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.

Bee pollen as a dietary supplement for fish: Effect on the reproductive performance of zebrafish and the immunological response of their offspring.

Bee pollen, a natural resource collected by bees, is rich in many nutrients, therefore it may represent a useful dietary supplement. Different uses of bee pollen are proposed due to its beneficial health properties, which includes the capacity to improve animal performance and promote immunostimulation. Animal nutrition can directly affect adults and their offspring, and larval stage is a critical moment for fish due to high mortality related to immune challenges. Thus, the present study attempted to evaluate the effects of adding bee pollen to a zebrafish diet, specifically, analyzing the effects on reproduction and immunity transference to descendants. Zebrafish adults received control diets based on commercial flakes and live food Artemia sp. nauplii or bee pollen-supplemented diets, administered three times a day, at the same time. The animals received the diets over 60 d, and throughout this period, they were tested for: egg production per female, total number of eggs, embryo viability rate, larval survival rate after exposure to spring viremia of carp virus and to Salmonella enterica serovar Typhimurium, and larval neutrophil recruitment after tail wounding. Bee pollen supplementation failed to improve egg production and embryo viability, and was unable to substitute flakes in zebrafish breeders. Instead, the offspring of breeders fed with bee pollen supplemented diets showed longer survival upon virus exposure and higher neutrophil migration to wounds. These results indicate that bee pollen can influence vertical immunity through important mechanisms related to offspring immunity in the early stages, when larval immune system is not fully developed.

Seminal plasma features of Prochilodus lineatus and Brycon orbignyanus throughout two consecutives spawning seasons.

This study aims to describe seminal plasma characteristics, detect changes during and between two consecutive spawning seasons (SS), and compare plasma features between two important South American fish species. Prochilodus lineatus and Brycon orbignyanus sperm was collected over two (SS1; SS2). Each season was divided into first and second sampling periods (P1; P2). Thus, the four experimental periods were referred to as SS1P1, SS1P2, SS2P1, and SS2P2. Seminal plasma was analyzed for osmolality, pH, and Na+ , K+ , and Ca2+ concentration. Additionally, sperm concentration, motility rate, and velocities (curvilinear = VCL; straight line = VSL) were determined and correlated with plasma features. In P. lineatus, plasma osmolality was lower in SS1P2, pH was higher in SS2P2, Na+ was higher and K+ and Ca2+ were lower in SS2P1 compared with other experimental periods. Positive correlations were observed between motility and plasma osmolality, motility and Na+ , and VCL and Na+ . In B. orbignyanus, plasma osmolality was higher in SS2P1 and SS2P2 and K+ concentration was higher in SS1P1 compared with other experimental periods; no correlation was observed. Seminal plasma parameters change during SS; therefore, the composition of a sperm extender and artificial fertilization methods should be adapted to maximize fertilization rates.

Sperm quality and its freezing ability throughout the spawning season in Prochilodus lineatus and Brycon orbignyanus.

The aim of this study was to determine fresh and frozen sperm quality evaluated over two spawning seasons (2013-2014; 2014-2015) in Prochilodus lineatus and Brycon orbignyanus. The spawning seasons were divided into two sampling periods: November to December and January to February. Males were hand-stripped after carp pituitary treatment. Fresh sperm motility rate, velocities (curvilinear = VCL; straight-line = VSL; average path = VAP), and the beat cross frequency (BCF) were determined using a Computer-Assisted Sperm Analyzer (CASA). Sperm of each species was frozen using methyl glycol as cryoprotectant and a glucose solution for P. lineatus or a NaCl solution for B. orbignyanus as extender. Diluted sperm was loaded into 0.25 mL straws, frozen in a nitrogen vapor vessel (dry shipper) and stored in a liquid nitrogen vessel. Six months later, straws were thawed in a water bath at 60 °C for 3 s and sperm quality was determined, as described for fresh sperm. No significant difference was observed for any of the fresh and frozen sperm features between the two spawning seasons or the two sampling periods in P. lineatus and in B. orbignyanus. Motility rate and velocities, but not BCF, was always higher in fresh sperm when compared with frozen sperm. Comparing both species, higher motility in frozen sperm and higher VCL and VAP in both fresh and frozen sperm were observed for P. lineatus, while higher VSL in fresh sperm and higher BCF in both fresh and frozen sperm were observed for B. orbignyanus. Sperm quality and its freezing ability of both species were sustained over the spawning season and thus fish farmers can reproduce these species and freeze their sperm in any time throughout the spawning season. P. lineatus sperm is more resistant to the cryopreservation process than B. orbignyanus.

Storage and transportation of Prochilodus lineatus (Characiformes) sperm prior to cryopreservation.

In this study we compared post-thaw quality of P. lineatus sperm frozen shortly after collection, with sperm frozen after dilution and transportation, and up to 6h from collection. From each sperm sample (n=10 males) five aliquots were taken. One aliquot was diluted in the freezing medium (1 sperm:8 glucose:1 methyl glycol) and frozen ∼20min after collection in the field (control), while the other four aliquots were transported to the laboratory where freezing took place 3 or 6h after collection. From the transported aliquots, two were diluted 1:4 in glucose solution before transportation (diluted samples), while the other two were kept undiluted until freezing (undiluted samples). Thus the five treatments were: control, undiluted-3h, diluted-3h, undiluted-6h and diluted-6h. Post-thaw sperm was evaluated for membrane integrity, motility rate and velocities (curvilinear=VCL; average path=VAP; straight line=VSL). Post-thaw membrane integrity did not differ among the five treatments (48-60% intact sperm). Sperm motility rate was similar (P>0.05) between control (64%) and undiluted samples (60-62%) and higher (P<0.05) than that in diluted samples (35-45%), regardless the time after collection when freezing took place. Velocities were higher in control and in undiluted-3h samples (VCL of 254-265µm/s, VAP of 219-244µm/s and VSL of 134-147µm/s) than in diluted samples or samples frozen 6h after collection. P. lineatus sperm can be transported/shipped to the laboratory without decreasing its suitability for cryopreservation. Sperm should be kept undiluted during storage and be frozen within 3h.

Gamete quality of streaked prochilod Prochilodus lineatus (Characiformes) after GnRHa and dopamine antagonist treatment.

The efficiency of Ovaprim™ salmon gonadotropin-releasing hormone agonist (GnRHa) and dopamine antagonist on the induction of spawning and spermiation in Prochilodus lineatus in comparison with the commonly used method using pituitary extract (PE) was evaluated. Females received PE at 0.5 + 5.0 mg/kg and Ovaprim™ at 0.05 + 0.45 ml/kg or at 0.125 + 0.375 ml/kg. All males received a first dose of PE at 0.4 mg/kg and then PE at 4.0 mg/kg or Ovaprim™ at 0.25 ml/kg. Oocyte, egg, larvae and sperm quality were evaluated. All females spawned and oocyte, egg and larvae quality were similar between Ovaprim™-treated (both doses) and PE-treated females. Data from females were pooled and the mean values were: 242 g ova weight, 15% ova index, 1209 oocytes/g ova, 284,539 oocytes/female, 183 oocytes/g body weight, 1.18 mm oocyte diameter, 49% fertilization rate, 43% hatching rate and 89% normal larvae. Sperm quality was similar between Ovaprim™-treated and PE-treated males. Data from males were pooled and the mean values of semen were: volume of 3.0 ml, 14.9 × 109 sperm/ml, osmolality of 283 mOsm/kg, pH of 7.4, 71% motile sperm, 217 µm/s curvilinear velocity, 102 µm/s straight-line velocity and 189 µm/s average path velocity. Ovaprim™ treatment can be used for commercial reproduction of P. lineatus, without any loss of gamete quality in comparison with PE treatment.