Reproductive strategies and chromosomal aberrations affect survival in the Rivuliid fish Hypsolebias sertanejo.

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.

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.

Chilling curves for Piaractus mesopotamicus (Holmberg, 1887) embryos stored at -8°C.

The present study investigates the effect of different slow chilling curves on the storage of pacu (Piaractus mesopotamicus) embryos submitted to chilling at -8°C. Embryos at the blastopore closure stage were divided into two groups: G1 - embryos exposed to cryoprotectant solution containing methanol (10%) and sucrose (0.5 M), treated as follows: (T1) taken directly from room temperature to the refrigerator without being submitted to the curve; (T2) chilling curve of 0.5°C/min; and (T3) chilling curve of 1°C/min; and G2 - the cryoprotectant solution alone was submitted to these same temperatures, receiving the embryos only after temperature had decreased, corresponding to treatments T4, T5 and T6, respectively. Treatments were kept at -8°C for a period of 6 h. Embryo development was evaluated for each treatment, with six replicates in an entirely randomized design. Survival among embryos not submitted to refrigeration was 94.3 ± 8.05%. Percentage of total larvae (TL) and addled eggs (AE) did not differ statistically between the groups, although percentage of swimming larvae (SL) exhibited higher values in G1 for the 1°C/min curve. Furthermore, when comparing the three chilling curves, a decrease of 1°C/min resulted in the highest TL percentage (90.85%), followed by the 0.5°C/min curve (78.52%). Thus, the use of 1°C/min chilling curves is recommended for P. mesopotamicus embryos stored for 6 h at -8°C.

Erratum to "Chilling sensitivity of Steindachneridion parahybae (Siluriformes: Pimelodidae) oocytes in different cryoprotectants" [7C (June 2019) 100046].

[This corrects the article DOI: 10.1016/j.vas.2019.100046.].

Chilling sensitivity of Steindachneridion parahybae (Siluriformes: Pimelodidae) oocytes in different cryoprotectants.

The viability of post-thaw fish oocytes can be affected by different stages of the freezing process, such as cryoprotectant toxicity, cold sensitivity, freezing curves and thawing. Therefore, these steps need to be investigated for the development of a protocol. In the present study, the aim was to investigate chilling sensitivity at different oocyte stages of Steindachneridion parahybae. Immature and mature oocytes were incubated in Hanks' or 90% L15 solutions containing different CPAs (cryoprotectant solutions) per experiment: (1) 0.1-0.4 M sucrose + 1-2 M methanol and (2) 1-4 M methanol X 1-4 M propylene glycol X 1-4 M DMSO for mature oocytes; (3) 0.5 M sucrose or fructose + 2 M methanol or PG or DMSO and (4) 0.25-1 M fructose + 1-4 M DMSO for immature oocytes. All treatments were kept for 120 min at -5.9 ±â€¯2.8°C. For the control treatment, only Hanks' or 90% L15 solutions were carried out. Evaluations were made by viability tests: membrane integrity staining in 0.4% Trypan blue (TB) and fertilization rate (%F) sole for mature oocytes. Results presented that mature oocytes were the most sensitive to lower temperatures, because there was no %F. All cryoprotectants tested in the different concentrations can be used for immature oocytes, however the statistically superior cryoprotectant was CPA with fructose and DMSO, with the low concentration of this CPA being was the best statistically. This may indicate that for this species the immature stages have presented a lower chilling sensitivity than the mature stages.

Colossoma macropomum females can reproduce more than once in the same reproductive period.

The aim of the present study was to evaluate induced reproduction in Colossoma macropomum females at the beginning of the reproductive period and 75 days after the first spawning in which reproduction was induced. The experiment was conducted in Nova Mutum, MT, Brazil. Eight 4-year-old C. macropomum females with an average body weight of 6.7 ± 2.4 kg were used. Hormonal induction was performed at the beginning of the reproductive period and repeated 75 days after the first spawning. The following variables were then evaluated: weight of released oocytes, production index, absolute fecundity, oocyte diameter, fertilization rate, and hatching rate. Of the eight females that spawned during the first hormonal induction, three (37.5%) spawned again 75 days after the first spawning. Two females died after the first induced spawning. None of the means of the evaluated variables differed between the two induced spawnings, except for fertilization rate, which was greater (P < 0.05) with the first spawning (88.8 ± 6.1%) than in the second (74.1 ± 10.4%). The results of the present study indicate that C. macropomum females can reproduce again 75 days after a first induced spawning.

