Ana Paula Ribeiro Rodrigues

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.

Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries.

The preantral follicles are the major source of oocytes and its utilization has been investigated as an important tool to store large numbers of female gametes for further utilization in reproductive programs. The aim of the present study was to perform quantitative and qualitative analyses of the efficacy of a mechanical method for isolating of preantral follicles from the ovaries of fetuses and from nonpregnant and pregnant ewes, using as reference the population of preantral follicles in situ. In the isolation method the ovaries were cut into fragments in the tissue chopper. Then, the suspension was filtered through nylon mesh filters. The number of isolated follicles per ovary was 1655, 4735 and 4770, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The number of in situ preantral follicles per ovary was 32961, 16627 and 17794, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The follicle recovery rate (number of isolated preantral follicles/number of in situ preantral follicles x 100) was higher in adult ewes (26 and 28%, respectively, for nonpregnant and pregnant ewes) than in fetuses (5%). Histological analysis showed that very few preantral follicles (less than 0.26% in situ and 0.46% after the isolation procedure) were degenerated. In conclusion, this study showed that a mechanical method could be used effectively to isolate a large number of intact ovine preantral follicles. In the future, with improvements in culture systems, the isolation of a great number of oocytes enclosed in preantral follicles will make a valuable contribution to the rare breeds and endangered species, agricultural efficiency and basic research in folliculogenesis.

Isolated ovine primordial follicles cryopreserved in different concentrations of ethylene glycol.

Cryopreservation of primordial follicles represents an opportunity to preserve female gametes, and consequently to protect the reproductive capacity of humans and animals, as well as to safeguard genetic material from endangered animal species or rare breeds. The aim of this work was to assess the toxicity of different concentrations of ethylene glycol (EG) to primordial follicles, and verify the viability of these follicles after the freezing-thawing procedure. Primordial follicles were isolated from ovine ovaries and exposed to different EG concentrations to evaluate the cryoprotectant (CPA) toxicity before and after cryopreservation. After isolation of primordial follicle (control), the number (mean+/-S.E.M.) of viable primordial follicles/ml was 3764+/-795.21. The number of viable follicles in the toxicity test using EG at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 1096+/-447.9, 960+/-446.67, 948+/-366.14, 832+/-313.59, 856+/-280.67, and 700+/-255.02, respectively. The number of viable follicles at concentrations of 2.5 M was less than for controls. After cryopreservation, the numbers decreased to 0+/-0, 148+/-85.46, 764+/-246.69, 824+/-291.9, 844+/-296.27, and 588+/-200.65, respectively for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M EG. The number of viable follicles at 0, 0.5, and 2.5 M was less than for controls. In conclusion, after the freezing and thawing procedure, concentrations of 1.0, 1.5, and 2.0 M EG can be successfully used for the cryopreservation of isolated follicles in sheep.

Thymol and eugenol derivatives as potential antileishmanial agents

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.

Cryopreservation of ovine primordial follicles using dimethyl sulfoxide.

OBJECTIVE To verify the viability of isolated primordial follicles after exposure to different concentrations of dimethyl sulfoxide (DMSO) and after cryopreservation. DESIGN Randomized control trial. SETTING Laboratorio Renzo Giuliani, University of Florence, Italy. ANIMAL(S) Thirty- to 40-day-old lambs. MAIN OUTCOME MEASURE(S) Isolated primordial follicles were stained with trypan blue to evaluate the effect of different DMSO concentrations before and after the cryopreservation. Histological structure and follicular mortality were evaluated. RESULT(S) After the isolation procedure (control), a mean (+/-SE) of 800 +/- 203.86 live primordial follicles/mL were obtained. The number of live follicles in the toxicity test using the DMSO at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 782 +/- 193.96, 754 +/- 172.03, 778 +/- 191.58, 736 +/- 191.92, 476 +/- 122.9, and 316 +/- 83.52, respectively. The number of live follicles at 2.5 M was lower than that in the control procedure. After cryopreservation, the numbers decreased to 0 +/- 0, 232 +/- 44.20, 636 +/- 161.82, 628 +/- 181.28, 208 +/- 11.57, and 184 +/- 47.07, respectively at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M. The number of live follicles at 0, 0.5, 2.0, and 2.5 M were lower than that in the control procedure. CONCLUSION(S) After cryopreservation, only DMSO concentrations of 1.0 and 1.5 M showed a number of live follicles similar to that of the control procedure.

