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.

Embryonic development and larval growth of Brycon nattereri Günther, 1864 (Characidae) and its implications for captive rearing.

The aim of this study was to describe, for the first time, the embryogenesis and larval growth of the Paraitinga Brycon nattereri Günther, 1864 reared in captivity. After artificial fertilization, eggs were incubated at constant temperature (~19°C) and collected every 15 min during the first 3 h and then every 3 h until hatching. Five larvae were collected daily over 15 days for evaluation of the length, yolk sac volume and specific growth rate. The following stages of embryonic development were identified: zygote, cleavage, gastrula, segmentation and larval. The hatching occurred after 50-54 h, with larvae poorly developed and fully depigmented, devoid of mouth and swimming capacity, presenting 6.32 mm total length and 3.64 mm3 yolk sac volume. The mouth opening was observed between days 3-4 after hatching. The yolk sac absorption was slow during the first 3 days, increasing sharply after this period, being completed on the day 11. During this period there was a decrease in the larval growth rate. After yolk sac absorption, an increase in the growth rate was observed that coincided with the start of exogenous feeding. Cannibalism was not observed during the 15 days of evaluation. The initial development of B. nattereri was slow and poorly developed larvae in relation to other Brycon species, certainly due to the lower temperature required for egg incubation and larval rearing. Other studies are needed in order to develop techniques to improve the methods of incubating eggs and feeding larvae.

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.

Sperm dilution ratio affects post-thaw motility rate and velocity of Prochilodus lineatus (Characiformes) sperm.

There is a lack of standardization in sperm cryopreservation of aquatic organisms and, thus, a necessity of more accurate investigations in all steps of this process. In this study, the effects of sperm dilution ratio on post-thaw sperm quality of Prochilodus lineatus were evaluated. Sperm was diluted in a standard freezing medium (glucose and methyl glycol) at four different ratios (sperm to final volume = 1:5, 1:10, 1:50 or 1:100), frozen in a nitrogen vapour vessel at -170°C and then stored in liquid nitrogen vessel at -196°C. Post-thaw motility rate and velocities (curvilinear = VCL; average path = VAP; straight line = VSL) were determined using a Computer-Assisted Sperm Analyzer (CASA) at 10 and 40 s post-activation. The highest motility rates were observed when sperm was frozen at a ratio of 1:5 (76%) and 1:10 (75%). The highest VCL (225 µm/s) and VAP (203 µm/s) were observed at a ratio of 1:10, while VSL was similar among samples frozen at 1:5, 1:10 and 1:50 (97-124 µm/s). When those parameters were evaluated again 30 s later, motility decreased significantly in samples frozen at a ratio of 1:5 (57%) and 1:10 (61%), while velocities decreased significantly in all samples regardless of dilution ratio (75-85 µm/s of VCL, 38-53 µm/s of VAP and 25-39 µm/s of VSL). P. lineatus sperm should be frozen at a ratio of 1:10, where both the number of loaded sperm per straw and the post-thaw quality are maximized.

Fresh, equilibrated and post-thaw sperm quality of Brycon orbignyanus (Valenciennes, 1850) and Prochilodus lineatus (Valenciennes, 1837) treated with either salmon GnRHa and domperidone or pituitary extract

The effects of reduced doses of Ovaprim™ (GnRHa + domperidone) on sperm release of Brycon orbignyanus and Prochilodus lineatus were evaluated. Furthermore, sperm quality was compared among fresh, equilibrated and post-thaw samples. Males received a single and reduced dose of Ovaprim™ (0.125 or 0.25 ml/kg); control males received pituitary extract (cPE; 3 mg/kg). Fresh sperm was evaluated for volume, concentration, seminal plasma osmolality and seminal plasma pH. Then sperm was diluted in a freezing medium, equilibrated for 15-20 min and frozen in nitrogen vapor vessel (dry-shipper). Sperm motility was analyzed during 60 s post-activation in fresh, equilibrated and post-thaw samples. Sperm quality of males treated with Ovaprim™ (both doses) were not different from that of cPE-treated males, thus these data were pooled. In B. orbignyanus, motility was higher in fresh (99%) than in equilibrated sperm (81%); post-thaw motility dropped to 42%. In P. lineatus, motility was similar in fresh (99%) and equilibrated sperm (92%); post-thaw motility was 73%. Motility decreased as a function of time post-activation, and this decrease was significant after 60 s in fresh and equilibrated sperm, and as soon as 30 s in post-thaw sperm, in both species. Ovaprim™ at 1/4 of the recommended dose can successfully replace cPE.