Ovarian development and the reproductive profile of Astyanax altiparanae (Teleostei, Characidae) over one year: Applications in fish farming.

Astyanax altiparanae is a Brazilian species of substantial commercial, environmental and scientific importance; however, existing studies on its reproduction do not seem to provide enough details. In light of the increasing use of this species in fish farming and the need for basic studies for the development of new production technologies, we describe the structural and ultrastructural characteristics of the ovaries of A. altiparanae, and characterize the species' reproductive cycle. Females were collected monthly from March 2013 to February 2014, and reproductive management began in October 2013. The ovaries were removed, fixed in Karnovsky's fixative, and prepared for light microscopy, transmission electron microscopy and immunohistochemistry anti-PCNA. These techniques enabled us to characterize the ovaries, the germ cells, and the somatic cells in detail, as well as their changes over time. The reproductive cycle was characterized based on the monthly variation in the gonadosomatic ratio, the proportion of germ cells, and the rate of oogonium proliferation. The macroscopic analysis of the ovaries suggests that the vascularization pattern and color of the ovaries vary according to development. There are new types of analyses that can be applied even in the fish farming industry, such as a comparison between ovaries staining and weight or the frequency distribution of these colors throughout the year. This study also provides details on microscopic characteristics that have never before been reported for species of Astyanax, such as the presence of annulate lamellae in oogonia, the development of the zona pellucida from oocytes in the one-nucleolus step, and the development of the micropylar apparatus in oocytes in the cortical alveolar step. When the reproductive cycle was analyzed, this species was found to have a long period of spawning, with a reproductive peak from October to February and multiple spawning events, confirming the period already described in the literature. Variations in reproductive periods and the ability to reproduce in lentic environments suggest that A. altiparanae has the ability to respond quickly to environmental changes and exhibits high reproductive flexibility. All of these characteristics confirm the great potential of this species in the fish farming industry.

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes).

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli-germ and germ-germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli-germ and germ-germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.

Chilling of Steindachneridionparahybae (siluriformes: pimelodidae) embryos.

The objective of this study was to assess the viability of Steindachneridion parahybae embryos after chilling using different cryoprotectant solutions, stages of embryonic development, chilling curves, and storage periods at temperatures between -10 °C and 0 °C. Three experimental tests were conducted, and the following aspects were evaluated: (1) the toxicity of six cryoprotectant solutions (10% methanol, ethylene glycol, or DMSO combined with 0.5-M sucrose or lactose); (2) viability of embryos submitted to cooling with two cryoprotectant solutions (10% or 20% methanol combined with 10-M sucrose) at three different stages of development (closure of blastoporus, appearance of the optic vesicle and the moment when the tail began to straighten out), and two chilling periods (6 and 12 hours); (3) viability of embryos submitted to cooling with three chilling curves (directly to the freezer without a curve, 0.5 °C/min and 1.0 °C/min) and two chilling periods (6 and 12 hours). After the tests, it was concluded that the protocol which presented the most positive results after chilling, with a hatching rate of 63.50 ± 9.98% of the embryos and 12.32 ± 3.85% normal hatched larvae, was the one with embryos at the free-tail stage, the cryoprotectant solution with 10% methanol and 10-M sucrose, a chilling curve of 0.5 °C/min, stored for a maximum of 6 hours at subzero temperatures (temperature ranging between -5.05 °C and -7.83 °C).

Probiotic supplementation causes hematological changes and improves non-specific immunity in Brycon amazonicus

