineatus. The cryoprotective medium was supplemented with 2.00, 2.75, 3.50, and 4.25 mM melatonin, and the control group (without melatonin). Sperm motility parameters, membrane integrity, sperm morphology, oxidative stress (lipid peroxidation and enzymatic activity), and fertilization capacity were analyzed in the post-thaw sperm. Samples cryopreserved with 2.00 mM melatonin yielded higher sperm motility rate than other treatments with the addition of melatonin. Sperm curvilinear velocity (VCL) and average path velocity (VAP) were higher in samples containing 2.00 mM melatonin than in other treatments. Samples from control and with 2.00 mM melatonin presented higher membrane integrity and morphological normality than samples containing 4.25 and 2.75 mM melatonin, respectively. Regarding oxidative stress, lower lipid peroxidation (LPO) occurred in 2.75 and 3.50 mM melatonin, compared to control. While higher enzyme activity of catalase (CAT) occurred in the control than in other treatments, no differences were observed in the activity of superoxide dismutase (SOD). Higher fertilization and hatching rates occurred at 2.75 mM melatonin compared to 4.25 mM. Although no significant differences were observed in LPO between the control and samples supplemented with 2 mM melatonin, it was observed that this dosage of melatonin allowed higher VCL and VSL and reduced values of CAT. However, as there are no differences in motility and fertilization rates between the control and the 2 mM concentration, it is suggested that further studies be carried out with lower concentrations of melatonin in order to determine its effectiveness as an antioxidant in the sperm of this species.

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Melatonin plays a fundamental homeostatic role in basic biological functions, and an anti-stress role has been also proposed for this hormone. This study aimed to evaluate hormonal, enzymatic and behavioral parameters of zebrafish that received administration of melatonin and were submitted to acute stress. A total of 120 wild-type zebrafish were divided into five groups: naïve control (N), negative control group (Stress/C), positive control treated with diazepam (Stress/Diaz), treatment with melatonin at dose 1 (Stress/Melt. 1) and treatment with melatonin at dose 2 (Stress/Melt. 2). The exposure to treatments (diazepam or melatonin) was performed prior to the acute stress protocol, based on a chase by a fishing net during 5 min followed by exposure to the air for 1 min. The body cortisol levels were assessed, as well as oxidative stress (thiobarbituric acid reactive substances, reactive species of oxygen and antioxidant activity), and fish behavior (open field test). Melatonin was able to modulate acute stress effects on zebrafish by inhibiting cortisol increasing levels, reducing locomotor parameters, inducing a sleep state, reducing lipid peroxidation and stimulating antioxidant enzymatic activity.

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The aim of the present study is to verify the viability of frozen B. orbignyanus sperm cells after freezing with dilution media containing different concentrations of the melatonin and after different freezing times. Semen from 15 males was collected and pooled as five pools from three random animals. Oocytes (100) from three females were separately used for fertilization. There were three treatments: (C) Control Medium: 90% of extender Beltsville thawing solution (5% concentration) + 10% methylglycol (MG); (M1) Control Medium + 1 mM melatonin; and (M2) Control Medium + 2 mM melatonin. Sperm samples were diluted in media at a final proportion of 1:4 [125 µl sperm (25% V/V) + 337.5 µl BTS (65% V/V) + 37.5 µl MG (10% V/V)]. Melatonin was added at final solution. Three Dry shipper freezing times were used: T1 (15 min), T2 (12 h) and T3 (24 h). The samples were transferred, stored in a cryobank and thawed in a water bath at 60 °C for 5 s and evaluated concerning viability, morphology and fertilization rate. B. orbignyanus semen frozen in M2 presented the highest fertilization rate (8.40 ± 2.54%). The highest vitality (85.2 ± 2.8%), motility (64.63 ± 8.3%), motility duration (84.22 ± 11.4 s) and progressive motility (17.01 ± 1.2%) rates were maintained for M2. The highest number of altered cells was observed in C (57.4 ± 5.9%). Melatonin at 2 mmol L-1 associated with the cryoprotectant methylglycol in cryopreservation could be used to improve a cryobank for endangered Brycon orbignyanus populations.

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