Carolina Madeira Lucci

Effect of the interval of serial sections of ovarian tissue in the tissue chopper on the number of isolated caprine preantral follicles.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.

Light microscopical and ultrastructural characterization of goat preantral follicles.

Goat ovarian preantral follicles were morphologically and ultrastructurally described in this work. Primordial follicles are oocytes surrounded by one layer of squamous or squamous-cuboidal granulosa cells; primary follicles have a single layer of cuboidal granulosa cells, and secondary follicles are oocytes surrounded by two or more layers of cuboidal granulosa cells. At all developmental stages a thick layer of glycoproteins, the basement membrane, surrounded the preantral follicles. The quiescent oocyte is spherical or oval and it has a large eccentrically located nucleus with a conspicuous nucleolus. The organelles were uniformly distributed in the cytoplasm. A large number of vesicles were spread throughout the cytoplasm in all the oocytes. The cytoplasm of oocytes also contains numerous rounded mitochondria besides the usual organelles. As the follicle develops, the mitochondria become elongated. The communication between the oocyte and the granulosa cells is apparently mediated through endocytosis as indicated by the abundant coated pits and vesicles noted in the cortical cytoplasm of the oocyte. The oocyte plasma membrane presented projections that penetrated between adjacent granulosa cells and a few short microvilli lying parallel to the oocyte surface. In secondary follicles, patches of zona pellucida material were observed. Overall, the results indicate that the morphological and ultrastructural organization of caprine preantral follicles resembles that of other mammals. However, some particularities were observed, and that may indicate species specific differences.

Study of preantral follicle population in situ and after mechanical isolation from caprine ovaries at different reproductive stages.

The purposes of this study were to estimate the population of caprine preantral follicles, and to evaluate quantitatively and qualitatively the efficiency of a specific mechanical method for the isolation of preantral follicles from mixed breed goats at different reproductive stages. On average, 37,646+/-4277 preantral follicles were present in goat ovaries, and 13,631+/-2399 preantral follicles were obtained after isolation. The number of preantral follicles isolated or in situ was not significantly affected by the reproductive stage. The mean recovery rate per ovary ([number of isolated follicles/number of in situ follicles] x 100) of isolated follicles was 36.2%. The distribution of follicles in situ was 67.8% primordial, 25.8% primary and 6.4% secondary; the respective distribution after isolation was 93.8%, 5.2% and 1.0%. In this study, many polyovular follicles were also observed, mainly in prepubertal goat ovaries. Histological analysis showed that few preantral follicles were atretic in situ (4.83%+/-0.35) or after the isolation procedure (4.67%+/-0.65) in the three reproductive stages. The percentage of atretic follicles was not affected either by the mechanical method or by the reproductive stage. It is concluded that a large number of preantral follicles can be successfully isolated mechanically, with a high recovery rate and a low rate of follicular atresia, irrespective of the reproductive stage of the caprine female.

Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles.

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control - Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 degrees, 20 degrees or 39 degrees C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 degrees or 39 degrees C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 degrees C for 4 h and in both solutions at 4 degrees C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 degrees C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 degrees C may explain why the best preservation of preantral follicles was at 4 degrees C, which may suggest a useful method for ovary transport in the future.

New approach to improve encapsulation and antitumor activity of doxorubicin loaded in solid lipid nanoparticles

This work aimed to develop solid lipid nanoparticles (SLNs) loaded with doxorubicin evaluating the influence of docosahexaenoic acid (DHA), a polyunsaturated fatty acid that enhances the activity of anticancer drugs, on drug encapsulation efficiency (EE). The SLN were characterized for size, zeta potential, entrapment efficiency (EE) and drug release. Studies of in vitro antitumor activity and cellular uptake were also conducted. The reduction in particle size (from 127 ± 14 to 94 ± 1 nm) and negative charges were obtained for SLN loaded with DHA and triethanolamine (TEA), amine used to increase the solubility of doxorubicin in melted lipid. The EE was significantly improved from 36 ± 4% to 99 ± 2% for SLN without and with DHA at 0.4%, respectively. The doxorubicin release in a slightly acid medium (pH 5.0) was higher than that observed at physiological pH. The in vitro studies clearly showed the higher cytotoxicity of doxorubicin-DHA-loaded SLN than free doxorubicin+DHA on human lung tumor cell line (A549) and the improved cellular uptake achieved with the drug encapsulation can be an explanation. These findings suggest that DHA-doxorubicin-loaded SLN is a promising alternative for the treatment of cancer.

