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Piracanjuba (Brycon orbignyanus) is an endangered fish species from the Neotropical region. The establishment of a cryobank using spermatogonial stem cells (SSCs) and subsequent production of a germline chimera is thus a promising strategy for such species. In the present work, procedures for the isolation and cryopreservation of piracanjuba SSCs and subsequent transplantation into sterile recipients were established. The piracanjuba SSCs were obtained by Percoll density gradient centrifugation and differential plating. SSC fractions were evaluated by relative ddx4 expression, alkaline phosphatase activity, and light microscopy. SSC cryopreservation was performed using five cryoprotectants at three different concentrations. The mix of the cells from the 20% and 30% Percoll density gradients showed 58.35 ± 0.03% purity of SSCs. The purity of SSCs increased to 66.00 ± 0.01% after differential plating. The relative ddx4 expression was 3.5 times higher in cells from the Percoll density gradient centrifugation than in the gonad and cells after differential plating. Propanediol (1 M) was the most effective cryoprotector evaluated (P = 1.000), showing 90.75 ± 1.85% cell viability. Freshly isolated and cryopreserved cells from the Percoll density gradient centrifugation were transplanted into a sterile male adult triploid hybrid with germ cell-less gonads. SSCs were observed in the germinal epithelium of the testes of recipients 20 days after transplantation. The results are promising for obtaining functional germline chimeras in Neotropical fish. Consequently, although the number of males used for the experiment was borderline, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction., Competing Interests: Declarations Ethics approval The experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA #02031.000088/2021–61). The authenticity of the document can be verified on the website https://sei.icmbio.gov.br/autenticidade informing the verification code: 9643544 and the code CRC: EC53598A. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, and therefore, PGCs are a genetic source for germplasm conservation through cryobanking and the generation of germline chimeras. The knowledge of PGC migration routes is essential for transplantation studies. In this work, the mRNA synthesized from the ddx4 3'UTR sequence of Pseudopimelodus mangurus, in fusion with gfp or dsred, was microinjected into zygotes of three neotropical species (P. mangurus, Astyanax altiparanae, and Prochilodus lineatus) for PGC labeling. Visualization of labeled PGCs was achieved by fluorescence microscopy during embryonic development. In addition, ddx4 and dnd1 expressions were evaluated during embryonic development, larvae, and adult tissues of P. mangurus, to validate their use as a PGC marker. As a result, the effective identification of presumptive PGCs was obtained. DsRed-positive PGC of P. mangurus was observed in the hatching stage, GFP-positive PGC of A. altiparanae in the gastrula stage, and GFP-positive PGCs from P. lineatus were identified at the segmentation stage, with representative labeling percentages of 29% and 16% in A. altiparanae and P. lineatus, respectively. The expression of ddx4 and dnd1 of P. mangurus confirmed the specificity of these genes in germ cells. These results point to the functionality of the P. mangurus ddx4 3'UTR sequence as a PGC marker, demonstrating that PGC labeling was more efficient in A. altiparanae and P. lineatus. The procedures used to identify PGCs in P. mangurus consolidate the first step for generating germinal chimeras as a conservation action of P. mangurus., Competing Interests: Declarations Ethics approval Pseudopimelodus mangurus were collected from the Moji Iguaçu River (Pirassununga–SP, Brazil) according to Brazilian law (Sisbio #31836-10), and the experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA #010/02031.000114/2022-31). Competing interests The authors declare no competing interests., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Herbicides are used in agriculture to control harmful crop weeds, prevent algae proliferation, and enhance macrophyte growth. Herbicide contamination of water bodies might exert toxic effects on fish in different development stages. Sperm, embryos, and adults of Astyanax altiparanae were used as a model to examine the detrimental effects of the following herbicide formulations: Roundup Transorb® (glyphosate), Arsenal® NA (imazapyr), and Reglone® (diquat). The lethal concentration 50 (LC 50 ) values for adults using glyphosate and imazapyr were 3.14 mg/L and 4.59 mg/L, respectively, while the LC 50 was higher than 28 mg/L for diquat. For the initial stages of embryo development, LC 50 values were 16.52 mg/L glyphosate, 9.33 mg/L imazapyr, and 1084 mg/L diquat. Inhibition of sperm motility was noted at 252 mg/L glyphosate, 137 mg/L imazapyr, and 11,300 mg/L diquat, with an average sperm viability of 12.5%, 73.2%, and 89.3%, respectively, compared to 87.5% detected to control. A. altiparanae exhibited different sensitivities to the herbicide formulations investigated in the developmental stages evaluated. Roundup Transorb® exposure was more toxic for adults, while Arsenal® NA was most harmful for early embryonic development and inhibited sperm motility. Reglone® demonstrated low toxicity for A. altiparanae compared to Roundup Transorb® and Arsenal® NA. A. altiparanae may be considered an emerging fish model for toxicological studies for the neotropical region due to its wide distribution and biological characteristics.

