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We aimed to vitrify embryos of Prochilodus lineatus in a high-osmolarity cryoprotectant solution, evaluating, after the vitrification-thawing process, their morphological changes. Thus, 240 embryos in the 20-somite phase (20S) were exposed for 20 min to one main internal cryoprotectant solution (1,2-propanediol-PROP), divided into four immersion sequence steps of five minutes each. The first three steps were performed in solutions containing only a main internal cryoprotectant (PROP-2, 3 and 4 M), and the fourth step in a high-osmolarity solution combining internal (PROP + dimethyl sulphoxide-Me 2 SO) and external cryoprotectants (sucrose-SUC). The final concentration of vitrification was PROP 5 M + Me 2 SO 5 M + SUC 0.2 M. During vitrification, the straws exhibited a translucent solid appearance; however, during thawing, their structure became totally opaque and white. After thawing, the embryos suffered an increase in volume and presented morphological changes including protrusions on the surface of the yolk sac, yolk sac rupture, and optical vesicle degradation. On the inside, we observed intercellular spaces and a yolk syncytial layer (YSL) with altered chromatin. Yet, structures such as somites, neural tube, endoderm and epidermis presented cells with a nucleus and integral mitochondria. We conclude that the use of the tested cryoprotectant solution permits the formation of a vitreous solid and preserves part of the cells of the blastoderm. Yet, the heating protocol does not control recrystallization, resulting in the formation of serious morphological anomalies that prevent the preservation of the embryonic unit., (© 2018 Blackwell Verlag GmbH.)

This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered "ideal hosts". However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae. Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte-1 ml-1). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 µm), large micropyle diameter (7.57 µm), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.

Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me2SO); at different temperatures (0.0±2.0°C and 8.0±2.0°C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10h and the temperature decreased from 8.0±2.0°C to 0.0±2.0°C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Juvenile fish trade monitoring is an important task on Brazilian fish farms. However, the identification of juvenile fish through morphological analysis is not feasible, particularly between interspecific hybrids and pure species individuals, making the monitoring of these individuals difficult. Hybrids can be erroneously identified as pure species in breeding facilities, which might reduce production on farms and negatively affect native populations due to escapes or stocking practices. In the present study, we used a multi-approach analysis (molecular and cytogenetic markers) to identify juveniles of three serrasalmid species (Colossoma macropomum, Piaractus mesopotamicus and Piaractus brachypomus) and their hybrids in different stocks purchased from three seed producers in Brazil. The main findings of this study were the detection of intergenus backcrossing between the hybrid ♀ patinga (P. mesopotamicus×P. brachypomus)×♂ C. macropomum and the occurrence of one hybrid triploid individual. This atypical specimen might result from automixis, a mechanism that produces unreduced gametes in some organisms. Moreover, molecular identification indicated that hybrid individuals are traded as pure species or other types of interspecific hybrids, particularly post-F1 individuals. These results show that serrasalmid fish genomes exhibit high genetic heterogeneity, and multi-approach methods and regulators could improve the surveillance of the production and trade of fish species and their hybrids, thereby facilitating the sustainable development of fish farming.

In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.

This paper reports the triiodothyronine's (T3) effects on the early growth and survival of piracanjuba (Brycon orbignyanus) produced from fertilized eggs hormone exposed. The study was carried out in two phases. In the first phase, eggs divided in 6 batches were immersed in T3 solutions: 0.01; 0.05; 0.1; 0.5 ppm; 1 ppm and control (no T3). After a 15-min immersion, eggs were transferred to incubators where larvae were kept up to 72 h after hatching. Larval weight, length and yolk sac volume were determined every 12 h. Sixty and 72 h after hatching, larvae exposed to 0.5 ppm T3 were significantly heavier than the others, and those exposed to 1 ppm T3 showed the lowest weight. The yolk sac absorption was not affected. In the second experimental phase, the resulting fry from the first phase were stocked into 3 boxes per treatment (5 larvae L(-1)) and fed with plankton, fish larvae and feed prepared in the hatchery (48% CP) in the first 3 days, plankton and feed from the 4th to the 10th day and only feed in the next (last) 5 days. Fry weight, length and specific growth rate were determined at 1, 5, 10 and 15 days. Survival was calculated in the last day. In the 15th day, fry length did not differ among treatments but the weight of the control group was higher. Higher survival in the T3-treated groups suggested lower predation among fry. The results allowed us to conclude that there was no expressive effect of T3 on the growth, but it improved the survival of the piracanjuba progeny.

