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Universidade Estadual de Londrina (2018)

Aspectos moleculares de oócitos de fêmeas bos indicus e bos taurus submetidos ao estresse térmico

Mirela Brochado Souza Cáceres

Titre : Aspectos moleculares de oócitos de fêmeas bos indicus e bos taurus submetidos ao estresse térmico

Molecular aspects of gametes and embryos submitted to thermal stress

Auteur : Mirela Brochado Souza Cáceres

Université de soutenance : Universidade Estadual de Londrina

Grade : Doutor em Ciência Animal 2018

Résumé
Aiming to evaluate the effects of in vivo and in vitro thermal stress on oocyte competence and early embryonic development as well as on physiological parameters of cows of different breeds, they were conducted three experiments. In the experiment I, the cumulus-oocyte complex (COCs) of Simmental cows matured in vitro at different temperatures (37°C, 38.5°C and 40°C) were evaluated. Before maturation the oocytes were stained with Brilliant Cresilblue (BCB) and then categorized according to staining in BCB + and BCB-. The labeled COCs (BCB +) were considered to be of better quality. After maturation, embryos were produced in vitro and oocytes were evaluated by real-time PCR and immunofluorescence, for the evaluation of sirtuins, BCL11A and p53. The change in temperature (±1,5°C) of oocyte maturation adversely affects the blastocyst rate after in vitro fertilization, in addition to influencing gene expression important for cell protection. Expression of the SIRT1, SIRT2, SIRT3, SIRT5, BCL11a and p53 genes were influenced by the maturation temperature in oocytes and granulosa cells. In the experiment II, cumulus-oocyte complexes (COCs) of Nellore (Bos Taurus indicus) females were evaluated in vitro at different temperatures (37°C, 38,5°C and 40°C). After this period part of the COCs were submitted to embryo fertilization and culture and the other part, after determining the maturation rate, were fixed in paraformoldeído for analysis of HSP70 and HSP90 by western blot. Thermal stress did not influence the maturation rate of oocytes, but influenced the rate of cleavage and blastocyst. The signal intensity of HSP70 was increased in stressed oocytes compared to the control group, and cumulus cells showed a significant increase in the signal intensity of this protein at 40°C. HSP90 showed higher numbers in oocytes matured at 37 ° C, but remained stable in the other temperatures. The lower rate of blastocysts and the parallel action of HSPs demonstrate that the in vitro production of embryos is affected when in vitro maturation occurs in situations of chronic stress (24 hours of IVM) by cold and heat. In the experiment III, cumulative oocytes (COCs) recovered through ultrasound-guided follicular aspiration (OPU), Bos taurus x Bos indicus (Girolando) and Bos taurus (Pantaneira) cows were evaluated. Part of the viable COCs were submitted to immunofluorescence analysis under confocal microscopy for identification of HSP70 and HSP90 proteins. Before each OPU, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. Thermal stress was estimated by the Black Globe Humidity Index (BGHI) which was calculated according to the formula described by Buffington et al. (1981) classified according to the National Weather Service, 90 days before each OPU. The rusticity of the Girolando and Pantaneira breeds can be confirmed by the maintenance of the RT and RF in physiological levels in BGHIs of up to 94. The HSP in the oocytes was influenced by the BGHIs for both breeds, but with opposite behaviors. However, the Pantaneira breed had negative effects on BGHIs <78 demonstrating stress at warmer temperatures. On the other hand, it is observed that BGHIs >78 have a deleterious effect on the quality of Girolando cow oocytes. It appears that the Pantaneira breed reacts negatively at lower temperatures, since it presented lower oocyte viability with BGHI <78. On the other hand it is observed that ITGUs of> 79 at 90 days before OPU have a deleterious effect on the quality of Girolando cow oocytes.

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Page publiée le 29 novembre 2020