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.

Osmolality and composition of the activating solution affects motility of fresh and frozen Prochilodus lineatus sperm differently.

Osmolality and composition of the activating solution on motility of fresh and frozen Prochilodus lineatus sperm were evaluated. Sperm was triggered in 11 solutions prepared with reverse osmosis (RO) water (∼0mOsmkg(-1)), and glucose or NaCl adjusted to 50, 100, 150, 200 and 250mOsmkg(-1). Sperm motility rate and velocities (curvilinear=VCL, among others) were evaluated in fresh sperm at 10, 30 and 50s post-activation (spa), and in frozen sperm at 10 spa only. Sperm was frozen under a standardized methodology for this species. Fresh sperm motility was higher in samples triggered in RO (91%), in glucose at all osmolalities (90-92%) and in 50-150mOsmkg(-1) NaCl (88-91%) than that in 200-250mOsmkg(-1) NaCl (74-80%). Motility decreased (P<0.05) as a function of time after activation in samples activated in RO and in NaCl but not in glucose. Samples activated in 100-250mOsmkg(-1) glucose yielded motility above 80%, at 50 spa. Curvilinear velocity was higher (P<0.05) in glucose-activated samples (322-357µms(-1)) compared to that activated in NaCl (192-283µms(-1)) and in RO (298µms(-1)). Frozen sperm motility and velocities were similar when triggered in RO, glucose or NaCl and were higher at 0-150 mOsm kg(-1) (69-78% motility; 163-208µms(-1) VCL) than at 200-250mOsmkg(-1) (34-59% motility; 127-168µms(-1) VCL). High sperm motility with fast velocity for a long period is achieved at 100-150mOsmkg(-1), in glucose solution for fresh sperm and in glucose or NaCl for frozen sperm.