Ana Tereza de Mendonça Viveiros

A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen

The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 degrees C water bath for 8s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO(3)). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 degrees C for 8s and the other half at 30 degrees C for 16s. The 4.0-mL straws were thawed at 60 degrees C for 24s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 degrees C for 8s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

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.

Sucesso do resfriamento e congelamento de sêmen de pirapitinga Brycon nattereri

Cooling and freezing protocols of pirapitinga (Brycon nattereri) semen were evaluated using semen diluted in 154mM NaCl, 200mM NaCl, Saad or BTS™, and cooled for seven days. Sperm motility was daily evaluated. Five extenders (277mM glucose, 154mM NaCl, 200mM NaCl, Saad and BTS™) were combined with two cryoprotectants (DMSO - dimethyl sulphoxide and methylglycol) to produce 10 cryosolutions. Semen was diluted in each cryosolutions, aspirated into 0.5ml straws and frozen. Sperm motility was evaluated after thawing (60°C, 8 sec). Then, semen was frozen in straws with different volumes (0.25 and 0.5ml), and thawed under different water-bath temperatures (50° and 60°C). Higher sperm motility (48%) was observed when semen was cooled in BTS™ for seven days. Post-thawing sperm motility above 68% was observed when semen was frozen in 154mM NaCl-methylglycol, BTS™-methylglycol, 200mM NaCl-DMSO or Saad-DMSO. There was no difference on sperm motility when semen was frozen in 0.25 or 0.5ml straws and thawed in 50° or 60°C water-bath. Thus, pirapitinga semen can be successfully cooled in BTS™ for seven days or frozen in 154 mM NaCl-methylglycol, BTS™- methylglycol, 200mM NaCl-DMSO and Saad-DMSO.

Out-of-season sperm cryopreserved in different media of the Amazonian freshwater fish pirapitinga (Piaractus brachypomus)

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P<0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality.

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian fish Brycon insignis (Characiformes)

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.

Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes)

The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide=dimethyl sulfoxide (DMSO) and methyl glycol=methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60 °C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at -170 °C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60 °C for 8 s, of regardless straw size: 74±7% motile sperm, 47±4 s of motility duration, 69±3% intact sperm, 64±4% fertilization and 63±3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.

Sensibilidade dos espermatozoides de dourado (Salminus brasiliensis) a diferentes soluções crioprotetoras

The sensitivity of dourado (Salminus brasiliensis) spermatozoa to different cryoprotectant solutions was evaluated in three experiments. In experiment 1, semen was diluted, 1:10, in 12 solutions (four extenders x three cryoprotectants - dimethylsulphoxide (DMSO), methyglycol, or glycerol). Half of each sample was refrigerated for one hour while the other half was cryopreserved. Sperm motility was immediately assessed after dilution and after refrigeration in all samples, and after thawing in those cryopreserved in DMSO. In experiment 2, semen was diluted, 1:5, in five solutions containing DMSO, refrigerated, cryopreserved, and analyzed as in experiment 1. In experiment 3, semen was diluted, 1:5, in five solutions containing DMSO, cryopreserved and evaluated for motility and fertility. When semen was diluted 1:10, motility higher than 58% was observed in all samples refrigerated in DMSO and in NaCl-tris-methylglycol. Low motility was observed in samples refrigerated in the other combinations of methylglycol (5-32%) or glycerol (0-8%) and in those cryopreserved (16-20%). All samples diluted 1:5 yielded motility of 65-72% after refrigeration, and 45-66% after thawing (experiments 2 and 3). The hatching rates produced with cryopreserved semen, however, were lower (17-23%) compared to fresh semen (60%).

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.

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 Kupffers 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.

Efeito do peso de abate nos rendimentos do processamento da piracanjuba (Brycon orbignyanus, Valenciennes, 1849)

Objetivou-se, com o presente trabalho, avaliar o efeito do peso de abate sobre os rendimentos do processamento do peixe teleosteo piracanjuba (Brycon orbignyanus, Valenciennes, 1849). Inicialmente, alevinos com peso medio de 12 g, oriundos da Estacao Experimental da Usina Hidreletrica de Itutinga, foram estocados e cultivados em um viveiro de terra na Estacao de Piscicultura do Departamento de Zootecnia da Universidade Federal de Lavras. Ao final de 18 meses de cultivo, 121 peixes foram insensibilizados, abatidos, pesados e dissecados, para a determinacao das porcentagens de cabeca (%CAB), nadadeiras (%NAD), visceras (%VIS), pele com escamas (%PE), e residuos da filetacao (%RF) e dos rendimentos de carcaca (RCAR) e de file sem pele (RFSP), em relacao ao peso de abate. Para as analises de variância, os dados obtidos foram agrupados em 4 classes de peso (CP1=515 g a 629 g; CP2=630 g a 744 g; CP3=745 g a 859 g; e CP4=860 g a 975 g) e as equacoes de regressao estimadas em funcao do peso medio de cada classe. Nao foi constatado (P>0,05) efeito de classe de peso sobre RCAR, %NAD e %PE. Por outro lado, observaram-se efeito linear do peso de abate sobre %CAB (P<0,01), %RF (P<0,01) e RFSP (P<0,09), e efeito quadratico sobre %VIS (P<0,05). As variaveis %CAB e %RF diminuiram, ao passo que o RFSP e %VIS aumentaram, com o aumento do peso de abate. Pode-se concluir que, nas condicoes em que o experimento foi realizado, piracanjubas com maiores pesos de abate proporcionam maior RFSP e menores %CAB e %RF. Entretanto, a reducao da %CAB nao foi suficiente para aumentar o RCAR como seria esperado, em razao provavelmente de um maior aumento da %VIS.