A commercialprobioticcontaining Bacillussubtilis(109 CFU g-1) was evaluated incaged matrinxã,Bryconamazonicus,by measuring hematological parametersand macrophage activity after 42 and 84 days after feeding. The product wasadded tocommercial feed using 2%soybean oil as a protectant. A randomized three-treatmentexperiment was performed using fourreplicates per treatment. The groups included: (a) control without probiotic, (b) 5 g kg-1probiotic, and (c) 10 g kg-1probiotic. Forhematological analysis,eightfishper treatmentwere used to determinetotal cell count (RBC); thrombocytes, differential, and total leukocyte count (TLC); hematocrit (Htc); hemoglobin tax; mean corpuscular volume (MCV); and mean corpuscular hemoglobin concentration (MCHC). Furthermore, plasma cortisol and glucose levels were measured in blood samples. Macrophage phagocytic activity was evaluated by injecting Saccharomyces cerevisiae(11,000 cells in a 3 mLvolume) into the coelomic cavity incubating for 8hours.Addition of probiotics to the diet of caged matrinxã altered the Htc, RBC, MCV, MCHC, TLC, lymphocyte, and eosinophil values. We observed increased cortisol and glucose levels and phagocytic activity, but no increase in the phagocytic index. We thus conclude that supplementing caged Brycon amazonicuswith probiotics improves their non-specific immunity and alters blood profiles.

Storage of Steindachneridion parahybae oocytes at different temperatures.

The objective of this study was to assess the influence of temperature and time on the storage of fresh Steindachneridion parahybae oocytes. Two experiments were carried out: (1) the fertilization rates of oocytes exposed to temperatures of 5, 15, 28 (room temperature) and 35°C were assessed 15min (control), 115, 235 and 355min after release; (2) the fertilization and hatching rates, as well as the percentage of normal larvae of oocytes exposed to 14, 17 or 20°C, 20min (control) were assessed 50, 80 and 110min after stripping. In the first experiment, the highest fertilization rates (P<0.05) were obtained in the control treatment (15min, 28°C), with 74.34±5.48% oocytes showing loss of viability over time. In the second experiment, there was a reduction (P<0.05) in the fertilization rates at the temperatures and times tested. The artificial fertilization of S. parahybae oocytes is recommended immediately after collection, and if storage is necessary, it should be conducted at temperatures between 17 and 20°C.

Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants / Viabilidade de oócitos de surubim do Paraíba após exposição a diferentes crioprotetores

ABSTRACT: To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter <1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.

RESUMO: Conhecer os crioprotetores não tóxicos aos oócitos de peixes é de extrema importância para testes que visam aumentar a resistência dos oócitos ao frio, permitindo, assim, estudos mais avançados em criopreservação. Desta forma, crioprotetores comumente utilizados como o metanol, dimetil sulfóxido, etilenoglicol, propilenoglicol, sacarose e frutose foram estudados. Os oócitos imaturos, nos estágios inicial até vitelogênico (diâmetro <1,7mm), e maduros (diâmetro >1,8mm) de Steindachneridion parahybae foram avaliados. Quatro experimentos distintos foram realizados, sendo três destes utilizando oócitos imaturos, e um usando oócitos no estágio maduro. Para cada estágio oocitários foram avaliados, considerando qual a melhor solução de manutenção a ser utilizada: Hank ou 50% L15 e; viabilidade após banhos por 30min (temperatura ambiente) em concentrações de crioprotetores, variando de 0,25 a 4M. Diferentes testes foram utilizados para avaliar a viabilidade dos oócitos: maturação in vitro seguido por observação da quebra da vesícula germinativa (somente para oócitos imaturos), coloração por Azul de Tripan (todos os estágios) e taxas de fertilização e eclosão (somente no estágio maduro). Os resultados mostraram que o efeito tóxico dos crioprotetores em oócitos geralmente crescem com o aumento das concentrações. A sensibilidade dos oócitos a crioprotetores aumentam de acordo com o estágio de desenvolvimento, com oócitos maduros sendo mais sensíveis. Sacarose, frutose, metanol, propileno glicol e dimetil sulfóxido podem ser usados como crioprotetores para oócitos de S. parahybae.

Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants / Viabilidade de oócitos de surubim do Paraíba após exposição a diferentes crioprotetores

To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter 1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.(AU)

