Effects of 17ß-estradiol on early gonadal development and expression of genes implicated in sexual differentiation of a South American teleost, Astyanax altiparanae.

Gonadal sex differentiation in teleost fish shows greater plasticity as compared to other vertebrates, as it can be influenced by a variety of factors such as exogenous sex steroids. Exogenous estrogens, such as 17ß-estradiol (E2), can induce feminization when administered during early embryonic development. However, the mechanisms underlying the E2-induced feminization are not fully understood, especially in Neotropical species. Therefore, the aim of this study was to evaluate the effects of E2 administration on the phenotypic sex characteristics, histological assessment of the gonads, and the expression of selected genes in Astyanax altiparanae exposed to dietary E2 prior to gonadal differentiation. At 4 days post-hatch (dph), groups of 30-40 undifferentiated larvae were fed with a diet containing varying amounts of E2 for 28 days, and fish were sampled at 90 dph. Previous studies revealed that ovary formation in A. altiparanae occurred at 58 dph, whereas the first sign of testis formation was found at 73 dph. In relation to the control, E2 exposure increased the proportion of phenotypic females in 120% and 148.4% for 4 and 6 mg E2/Kg, respectively. However, histological analysis revealed that treatments did not affect gonadal sex ratio between males and females, but induced intersex (testis-ova) in the group treated with 6 mg E2/Kg food. Treatment with E2 also altered gonadal transcript levels of a selected number of genes implicated in sexual differentiation. Males overexpressed dmrt1, sox9 and amh following E2 treatment as compared to control. Females showed increased mRNA levels of dmrt1 and sox9, which might be related to the down-regulation of cyp19a1a after E2 exposure. In summary, E2 exposure during early gonadal development affected male secondary characteristics without changing the gonadal sex ratio, and altered expression of genes implicated in sexual differentiation.

Ultrastructure aspects of Brycon gouldingi (Teleostei, Characidae) related to swimming ability and feeding during larval development.

The larval ultrastructure of Brycon gouldingi related to swimming and feeding from hatching to total yolk absorption is described from scanning electron micrographs. Newly hatched larvae (time zero) had no mouth opening, undefined optic vesicles, an olfactory plate visible as a shallow depression, rudimentary gill arches, neural groove, embryonic fin and a primary neuromast in the dorsal region of the head. At the time of yolk absorption, 55 h post hatching, the larvae presented an optic vesicle comprising an optic cup and crystalline lens; a mouth with tongue, tapered teeth and taste buds; a ciliated olfactory cavity; branched gill arches; filled neural groove signalling central nervous system development; caudal, pectoral, dorsal and anal fins; and neuromasts distributed throughout the head and body. These characters are related to prey capture and swimming ability, key aspects of survival during the larval stage. The results of this study provide important information for exploitation and aquaculture of B. gouldingi.

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish.

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.

Morfologia e quantificação da microbiota intestinal do curimbatá (Prochilodus lineatus) e do cascudo cinza (Pterygoplichthys anisitsi) cultivados em cativeiro / Morphology and quantification of intestinal microbiota of curimbatá (Prochilodus lineatus) and the gray armored catfish (Pterygoplichthys anisitsi) reared in captivity

Todos os animais vivem em íntima associação com micro-organismos que desempenham importantes funções em seu desenvolvimento normal. Nos vertebrados, a mais populosa e complexa comunidade de micro-organismos reside no trato intestinal. O intuito do estudo foi quantificar, classificar e verificar morfologicamente a população microbiana intestinal de duas importantes espécies de peixes de água doce, o curimbatá (Prochilodus lineatus) e o cascudo cinza (Pterygoplichthys anisitsi). As amostras foram coletadas por meio de raspagens da mucosa intestinal, diluídas seriadamente até 10-4, semeadas em placas contendo ágar soja tripticaseína (TSA) e ágar chocolate (AC) para contagem de bactérias totais e identificação morfológica por Gram, em aerobiose e em anaerobiose facultativa, respectivamente. As contagens de bactérias totais mostraram resultados que variaram entre 10³ e 10(4)ufc.mL-1. Os tipos morfológicos encontrados foram cocos, leveduras e bastonetes Gram negativos e positivos. Estudos adicionais sobre os padrões de colonização microbiana e a morfologia dos micro-organismos aderidos à mucosa intestinal foram possíveis com o uso da microscopia eletrônica de varredura (MEV), sendo encontradas formas variadas de micro-organismos, tais como leveduras, formas cocoides e bacilares flageladas e não flageladas. A microbiota intestinal do curimbatá e a do cascudo cinza provaram ser bastante diversas e populosas, com o predomínio de micro-organismos Gram negativos.

