Indução à ovulação pelo uso de LHRH análogo e fertilização artificial em rã-touro (Rana catesbeiana)

Este trabalho teve por objetivo aperfeiçoar a técnica de reprodução induzida existente para rã-touro, com o intuito de aumentar a taxa de fecundidade e viabilizar seu uso pelo produtor. As doses hormonais para a indução da ovulação e espermiação seguiram as propostas de FALCON e CULLEY (1995) e ALONSO (1997); entretanto, a técnica de fertilização artificial foi adaptada da metodologia para reprodução artificial de peixes com ovos não-aderentes (WOYNAROVICH e HORVÁTH, 1983). A técnica proposta apresenta as seguintes etapas: I) sincronização da ovulação e da espermiação, por meio de hormônio liberador de gonadotropina ((Des-Gli10, D-His(Bzl)6, Pro-NHEt9)-LHRH)); II) extração dos óvulos de cada fêmea (1 a 2 minutos); III) fertilização dos óvulos (2 minutos) com líquido espermático diluído em 100 mL de água; IV) hidratação dos ovos em 10 a 20 litros de água; e V) incubação dos ovos em quadros de tela de 1x 0,70 m, com malha de 1 mm. As taxas de fertilização obtidas com as modificações propostas foram superiores a 60%. Ressalta-se ainda que a técnica propiciou a obtenção, a partir de um mesmo animal, de várias desovas, sendo que cada fêmea pode ovular em intervalos de, aproximadamente, 45 dias.

Efeito da temperatura sobre o desempenho da rã-touro (Rana catesbeiana Shaw, 1802)

Este estudo foi realizado para avaliar os efeitos do ambiente sobre a performance de rã-touro (Rana catesbeiana Shaw, 1802), criada em gaiolas de fibra de vidro instaladas no interior de estufas climatizadas. Após um período inicial de 15 dias de adaptação às instalações, à temperatura constante de 25,0ºC, os seguintes tratamentos foram aplicados: temperaturas de 23,0; 26,0; 29,0; 32,0; e 35,0ºC, por 30 dias, para rãs com 100 g PV inicial; 24,5; 26,0; 27,5; 29,0; 30,5; e 32,0ºC, também por 30 dias, para rãs com 20 g PV inicial; e a combinação das temperaturas de 26,0 e 29,0ºC com os fotoperíodos de 8, 12 e 16 h de luz a cada 24 horas, para rãs com 100 g PV inicial. Derivando-se as equações de regressão que explicam os efeitos de temperatura sobre o desempenho das rãs, estimaram-se melhores ganhos de peso à temperatura de 27,6e30,1ºC, para rãs com 100 e 20 g PV inicial, respectivamente, com melhor crescimento a 28,2ºC, para as rãs de 100 g PV inicial, e a 29,7ºC, para as rãs de 20 g PV inicial. A temperatura interagiu com fotoperíodo nos seus efeitos sobre ganho de peso e crescimento corporal, peso e rendimento de carcaça, consumo de alimentos e conversão alimentar.

Efeito da temperatura e do fotoperíodo sobre o desenvolvimento do aparelho reprodutor de rã-touro (Rana catesbeiana Shaw, 1802)

Foram avaliados os efeitos da temperatura e do fotoperíodo sobre a maturação sexual de rãs-touro pesando 94,22 g ± 12,03, mantidas durante trinta dias em temperaturas de 20, 23, 26, 29, 32 e 35°C, com fotoperíodo de 12/12 horas de luz/horas de escuridão (h L/E). A temperatura afetou os pesos do corpo gorduroso e do fígado, os quais variaram de acordo com modelos quadráticos, estimando-se maiores pesos de corpo gorduroso a 27,27°C e de fígado a 26,81°C. Estimaram-se ovários mais pesados a 28,36°C e ovidutos mais pesados a 28,77°C. Temperatura afetou a maturação sexual das rãs, avaliada por índices numéricos. Num experimento mais longo, rãs com peso médio inicial de 95,31 ± 8,46 g foram submetidas à combinação das temperaturas de 26 e 29°C com os fotoperíodos de 8/16, 12/12 e 16/8 h L/E, até atingirem a maturidade gonadal. Temperatura interagiu com fotoperíodo em seus efeitos sobre o desenvolvimento dos órgãos reprodutivos de rã-touro. Temperatura afetou a relação diâmetro do abdômen/distância entre os olhos, com maiores valores calculados para 26°C. Verificou-se que os maiores diâmetros dos ovócitos são obtidos a uma temperatura de 26°C, com fotoperíodo de 12,6/11,4 h L/E.