Conhecer os crioprotetores não tóxicos aos oócitos de peixes é de extrema importância para testes que visam aumentar a resistência dos oócitos ao frio, permitindo, assim, estudos mais avançados em criopreservação. Desta forma, crioprotetores comumente utilizados como o metanol, dimetil sulfóxido, etilenoglicol, propilenoglicol, sacarose e frutose foram estudados. Os oócitos imaturos, nos estágios inicial até vitelogênico (diâmetro 1,7mm), e maduros (diâmetro >1,8mm) de Steindachneridion parahybae foram avaliados. Quatro experimentos distintos foram realizados, sendo três destes utilizando oócitos imaturos, e um usando oócitos no estágio maduro. Para cada estágio oocitários foram avaliados, considerando qual a melhor solução de manutenção a ser utilizada: Hank ou 50% L15 e; viabilidade após banhos por 30min (temperatura ambiente) em concentrações de crioprotetores, variando de 0,25 a 4M. Diferentes testes foram utilizados para avaliar a viabilidade dos oócitos: maturação in vitro seguido por observação da quebra da vesícula germinativa (somente para oócitos imaturos), coloração por Azul de Tripan (todos os estágios) e taxas de fertilização e eclosão (somente no estágio maduro). Os resultados mostraram que o efeito tóxico dos crioprotetores em oócitos geralmente crescem com o aumento das concentrações. A sensibilidade dos oócitos a crioprotetores aumentam de acordo com o estágio de desenvolvimento, com oócitos maduros sendo mais sensíveis. Sacarose, frutose, metanol, propileno glicol e dimetil sulfóxido podem ser usados como crioprotetores para oócitos de S. parahybae.(AU)

Crude protein in diets for Nile tilapia broodfish / Proteina bruta em dietas para reprodutores de tilápia do Nilo

The effect of different levels of crude protein (32A, 32B, 36, 38, 44 and 50% CP; 3,500 kcal digestible energy) on Nile tilapia broodstock was assessed. After 30 experimental weeks (Sept./14 to Mar./15), 91.0% of eggs from fish fed 44% CP hatched and produced 16.4% more viable larvae than the treatment with 32% CP. Egg production and absolute fecundity were similar between treatments (p>0.05). Sperm motility, average path, straight line and curvilinear velocities showeds atisfactory values with 44% CP, unlike 36% CP. Lower profitability was observed with 32% CP; profit increased as protein level upped. Statistically significant responses were not found for reproductive performance of females. Results were satisfactory for commercial-scale production as crude protein increased. The initial hypothesis was demonstrated for most parameters assessed in males, larvae growth and economic viability. Therefore, it is recommend the use of diets with 44% CP for Nile tilapia brood fish.(AU)

O efeito de diferentes níveis de proteína bruta (32A, 32B, 36, 38, 44 e 50% PB; 3500 kcal energia digestível) em reprodutores de tilápia do Nilo foi avaliado. Após 30 semanas experimentais (set./14 a mar./15), 91, 0% dos ovos de peixes alimentados com 44% PB, eclodiram e produziram 16,4% de larvas viáveis a mais do que o tratamento que recebeu 32% PB. A produção de ovos e a fecundidade absoluta foram semelhantes entre os tratamentos (p>0,05). A motilidade e a velocidade médiasdo esperma, em linha reta e curvilínea, apresentaram valores satisfatórios no tratamento em 44% CP, ao contrário de 36% PB. Observou-se baixa rentabilidade com 32% PB; todavia, o lucro aumentou com o nível de proteínas na dieta. Respostas estatisticamente significativas não foram encontradas para o desempenho reprodutivo de fêmeas. Os resultados foram satisfatórios para a produção em escala comercial com o aumento da proteína bruta. A hipótese inicial foi demonstrada para a maioria dos parâmetros avaliados em machos, crescimento de larvas e viabilidade econômica. Portanto, recomenda-se dieta com 44% PB para reprodutores de tilápia do Nilo.(AU)

Crude protein in diets for Nile tilapia broodfish / Proteina bruta em dietas para reprodutores de tilápia do Nilo

The effect of different levels of crude protein (32A, 32B, 36, 38, 44 and 50% CP; 3,500 kcal digestible energy) on Nile tilapia broodstock was assessed. After 30 experimental weeks (Sept./14 to Mar./15), 91.0% of eggs from fish fed 44% CP hatched and produced 16.4% more viable larvae than the treatment with 32% CP. Egg production and absolute fecundity were similar between treatments (p>0.05). Sperm motility, average path, straight line and curvilinear velocities showeds atisfactory values with 44% CP, unlike 36% CP. Lower profitability was observed with 32% CP; profit increased as protein level upped. Statistically significant responses were not found for reproductive performance of females. Results were satisfactory for commercial-scale production as crude protein increased. The initial hypothesis was demonstrated for most parameters assessed in males, larvae growth and economic viability. Therefore, it is recommend the use of diets with 44% CP for Nile tilapia brood fish.