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development. In vertebrates, the most populous and complex community of microbes resides in the digestive tract.The aim of this research was to morphologically quantify, classify and verify the composition of intestinal microbiota of two species of freshwater fish, the Prochilodus lineatus and the Pterygoplichthys anisitsi. The samples were collected by scrapings of intestinal mucosa, serially diluted to 10-4, plated on tryptic soy agar (TSA) and chocolate agar (CA) for total bacterial counting and morphological identification by Gram, in aerobiosis and facultative anaerobiosis conditions, respectively. In the total bacterial counting results ranged between 10³ to 10(4) cfu.mL-1. The morphological types found were cocci, yeasts and Gram negative and positive rods. Additional studies about patterns of microbial colonization and the morphology of the adhered microorganisms to the intestinal mucosa were possible using the scanning electron microscopy (SEM) and several forms of microorganisms, such as yeasts, cocci and bacillary shapes flagellated and non-flagellated were found. The intestinal microbiota of P. lineatus and P. anisitsi was diverse and populous, with a predominance of Gram negative microorganisms.

Efeito da adição de agentes tróficos na dieta de leitões desmamados sobre a estrutura e ultraestrutura do intestino delgado e sobre o desempenho / The effect of the addition of trofic agents in weaned piglet diets over the structure and ultra-structure of small intestine and over performance

Foram estudados os efeitos da glutamina, dos ácidos graxos poli-insaturados e da parede celular de levedura (PCL) sobre a estrutura e ultraestrutura do intestino delgado e o desempenho de leitões. Foram utilizados 45 leitões, desmamados aos 21 dias de idade, para testar os seguintes tratamentos: T1 - dieta basal; T2 - dieta basal + 1 por cento de glutamina; T3 - dieta basal + 0,2 por cento de PCL; T4 - dieta basal + 5 por cento de óleo de peixe. Nos dias sete e 14 pós-desmame, foram abatidos cinco leitões de cada tratamento. Os aditivos testados não alteraram a altura e a densidade dos vilos nem a profundidade das criptas do intestino delgado. Foi observado efeito de idade, mostrando redução na altura e na densidade dos vilos e na profundidade das criptas após o desmame. No duodeno e jejuno, foram observados maiores valores de relação vilo:cripta, que aumentaram com a idade pós-desmame. Ocorreram redução da altura dos microvilos do duodeno aos sete dias e aumento da largura dos microvilos do jejuno aos 14 dias pós-desmame. A área de superfície apical dos enterócitos não foi alterada pelos fatores estudados. Os aditivos estudados não foram eficientes em prevenir a atrofia da mucosa intestinal do jejuno, ao não interferir na sua ultraestrutura. Os aditivos incluídos na dieta não influenciaram o desempenho dos leitões no pós-desmame.

The effects of glutamine, poliunsatured fatty acids and cellular wall of yeast (CWY) under the structure and ultra structure of the small gut and the performance of the piglets were studied. Forty five piglets weaned at 21 days were used to test the following treatments: T1 - basal diet; T2 - basal diet + 1 percent of glutamine; T3 - basal diet + 0,2 percent of CWY; T4 - basal diet + 5 percent of fish oil. At seven and 14 post weaning days, five piglets of each treatment were slaughtered. The height, density of villus and depth of small gut crypts were not altered by the inclusion of additives. The effect of age was observed, showing a reduction in the height and density of villus and depth of crypts after weaning. In duodenum and jejunum higher values were observed in the relation villus:crypt, which increased with the post wean age. There was a decrease in the height of microvillus of the duodenum at 7 days and an increase of the width of the microvillus of jejunum at 14 days after wean. The area of the apical surface of the enterocytes was not altered by the studied factors. The studied additives were not efficient to prevent the atrophy of the intestinal mucosa of the jejunum, since they did not interfere on its ultra structure. Piglet performance was not affected by the additives included in the diet.

