Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract

RESUMO.-[Vitrificação de folículos pré-antrais bovinos com dimetilxulfoxido e sacarose adicionado de α-tocopherol.] Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles. tamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.

Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent

“…; Jimenez et al. ). For every 100 μl of suspension medium containing the isolated preantral follicles, 5 μl trypan blue stain was added (0.4%; Vetec‐Sigma, Brazil).…”

“…The fragments formed were suspended, with the aid of 1000-lm and 600-lm Pasteur pipettes, for 80 times so that the preantral follicles could be separated from the stroma. The material obtained went through a 100-lm nylon mash, resulting in a suspension containing follicles smaller than 100 lm in diameter (Figueiredo et al 1994;Jimenez et al 2016c). For every 100 ll of suspension medium containing the isolated preantral follicles, 5 ll trypan blue stain was added (0.4%; Vetec-Sigma, Brazil).…”

“…For every 100 ll of suspension medium containing the isolated preantral follicles, 5 ll trypan blue stain was added (0.4%; Vetec-Sigma, Brazil). The isolated follicles were observed under an inverted microscope (Leica, DFC295, Jena, Germany), with 1009 magnification, and classified as viable or non-viable, when not stained or stained in blue, respectively (Van Den Hurk et al 1998;Jimenez et al 2016c). 450 isolated preantral follicles were classified (50 follicles per treatment).…”

Sequential medium with GH and IGF‐1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue

“…Five micro litre of Trypan Blue stain (0.4%; Vetec/Sigma, Brazil) was added to every 100 µl of suspension medium containing isolated preantral follicles. They were then observed under the inverted microscope (Leica DFC295, Wetzlar, Germany), with 100X magnification, and were then evaluated as either viable (not stained) or nonviable (stained) in Trypan Blue (Jimenez, Penitente‐Filho, Torres, Medeiros, & Silva, ). Six hundred and fifty preantral follicles were evaluated: 50 of the fresh control and 200 per day of culture.…”

Effect of α-Tocopherol on Viability, Lipid Peroxidation and Oxidative Stress of Cryopreserved Ovine Preantral Follicle

“…Since vitrification increase the oxidative stress and decrease the cellular enzymatic antioxidant SOD activity, the supplementation of antioxidants may contribute for the improvement of vitrification of preantral follicles. It has been reported that antioxidants like α-tocopherol reduce the generation of free radicals and protect the cell membrane from lipid peroxidation and avoid irreversible damages and cellular death (Jimenez et al, 2016). Considering the advantage of an antioxidant, we hypothesized that the addition of α-tocopherol in the vitrification solution may…”