O efeito de diferentes níveis de proteína bruta (32A, 32B, 36, 38, 44 e 50% PB; 3500 kcal energia digestível) em reprodutores de tilápia do Nilo foi avaliado. Após 30 semanas experimentais (set./14 a mar./15), 91, 0% dos ovos de peixes alimentados com 44% PB, eclodiram e produziram 16,4% de larvas viáveis a mais do que o tratamento que recebeu 32% PB. A produção de ovos e a fecundidade absoluta foram semelhantes entre os tratamentos (p>0,05). A motilidade e a velocidade médiasdo esperma, em linha reta e curvilínea, apresentaram valores satisfatórios no tratamento em 44% CP, ao contrário de 36% PB. Observou-se baixa rentabilidade com 32% PB; todavia, o lucro aumentou com o nível de proteínas na dieta. Respostas estatisticamente significativas não foram encontradas para o desempenho reprodutivo de fêmeas. Os resultados foram satisfatórios para a produção em escala comercial com o aumento da proteína bruta. A hipótese inicial foi demonstrada para a maioria dos parâmetros avaliados em machos, crescimento de larvas e viabilidade econômica. Portanto, recomenda-se dieta com 44% PB para reprodutores de tilápia do Nilo.

Cooling of pacu (Piaractus mesopotamicus) embryos at various stages of development for 6 or 10 hours.

The objective of this research was to verify the effects of cooling embryos of pacu, Piaractus mesopotamicus, in four stages of development during two stocking periods. The stages of embryo development were at: blastoderm, ∼ 64 cells-1.4 h after fertilization (haf); 25% of the epiboly movement--5.2 haf; blastoporous closing--8.0 haf; and optical vesicle appearing--13.3 haf. Embryos were exposed to a cryoprotectant solution containing methanol (10%) and sucrose (0.5 M). Thereafter, embryos were submitted to a cooling curve until they reached -8 °C, and then kept cooled for 6 or 10 h. In addition, for each stage of embryonic development, a control group with uncooled embryos was used to compare hatching rates. The total number of larvae from the first two stages of ontogenetic development (1.4 and 5.2 haf) was lower compared to the other stages (0.0 and 8.0 haf). There was no significant difference between stages 8.0 and 13.3 haf for the total number of larvae (49.9 ± 6.7% and 55.2 ± 6.7%, respectively). Embryo diameter varied according to embryonic stage, providing evidence of differences in membrane permeability. There was a negative correlation between embryo diameter and the total number of larvae (r = -0.372). In conclusion, use of embryonic stages 8.0 and 13.3 haf were recommended for maintaining cooled pacu embryos at -8 °C for 6 or 10 h.

Inseminating dose and water volume applied to the artificial fertilization of Steindachneridion parahybae (Steindachner, 1877) (Siluriformes: Pimelodidae): Brazilian endangered fish

The Steindachneridion parahybae is an endangered catfish from Brazil and strategies applied for gametes optimization are necessary. The aim of this study was to assess inseminating doses and water volume upon the fertilization, hatching rates and percentage of normal larvae in S. parahybae . Was used a randomized design in factorial scheme (4×4) with four inseminating doses: 1.0×104, 1.0×105, 1.0×106, 1.0×107spermatozoa oocyte-1 and four volumes of water: 1, 35, 65 and 95mL of water g-1 of oocytes. The combination of doses and volumes were performed in triplicates (n=48). Each incubator (1.5L of useful volume) with 1g of oocytes was considered as an experimental unit. Significant interaction between inseminating doses and volumes of water to the values of the fertilization rates and quadratic effect of doses and volume for the values of hatching rates were observed. The doses and volumes did not influence the percentage of normal larvae (87.70±5.06%). It is recommended the use of 5.5×106 spermatozoa oocyte-1 and 1mL of water g-1 of oocytes during in vitro fertilization procedure. These results allowed us to develop new biotechnological strategies applied to the conservation of S. parahybae.(AU)