O efeito de doses reduzidas de Ovaprim® (GnRHa + domperidona) na liberação do sêmen de Brycon orbignyanus e Prochilodus lineatus foi avaliado. Além disso, a qualidade do sêmen foi comparada entre as amostras frescas, equilibradas e descongeladas. Os machos receberam dose única e reduzida de Ovaprim® (0,125 ou 0,25 ml/kg); os machos-controle receberam extrato de hipófise (cPE; 3 mg/kg). O sêmen fresco foi avaliado quanto ao volume, concentração, e osmolalidade e pH do plasma seminal. Em seguida, o sêmen foi diluído num meio de congelamento, equilibrado por 15-20 min e congelado em botijão de vapor de nitrogênio (dry-shipper). A motilidade espermática foi analisada durante 60 s pós-ativação no sêmen fresco, equilibrado e descongelado. A qualidade do sêmen não diferiu entre os machos tratados com Ovaprim® (ambas as doses) ou cPE, assim foi feito um pool desses dados. Em B. orbignyanus, a motilidade foi maior no sêmen fresco (99%) do que no equilibrado (81%); a motilidade do sêmen descongelado caiu para 42%. Em P. lineatus, a motilidade foi semelhante entre o sêmen fresco (99%) e equilibrado (92%); a motilidade do sêmen descongelado foi 73%. A motilidade caiu em função do tempo pós-ativação, e essa queda foi significante após 60 s no sêmen fresco e equilibrado, e tão precoce quanto 30 s no sêmen descongelado, em ambas as espécies. Ovaprim® a 1/4 da dose recomendada pode substituir o cPE com sucesso.

Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm.

The aim of this study was to use more accurate techniques to investigate the effects of cryoprotectants (CPAs) and extenders on post-thaw sperm quality of Brycon orbignyanus and Prochilodus lineatus. Six freezing media comprising the combination of three CPAs (DMSO, methanol and methyl glycol) and two extenders (BTS and glucose) were used. Sperm was diluted in each medium, loaded into 0.5-mL straws, frozen in a nitrogen vapor vessel (dry-shipper), and stored in liquid nitrogen at -196 °C. Post-thaw sperm motility rate and velocities (curvilinear = VCL; straight line = VSL; average path = VAP) were evaluated using a computer-assisted sperm analyzer. Membrane integrity and mitochondrial function were determined using fluorochromes. Post-thaw quality was considered high when samples presented the following minimum values: 60 % motile sperm, 140 µm/s of VCL, 50 % intact sperm membrane and 50 % mitochondrial function integrity. High post-thaw quality was observed in B. orbignyanus sperm frozen in BTS-methyl glycol and in P. lineatus sperm frozen in BTS-methyl glycol, glucose-methyl glycol and glucose-methanol. All samples frozen in DMSO yielded low quality. The presence of ions in the BTS extender affected post-thaw sperm quality positively in B. orbignyanus and negatively in P. lineatus. Methyl glycol was the most suitable CPA for both fish species, leading to a good protection of cell membrane, mitochondrial function and motility apparatus during the cryopreservation process. For an improved protection, B. orbignyanus sperm should be frozen in an ionic freezing medium.

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes).

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 µm in thickness with eight pore canals/µm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

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.

