Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 degrees C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.

Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score-arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 μm/sec of curvilinear velocity, 87 μm/sec of straight-line velocity and 103 μm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring.

The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution™ and Merck III™), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO3) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at −170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO3 (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO3 (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.

Piracanjuba, Brycon orbignyanus, is an endangered Brazilian freshwater fish species. Refrigerated storage of semen is a simple and useful tool for artificial reproduction, especially when the number of brood fish is reduced. In our previous study, some extenders containing gentamycin were tested during refrigerated storage of piracanjuba semen. It was observed that semen stored in extender containing gentamycin did not retain sperm motility for longer periods compared to samples diluted in extender only, as expected. We hypothesized that the bacterial population present in piracanjuba semen was not susceptible to gentamycin, the gentamycin concentration tested did not control bacterial growth, or extender pH was not optimum for sperm storage. To test these hypothesis we (a) stored semen in NaCl‐tris solution with the pH adjusted to different values; (b) quantified bacterial growth during the refrigerated storage of semen; (c) submitted some colony‐forming units of different strains to susceptibility profile to penicillin, streptomycin, lincomicin, ampicillin, and gentamycin; and (d) evaluated the effects of gentamycin (0, 0.01, 0.1, 0.5, and 1.0 mg/mL) on bacterial growth, sperm motility and fertilization rate. Higher sperm motilities were yielded when extender pH was adjusted to 7.6. A progressive increase in bacterial population and a gradual reduction in sperm motility were observed during the 8 d of storage. Ninety‐two percent of the bacterial colonies tested were susceptible to gentamycin. Semen diluted in NaCl‐tris containing gentamycin at 0.1 mg/mL yielded higher motility and fully inhibited bacterial growth during refrigerated storage, and did not affect the fertilization process. Thus, the addition of gentamycin at 0.1 mg/mL of NaCl‐tris pH 7.6 can be used as a routine when storage of semen for 4–6 d is necessary. The refrigerated storage of semen improves the efficiency of artificial reproduction and consequently can promote a better recovery of this species.

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.

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater ¢sh pirapitinga Brycon nattereri. Extenders (BTS TM and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 1C) and activating agents (0.29% NaCl and 1% NaHCO 3 ) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry-shipper). Post-thawed sperm motility rate, motility quality (score 0 5 no movement; 5 5 rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 mm long, the head was ovoid (2.00 Â 1.22 mm) with no acrosome, the midpiece was 2.15 mm long and the £agellum was 30.90 mm long with the typical 912 axoneme arrangement. Post-thawed sperm motility rate (70^79% motile sperm), motility quality (score 3.1^3.7) and morphology (9.3^11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO 3 (392^1031s) compared with 0.29% NaCl (144^338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.

The aims of this study were to evaluate the e⁄ciency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted-cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4^6 1C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0^26% sperm motility, whereas sperm diluted in complex extenders (BTS TM , M III TM and Androstar TM ) yielded 62^81% sperm motility on day 4 after cold storage. When Androstar TM was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26^61% fertility rate; and 22^60% hatching rate. The use of Androstar TM improves the sperm fertility of the streaked prochilod during a 4-day storage period and can therefore be used to facilitate arti¢cial reproduction.

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