Testosterone-immunopositive primordial germ cells in the testis of the bullfrog, Rana catesbeiana

In amphibia, steroidogenesis remains quiescent in distinct seasonal periods, but the mechanism by which spermatogenesis is maintained under low steroidogenic conditions is not clear. In the present study, testosterone location in the testes of Rana catesbeiana was investigated immunohistochemically during breeding (summer) and nonbreeding (winter) periods. In winter, the scarce interstitial tissue exhibited occasional testosterone immunopositivity in the interstitial cells but the cytoplasm of primordial germ cells (PG cells) was clearly immunopositive. By contrast, in summer, PG cells contained little or no immunoreactivity whereas strong immunolabelling was present in the well-developed interstitial tissue. These results suggest that PG cells could retain testosterone during winter. This androgen reservoir could be involved in the control of early spermatogenesis in winter and/or to guarantee spermiogenesis and spermiation in the next spring/summer. The weak or negative immunoreaction in the summer PG cells might reflect consumption of androgen reservoir by the intense spermatogenic activity from spring to summer. Thus, besides acting as stem cells, PG cells of R. catesbeiana could exert an androgen regulatory role during seasonal spermatogenesis.

Apoptosis during the seasonal spermatogenic cycle of Rana catesbeiana

In the bullfrog Rana catesbeiana, testicular weight is constant throughout the year, but the volume densities of germinative and interstitial compartments undergo inverse changes from winter (non-breeding) to summer (breeding). The occurrence of apoptosis in the seminiferous lobules of bullfrogs was investigated in these two periods using sections stained with haematoxylin and eosin (H&E), the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) method and transmission electron microscopy. TUNEL-positive cells were observed in the seminiferous lobules, and ultrastructural morphological details confirmed the occurrence of cell death by apoptosis. In summer, the occurrence of several spermatogenic processes (in addition to spermiogenesis and spermiation), and then the overconsumption of Sertoli cell-derived pro-survival factors, could be responsible for the increased density of apoptotic cells. Alternatively, the low apoptotic frequency in winter could be related to the constant homeostasis in the germinative compartment given that most lobules are filled with primary spermatocytes. As volume densities of interstitial and germinative compartments undergo inverse seasonal variations through the year, the incidence of apoptosis (in summer) could play a part in controlling the spermatogenic process, maintaining the lobular size when interstitial tissue is maximally developed. In winter, the low apoptotic cell density leads to spermatogenic recrudescence and, thereby, the production of an adequate quantity of spermatozoa for the next breeding period. Thus, apoptosis may participate not only in the maintenance of spermatogenic homeostasis, but also in the cyclical control of the different spermatogenic processes according to seasonal changes of the testicular compartments as a whole.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.

Efeito do alecrim na defumação da carne de rã (Rana catesbeiana): características sensoriais, composição e rendimento

Este trabalho avaliou o efeito do alecrim na qualidade da carne de rã (Rana catesbeiana) defumada, por meio da análise das características sensoriais, da composição centesimal e do rendimento. Após o abate e a evisceração, as carcaças foram imersas em solução de salmoura (20%) numa proporção de 2:1 (volume da salmoura/peso). Posteriormente, elas foram banhadas em azeite e defumadas a uma temperatura de 50 a 90 °C. O delineamento experimental foi inteiramente casualizado, com 2 tratamentos (T1 = Carcaça de rã defumada com alecrim; T2 = Carcaça de rã defumada sem alecrim) com 16 repetições. Na análise estatística, foi utilizado o programa SAEG 2004, com 5% de significância. As carcaças defumadas apresentaram valores médios de proteína bruta (28,39%), lipídios totais (5,13%) e cinzas (2,79%) superiores comparados aos das carcaças in natura (23,41; 2,29 e 0,85%). Não houve efeito significativo do alecrim na composição centesimal. A carcaça de rã defumada com alecrim apresentou melhor aceitação quanto ao aroma. Nas demais características sensoriais, a presença do alecrim não interferiu nos resultados. A carne de rã pode ser defumada com e sem alecrim, sem interferir na aceitabilidade do produto.

Suplementação com vitamina C e E sobre o desenvolvimento e sobrevivência de girinos de rã-touro (Rana catesbeiana Shaw, 1802)

O presente trabalho teve como objetivo avaliar os efeitos da suplementação de vitamina C e E sobre o desenvolvimento e sobrevivência de girinos de rã-touro. Os animais receberam 50, 250 e 500mg de vitamina C ou E/kg de ração, distribuídos segundo delineamento inteiramente casualizado com 3 repetições cada. Na dieta controle, não foram adicionadas vitaminas. Os girinos suplementados com vitamina C ou E não apresentaram diferença significativa no crescimento, comparados ao controle. Não obstante, os suplementados com 500mg de vitamina C apresentaram melhor sobrevivência (62,83%) do que os animais controle (40,48%; p<0,05), nos demais níveis desta vitamina e a adição de vitamina E não alteraram a sobrevivência. O presente trabalho sugere que 500mg de vitamina C/kg de ração constitui uma opção para melhorar a sobrevivência de girinos de rã-touro.

