Sônia Nair Báo

Chilling ovarian fragments during transportation improves viability and growth of goat preantral follicles cultured in vitro

The aim of the present study was to evaluate the effects of storage of goat ovarian fragments at different temperatures and for different incubation times on the viability and growth of cultured preantral follicles in vitro. Caprine ovaries were collected and divided into 19 fragments, with one fragment being fixed immediately (fresh control). The remaining fragments were placed in minimal essential medium (MEM) and maintained at 4, 20 or 35 degrees C for 2 or 4 h. After each incubation period, some of the fragments were fixed (non-cultured), whereas others were cultured in vitro for 1 or 7 days. Fragments were processed to enable routine histological and transmission electron microscopic examination. After 7 days of culture, only ovarian fragments stored at 4 degrees C for 4 h maintained a percentage of morphologically normal follicles similar to that in the fresh control. For all other treatments groups, there was a significant increase in follicular activation observed. In addition, there was an increase in oocyte and follicular diameter after culture of ovarian cortex that had been chilled previously at 4 degrees C for 2 or 4 h. In conclusion, the present study demonstrated that chilling ovarian fragments at 4 degrees C during transportation is best for maintaining follicle viability and to increase follicular growth during in vitro culture.

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.

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.

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.

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.

Short-term preservation of canine preantral follicles: effects of temperature, medium and time.

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 degrees C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 degrees C for up to 6h and at 38 degrees C for 2 h. Using MEM, such preservation was possible for 12h at 4 or 20 degrees C, and for 6h at 38 degrees C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 degrees C) storage in MEM, which ensures maintenance of morphology and viability for up to 12h.

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.

Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 degrees C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 degrees C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 degrees C for up to 18 h and at 20 degrees C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 degrees C. In conclusion, zebu cow ovaries were successfully stored at 4 degrees C for up to 18 h with no morphological damage to preantral follicles. However, at 20 degrees C, ovaries could only be stored for 6 h.

Morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro

The present work has investigated the morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro and showed quantitative data about the distribution of follicular degeneration types in the control and after preservation in coconut water solution or Braun-Collins solution at different temperatures (4, 20 or 39 degrees C) and incubation times (4, 12 or 24h). At the slaughterhouse, the pair of ovaries of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (control: Time 0). The other 18 fragments were randomly distributed in tubes containing 2ml of coconut water or Braun-Collins solution at 4, 20 or 39 degrees C and stored for 4, 12 or 24h. Normal preantral follicles exhibited a healthy oocyte surrounded by one or more well-organized layers of granulosa cells. The ooplasm contained numerous rounded or elongated mitochondria with continuous mitochondrial membranes. Golgi complexes were rare. Both smooth and rough endoplasmic reticulum were observed, either as isolated aggregations or complex associations with mitochondria and vesicles. Degenerated preantral follicles in the control tissue exhibited pycnotic nuclei of the oocyte, vacuolated ooplasm and normal granulosa cells. This kind of degeneration also predominated significantly (P<0.05) after preservation at 4 degrees C. In contrast, after preservation at 20 or 39 degrees C a significant predominance (P<0.05) of preantral follicles showing a retracted oocyte and swollen granulosa cells was observed. These follicles showed large irregularity of the oocyte and nuclear outlines. The ooplasm exhibited moderate proliferation of the endoplasmic reticulum and mitochondria showed disappearance of most of the cristae and damage to the mitochondrial membrane. Some follicles had numerous vacuoles in the ooplasm. Granulosa cells were spread and a low density of organelles was observed. The alterations in follicular structure progressed with an increase of temperature from 20 to 39 degrees C as well as with an increase of the incubation time from 4 to 12, or 24h. In conclusion, the present study shows for the first time that initial proliferation of the endoplasmic reticulum and damage to mitochondria are the first signs of degeneration in goat preantral follicles during storage in vitro.

Effect of Braun-Collins and Saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ

The present work has investigated the efficiency of Braun-Collins and saline (0.9%) solutions in the conservation of goat preantral follicles in situ, at different temperatures and incubation times. For each animal the ovarian pair was divided into 19 fragments. One ovarian fragment was taken randomly and immediately fixed (control). The other 18 ovarian fragments were randomly distributed in tubes containing Braun-Collins or saline (0.9%) solutions at 4, 20 or 39 degrees C for 4, 12 or 24h. A total of 3385, 372 and 191 primordial, primary and secondary follicles were examined, respectively. The quality of preantral follicles was evaluated by histology and transmission electron microscopy. The storage of ovarian fragments in saline (0.9%) or Braun-Collins solutions at 4 degrees C did not reduce significantly the percentage of morphologically normal follicles when compared with the control. The histological analysis revealed a morphological integrity of goat preantral follicles stored at 4 degrees C for up to 24h in both solutions, but these results were not confirmed by ultrastructural analysis. The transmission electron microscopy revealed that only preantral follicles stored at 4 degrees C for a maximum of 12h in both solutions were ultrastructurally normal. In conclusion, this study shows for the first time that goat preantral follicles can be stored in situ successfully at 4 degrees C in saline (0.9%) or Braun-Collins solution for up to 12h.