O Steindachneridion parahybae é um bagre ameaçado de extinção no Brasil e estratégias aplicadas para a otimização de gametas são necessárias. O objetivo deste estudo foi avaliar doses inseminantes e volume de água sobre os valores das taxas de fertilização, eclosão e larvas normais em S. parahybae. Utilizando-se um delineamento experimental casualizado em esquema fatorial (4×4), com quatro doses inseminantes: 1,0×104; 1,0×105; 1,0×106; 1,0×107 espermatozóides ovócito-1 e quatro volumes de água: 1; 35; 65 e 95mL de água g-1 de ovócitos. As combinações de doses e volumes foram realizadas em triplicatas (n=48). Cada incubadora (1,5L de volume útil) contendo 1g de ovócitos foi considerada como uma unidade experimental. Interações significativas entre doses inseminantes e volumes de água para os valores das taxas de fertilização e efeito quadrático das doses e do volume para os valores das taxas de eclosão foram verificadas. As dosagens e os volumes aplicados não influenciaram no percentual de larvas normais (87,70±5,06). Recomenda-se a aplicação de 5,5×106 espermatozoides ovócito-1 e a utilização de 1mL de água.g-1 de ovócitos no procedimento de fertilização artificial in vitro. Estes resultados permitiram desenvolver novas estratégias biotecnológicas aplicadas na conservação do S. parahybae.(AU)

Inseminating dose and water volume applied to the artificial fertilization of Steindachneridion parahybae (Steindachner, 1877) (Siluriformes: Pimelodidae): Brazilian endangered fish

The Steindachneridion parahybae is an endangered catfish from Brazil and strategies applied for gametes optimization are necessary. The aim of this study was to assess inseminating doses and water volume upon the fertilization, hatching rates and percentage of normal larvae in S. parahybae . Was used a randomized design in factorial scheme (4×4) with four inseminating doses: 1.0×104, 1.0×105, 1.0×106, 1.0×107spermatozoa oocyte-1 and four volumes of water: 1, 35, 65 and 95mL of water g-1 of oocytes. The combination of doses and volumes were performed in triplicates (n=48). Each incubator (1.5L of useful volume) with 1g of oocytes was considered as an experimental unit. Significant interaction between inseminating doses and volumes of water to the values of the fertilization rates and quadratic effect of doses and volume for the values of hatching rates were observed. The doses and volumes did not influence the percentage of normal larvae (87.70±5.06%). It is recommended the use of 5.5×106 spermatozoa oocyte-1 and 1mL of water g-1 of oocytes during in vitro fertilization procedure. These results allowed us to develop new biotechnological strategies applied to the conservation of S. parahybae.

O Steindachneridion parahybae é um bagre ameaçado de extinção no Brasil e estratégias aplicadas para a otimização de gametas são necessárias. O objetivo deste estudo foi avaliar doses inseminantes e volume de água sobre os valores das taxas de fertilização, eclosão e larvas normais em S. parahybae. Utilizando-se um delineamento experimental casualizado em esquema fatorial (4×4), com quatro doses inseminantes: 1,0×104; 1,0×105; 1,0×106; 1,0×107 espermatozóides ovócito-1 e quatro volumes de água: 1; 35; 65 e 95mL de água g-1 de ovócitos. As combinações de doses e volumes foram realizadas em triplicatas (n=48). Cada incubadora (1,5L de volume útil) contendo 1g de ovócitos foi considerada como uma unidade experimental. Interações significativas entre doses inseminantes e volumes de água para os valores das taxas de fertilização e efeito quadrático das doses e do volume para os valores das taxas de eclosão foram verificadas. As dosagens e os volumes aplicados não influenciaram no percentual de larvas normais (87,70±5,06). Recomenda-se a aplicação de 5,5×106 espermatozoides ovócito-1 e a utilização de 1mL de água.g-1 de ovócitos no procedimento de fertilização artificial in vitro. Estes resultados permitiram desenvolver novas estratégias biotecnológicas aplicadas na conservação do S. parahybae.

Danos causados a embriões de Piaractus mesopotamicus, em diferentes estádios de desenvolvimento, após técnica de resfriamento / Post-cooling damage to Piaractus mesopotamicus embryos at different stages of development

The present study investigated the effects of 6h- and 10h-storage at -8ºC on the quality and hatch rate of Piaractus mesopotamicus embryos at 4 stages of development. Embryos were exposed to a cryoprotectant solution, cooled down at a rate of 1ºC.min-1 to -8ºC, and stored at this temperature for 6h and 10h, respectively. For control treatment, viable embryos at the 4 developmental stages studied, were selected and taken immediately to the incubator, without going through cooling. The results were evaluated using a multivariate statistical technique (factor analysis). Damage was characterized according to the following variables: uniformity, adhesion, symmetry, margins, and inclusion. Two factors that best explained the variance of each parameter were defined. The control group had the highest hatch rates, and a weak relationship with embryo damage. Although treatments involving 6h and 10h cooling exhibited lower hatch rates and a higher association to damage. The information obtained in this study is useful in promoting improved cryopreservation techniques for fish embryos, indicating the probable conditions under which certain injuries are more frequent.