Morfologia e histologia do oviduto de marrecas Ana boschas / Morphology and histology of Ana Boschasfemale ducks oviduct

Avaliaram-se o comprimento do infundíbulo, do magno, do istmo, do útero e da vagina e o número de pregas do magno e do istmo do oviduto de 20 marrecas Ana boschas na fase reprodutiva. O infundíbulo apresenta mucosa com pregas longitudinais e baixas, revestidas por epitélio pseudoestratificado cilíndrico ciliado, com células caliciformes. O magno, compartimento mais longo do oviduto, 25,38cm±3,20, encontra-se constituído por uma camada mucosa com pregas altas e espessas revestidas por células cilíndricas ciliadas e abundantes células caliciformes. O istmo é formado por uma mucosa com pregas estreitas e curtas e numerosas glândulas tubulares que se estendem para o interior da lâmina própria. O útero, região curta do oviduto, 5,25cm±1,26, apresenta parede com pregas e cristas baixas e numerosas glândulas tubulares enoveladas, dirigidas para o interior da lâmina própria. A vagina, um estreito tubo muscular, está constituído por oito anéis circulares, em média, e uma camada muscular altamente desenvolvida e espessa. A morfologia do oviduto da marreca apresenta características morfológicas e histológicas distintas dos galiniformes, observando-se que a vagina e a porção cranial do infundíbulo apresentam pregas e células caliciformes, respectivamente, sendo estas últimas estruturas ausentes nos galiniformes.

The length of infundibulum, magnum, isthmus, uterus, vagina, and the number of oviduct, magnum, and isthmus folds were evaluated in 20 Ana boschas female ducks in the reproductive phase. The infundibulum presented mucous membrane with longitudinal and short folds, covered by ciliated columnar pseudostratified epithelium, with goblet cells. Magnum, the longest oviduct compartment, 25.38cm±3.20, is constituted by mucous membrane with high and thick folds that are covered by ciliated columnar cells and many goblet cells. Isthmus is formed by mucous membrane with narrow and short folds, and many tubular glands that extend inside lamina propria. Uterus, an oviduct short region, 5.25cm±1.26, presented surface with short folds and crests, and numerous reeled tubular glands, that are directed inside lamina propria. Vagina, a strait muscular tube, is constituted by, approximately, eight circular rings, and a very developed and thick muscular layer. The oviduct morphology of female ducks presented different morphological and histological characteristics from Galliformes, because vagina and infundibulum cranial portion present folds and goblet cells, respectively, and the last cited structures are absent in Galliformes.

Sexagem histológica e desempenho de Oreochromis niloticus testando diâmetros de ração de acordo com o aparato bucal / Histological gender diagnosis and performance of Oreochromis niloticus testing diameters of crumble diet according to oral apparatus

Estudou-se o tamanho da boca de larvas de tilápia-do-nilo e testou-se o efeito de diferentes granulometrias da ração sobre o ganho de peso, comprimento e sobrevivência das larvas aos 30 e 60 dias de arraçoamento. Avaliou-se também o método de sexagem por meio de microscopia de luz aos 35 dias de idade. A medida da boca das larvas apresentou valores médios de 918,2±152,9μm aos cinco dias de idade. De acordo com esse dado, testaram-se três granulometrias: 0,25, 0,35 e 0,50mm. Aos 30 e aos 60 dias de arraçoamento, 10 por cento das larvas foram medidas, pesadas e contadas para cálculo da taxa de sobrevivência. O tamanho dos grânulos testados não afetou o desempenho das larvas de tilápia nilótica com alimentação iniciada aos cinco dias pós-eclosão. Quanto à sexagem histológica aos 35 dias de idade, as gônadas apresentaram-se, em sua maioria, indiferenciadas. Recomenda-se que essa análise deva ser realizada de acordo com o tamanho dos animais e não com a idade.

The mouth size of Nile tilapia larvae and the effect of different diameters of crumble fish food over weight gain, total length, and survival of larvae after 30 and 60 days of feeding were studied. The method of gender diagnosis based on light microscopy at 35 days of age was also evaluated. The larval mouth measurement presented average values of 918.2±152.9μm at five days of age. Based on this information, three granule sizes were tested: 0.25, 0.35, and 0.50mm. At 30 and 60 days of feeding, 10 percent of larvae were measured, weighed, and counted to calculate the survival rate. It was verified that the crumble size did not affect the performance of Nile tilapia larvae when feeding was initiated five days after hatching. In relation to the histological gender diagnosis at 35 days of age, most of gonads were undifferentiated. Therefore, it is recommended that this analysis should be carried out according to the size of animals instead of their age.