Cryopreservation of swine ovarian tissue: Effect of different cryoprotectants on the structural preservation of preantral follicle oocytes ☆

The present study aimed to test different cryoprotectants on cryopreservation of pig ovarian tissue. Pig ovaries (n=3) were collected at a local slaughterhouse. From each ovary, ten cortex samples were taken. One was immediately fixed (control) and another placed in short-term tissue incubation (STTI control). The other 8 samples were cryopreserved, in pairs, using 4 different cryoprotectants: dimethyl sulphoxide (Me2SO - 1.5M), ethylene glycol (EG - 1.5M), propanediol (PROH - 1.5M) and glycerol (GLY - 10%), all with 0.4% sucrose. Samples were slow cooled and stored in liquid nitrogen for 7 days. After thawing and cryoprotectant removal, one sample from each treatment was immediately fixed and the other was placed in short-term tissue incubation (STTI) for 2h and then fixed. Samples were processed for histology and transmission electron microscopy. The percentages of morphologically normal follicles (MNF) in cryopreserved tissue using Me2SO (67.0+/-4.9), EG (81.8+/-1.4) and PROH (55.9+/-9.9) were significantly lower (P<0.05) than observed in fresh control tissue (97.7+/-1.2). When ovarian tissue was cryopreserved with GLY, no morphologically normal follicles could be found (0%). After STTI, PROH showed a significantly lower percentage of MNF when compared with all other treatments and the control. After ultrastructural analysis, follicles cryopreserved with Me2SO and EG showed some small alterations, but no signs of advanced degeneration. Overall, these were similar to follicles from the control group. In conclusion, it is possible to cryopreserve preantral follicles from pig ovarian tissue using Me2SO or EG.

Zebu (Bos indicus) ovarian preantral follicles : morphological characterization and development of an efficient isolation method

Preantral follicles are a major source of oocytes, and their utilization as an important tool to store large number of female gametes for future use in reproductive programs has been investigated. The increasing importance of studies in this subject, together with the important role of Zebu cattle in the economy of tropical and subtropical countries as well as their well-known differences from European cattle, led to this research. The present study aims to determine the best size interval for sectioning ovarian tissue to isolate preantral follicles from Zebu cows using a tissue chopper and to evaluate the follicular quality after isolation. Furthermore, it aims to provide information about the Zebu cow preantral follicle population and use this data (as a control) to evaluate the effectiveness of the tested isolation method. Testing eight different tissue sectioning size intervals, it was possible to conclude that the 125-microm-section interval is shown to be better than the intervals of 25, 50, 175 and 200 microm to isolate preantral follicles from Zebu cow ovaries. The 125-microm interval allowed the recovery of 26,050+/-1611 (mean +/- S.E.M.) preantral follicles per one-half ovary, while the number of preantral follicles in situ estimated by evaluation of histological sections was 35,288+/-2342 per one-half ovary. Thus, the mean (+/-S.E.M.) recovery rate (=[number of preantral follicles isolated/number of preantral follicles in situ in the same ovary] x 100) was 74.3+/-4.3%. The morphometrical analysis showed that Bos indicus preantral follicles are similar to B. taurus preantral follicles based on previous reports. In conclusion, this study showed that a simple, mechanical method can be used effectively to isolate a large number of intact preantral follicles from Zebu cow ovaries, with a high recovery rate.

Effects of Fibroblast Growth Factor-2 on the in vitro Culture of Caprine Preantral Follicles

The aims of the present study were to evaluate the effects of fibroblast growth factor-2 (FGF-2) on survival, activation and growth of caprine early-staged (preantral) follicles using histological and ultrastructural studies. Fragments of caprine ovarian cortex were cultured for 1 or 5 days in an enriched minimum essential medium, supplemented or not with different concentrations of FGF-2 (10, 50 or 100 ng/ml). Fragments from non-cultured ovarian tissue (control) and from tissues cultured for 1 or 5 days in a specific medium were processed for transmission electron microscopy (TEM) or classical histology to evaluate the morphological quality of caprine preantral follicles and to calculate the percentages of normal follicles. Additionally, effects of FGF-2 on oocyte and follicle diameter of cultured preantral follicles were investigated. Our results showed that, although the percentages of histologically normal follicles were lower in cultured than in non-cultured ovarian tissue fragments, there were no differences in this regard among treatments, neither on day 1 nor on day 5 of culture. After 1 and 5 days of culture, a significantly higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FGF-2. This FGF-2 treatment furthermore resulted in an increase in diameter of both oocytes and follicles that were cultured for 5 days. TEM showed that the ultrastructural integrity of caprine preantral follicles was maintained during their 5-day culture in the presence of 50 ng/ml FGF-2. In conclusion, this study demonstrated that at a concentration of 50 ng/ml FGF-2 not only maintains the morphological integrity of caprine preantral follicles cultured for 5 days, but also stimulates the activation of primordial follicles and the growth of activated follicles.

Características corporais associadas com a adaptação ao calor em bovinos naturalizados brasileiros

The aim of this work was to evaluate physical aspects of heat tolerance in cattle. Sixty-four animals from five naturalized breeds were used, including Curraleiro (15 animals), Mocho Nacional (7), Crioulo Lageano (17), Pantaneira (14) and Junqueira (11), as well as twenty-six animals from two commercial breeds: Nellore (15) and Holstein (11). Measurements on shoulder height, girth, body length, cannon bone circumference, skin and hair thickness were analysed as well as hair density and hair and skin pigmentation. Girth in Curraleiro was significantly smaller than in other breeds, especially Mocho Nacional. Crioulo Lageano and Pantaneira had the thickest hair and Mocho Nacional the thickest skin. Curraleiro and Junqueira were shown to be the most heat tolerant of the naturalized breeds.