Osmolality and composition of the extender during the cold storage of Prochilodus lineatus (Characiformes: Prochilodontidae) sperm

This study evaluates the effects of extender osmolality and composition on the cooling of Prochilodus lineatus sperm. Sperm was diluted in six extenders: two compositions (powdered coconut water(tm) = ACP; Beltsville Thawing Solution(tm) = BTS) x three osmolalities (285, 325, and 365 mOsm/kg) plus an undiluted control, and stored at 6-8°C. Motility rate and velocities (curvilinear, straight line, and average path) were determined every other day. Osmolality did not affect the quality of cooled sperm, thus data were pooled. Motility was higher on d 0 compared to the other days and diluted samples (85-90%) yielded higher motility than control (75%). On d 2, motility was higher in BTS-diluted samples and control, but on d 4 and 6, control yielded the highest motility. Velocities decreased from d 0 to 6 in diluted samples, but not in control. On d 0, velocities were higher in BTS-diluted sperm, but, on d 2, 4, and 6, control yielded higher velocities despite of the large variation among males. Thus P. lineatus sperm should be stored in BTS or without dilution, for a maximum of two days at 6-8°C. Extender osmolality between 285 and 365 mOsm/kg does not affect sperm quality during cold storage...

Neste trabalho avaliou-se os efeitos da osmolalidade e da composição do diluidor no sêmen de Prochilodus lineatus, após o resfriamento. O sêmen foi diluído em seis diluidores: duas composições (água de coco em pó(r) = ACP; Beltsville Thawing Solution(r) = BTS) x três osmolalidades (285, 325 e 365 mOsm/kg) mais uma alíquota sem diluição como controle e armazenadas a 6-8°C. A taxa de motilidade e velocidades (curvilinear, retilinear e média de percurso) foram determinadas a cada dois dias. A osmolalidade não afetou a qualidade do sêmen resfriado, dessa forma foi feito um 'pool' desses dados. A motilidade foi maior no d 0 comparado aos outros dias e as amostras diluídas (85-90%) apresentaram as maiores motilidades do que o controle (75%). No d 2, a motilidade foi maior nas amostras diluídas em BTS e controle, mas nos d 4 e 6, o sêmen controle apresentou as maiores motilidades. As velocidades diminuíram do d 0 para o d 6 nas amostras diluídas, mas não no controle. No d 0, as velocidades foram maiores nas amostras diluídas em BTS, mas, nos d 2, 4 e 6, o controle apresentou as maiores velocidades apesar da grande variação entre os machos. Assim, o sêmen de P. lineatus deve ser resfriado em BTS ou sem diluição (controle), por no máximo dois dias a 6-8°C. A osmolalidade do diluidor entre 285 e 365 mOsm/kg não afeta a qualidade do sêmen durante o resfriamento...

Structural analysis of oocytes, post-fertilization events and embryonic development of the Brazilian endangered teleost Brycon insignis (Characiformes).

The aim of this study was to evaluate the oocytes, post-fertilization events and embryonic development in Brycon insignis, under both scanning electron microscopy and stereomicroscopy. Oocytes and embryos were sampled from spawning up to hatching. Stripped oocytes were spherical, non-adhesive, greenish-brown, possessed a single micropyle, pore-canals and had a mean diameter of 1.46 mm. In 63% of oocytes the germinal vesicle was peripheric. The main post-fertilization events were the fertilization cone formation (20 s), micropyle closure (100-180 s) and agglutination of supernumerary spermatozoa (100-180 s). Embryonic development lasted 30 h at ~24 °C and was characterized by seven stages. Zygote, cleavage, blastula and gastrula stages were first observed at 0.25, 1, 3 and 6 h post-fertilization, respectively. Fertilization rate was determined at the moment of blastopore closure, 10-11 h post-fertilization. The segmentation stage began at 11 h post-fertilization and comprised the development of somites, notochord, optic, otic and Kupffer's vesicles, neural tube, primitive intestine, and development and release of the tail. The larval stage began 21 h post-fertilization and was characterized by the presence of somites, growth and elongation of the larvae. At the hatching stage, embryos presented vigorous contractions of the tail and body leading to chorion rupture (30 h). The morphological characteristics described for B. insignis were similar to that described for other teleost species, and such knowledge is important for a better understanding of reproductive features of a species and useful for ecological and conservational studies.

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.).

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.