The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 μm) to the secondary follicular stages (38.0 ± 14.9 μm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.
ABSTRACT:The purpose of this study was to evaluate by ultrasound and breeding soundness examination (BSE) the major diseases affecting the reproductive tract of Nelore bulls, bred extensively in the state of Para, Brazil. Fifty-nine pure Nelore bulls were used, aged between 5 to 10 years, raised extensively in a commercial farm in the municipality of Paragominas, Pará state, Brazil. Scrotal circumference, testicular length and width were measured. Semen collection for evaluation of ejaculate volume, wave motion, motility, vigor, concentration and sperm pathologies was performed. Ultrasound examination was performed by equipment type Ultrasonic Transducer -CHISON/D600vet, linear transducers, where the frequency used was 5 MHz, being held two images of each testis, the longitudinal-lateral, lateral and transverse planes. The images were processed using the program Image J. The data were analyzed using the statistical program SAS (2000) and means were compared using Tukey's test (p<0.05). Among the fifty-nine bulls evaluated, 39 (66.1%) were classified as suitable for breeding and 20 (33.8 %) were classified as unsuitable. Throughout the use of ultrasonography changes as testicular degeneration one case (1.6%), one of orchitis (1.6%), one of hydrocele (1.6%), nine of calcification (15.2%) and two cysts in the testicular region parenchyma (3.3%) were found. The average scrotal circumference was 38.6±2.4 and 40.4±3.6 cm for the animals fit and unfit for reproduction categories respectively, observing statistical difference (P < 0.05). The average physical characteristics of semen for breeding animals was 74.2±18.8% for motility, 2.9±1.62% for wave motion, 3.2±1.08% for vigor, 4.7 ± 2.58 mL for volume, 0.84 ± 0.56 x106 sperm/mm 3 concentration and 9.4±4.7% of total sperm pathologies. For the unfit animals data was 73.2±24.6% for motility, 2.8±1.78% for wave motion, 3.3±1.17 vigor, 4.5 ± 2.16 mL volume, 27±0.14% of total sperm pathologies and 0.85±0.51 x 10 6 sperm/mm 3 for concentration. Statistical differences in the percentage of total sperm pathologies between fit and unfit animals was observed (P<0.05). The mean pixel intensity of testicular parenchyma was 45.35 to 45.25 for fit for animals unfit animals, without any statistical difference (P>0.05). Thus, it can be concluded that ultrasonography is an essential complementary tool in the diagnosis of reproductive disorders in animals subjected to BSE and its use should be recommended.
Based on these data, we demonstrated the beneficial effects of TES-TRIS for post-thaw buffalo sperm quality; however, no protective effect was observed for buffalo sperm cryopreserved with the different tested concentrations of Lippia origanoides extract oil. Key words: Powdered Coconut Water. Lippia. Cryopreservation. Spermatozoa. Buffalo. ResumoPara a implantação da inseminação artificial é indispensável à utilização de sêmen congelado, que pode provocar mudanças deletérias na estrutura e na integridade das membranas espermáticas, comprometendo sua função. Para evitar estes danos celulares, há a necessidade de se utilizar meios diluidores e substratos adequados que recuperem o maior número possível de células viáveis pós-descongelação. Para isso, foram avaliados, no experimento I, três diferentes diluidores, o diluidor TES-TRIS, bastante utilizado para bubalinos, e um diluidor a base de água de coco em pó (ACP-112 ), associado ou não ao leite (ACP-112 -Leite), na congelação do sêmen de bubalinos; e no experimento II, foi avaliado o efeito do óleo extraído da Lippia origanoides na proteção dos espermatozóides contra as crioinjúrias decorrentes da congelação do sêmen bubalino. Foram utilizados 10 touros bubalinos para as colheitas de sêmen (10 ejaculados/touro), sendo os ejaculados diluídos em TES-TRIS (controle), ACP-112 e ACP-112 -Leite no experimento I; e no experimento II, os ejaculados foram diluídos no melhor diluidor obtido no experimento I, acrescido de 2.5 µg mL -1 , 5 µg mL -1 e 10 µg mL -1 da planta e o grupo controle, constituído somente do diluidor. O sêmen recém colhido foi analisado quanto as características convencionais, tais como, motilidade, concentração, morfologia e viabilidade. Após a descongelação das amostras foram avaliados novamente, motilidade e viabilidade espermática, e posteriormente, foram realizados os testes de termo-resistência, hiposmótico e de avaliação das membranas dos espermatozóides, através das sondas fluorescentes PI, FITC-PSA e JC-1, utilizando a citometria de fluxo. Os dados obtidos foram submetidos à ANOVA e ao Teste de Tukey a 5%. No experimento I, o diluente TES-TRIS apresentou melhores resultados para as várias características avaliadas quando comparado com o ACP-112 e ACP-112 -Leite (P < 0.05), demonstrando maior proteção deste diluidor às estruturas espermáticas durante a criopreservação do sêmen de bubalinos. No experimento II, as diferentes concentrações do óleo extraído da Lippia origanoides não demonstraram nenhuma diferença (P > 0.05) entre as variáveis avaliadas, quanto à proteção das estruturas espermáticas durante a criopreservação. Com base nestes dados, pôde-se demonstrar os efeitos benéficos do diluidor TES-TRIS na qualidade pós-descongelação dos espermatozóides bubalinos; entretanto, nenhum efeito protetor foi observado sobre os espermatozóides bubalinos criopreservados em associação com o óleo extraído da Lippia origanoides, nas diferentes concentrações utilizadas. Palavras-chave: Água de coco em pó. Lippia. Criopreservação. Espermatozóid...
Buffalo farming in Brazil is increasing, as is the challenge of identifying molecular markers that will improve productivity. Therefore, the aim of this study was to analyze single nucleotide polymorphisms of the receptor gene for the hormone melatonin in buffaloes from northern Brazil by polymerase chain reactions (PCRs) and restriction fragment length polymorphism assays. The PCR products exhibited a cutting point for HpaI at the 318th position of the gene, indicating a transition substitution (T↔C). This substitution was synonymic, and did not alter the stability of the mRNA structure. Allelic and genotypic frequencies differed between the populations studied, and all of the populations demonstrated endogamy and were in Hardy-Weinberg equilibrium. Therefore, the HpaI restriction marker in the melatonin receptor gene cannot be used for genetic improvement, but is an excellent marker for population genetic studies.
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