This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra Astyanax altiparanae , both females (three diploids and three tetraploids) and males ( n = 3 diploids). Breeders were subjected to hormonal induction with pituitary gland extract from common carp fish ( Cyprinus carpio ). Females received two doses at concentrations of 0.3 and 3.0 mg/kg -1 body weight and at intervals of 6 h. Males were induced with a single dose of 3.0 mg/kg -1 applied simultaneously with the second dose in females. Oocytes from each diploid and tetraploid female were fertilized with semen from the same male, resulting in two crosses: cross 1 (diploid male and diploid female) and cross 2 (diploid male and tetraploid female). The procedures were performed with separate females (diploid and tetraploid) and diploid males for each repetition ( n = 3). For ploidy determination, 60 larvae from each treatment were analyzed using flow cytometry and cytogenetic analyses. As expected, flow cytometry analysis showed that progenies from crosses 1 and 2 presented diploid and triploid individuals, respectively, with a 100% success rate. The same results were confirmed in the cytogenetic analysis, in which the larvae resulting from cross 1 had 50 metaphase chromosomes and those from cross 2 had 75 chromosomes. The oocytes have a slightly ovoid shape at the time of extrusion. Diploid oocytes had a size of 559 ± 20.62 μm and tetraploid of 1025.33 ± 30.91 μm. Statistical differences were observed between eggs from crosses 1 and 2 ( P = 0.0130). No significant differences between treatments were observed for survival at the 2-cell stage ( P = 0.6174), blastula ( P = 0.9717), gastrula ( P = 0.5301), somite ( P = 0.3811), and hatching ( P = 0.0984) stages. In conclusion, our results showed that tetraploid females of the yellowtail tetra A. altiparanae are fertile, present viable gametes after stripping and fertilization using the 'dry method', and may be used for mass production of triploids. This is the first report of these procedures within neotropical characins, and which can be applied in other related species of economic importance.

Biotechniques, including surrogate propagation derived from primordial germ cell (PGC) transplantation, are valuable tools for the reconstitution of endangered fish species. Although promising, there are no previous studies reporting such approaches using neotropical fish species. The aim of this study was to establish germline chimeras in neotropical fish by using the yellowtail tetra Astyanax altiparanae as a model species of the order Characiformes. Germline chimeras were obtained after transplantation of PGCs cultivated under different conditions: saline medium and supplemented with DMEM, amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, and subsequently transplanted into A. altiparanae triploids and triploid hybrids from the cross between A. altiparanae (♀) and A. fasciatus (♂). The results indicate ectopic migration in host embryos after transplantation of PGCs cultivated in saline medium. However, PGCs cultivated in supplemented medium migrated to the region of the gonadal ridge in 4.5% of triploid and 19.3% in triploid hybrid. In addition, the higher expression of dnd1, ddx4 and dazl genes was found in PGCs cultivated in supplemented culture medium. This indicates that the culture medium influences the maintenance and development of the cultivated cells. The expression levels of nanos and cxcr4b (related to the differentiation and migration of PGCs) were decreased in PGCs from the supplemented culture medium, supporting the results of ectopic migration. This is the first study to report the transplantation of PGCs to obtain germline chimera in neotropical species. The establishment of micromanipulation procedures in a model neotropical species will open new insights for the conservation of endangered species.

Primordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus . Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 10 9 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 10 9 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 10 9 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol., Competing Interests: Declaration of conflict of interest The authors declare no conflicts of interest with regard to the study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish ( Danio rerio ), the medaka ( Oryzias latipes ), and loach species ( Misgurnus spp . ). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class., Competing Interests: GY and JS were employed by Peixetec Biotecnologia Em Organismos Aquáticos LTDA. NV was employed by China Three Gorges Corp. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yasui, Ferreira do Nascimento, Pereira-Santos, Santos Silva, Coelho, Visintin, Porto-Foresti, Okada Nakaghi, Vianna, Carvalho, Monzani, López and Senhorini.)

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Genome size (GS) or DNA nuclear content is considered a useful index for making inferences about evolutionary models and life history in animals, including taxonomic, biogeographical, and ecological scenarios. However, patterns of GS variation and their causes in crustaceans are still poorly understood. This study aimed to describe the GS of five Neotropical Synalpheus non-gambarelloides shrimps ( S. apioceros, S. minus, S. brevicarpus, S. fritzmueller , and S. scaphoceris ) and compare the C-values of all Caridea infraorder in terms of geography and phylogenetics. All animals were sampled in the coast of São Paulo State, Brazil, and GS was assessed by flow cytometry analysis (FCA). The C-values ranged from 7.89 pg in S. apioceros to 12.24 pg in S. scaphoceris . Caridean shrimps had higher GS than other Decapoda crustaceans. The results reveal a tendency of obtaining larger genomes in species with direct development in Synalpheus shrimps. In addition, a tendency of positive biogeographical (latitudinal) correlation with Caridea infraorder was also observed. This study provides remarkable and new protocol for FCA (using gating strategy for the analysis), which led to the discovery of new information regarding GS of caridean shrimps, especially for Neotropical Synalpheus , which represents the second-largest group in the Caridea infraorder.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

B chromosomes are additional dispensable elements to the standard chromosomal set of an organism. In most cases, their transmission differs from Mendelian patterns, leading to their accumulation or extinction. The present study aimed to describe, for the first time, the transmission pattern of B chromosome in a population of Psalidodon paranae through directed crosses, as well as to analyze the populational dynamics of B chromosome. Our results revealed the possible elimination of B chromosome in crossings where only females were B-carriers, with a mean transmission rate ( k B ) of 0.149; however, k B was significantly higher in crossings involving male B-carriers ( k B  = 0.328-0.450). Moreover, we observed an increase in the frequency of B chromosomes in the natural population of P. paranae in the last two decades. These apparently contradictory results can make sense if the B chromosome provides adaptive advantages to their carriers. Here, we observed a differential transmission of B chromosomes in each sex of parental individuals, with higher transmission rates in crossing involving males B-carriers, in addition to describe the temporal changes of B chromosome frequency in P. paranae .