O presente estudo investigou o efeito da estocagem de embriões de Piaractus mesopotamicus em quatro diferentes estádios de desenvolvimento, durante 6 e 10 horas a -8ºC na qualidade e taxa de eclosão. Os embriões foram expostos à solução crioprotetora e passaram por curva de resfriamento de 1ºC.min-1 até atingir -8ºC, onde foram mantidos por 6 e 10 horas, respectivamente. Para o tratamento controle, embriões viáveis nos quatro estádios de desenvolvimento estudados, foram selecionados e levados a incubadoras, sem passar por resfriamento. Os resultados foram avaliados usando estatística multivariada (análise de fatores). Os danos causados pelo resfriamento foram caracterizados de acordo com as variáveis: uniformidade, adesão, simetria e bordas das células, além de inserção no vitelo. Foram definidos dois fatores que conseguiram reter maior variância contida nos dados. O grupo controle apresentou alta taxa de eclosão e baixa relação aos danos verificados nos embriões. Enquanto os tratamentos com 6 e 10 horas após resfriamento tiveram taxas de eclosão mais baixas e alta associação aos danos. Os resultados encontrados são importantes, pois indicam condições prováveis em que ocorrem lesões durante o processo de resfriamento das células, contribuindo assim para o aperfeiçoamento da técnica de criopreservação de embriões.

Danos causados a embriões de Piaractus mesopotamicus, em diferentes estádios de desenvolvimento, após técnica de resfriamento / Post-cooling damage to Piaractus mesopotamicus embryos at different stages of development

The present study investigated the effects of 6h- and 10h-storage at -8ºC on the quality and hatch rate of Piaractus mesopotamicus embryos at 4 stages of development. Embryos were exposed to a cryoprotectant solution, cooled down at a rate of 1ºC.min-1 to -8ºC, and stored at this temperature for 6h and 10h, respectively. For control treatment, viable embryos at the 4 developmental stages studied, were selected and taken immediately to the incubator, without going through cooling. The results were evaluated using a multivariate statistical technique (factor analysis). Damage was characterized according to the following variables: uniformity, adhesion, symmetry, margins, and inclusion. Two factors that best explained the variance of each parameter were defined. The control group had the highest hatch rates, and a weak relationship with embryo damage. Although treatments involving 6h and 10h cooling exhibited lower hatch rates and a higher association to damage. The information obtained in this study is useful in promoting improved cryopreservation techniques for fish embryos, indicating the probable conditions under which certain injuries are more frequent.(AU)

O presente estudo investigou o efeito da estocagem de embriões de Piaractus mesopotamicus em quatro diferentes estádios de desenvolvimento, durante 6 e 10 horas a -8ºC na qualidade e taxa de eclosão. Os embriões foram expostos à solução crioprotetora e passaram por curva de resfriamento de 1ºC.min-1 até atingir -8ºC, onde foram mantidos por 6 e 10 horas, respectivamente. Para o tratamento controle, embriões viáveis nos quatro estádios de desenvolvimento estudados, foram selecionados e levados a incubadoras, sem passar por resfriamento. Os resultados foram avaliados usando estatística multivariada (análise de fatores). Os danos causados pelo resfriamento foram caracterizados de acordo com as variáveis: uniformidade, adesão, simetria e bordas das células, além de inserção no vitelo. Foram definidos dois fatores que conseguiram reter maior variância contida nos dados. O grupo controle apresentou alta taxa de eclosão e baixa relação aos danos verificados nos embriões. Enquanto os tratamentos com 6 e 10 horas após resfriamento tiveram taxas de eclosão mais baixas e alta associação aos danos. Os resultados encontrados são importantes, pois indicam condições prováveis em que ocorrem lesões durante o processo de resfriamento das células, contribuindo assim para o aperfeiçoamento da técnica de criopreservação de embriões.(AU)