Avaliação de métodos de coleta de fezes para determinação da digestibilidade protéica em rã-touro (Rana catesbeiana)

O presente trabalho teve como objetivo avaliar diferentes metodologias de coleta de fezes para determinação da digestibilidade protéica de alimentos para a rã-touro (Rana catesbeiana). As metodologias estudadas foram o método de coleta total das fezes; o método de coleta parcial das fezes usando indicador; o método de dissecação intestinal e o método por decantação de fezes com indicador, utilizando aquários para ensaios de nutrição com peixes. Os resultados dos CDA PB da ração obtidos para avaliação dos métodos de coleta foram: 62,1%, 65,0%, 61,3% e 70,3%, respectivamente. Todos os métodos de coleta de fezes são adequados para a realização de ensaios de digestibilidade com rãs.

Caracterização morfológica dos estádios de desenvolvimento do aparelho reprodutor masculino da rã-touro, Rana catesbeiana, no sistema anfigranja de criação intensiva

The objective of this work was to caracterize the morphological characterization of the development stages of male reproduction apparel of bullfrog Rana catesbeiana from intensive Anfigranja system. During the period of one year, 657 bullfrog males were removed from regrowing and reproduction sections in Anfigranja system to study basic aspects of the reproduction of this specie. The collections were realized monthly and, in that occasion, the body and testicles weights for each animal were recorded and their macroscopic gonadal characteristics, registred. Testicles from 274 males were removed and histologically analyzed. From the macro and microscopic gonadal characteristics, analysis set of the testicles, five stages of gonadal maturation for the male of Rana catesbeiana were determined: juvenile, beginning of maturation, intermediate maturation, advanced maturation and spent.

Estimativa do Tamanho da Primeira Maturação Sexual da Rã - Touro, Rana catesbeiana, no Sistema Anfigranja de Criação Intensiva

The objective of this research was to study the basic aspects of bullfrog reproduction in Anfigranja system from June 1989 to July 1990. For the 1493 animals involved in this experiment (657 males and 836 females) biometrical data were obtained (weight, length and tympanum diameter) and developmental stages of gonads were determined. Female sizes at first maturation were 10,33 cm and 109,78 g and for males it was around 8,09 cm; all males above 45 g were already in the maturation process. The relationship between tympanum diameter and body length (RTC) was considered a good index for male maturation.

Liver lesions produced by aflatoxins in Rana catesbeiana (bullfrog)

This study describes alterations induced in Rana catesbeiana (bullfrog) liver after extended dietary exposure to aflatoxins (AFs). Bullfrogs of both sexes were fed for 120 days a commercial chow blended with a rice bran-based mixture of Al's containing 667.0, 11.65, 141.74, and 3.53 mg/kg of AFs B1, B2, G1, and G2, respectively. Animals were sacrificed on study days 45, 90, and 120. Severe and progressive liver lesions with structural collapse, increased hepatocyte and biliary duct cell proliferation, appearance of basophilic hepatocytes, and diffuse scarring, were observed at all time points. There were no quantitative alterations in the liver melanomacrophage centers of the AFs-exposed animals. Increased amounts of lipid hydroperoxides, indicative of ongoing oxidative stress, were more evident in the Addutor magnum muscle than in the AFs-damaged livers. No tumors were found in the R. catesbeiana livers after 120 days of exposure to relatively high doses of AFs. (c) 2006 Elsevier B.V. All rights reserved.

Effect of oxytocin on transepithelial transport of water and Na+ in distinct ventral regions of frog skin (Rana catesbeiana)

Thoracic, abdominal, and pelvic fragments of ventral skin of Rana catesbeiana were analysed regarding the effect of oxytocin on: (1) transepithelial water transport; (2) short-circuit current, (3) skin conductance and electrical potential difference; (4) Na+ conductance, the electromotive force of the Nat transport mechanism, and shunt conductance; (5) short-circuit current responses to fast Na+ by K+ replacement in the outer compartment, and (6) epithelial microstructure. Unstimulated water and Na+ permeabilities were low along the ventral skin. Hydrosmotic and natriferic responses to oxytocin increased from thorax to pelvis, Unstimulated Na+ conductance was greater in pelvis than in abdomen, the other electrical parameters being essentially similar in both skin fragments. Contribution of shunt conductance to total skin conductance was higher in abdominal than in pelvic skin. Oxytocin-induced increases of total skin conductance, Na+ conductance, and shunt conductance in pelvis were significantly larger than in abdomen, An oscillatory behaviour of the short-circuit current was observed only in oxytocin-treated pelvic skins. Decrease of epithelial thickness and increase of mitochondria-rich cell number were observed from thorax to pelvis, Oxytocin-induced increases of interspaces were more conspicuous in pelvis and abdomen than in thorax.