“…After the collection, the ovaries were washed in alcohol 70% and in TCM199, supplemented with 20 mM HEPES, 2,200 mg/L sodium bicarbonate, 25 µg/ml pyruvic acid, 75 µg/ml penicillin and 50 µg/ml streptomycin, named transport TCM199. After the washes, the ovaries were transported in transport TCM199 at 4°C (Jimenez, Araújo, et al, ; Jimenez et al, ). "The ethics committee statement is not required for this research, due we do not have any kind of contact with animals, we worked with slaughterhouse ovary."…”
Section: Methodsmentioning
confidence: 99%
“…In the laboratory, under sterile conditions, thin cortical strips (~9 m 3 ) were cut with the aid of a scalpel blade. Once obtained, the fragments were individually cultured in a 24‐well plate containing αMEM, supplemented with 20 mM HEPES, 3 mM glutamine, 10 ng/ml insulin, 2.5 µg/ml transferrin, 4 ng/ml selenium, 0.1% bovine serum albumin (BSA), 0.1 mg/ml penicillin and 0.1 mg/ml streptomycin, named αMEM + (Jimenez, Araújo, et al, ). The treatments were distributed in the following way: (a) αMEM + during the 14 days (αMEM + ), or αMEM + supplemented with (b) GH during 7 days, followed by 7 days with IGF‐1 (GH → IGF‐1), (c) IGF‐1 during 7 days, followed by 7 days with GH (IGF‐1 → GH) and GH plus IGF‐1 during 14 days of culture (GH + IGF‐1; Figure ).…”
The growth hormone (GH) and growth insulin-like factor-1 (IGF-1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF-1 in the long-duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1-7), second half (days 7-14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF-1; IGF-1 → GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO . The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF-1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF-1 → GH and GH + IGF-1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF-1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.
“…After the collection, the ovaries were washed in alcohol 70% and in TCM199, supplemented with 20 mM HEPES, 2,200 mg/L sodium bicarbonate, 25 µg/ml pyruvic acid, 75 µg/ml penicillin and 50 µg/ml streptomycin, named transport TCM199. After the washes, the ovaries were transported in transport TCM199 at 4°C (Jimenez, Araújo, et al, ; Jimenez et al, ). "The ethics committee statement is not required for this research, due we do not have any kind of contact with animals, we worked with slaughterhouse ovary."…”
Section: Methodsmentioning
confidence: 99%
“…In the laboratory, under sterile conditions, thin cortical strips (~9 m 3 ) were cut with the aid of a scalpel blade. Once obtained, the fragments were individually cultured in a 24‐well plate containing αMEM, supplemented with 20 mM HEPES, 3 mM glutamine, 10 ng/ml insulin, 2.5 µg/ml transferrin, 4 ng/ml selenium, 0.1% bovine serum albumin (BSA), 0.1 mg/ml penicillin and 0.1 mg/ml streptomycin, named αMEM + (Jimenez, Araújo, et al, ). The treatments were distributed in the following way: (a) αMEM + during the 14 days (αMEM + ), or αMEM + supplemented with (b) GH during 7 days, followed by 7 days with IGF‐1 (GH → IGF‐1), (c) IGF‐1 during 7 days, followed by 7 days with GH (IGF‐1 → GH) and GH plus IGF‐1 during 14 days of culture (GH + IGF‐1; Figure ).…”
The growth hormone (GH) and growth insulin-like factor-1 (IGF-1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF-1 in the long-duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1-7), second half (days 7-14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF-1; IGF-1 → GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO . The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF-1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF-1 → GH and GH + IGF-1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF-1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.
“…; Jimenez et al. ,b. 30 follicles were evaluated through repetition (n = 6) for the total of 180 preantral follicles per treatment on day one and on day 7 of in situ culture.…”
Section: Methodsmentioning
confidence: 99%
“…It is known that several factors may interfere in this process, such as the concentrations of substances added to culture media (growth factors, hormones and so forth) as well as breed and culture system (Jimenez et al. ).…”
The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.
“…The PFs are a great source of female germplasm and represents about 95% of the ovarian follicles, being possible the use of this source in many assisted reproductive techniques (Jimenez et al 2016). Once ovary of the hystricomorphs has marked interspecific variation (Weir & Rowlands 1974), it is important to quantify and characterize the D. leporina PFs and it will be useful for the development of future technologies to preserve this species and its preservation would contribute to the efforts to form cryobanks (Santos et al 2010).…”
The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40μm, 12.28±2.37μm and 6.10±0.93μm for primordial, 23.75±5.70μm, 14.22±3.00μm and 6.70±1.24μm for primary and 88.55±17.61μm, 52.85±17.56μm and 22.33±17.61μm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.
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