Impact of activation solutions on fresh and frozen-thawed sperm motility and fertilization success for two species of migratory freshwater fishes.

Extracellular environment conditions, ionic concentration, pH, osmolality, and temperature influence sperm activation and sperm quality. The aim of this study was to evaluate the effects of different activating solutions on sperm quality and fertilization rate of fresh and post-thaw sperm in Brycon orbignyanus and Prochilodus vimboides. Activation solutions with ions: NaCl, KCl, CaCl2 (150 mOsm kg-1), without ions: glucose (150 mOsm kg-1) and water: reverse osmosis (RO) (∼0 mOsm kg-1) and tank water (TW) were tested. In experiment 1, fresh sperm motility was activated in each activating solution and motility rate (%), motility quality score (0-5), and motility duration (seconds) were subjectively evaluated using a microscope. In experiment 2, sperm was cryopreserved and post-thaw sperm quality was assessed in each activating solution. Methyl glycol was used as cryoprotectant and for B. orbignyanus a solution of BTS® 5% in water reverse osmosis - 325 mOsm kg-1 was used as extender, while for P. vimboides it was used a solution of glucose 5% in water reverse osmosis - 325 mOsm kg-1. In straw, cryoprotectant, extender, sperm were respectively 10%, 80% 10% (V/V). B. orbignyanus fresh sperm activated in NaCl, KCl, glucose solutions, TW and RO yielded higher averages for all the subjective parameters analysed. In fresh sperm of P. vimboides the highest values were observed when glucose solution was used for sperm motility activation, and the highest fertilization rates were observed in samples activated in glucose or RO solutions. B. orbignyanus post-thaw sperm activated in TW (45.1%) or RO (39.7%) presented the highest values for motility. The highest values of curvilinear velocity (VCL) were observed using glucose (69.5 µm s-1), NaCl (67 µm s-1) and KCl (68.4 µm s-1), but the highest fertilization rates were observed when glucose (3.6%), RO (3.5%) and TW (2.5%) were used. P. vimboides post-thaw sperm activated in glucose solution presented the highest motility rate (41%), VCL (43.7 µm s-1), fertilization rate (18.2%) and hatching rate (13.7%). In order to achieve the best seminal quality, fertilization and hatching rates in both fresh and post-thaw sperm, the glucose solution, TW and RO are indicated for use as sperm motility activators in B. orbignyanus, whereas for P. vimboides the glucose solution and RO are indicated.

Molecular and functional regionalization of bicarbonate secretion cascade in the intestine of the European sea bass (Dicentrarchus labrax).

In marine fish the intestinal HCO3- secretion is the key mechanism to enable luminal aggregate formation and water absorption. Using the sea bass (Dicentrarchus labrax), the present study aimed at establishing the functional and molecular organization of different sections of the intestine concerning bicarbonate secretion and Cl- movements. The proximal intestinal regions presented similar HCO3- secretion rates, while differences were detected in the molecular expression of the transporters involved and on regional HCO3- concentrations. The anterior region presented significantly higher Na+/K+-ATPase activity, Cl- transepithelial transport and basolateral slc4a4, apical slc26a6 and slc26a3 expression levels. In the mid intestine, the total HCO3- content was significantly increased in the fluid as in the carbonate aggregates. In the rectum no HCO3- secretion was observed and was characterized by the diminished HCO3- total content, residual molecular expression of slc4a4, slc26a6 and slc26a3, higher H+-ATPase activity and expression, suggesting the existence of a different bicarbonate handling mechanism. The possible regulation of HCO3- secretion by extracellular HCO3- and increased intracellular cAMP levels were also investigated. cAMP did not affect HCO3- secretion, although Cl- secretion was enhanced by cftr. HCO3- secretion rise due to the HCO3- basolateral increment showed that at resting levels slc4a4 was not a limiting step for secretion. The transcellular/intracellular dependence of apical HCO3- secretion differed between the proximal regions. In conclusion, intestinal HCO3- secretion has a functional region-dependent organization that was not reflected by the anterior-posterior regionalization on HCO3- secretion and expression profiles of chloride/water absorption related genes.