Prochilodus lineatus is widely used in pisciculture projects, mainly in the south of Brazil. It shows a basic karyotype composed of 2n = 54 chromosomes, in addition to as many as seven supernumerary chromosomes, also known as B-chromosomes. These additional small chromosomes are frequently heterochromatic, vary in number and morphology, and generally have no homology with the A complement. Intensive studies have investigated the function, origin and inheritance of these supernumerary chromosomes. The present study aimed to determine the inheritance pattern of the B-chromosomes resulting from directed cross-fertilization of P. lineatus, Prochilodontidae isolated from the Mogi-Guaçu River, Pirassununga, SP. These cross-fertilization experiments were performed at CEPTA/ICMBio in Pirassununga. The transmission patterns of these micro-chromosomes agreed (K(B)= 0.48) with the expectation of regular meiotic behavior following a Mendelian transmission model (K(B)= 0.5). A non-accumulation process was observed for these B-chromosomes in filial generations. Together, our results indicate that the supernumerary chromosomes in P. lineatus samples from the Mogi-Guaçu River exist in a neutral state and follow a Mendelian inheritance pattern.

The embryogenesis of Brycon cephalus was established in seven stages: zygote, cleavage, blastula, gastrula, segmentation, larval and hatching, in an incubation period of 11 h (26 degrees C). The zygote phase was observed directly after fertilization and egg hydration. Cleavage began at 0.5 h of incubation and extended up to the morula phase (1.5 h; +100 blastomeres). Cleavage was meroblastic and underwent the following division pattern: the first five divisions were vertical and perpendicular to each other, following the model 2 x 2, 4 x 2, 4 x 4 and 4 x 8. The sixth division was horizontal and occurred at 1.25 h after fertilization, giving rise to two cell layers (4 x 8 x 2) with 64 blastomeres. At the blastula stage (1.25-1.5 h), an irregular space between the blastomeres, the blastocoele, could be detected and the periblast structure initiated. The gastrula (1.75-6.0 h) was characterized by the morphogenetic movements of epiboly, convergence and cell involution, and formation of the embryonic axis. The segmentation stage (7-9 h) comprised the development of somites, the notochord, optic, otic and Kupffer's vesicles, neural tube, primitive intestine and ended with the release of the tail. The larval stage (up to 10 h) was characterized by the presence of 30 somites and growth and elongation of the larvae. At the hatching stage, the embryos presented more than 30 somites and exhibited swimming movements and a soft chorion. The blastomeres presented euchromatic nuclei, indicating a high mitotic activity and many yolk globules in the cytoplasm. The periblast was constituted of a layer with several nuclei and many vesicles, which grew during the epiboly movement.

The genus Prochilodus includes individuals ranging in size from medium to large, being highly relevant for commercial and subsistence fishing. Prochilodus species have a diploid number of 2n = 54 chromosomes and up to seven supernumerary (B) microchromosomes. Previous research has shown that B frequency increased drastically in the Mogi-Guaçu river population of Prochilodus lineatus in the early 1980s, whereas it remained about constant in the 1990s. Here we analyses B frequency in this population during the 2003-2007 period and have found that frequency has not changed significantly since 1987, and that these B chromosomes do not show the intra-individual variation in number that characterized them in the 1980s. This indicates that these B chromosomes have been neutralized, after their invasion, through their mitotic stabilization.

Interspecific hybrids of fish can offer important advances in the zootechnical exploration of this animal group. However, hybrids can pose serious biological risks to the environment and natural populations, making genetic characterization and monitoring of hybrids produced in fish culture important concerns. In the present study, individuals of the hybrid 'Piaupara' were characterized by fluorescence in situ hybridization (FISH) using sex-specific probes to differentiate the respective parental contributions from female Leporinus macrocephalus (Piauçu) and male Leporinus elongatus (Piapara). The application of this technique demonstrated a difference in hybridization patterns between males and females of L. elongatus and L. macrocephalus, mainly due to the fact that the NOR-bearing chromosomes of L. elongatus contain some homology with the probe. This allowed us to detect a clear sex-specific distinction of the hybrid 'Piaupara', demonstrating that this technique is well-suited for the characterization of these hybrids. Moreover, this technique offers an important methodological advance for the study of diverse sex chromosome systems of Leporinus, supplying valuable information about the origin and establishment of a recently described sexual system in this genus., (Copyright 2009 S. Karger AG, Basel.)

Toxicity tests using early life stages of fish are of great importance in assessing risks to growth, reproduction and survival in polluted environments and are important tools for good environmental monitoring. However, a small number of standard bioassays of this type have been developed in Brazil. Curimbatá (Prochilodus lineatus) is an abundant South American characid fish of great commercial interest. It was chosen for testing different concentrations of 40% dimethoate, an organophosphate insecticide and acetylcholinesterase enzyme (AChE) inhibitor used widely in Brazil. The 48-h LC50 for eggs is higher than 16.0 microg.L-1, whereas for recently hatched larvae it was found to be significantly lower (11.81 microg.L-1, ranging between 10.23 microg.L-1 - 13.65 microg.L-1) and also significantly lower than that for eggs by a Student t-test for independent samples (p = 0.03). The 96-h LC50 for 3-day old larvae was 10.44 microg.L-1 (8.03 microg.L-1 - 13.57 microg.L-1), similar to that of recently-hatched larvae (p = 0.76). Larval mobility was also found to be reduced by this insecticide.