Emprego da microscopia eletrônica na avaliação pós-vacinal de epitélio traqueal de patos (Anas platyrhynchos) imunizados contra a doença de Newcastle / The use of scanning electron microscopy in post-vaccinal evaluation of tracheal epithelium in ducks (Anas platyrhynchos) imunized against Newcastle disease

Avaliou-se o emprego da microscopia eletrônica de varredura no estudo da reação respiratória pós-vacinal em epitélio traqueal de patos (Anas platyrhynchos) imunizados contra a doença de Newcastle. Foram utilizadas 48 aves, distribuídas em quatro grupos: T1 - grupo de aves-controle (não vacinadas), T2 - grupo de aves vacinadas com a estirpe Ulster 2C, T3 - grupo de aves vacinadas com a estirpe B1 e T4 - grupo de aves vacinadas com a estirpe LaSota. Independente do grupo experimental, as aves não apresentaram sinais clínicos detectáveis de reação respiratória pós-vacinal. Ao microscópio eletrônico de varredura, observou-se que os animais vacinados com as estirpes B1 e LaSota desenvolveram descamação epitelial da traqueia, enquanto os vacinados com a estirpe Ulster 2C não, mostrando um epitélio traqueal íntegro, semelhante ao do grupo-controle. Os patos vacinados com a estirpe B1 mostraram evidências de regeneração epitelial da traqueia decorridos 21 dias pós-vacinação, o que não ocorreu com os vacinados com a amostra LaSota.

Scanning electron microscopy was used in the study of the post-vaccinal respiratory reaction of the tracheal epithelium of ducks (Anas platyrhynchos) immunized against Newcastle disease. Forty-eight ducks were distributed into four groups: T1 - control birds (non-vaccinated); T2 - birds vaccinated with Ulster 2C strain; t3 - birds vaccinated with B1 strain; and t4 - birds vaccinated with LaSota strain. Regardless the experimental group, birds did not show detectable clinical signs of post-vaccinal respiratory reaction. Scanning electron microscopy showed that birds vaccinated with B1 and LaSota strains developed epithelial sloughing of the trachea, whereas those vaccinated with Ulster 2C strain did not develop this change, showing intact tracheal epithelium, similar to the control group. However, the birds vaccinated with B1 strain showed evidences of regeneration of tracheal epithelium 21 days post-vaccination, which did not happen with the ducks vaccinated with LaSota strain.

Efeito do resfriamento sobre a textura post-mortem da carne do peixe matrinxã Brycon cephalus / Chilling effect on the post-mortem texture of the matrinxã fish muscle Brycon cephalus

Os mecanismos que causam o amolecimento e a perda na textura post-mortem da carne de matrinxã foram determinados por meio das mudanças na microestrutura do músculo, imediatamente após a morte e depois de 12 horas de estocagem a -3ºC. As observações na microestrutura, realizadas com microscópio eletrônico de transmissão, foram semelhantes aos resultados obtidos na força de ruptura do músculo medidos com o texturômetro. Os valores da força da ruptura foram menores para a carne após o resfriamento. Observou-se que as fibras do colágeno do tecido conectivo pericelular se desintegraram e que as do colágeno do tecido conectivo do miocommata conservaram sua arquitetura e integridade. Houve pouca degradação da linha Z. Isso sugere que o amolecimento post-mortem da carne de mantrinxã, durante a estocagem a -3ºC, é causado pela degradação do tecido conectivo pericelular

In order to determine the mechanisms that cause the post mortem muscle softness of the matrinxã Brycon cephalus, changes in the micro structure of the muscle were observed immediately after death and after 12 hours of storage at -3º C, measuring the firmness of the flesh with test instruments. Observations by the transmission electron microscope were similar to the results obtained in the breaking strength of the muscle measured with a texturometer. The values of the breaking strength of the fish muscle were smaller after chilling. At the same time, it was observed that the collagen fibers of the pericellular connective tissue had disintegrated, while the collagen fibers of the miocommata connective tissue maintained their organization and integrity. No evident breakdown of Z-discs was observed. It is suggested that the post-mortem tenderization of the matrinxã muscle during chilled storage was due to the disintegration of the collagen fibers in the pericellular connective tissue and, in a smaller extent, to the weakening of Z-disk

Morphometry and morphology of nucleus of the Sertoli and interstitial cells of the tambaqui Colossoma macropomum (Cuvier, 1881) (Pisces: Characidae) during the reproductive cycle.

This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes.

Morphometry and morphology of nucleus of the Sertoli and interstitial cells of the tambaqui Colossoma macropomum (Cuvier, 1881) (Pisces: Characidae) during the reproductive cycle

This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes