Dynamics of ovarian maturation during the annual reproductive cycle of the endangered fish Steindachneridion parahybae (Siluriformes: Pimelodidae) in captivity.

To characterize the female reproductive biology of the endangered species Steindachneridion parahybae in captivity, the authors used the concentration of gonadal steroids and the oocyte development during the annual reproductive cycle (RC) and after artificial induced spawning (AIS) until 48 h. Three stages of gonadal maturation were identified, based on morphological and physiological features: early maturation or previtellogenic (PRV) oocyte, advanced maturation or vitellogenic (VTG) oocyte and regression (REG) or follicular atresia. They identified and characterized the following stages of germ cells: oogonia, perinucleolar and cortical alveoli, and VTG and atretic oocytes during RC. The oestradiol levels were higher in PRV than those in VTG and REG during the RC, whereas androgens showed higher levels of oestradiol in VTG than those in PRV and REG. The progestogen levels remained unchanged during the whole RC. The final oocyte maturation (FOM) was achieved after AIS and postovulatory follicles (POF) were identified. Plasma concentration of progestogens (17α,20ß-dihydroxy-pregnen-3-one and 17α-hydroxyprogesterone) increased considerably after AIS, remaining high up to 6 h after AIS, and progressively decreased over time after AIS. During RC, the lack of FOM and POFs reveals that captivity negatively impacts S. parahybae reproduction. Nonetheless, the VTG stage of oocytes, reached during RC, is suitable for ovulation induction with artificial hormone manipulation, enabling the reproduction of this species in captivity and being essential for the success of fish farming and/or fish conservation programmes (conservationist aquaculture).

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.].

Characterization of the gonadotropin-releasing hormone system in the Neotropical teleost, Steindachneridion parahybae during the annual reproductive cycle in captivity.

This study evaluated by immunohistochemical and Western blot methods, the distribution of two distinct gonadotropin-releasing hormones (GnRHs), corresponding to catfish GnRH (cfGnRH or GnRH1) and chicken-II GnRH (cGnRH-II or GnRH2), in Steindachneridion parahybae females in captivity, focusing these analyses on the reproductive cycle by semi-quantification of optical density (OD). Further, we found that the GnRH neuronal systems co-localized with their respective GnRH-associated peptides (GAPs). A group of neurons immunoreactive (ir) to GnRH1 were identified along the ventral region of the olfactory bulb (vOB) in the telencephalon (vTel) and in the main areas of the diencephalon (especially the medial basal hypothalamus, HBM), including fibers extending into the pituitary gland. In contrast, GnRH2 neurons were confined to the midbrain tegmentum, close to the ventricular surface, without projections to the pituitary gland. Moreover, a cfGAP (GnRH1)-specific band (9 kDa) was identified in the brain and pituitary gland, while a cGAP-II (GnRH2)-specific band (26 kDa) was observed only in the brain extract. During the reproductive cycle, GnRH1-ir presented greater OD values at the vitellogenic and regression stages than at the previtellogenic stage and after artificially induced to spawn. Larger GnRH2-ir neurons were observed during the reproductive cycle, but a higher OD was identified only in the regression stage compared with the other maturation stages. Finally, GnRH1 axons were found to be directed towards the pituitary, and this GnRH type, which is probably the hypophysiotropic form, can contribute to the reproductive dysfunction that occurs in S. parahybae females in captivity, whereas GnRH2 may act as a neuromodulator and/or neurotransmitter.

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.

Mobilization of energetic substrates in the endangered catfish Steindachneridion parahybae (Siluriformes: Pimelodidae): changes in annual reproductive cycle in captivity

This study aimed at analyzing the energetic substrate (ES) in the main storage tissues of Steindachneridion parahybae, throughout the reproductive cycle in captivity. Differently from wild, in captivity, feeding is not interrupted during the reproductive period, the females do not spawn spontaneously, and they are sedentary. Adult females were sampled monthly and based on their histology and gonadosomatic index (GSI), ovaries were classified into: previtellogenic (PRV), vitellogenic (VTG), and regression (REG) stages. Ovaries at the VTG stage showed higher protein and lipids levels than at the PRV stage with a positive correlation between these substrates and the GSI. Muscle was the main source of proteins transferred to the ovaries, according to the negative correlation between these organs. Lipids remained unchanged in the liver, which is an important supplier in vitellogenesis, a pattern that probably occurs due to the continuous feeding. Muscular glycogen levels were higher at the VTG and REG than at the PRV stages. Plasma triglycerides were also higher during REG, while glucose levels were more elevated during the VTG stage. These results suggest that with constant food supply, the pattern of deposition of ES in S. parahybae is different from that described for other wild potamodromous species.(AU)

O objetivo deste estudo foi analisar a composição do substrato energético (SE) nos principais tecidos de armazenamento de Steindachneridion parahybae, durante todo o ciclo reprodutivo em cativeiro. Diferentemente do ambiente natural, em cativeiro, a alimentação desses animais não é interrompida durante o período reprodutivo, as fêmeas não desovam espontaneamente, e são sedentárias. Fêmeas adultas foram amostradas mensalmente e baseada na histologia e no índice gonadossomático (IGS), os ovários foram classificados: estádios pré-vitelogênico (PRV), vitelogênico (VTG) e regressão (REG). Os ovários no estádio VTG apresentaram uma maior concentração de lipídeos e proteínas em relação ao estágio PRV. Esses substratos correlacionaram-se positivamente com o IGS. O músculo foi a principal fonte de proteína transferida aos ovários, como foi confirmado pela análise de correlação negativa entre esses órgãos. Os lipídeos mantiveram-se inalterados no fígado, considerado um importante órgão fornecedor de lipídeos para a vitelogênese, padrão que possivelmente ocorreu devido à contínua alimentação. A concentração do glicogênio muscular foi mais elevada durante os estágios VTG e REG em relação ao PRV. A concentração de triglicerídeos plasmática apresentou maiores valores no estádio REG enquanto a concentração de glicose no plasma foi maior durante os estádios VTG. Esses resultados sugerem que com alimentação constante, as fêmeas de S. parahybae apresentam um distinto padrão de mobilização dos substratos energéticos em relação ao que já foi descrito para outras espécies potamódromas de ambiente natural.(AU)

This study aimed at analyzing the energetic substrate (ES) in the main storage tissues of Steindachneridion parahybae, throughout the reproductive cycle in captivity. Differently from wild, in captivity, feeding is not interrupted during the reproductive period, the females do not spawn spontaneously, and they are sedentary. Adult females were sampled monthly and based on their histology and gonadosomatic index (GSI), ovaries were classified into: previtellogenic (PRV), vitellogenic (VTG), and regression (REG) stages. Ovaries at the VTG stage showed higher protein and lipids levels than at the PRV stage with a positive correlation between these substrates and the GSI. Muscle was the main source of proteins transferred to the ovaries, according to the negative correlation between these organs. Lipids remained unchanged in the liver, which is an important supplier in vitellogenesis, a pattern that probably occurs due to the continuous feeding. Muscular glycogen levels were higher at the VTG and REG than at the PRV stages. Plasma triglycerides were also higher during REG, while glucose levels were more elevated during the VTG stage. These results suggest that with constant food supply, the pattern of deposition of ES in S. parahybae is different from that described for other wild potamodromous species.(AU)

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.

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.

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.

A case of intersex occurrence in Steindachneridion parahybae (Steindachner, 1877) (Siluriformes: Pimelodidae) under captivity condition: a cytogenetic and morphological study

Little is known about reproductive biology of endangered Steindachneridion parahybae , a gonochoristic teleost species inhabiting the Paraíba do Sul River Basin, and herein is the first description of intersex in S. parahybae juvenile. The normal appearance of ovaries and testes in juvenile from the same lot of breeding were also described for comparison, even as cytogenetic analysis was performed in these juveniles. One specimen was a priori classified as female due to the macroscopic characteristic of ovaries, with small yellow oocytes, without fringes (a main characteristic of catfish male), and larger than testes; however the microscopic analysis revealed the presence of ovotestes, including the complete spermatogenesis. S. parahybae had diploid number, 2n = 56 chromosomes with no evidence of differentiated sex chromosomes or supernumerary chromosomes among them. These findings may be due to the result of exposure to endocrine disrupting compounds or may also be influenced by environmental conditions. The possibility of intersexes might also happen spontaneously and it cannot be ruled out. Therefore, the functional significance and reproductive consequences of this anomaly remain to be determined, suggesting that this species may be susceptible to endocrine disruption. These results contribute to gain expertise about reproductive biology of an endangered species in captivity.(AU)

Poco se sabe sobre la biología reproductiva de Steindachneridion parahybae , una especie de teleósteo gonocorístico en peligro de extinción que habita la cuenca del río Paraíba do Sul y en éste trabajo se describe por primera vez la aparición de individuo intersexo en juvenil de S. parahybae . También se describió el aspecto normal de los ovarios y de los testículos de individuos juveniles procedentes del mismo lote de cría para su comparación; se realizó además el análisis citogenético. Un espécimen fue clasificado a priori como hembra debido a las características macroscópicas de los ovarios, con pequeños oocitos amarillos, sin flecos (característica principal de los bagres macho) y más grande que los testículos; sin embargo el análisis microscópico reveló la presencia de un ovotestis, incluyendo una espermatogénesis completa. S. parahybae presentó un número diploide, 2n = 56 cromosomas, sin evidencia de cromosomas sexuales diferenciados o supernumerarios entre ellos. Estos hallazgos pueden deberse al resultado de la exposición de los individuos a desorganizadores endocrinos o estar influenciados por las condiciones ambientales. Sin embargo no se puede descartar la posibilidad de la presencia de intersexos de forma espontánea. Por lo tanto, la importancia funcional y las consecuencias reproductivas de estas anomalías permanecen aún sin ser determinadas, sugiriendo que esta especie puede ser susceptible a los disruptores endocrinos. Estos resultados contribuyen a ampliar el conocimiento de la biología reproductiva de esta especie en peligro de extinción en condiciones de cautiverio.(AU)

Gonadotropins and Growth Hormone Family Characterization in an Endangered Siluriform Species, Steindachneridion parahybae (Pimelodidae): Relationship With Annual Reproductive Cycle and Induced Spawning in Captivity.

The aim of this study was to identify and characterize pituitary cells of Steindachneridion parahybae females in captivity, highlighting the possible relationship with reproductive disorders at this level, since this species shows oocyte final maturation, ovulation and spawning dysfunction in captivity. The localization and distribution of growth hormone (GH), prolactin (PRL), somatolactin (SL), ß-luteinizing hormone (ß-LH), and ß-follicle stimulating hormone (ß-FSH) immunoreactive (-ir) cells in the adenohypophysis was studied by immunohistochemical and Western blot methods. In addition, cellular morphometric analyses and semi-quantification of ir-cells optical density (OD) during the annual reproductive cycle and after artificial induced spawning (AIS) were performed. Results showed that the distribution and general localization of pituitary cell types were similar to that of other teleost species. However, the morphometrical study of adenohypophysial cells showed differences along the reproductive cycle and following AIS. In general, females at the vitellogenic stage presented greater OD values for GH, PRL and SL than at other maturation stages (previtellogenic and regression stages), probably indicating an increased cellular activity during this stage. Conversely, ß-LH OD did not vary during the annual reproductive cycle. After AIS, ß-LH, SL and GH ir-cells showed an increase in OD values suggesting a possible involvement on oocyte final maturation, ovulation and spawning or a feedback control on the brain-pituitary-gonads axis. Reproductive dysfunction in S. parahybae females in captivity may be due to alteration of the synthesis pathways of ß-LH. In addition, GH family of hormones could modulate associated mechanisms that influence the reproductive status in this species.

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.

Embryonic development and larval stages of Steindachneridion parahybae (Siluriformes: Pimelodidae): implications for the conservation and rearing of this endangered Neotropical species

Steindachneridion parahybae is a freshwater catfish endemic to the Paraíba do Sul River and is classified as an endangered Neotropical species. An increasing number of conservation biologists are incorporating morphological and physiological research data to help conservation managers in rescue these endangered species. This study investigated the embryonic and larval development of S. parahybae in captivity, with emphasis in major events during the ontogeny of S. parahybae. Broodstocks were artificially induced to reproduce, and the extrusion occurred 200-255 degree-hours after hormonal induction at 24°C. Larval ontogeny was evaluated every 10 minutes under microscopic/stereomicroscopic using fresh eggs samples. The main embryogenic development stages were identified: zygote, cleavage, including the morula, blastula, gastrula phase, organogenesis, and hatching. The extruded oocytes showed an average diameter of 1.10 ± 0.10 mm, and after fertilization and hydration of eggs, the average diameter of eggs increased to about 1.90 ± 0.60 mm, characterized by a large perivitelline space that persisted up to embryo development, the double chorion, and the poles (animal and vegetative). Cell division started about 2 minutes after fertilization (AF), resulting in 2, 4, 8 (4 x 2 arrangement of cells), 16 (4 x 4), 32 (4 x 8) and 64 (2 x 4 x 8) cells. Furthermore, the blastula and gastrula stages followed after these cells divisions. The closed blastopore occurred at 11 h 20 min AF; following the development, the organogenetic stages were identified and subdivided respectively in: early segmentation phase and late segmentation phase. In the early segmentation phase, there was the establishment of the embryonic axis, and it was possible to distinguish between the cephalic and caudal regions; somites, and the optic vesicles developed about 20 h AF. Total hatching occurred at 54 h AF, and the larvae average length was 4.30 ± 0.70 mm. Gradual yolk sac reduction was observed during the first two days of larval development. The first feeding occurred at the end of the second day. During the larval phase, cannibalism, heterogeneous larval growth and photophobia were also observed. This information will be important in improving the artificial reproduction protocols of S. parahybae in controlled breeding programs.(AU)

Steindachneridion parahybae é um bagre de água doce, endêmico do rio Paraíba do Sul e é classificado como espécie neotropical ameaçada. Um número crescente de biólogos conservacionistas estão incorporando dados de pesquisas morfológicas e fisiológicas para ajudar os gestores de conservação no resgate destas espécies ameaçadas de extinção. Este estudo investigou o desenvolvimento embrionário e larval de S. parahybae em cativeiro, com ênfase nos principais eventos durante a ontogenia de S. parahybae. Reprodutores foram artificialmente induzidos à reprodução e a extrusão ocorreu com 200-255 horas-graus após a indução hormonal a 24°C. A ontogenia larval foi avaliada a cada 10 minutos sob microscópio/ estereomicroscópio, utilizando-se amostras de ovos recém coletados. Os principais estágios de desenvolvimento embrionário foram identificados: zigoto, clivagem, incluindo as fases, mórula, blástula e gástrula, organogênese e eclosão. Os ovócitos extrusados apresentaram uma média de diâmetro de 1,10 ± 0,10 mm e depois da fertilização e hidratação dos ovos, a média de diâmetro dos ovos aumentou para 1,90 ± 0,60 mm, caracterizado pelo grande espaço vitelínico que persistiu até o desenvolvimento do embrião, duplo córion e os polos (animal e vegetal). A divisão celular iniciou-se aproximadamente dois minutos após a fertilização (AF), resultando em 2, 4, 8 (4 x 2 arranjo das células), 16 (4 x 4), 32 (4 x 8) e 64 (2 x 4 x 8) células. Adicionalmente, seguiram as fases de blástula e gástrula depois das divisões celulares. O fechamento do blastóporo ocorreu às 11 h 20 min AF; seguindo o desenvolvimento, os estágios de organogênese foram identificados e subdivididos, respectivamente em: fase de divisão inicial e fase de divisão avançada. Na fase de divisão inicial, depois do estabelecimento do eixo do embrião, foi possível distinguir as regiões cefálica e caudal; os somitos e as vesículas ópticas se desenvolveram com aproximadamente 20 h AF. A eclosão total ocorreu com cerca de 54 h AF e o comprimento médio foi de 4,30 ± 0,70 mm. A redução gradual do saco vitelínico foi observada durante os primeiros dois dias de desenvolvimento larval. A primeira alimentação ocorreu no final do segundo dia. Durante a fase larval, canibalismo, crescimento larval heterogêneo e fotofobia também foram observados. Estas informações serão importantes para aperfeiçoar o protocolo de reprodução artificial em S. parahybae em programas de reprodução controlada.(AU)

