Sonia Helena Furtado Costa

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.

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.

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.

Effect of 0.9% saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles

O presente trabalho investigou a eficiencia da solucao salina 0,9% e tampao fosfato salina (PBS) na conservacao de foliculos pre-antrais caprinos in situ a diferentes temperaturas e tempos de incubacao. O par ovariano de cada animal foi dividido em 19 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado (controle). Os outros 18 fragmentos foram distribuidos aleatoriamente em tubos contendo solucao salina 0,9% ou PBS a 4, 20 ou 39 °C por 4, 12 ou 24 h. Um total de 5.921 foliculos pre-antrais foram analisados. A qualidade dos foliculos pre-antrais foi avaliada atraves de histologia classica. A incubacao de fragmentos ovarianos em solucao salina 0,9% ou PBS a 4 oC nao reduziu significativamente a percentagem de foliculos morfologicamente normais quando comparados com o controle, exceto apos a conservacao em solucao salina 0,9% por 24 h. A incubacao de fragmentos ovarianos a 20 ou 39°C reduziu a percentagem de foliculos pre-antrais normais quando comparados com o controle, exceto apos conservacao em PBS a 20°C por 4 h. Em conclusao, este estudo mostrou pela primeira vez que foliculos pre-antrais caprinos podem ser conservados in situ com sucesso a 4 oC em solucao salina 0,9% por 12 h e em PBS por 24 h, e a 20 oC em PBS por 4 h.

Preliminary study of short-term preservation of ovine ovarian tissue containing preantral follicles in saline solution or TCM199

PREANTRAL follicles represent 90 per cent of the follicular population of mammalian ovaries (Roy and Treacy 1993). However, the vast majority (99.9 per cent) become atretic during their growth and maturation in vivo (Carroll and others 1990). The maintenance of follicular quality in vitro by appropriate handling of the ovaries during their collection and transportation is important for providing healthy oocytes for cryopreservation and/or in vitro culture, as ovarian donors are frequently some distance from specialised laboratories. Several studies have successfully used saline solution to preserve bovine (Schernthaner and others 1997) and caprine (Lucci and others 1999) ovaries for a period of a few hours. Tissue culture medium 199 (TCMl99; Sigma) has also been used successfully in the transport of bovine ovaries (Figueiredo and others 1993) and bovine oocytes from antral follicles (Twagiramungu and others 1998). The aim of this study was to evaluate the effect of 0 9 per cent saline solution or TCM199 on the short-term preservation of sheep preantral follicles inside ovarian tissue, at different temperatures and over different periods of storage.