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

Triploid induction is a promising biotechnique that could be used to enhance aquaculture yields in the near future. However, studies conducted with several fish species have demonstrated that the presence of an extra set of chromosomes may result in deleterious health effects. Furthermore, studies of fish immune responses still need to be conducted before these specimens can be readily commercialized. In the study presented herein, we evaluated the effects of triploid induction on hematology, erythrocyte morphometry and morphology, phagocytosis, and the expression levels of IL-1β and TGF-β using specimens of the Neotropical species, Astyanax altiparanae. In general, the cell counts of erythrocytes, leukocytes, and neutrophils in triploid fish were lower than those in diploid fish. The erythrocytes of triploid fish were larger than those found in diploid fish, but also demonstrated considerably higher frequencies of cellular and nuclear abnormalities. Although not statistically significant, triploid induction resulted in a phagocytic capacity (PC) 20% lower than that found with diploid fish. No notable differences were observed in phagocytic index (PI). Gene expression levels for the cytokine IL-1 were lower in tissues from the head kidney, liver, and spleen of triploid fish with respect to diploid fish. Gene expression levels of TGF-β were lower only in the spleen of triploids compared to diploids. In conclusion, triploid induction resulted in A. altiparanae specimens with immune impairments and potentially lower resistances to disease and low-quality environments., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study interrogated factors which affect the appearance of secondary sexual characteristics, namely, fin spinelets (rigid dimorphic structure empirically associated with male sexual maturity in characids), in Astyanax altiparanae. Many variables such as the season of the year and several biotic components, including organism length, sex, phase of maturation, and the presence of gonads, were investigated. These factors were then associated with the physiological development of fin spinelets. The development of this trait is related to reproductive strategies but demonstrates considerable population variability as it is found throughout the year in some species but only during specific periods in others. Seventy-five specimens obtained from spontaneous spawn of farmed fish were arbitrarily grouped into small-, medium-, and large-sized groups in both summer and winter. Gonadal histology was performed to confirm each animal's sex and phase of maturation. Diaphanization of the fish was performed to visualize, count, and measure the fin spinelets. Finally, gonadectomization of some males was utilized to investigate the gonadal effect on the presence of fin spinelets. The present results show that the presence of fin spinelets is a secondary sexual characteristic of males which occurs independently of the season and is always present in males longer than 48 mm. However, in the summer, male specimens presented more rays with fin spinelets than during the winter. Furthermore, since fin spinelets were observed on immature males as well as spawning capable males, their presence cannot be directly associated with sexual maturity in male A. altiparanae, as previously supposed. Finally, gonadectomization resulted in an initial reduction in the length of fin spinelets. However, this trend was eventually normalized with time.

Supernumerary, or B, chromosomes are present in several eukaryotes, including characid fish of the genus Psalidodon. Notably, Psalidodon paranae carries the most studied B chromosome variant, a macro-B chromosome. The origin of this element was determined to be an isochromosome; however, data regarding its inheritance remain unavailable due to methodological barriers such as the lack of an efficient, non-invasive, and rapid protocol for identifying B-carrying individuals that would enable the design of efficient crossing experiments. Thus, in this study, we primarily aimed was to develop two non-invasive and fast (approximately 2 h) methods to identify the presence of B chromosomes in live specimens of P. paranae based on satellite DNA (satDNA) sequences known to be present in this element. The methods include fluorescence in situ hybridization in interphase nuclei and relative gene quantification of satDNAs using quantitative polymerase chain reaction. Our results reveal the efficiency of quick-fluorescence in situ hybridization and quantitative polymerase chain reaction for identifying B-carrying individuals using the proposed satDNA sequences and open up new possibilities to study B chromosomes.

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

The aim of this study was to evaluate different post-shock temperatures for tetraploid induction in the yellowtail tetra Astyanax altiparanae. Newly fertilized eggs were divided into four groups, three were submitted to heat shock (40°C for 2 min) at 24 min post-fertilization (mpf) and another group remained without shock (control). Groups submitted to temperature shock were further separated at the following temperatures: 22°C, 26°C and 28°C. Survival among embryonic development was counted and at hatching the ploidy was analyzed by flow cytometry. The results showed that the post-shock temperature affects the parameters analyzed and, therefore, must be considered for optimization of the production of tetraploid in A. altiparanae. Those data are innovative and could be used in future studies of basic biology in this species.

Rivulidae comprises a family of fish largely distributed in Brazil that includes 201 species, of which 125 are considered endangered. This fact emphasizes the need for development of conservation strategies including studies on genetics and reproduction. In this paper, we describe aspects of biology and reproduction of the rivuliid species Hypsolebias sertanejo. We outline the reproductive behaviour of this species under laboratory conditions, analyze ploidy status by flow cytometry, describe reproductive behaviour and performance and test dry and wet incubation of eggs. Although H. sertanejo showed well known patterns of reproductive behaviour, we verified many peculiarities inherent to its reproductive biology. As expected, most individuals were diploid (87.71%), however 14.29% were considered mosaics. Although no sterility was observed within mosaics, infertility of these fish was not fully evaluated. Hatching rate of the eggs collected was very low following both dry and wet incubation (5.04 and 3.79%, respectively). These results provide interesting information regarding the reproductive success of this species, and suggest that chromosomal abnormalities described may reduce the survival of H. sertanejo under natural conditions, limiting the perpetuation of this species, and emphasizing the need for more preservation efforts, including artificial propagation and gene banking.