Induced spawning of the endangered Neotropical species Steindachneridion parahybae (Siluriformes: Pimelodidae)

The "surubim do Paraíba" (Steindachneridion parahybae) is a freshwater catfish endemic to the Paraíba do Sul River basin, Brazil. This species has been seriously threatened by environmental disturbances in the last several decades. Wild Steindachneridion parahybae males and females were collected in 2003 and taken to the hatchery of a power plant of the Companhia Energética de São Paulo (CESP). Steindachneridion parahybae broodstocks were artificially induced to reproduce in December 2003 using a combination of carp pituitary extract (CPE) and human chorionic gonadotropin (hCG). Oocytes and milt were stripped; the fertilized eggs were transferred to 60-liter conical incubators and hatched larvae distributed in nine horizontal trays. Exogenous feed was started just after yolk sac absorption. A high rate of cannibalism and photophobia were observed during the larval period, resulting in a 26 percent survival rate from larvae to fingerlings.(AU)

O "surubim do Paraíba" (Steindachneridion parahybae) é um bagre de água doce, endêmico da bacia do rio Paraíba do Sul, Brasil. Esta espécie foi seriamente ameaçada por distúrbios ambientais nas últimas décadas. Machos e fêmeas selvagens de Steindachneridion parahybae foram coletados em 2003 e transferidos para a piscicultura da CESP (Companhia Energética de São Paulo). Reprodutores de S. parahybae foram induzidos à reprodução artificial em dezembro de 2003 usando uma combinação de extrato hipofisário de carpa (CPE) e gonadotropina coriônica humana (hCG). Após a extrusão dos óvulos e do sêmen, os ovos fertilizados foram transferidos para incubadoras cônicas de 60 litros e, em seguida, as larvas eclodidas distribuídas em nove incubadoras horizontais. Após a absorção do saco vitelino, a alimentação exógena foi iniciada. Uma alta taxa de canibalismo e fotofobia foram observados durante o período larval, resultando em uma taxa de sobrevivência de 26 por cento de larvas para os alevinos(AU)

Muscle biopsy technique for electrophoresis analysis of fish from the genus Brycon

Variabilidade apresentada por proteínas e pelo DNA mitocondrial tem sido utilizada em estudos populacionais de peixes. A obtençäo de tecido para análise eletroforética de proteína e DNA mitocondrial muitas vezes requer a morte dos indivíduos. No presente trabalho, nós apresentamos uma técnica modificada para biópsia de tecido muscular sem a morte dos animais. Oitenta Pirapitingas-do-sul (Brycon opalinus) foram biopsiadas pela presente técnica, näo tendo sido observado mortalidade. O DNA total foi extraído do tecido muscular e digerido com as enzimas de restriçäo ApaI e HpaI. Os fragmentos de DNA mitocondrial foram hibridizados com sondas de DNA mitocondrial de pirapitinga-do-sul marcadas com Pü². A técnica descrita pode ser útil em estudos genéticos em peixes.