Effect of sectioning on the number of isolated ovine preantral follicles

The aim of the present study was to test the effect of the interval of serial sections in the tissue chopper on the number of isolated ovine preantral follicles. Best results were obtained when the ovarian fragments were cut in the tissue chopper at interval of 87.5µm (1592 preantral follicles per treatment). Histochemical 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 92, 6, and 2%, respectively. The follicular sizes varied from 12.5 to 96.3µm in diameter. In conclusion, a greater number of isolated preantral follicles were obtained when the method of isolation used the tissue chopper adjusted at 87.5µm. Besides, this treatment does not affect the follicular integrity after the isolation procedure.

Evaluation of saline and coconut water solutions in the preservation of sheep preantral follicles in situ

This efficiency of saline and coconut water solutions in the preservation of sheep preantral follicles in situ at different temperatures and incubation periods was investigated. At the slaughterhouse, each pair of ovaries was divided into 19 fragments. One ovarian fragment was immediately fixed for histology (control time zero). The other 18 ovarian fragments were randomly distributed in tubes containing 2 ml of saline or coconut water solutions at 4, 20 or 39 °C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in saline solution at 20 °C for 12 and 24 h, in coconut water solution at 20 °C for 24 h and in both solutions at 39 °C reduced (P<0.05) the percentage of morphologically normal preantral follicles (MNPFs) when compared with the control. In contrast, the preservation at 4 °C in all incubation times and at 20 °C for 4 h, in both solutions, kept the percentage of MNPF similar to control values. There was a significant predominance of type 1 (only oocyte degeneration) and type 2 (oocyte and granulosa degeneration) degenerated follicles, respectively, in the fragments stored at 20 and 39 °C in both solutions. In conclusion, this study shows that sheep preantral follicles can be preserved in situ at low temperatures in saline or coconut water solutions.

Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation

Ovarian cortical fragments (3 x 3 x 1 mm) were exposed to dimethyl sulfoxide (DMSO) in different concentrations for further analysis of cryoprotectant perfusion by applying high-performance liquid chromatography (HPLC) and conventional cryopreservation. This simple perfusion test can predict the efficiency of the cryopreservation procedure.

Preservation of goat preantral follicles in saline or coconut water solution

O presente estudo investigou a eficiencia da solucao salina e solucao a base de agua de coco na preservacao de foliculos pre-antrais inclusos em tecido ovariano, em diferentes temperaturas e diferentes tempos de incubacao. No abatedouro, o par ovariano foi dividido em 19 fragmentos; um fragmento ovariano foi imediatamente fixado para histologia classica (controle-tempo zero). Os outros 18 fragmentos ovarianos foram conservados em ambas as solucoes a 4oC, 20oC ou 39oC por 4 h, 12 h ou 24 h. A analise histologica mostrou que a conservacao de fragmentos ovarianos em ambas as solucoes a 4oC por ate 24 h mantem a percentagem de foliculos pre-antrais normais similar aos valores do controle. Ao contrario, a conservacao a 20°C ou 39oC, em ambas as solucoes, reduziu significativamente a percentagem de foliculos pre-antrais normais comparado aos valores do controle, exceto em solucao salina a 20oC por 4 h ou em solucao a base de agua de coco a 20oC por 4 h e 12 h. Em conclusao, esse estudo mostrou que ambas as solucoes podem ser usadas com igual eficiencia para conservar foliculos pre-antrais caprinos a 4°C, independente do tempo de incubacao. No entanto, para conservar foliculos pre-antrais caprinos a altas temperaturas, a solucao a base de agua de coco e recomendada.