Sperm characterization and cryopreservation of the endangered freshwater fish Chirostoma estor (Atheriniformes).

The knowledge of the physiology of sperm of an endangered species allows the implantation of reproductive biotechnologies that aim at conservation. The aim of this study was to characterize fresh sperm and evaluate different cryopreservation solutions for sperm in Chirostoma estor. The characterization of Chirostoma estor fresh sperm (n = 22 males) was performed through analyzes of sperm concentration, membrane integrity, sperm morphology, motility rate, motility quality score, and motility duration. For cryopreservation (n = 42 males), 3 extenders (BTS™, MIII™, or Androstar Plus™) in combination with 2 permeable cryoprotectants (dimethyl sulfoxide (DMSO) or methyl glycol (Methyl)) were used. Analyzes of post-thaw sperm were performed as described for fresh sperm and additionally the fertilization rate analysis was performed. Fresh sperm presented a sperm concentration of 29.2 × 109 spermatozoa/mL, membrane integrity of 82.4%, and morphologically normal cells of 53%. After glucose activation (150 mM) a motility rate of 87.5%, sperm quality score of 5.0, and a duration of motility of 285 s were observed. For post-thaw sperm, MIII + Methyl and Androstar + Methyl solutions resulted in the highest motility rates of 40-48%. No differences were observed for motility duration, membrane integrity, and sperm morphology. Samples cryopreserved in Methyl (12-20%) showed a higher fertilization rate than DMSO, independently of the extender. In conclusion, the fresh sperm collected artificially from Chirostoma estor presents a compatible quality to carry out fertilization and can be cryopreserved in the commercial extenders MIII™ and Androstar Plus™ together with the cryoprotectant Methyl glycol.

Effects of melatonin supplementation on the quality of cryopreserved sperm in the neotropical fish Prochilodus lineatus.

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.

Cannibalism rate and mLeptin expression are influenced by photoperiod and diets in Piracanjuba, Brycon orbignyanus (Valenciennes, 1850) larvae.

Piracanjuba (Brycon orbignyanus) is a species with great productive potential, and during its larval phase, it presents intense cannibal activity. The photoperiod and diet are primary feed behaviour and cannibalism modulators to fishes. This experiment aimed to verify the effect of different photoperiods and diets in Piracanjuba larviculture. Larvae were kept under different photoperiods - 12 h light: 12 h dark (12 L: 12D); 24 h light:00 h dark (24hL: 00D) - Larvae were fed with Artemia nauplii and a formulated micro-diet in a factorial scheme for 10 days, and at the end of the experimental period, the influences of the treatments on performance and quantitative expression of mLeptin and mBmall1 were evaluated. In order to quantify the expression of mLeptin and mBmall1, qPCR adopting ß-actin and Elongation Factor 1 as endogenous genes was used. The primers for all the analysed transcripts were obtained through multiple sequences alignments of different fish species. It was observed that the diet and photoperiod influence the performance of Piracanjuba (B. orbignyanus) larvae in the initial phase of larviculture. Feeding with artemia nauplii and the photoperiod of 24 L:00D reduce cannibalism rates in intensive Piracanjuba larviculture. The results on the rate of cannibalism, rate of survival and the relative expression of mLeptin are related to the survival rate of the larvae, and it is inversely proportional to the cannibalism rate. The expression levels of mBmall1 showed a correlation with the final weight of the larvae. Piracanjuba Larvae under a photoperiod of 24 light and fed Artemia nauplii showed more significant levels of mLeptin expression.

Bee pollen in zebrafish diet affects intestinal microbiota composition and skin cutaneous melanoma development.

Bee pollen is recommended as dietary supplement due to immunostimulating functions including antioxidant, anti-inflammatory and anti-carcinogenic properties. Nevertheless, the effectiveness of such properties is still not well understood. As diet can be associated with animal performance, microbiota modulation and potentially factor for cancer, this study aimed to analyze if bee pollen could influence growth, gut microbial and skin cutaneous melanoma development in zebrafish. Control diets based on commercial flakes and Artemia were compared with the same diet supplemented with bee pollen. Fish weight gain, increased length, intestinal bacteria metagenomics analysis, serum amyloid A gene expression and cutaneous melanoma transplantation assays were performed. Bee pollen affected microbiota composition and melanoma development. Differential abundance revealed higher abundance in the control group for Aeromonadaceae family, Aeromonas and Pseudomonas genus, A. sobria, A. schubertii, A. jandaei and P. alcaligenes species compared with pollen diet group. Pollen group presented higher abundance for Chromobacterium genus and for Gemmobacter aquaticus, Flavobacterium succinicans and Bifidobacterium breve compared with control group. Unexpectedly, fish fed with bee pollen showed higher tumor growth rate and larger tumor size than control group. This is the first study to report intestinal microbial changes and no protective cancer properties after bee pollen administration.

Sex identification of the ornamental amazon fish Astronotus ocellatus by videoceloscopy and gonadal biopsy.

This study was conducted to evaluate and validate the efficacy and safety of videoceloscopy and gonadal biopsy as sexing methods for the A. ocellatus. A total of 31 adult individuals were used. Florfenicol (50 mg/kg) and morphine (5 mg/kg) were administered intramuscularly during the pre-surgical period. Animals were maintained in a supine position preceding a ventral midline incision and endoscope optics were then utilized for gonad visualization and sex identification. A gonadal fragment was collected using laparoscopic forceps and conditioned in 10 % formalin. To suture the cavity, polyamide yarn was used in a simple and continuous pattern. At 15 days subsequent to surgery, healing was evaluated, and the stitches were removed. Videoceloscopy accuracy and gonadal biopsy effectiveness were 97 % and 83 %, respectively. Total time devoted in the videoceloscopy, gonadal biopsy and surgery was longer for animals identified as males compared to females The survival rate was 100 %. There were differences regarding food consumption at 24 and 36 h post-surgery when compared to control specimens (pre-surgical) Regarding position in the water column, differences were observed at 24 and 72 h after surgery when compared individually to the control specimens. There were differences for interaction behavior at 24, 36 and 60 h, and regarding search for hiding places at 12 and 24 h after surgery in relation to the control specimens. The applied videoceloscopy and gonadal biopsy surgical techniques are, therefore, effective and safe for A. ocellatus sexing procedures.