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (-20 °C), ultra-low (-80 °C) and cryogenic temperatures (-196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (-20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (-80 °C) and cryogenic (-196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at -80 °C and -196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation., (Copyright © 2020. Published by Elsevier Inc.)

Astyanax Baird et Girard, 1854, is one of the largest genera in the family Characidae and comprises 177 valid species. This genus has been the focus of cytogenetic studies primarily owing to the presence of B chromosomes and high karyotypic diversity among different populations. The intense genetic variability in Astyanax is one of the factors responsible for the occurrence of species complexes, which are groups (1) with certain difficulties in establishing common genetic pools or (2) belonging to different cryptic species. To evaluate cytogenetic marker inheritance and the possibility of the identification of these hybrids, this study aimed to describe cytogenetic hybrids from three strains of species of the genera Astyanax and Hyphessobrycon Eigenmann, 1908. A. lacustris Lütken, 1875, A. schubarti Britski, 1964, A. fasciatus Cuvier, 1819, and H. anisitsi Eigenmann, 1907 were used to generate three hybrid lineages. The diploid number, heterochromatin sites, and ribosomal genes (18S and 5S rDNA) of the parental strains and the hybrids were analyzed. The results indicated that the three hybrid lineages had cytogenetic markers of both parents, presenting Mendelian inheritance. However, differences in distribution of heterochromatic blocks were observed between the hybrids and the parent strains. Our results allowed the identification of the hybrid strains based on the cytogenetic markers applied, reinforcing the efficiency of cytogenetic markers as tools for identification and indicating that such events may increase the karyotypic diversity in the genera Astyanax and Hyphessobrycon ., (Caio Augusto Gomes Goes, Sandro Natal Daniel, Lucas Henrique Piva, George Shigueki Yasui, Roberto Ferreira Artoni, Diogo Teruo Hashimoto, Fausto Foresti, Fábio Porto-Foresti.)

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

A histological characterization of gonadal development in the tetra Astyanax bimaculatus was performed, aimed at determining its reproductive cycle in streams localized inside the Amazonian forest. Collections were carried out monthly from August 2017 to July 2018 at the Zoobotânica Foundation of Marabá, PA. Collected specimens were weighed and measured, and their gonads and liver were removed and weighed to calculate gonadosomatic and hepatosomatic indexes. Gonads were fixed and treated for routine histology for light microscopy. Materials were stained with toluidine blue and haematoxylin and eosin. The Amazonian A. bimaculatus species presented two reproductive periods in the year, one at the end of the winter season and another during the summer. Females showed an asynchronous development of their oocytes and only two reproductive phases of development were observed during the whole period 'developing' and 'spawning capable'. Males presented cystic spermatogenesis, with an anastomosing tubular testis containing spermatogonia spread along the germinal epithelium (unrestricted spermatogonial). These morphological characteristics are considered phylogenetically more primitive. Male specimens were observed to have five different phases during the period: immature, initial maturation, mid maturation, final maturation and regression. The huge fluctuations in Amazonian streams was observed, in which water volumes varied considerably across seasons, culminating even in total drought. In spite of this, A. bimaculatus could be found during all seasons, showing its impressive reproductive adaptation to its conditions.

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3'UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

SummaryIn this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P &lt; 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Aflatoxins (AF) can be cumulative in fish tissues and can influence weight, length, feed intake and survival depending on the species. The aim of this work is to measure performance and aflatoxin levels in tissues of matrinxã (Brycon cephalus) fish chronically exposed to aflatoxin. Aflatoxin was incorporated into fish diets at the following levels: Control Feed + 0 μg AFB1 kg-1; A. Feed + 10 μg AFB1 kg-1; B. Feed + 20 μg AFB1 kg-1; C. Feed + 50 μg AFB1 kg-1. It was used one tank per treatment, each one with 150 juvenile fish, and three replicates within each tank were used for sampling, that was carried out monthly over a period of six months. Aflatoxin was quantified by HPLC in fish liver and muscle after clean up using immunoaffinity columns. Performance was evaluated by using weight, length, consumption and survival rate. Muscle and liver aflatoxin levels were below the limit of detection in all control samples. Aflatoxins B2, G1 and G2 were not detected in any tissues. Traces (values between limits of detection and quantification) of AFB1 were observed in liver tissue in treatment A from day 30 through 90, reaching 0.32 μg AFB1 kg-1 at 150 days of exposure. Treatment B presented traces up to day 60 and had, with a maximum level of 0.39 μg AFB1 kg-1 at 150 days of exposure. Treatment C had aflatoxin residues after day 30, with values ranging from 0.17 to 0.61 μg AFB1 kg-1 during exposure. Muscle samples only had traces of AFB1 in all treatments. Fish was affected by exposure to AFB1 with higher values (P<0.05) for weight and length in treatments A, B and C relative to controls. Therefore, results indicate that matrinxã do not accumulate AFB1 residues in edible tissues, but chronic exposure affects the species., Competing Interests: The authors have declared that no competing interests exist.

Environmental relevant concentrations of glyphosate-based herbicide as 50 µg l -1 , 300 µg l -1 and 1800 µg l -1 can affect sperm quality of yellowtail tetra fish Astyanax lacustris. Viability of sperm cells was impaired at 300 µg l -1 , a concentration that is within legal limits in U.S.A. waterbodies, while motility was impaired at 50 µg l -1 , which is the more stringent limit set in Brazilian law. Therefore, environment protection agencies must review regulations of glyphosate-based herbicides on water bodies., (© 2018 The Fisheries Society of the British Isles.)

SummaryThe aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22°C, 26°C, and 30°C, which resulted in fertilization rates of 96.95 ± 1.79%, 98.74 ± 0.76%, and 98.44 ± 0.19% for P. maculatus and 96.10 ± 1.58%, 98.00 ± 0.63%, and 94.60 ± 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22°C, 16 h 30 min at 26°C, and 11 h 20 min at 30°C, and the hatching rates were 43.87 ± 7,46%, 57.57 ± 17.49%, and 53.63 ± 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22°C and 17 h 30 min at 26°C with respective hatching rates of 45.4 ± 21.02% and 68.1 ± 12.67%. For this species, all embryos incubated at 30°C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22°C) and higher temperatures (30°C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26°C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species.

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, many factors can affect reproduction during in vitro fertilization, therefore determination of the factors that affect affecting gamete quality is needed. However, few studies have focused on gamete quality and the ploidy status. This study was conducted to elucidate whether oocyte storage can affect ploidy status, survival, and embryo viability in the characid species Astyanax altiparanae. Oocytes were stored in Dulbecco's phosphate-buffered saline (PBS) at 26°C, then aliquots were fertilized immediately after extrusion (control) and also after 60, 120, 180, and 240 min of storage. Fertilization and hatching rates were measured, and the developmental stages were analyzed at each stage before describing the main abnormalities. Ploidy status was analyzed by flow cytometry and blood smear. In the control group, 100% of the samples were diploid. After treatment for 60 min, 95.56 ± 4.44% samples were diploid and 4.44 ± 4.44% were triploid. After 120 min, 94.44 ± 9.62% of the samples was diploid and 5.56 ± 5.56% were triploid; 100% of the samples were diploid after 180 min and, after 240 min, there was no survival. In other treatments, the highest percentage of hatching was after 60 min (88.93 ± 5.15%; P = 0.015), and treatment with 180 min storage resulted in the highest percentage of abnormal larvae (95.76 ± 12.67%; P = 0.012). These results show that oocyte storage can affect ploidy status and may be an interesting parameter for analysis in studies on chromosome set manipulation and micromanipulation.

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.

Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30°C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26°C, and 6 mpf at 30°C. The duration of the 512-1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22°C, and 25 min at 26 and 30°C. Hatching occurred at 24 h 30 mpf at 22°C, 16 h post-fertilization (hpf) at 26°C, and 11 h 30 mpf at 30°C. The rate of morphologically normal larvae was 88.34% at 22°C, 90.49% at 26°C, and 73% at 30°C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.

The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100) at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5%) and sucrose (74 mM) and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at -20°C) and fixation in saturated NaCl solution, acetic methanol (1:3), ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl 2 .7H 2 0; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL -1 ; preservation procedure: somatic cells (dorsal fin samples) frozen at -20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.

The aim of this study was to verify the carryover of aflatoxin B 1 from feed to lambari fish. Aflatoxins (AF) were incorporated into feed, checking the levels by HPLC. Treatments were: Control, feed without toxin; A, feed + 10 µg AFB 1  kg - 1 ; B, feed + 20 µg AFB 1  kg - 1 ; and C, feed + 50 µg AFB 1  kg - 1 . Juveniles of lambari fish were placed in 12 aquariums at a density of 50 fish/m 2 . Fish were fed twice a day with extruded feed, at 5% of animal biomass. The unit sample was constituted by a pool of 10 fish. AFs B 1 , B 2 , G 1 , G 2 and M 1 were quantified by HPLC in fish muscle and liver after 30, 60, 90 and 120 days of experiment. There was accumulation of AFs is fish liver and muscle, mainly after 90 days. Fish from treatment C had higher levels of AFB 1 in muscle when compared with the others, and AFB 1 in muscle at 120 days was similar to the levels in feed. Therefore, when lambari fish is exposed on a daily and long-term basis to AFs in feed, the regulation limits for AFs in animal feed do not guarantee safety for consumers.

The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel® [(D-Ala6, Pro9-NEt) - mGnRH+metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1 ± 4.0%) or Ovopel® (79.5 ± 5.5%) when compared with the control group treated with 0.9% NaCl (55.6 ± 27.2%; P=0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4 ± 7.4%) when compared with untreated male fish (42.1 ± 26.1%; P=0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5°C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20°C when compared with fresh oocytes (P=0.0471), but considering only the stored samples, the optimum temperature was 15°C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.

Mexico is a biodiverse country in several taxa as reptiles, that include several species of freshwater and marine turtles. Eventhough most of this group species are under protection, Tabasco State has nine native freshwater turtles, like Kinosternon leucostomum, Trachemys scripta and Staurotypus triporcatus that are very important in traditional dishes. This has resulted in a critical level of their populations, together with little biological knowledge for their conservation. Therefore, this study was dedicated to turtle cytogenetics. The study was conducted using the conventional methods for cytogenetics. The results showed the modal diploid and haploid number for K. leucostomum of 2n = 56 (2n = 56+3 microchromosomes "B") and 1n = 28 chromosomes in mitosis and meiosis, respectively. In T. scripta 2n = 50 chromosomes (2n = 50+2 microchromosomes "B") and 1n = 25 chromosomes were also characterized. Whereas in S. triporcatus we only report the 2 = 54 chromosomes (2n = 54+2 microchromosomes "B"). The karyological formula for K. leucostomum was integrated by 12 metacentric-submetacentric chromosomes "msm"/"A"+22 subtelocentric-telocentric chromosomes "stt"/"B"+22 telocentric chromosomes "T"/"C" with fundamental number (FN) of 90 chromosome arms. While T. scripta karyotype was integrated by 32 "msm/"A"+10 "stt"/"B"+8"T/"C" chromosomes, with FN of 92 arms. S. triporcatus karyotype formula was built up by 20 chromosomes "msm"/"A"+34 chromosomes "T"/"C" with FN of 74. The variation in chromosome classification, the fundamental number and the presence of supernumerary microchromosomes "B" in the studied species, were evidence of a particular chromosome cytotypes in Tabasco. We considered that the presence of microchromosomes "B" probably has different origins, and they may be very important as a pattern for the formation or separation of new species. This study also showed the absence of heterologous chromosomes between the females and males karyotypes from the studied species.

An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes., (© 2011 American Society of Animal Science. All rights reserved.)

The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 degrees C for 10s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25mM NaCl and 50mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 degrees C for 10s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg(-1)mL(-1)). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully.