Abstract This study aimed to investigate the effect of growth and differentiation factor 9 (GDF-9) during the in vitro culture of isolated caprine early antral follicles. The isolated and selected early antral follicles were individually cultured for 18 days, and the following treatments were tested: α-MEM+ (control treatment) or α-MEM+ supplemented with 200 ng/mL GDF-9. The following endpoints were evaluated: follicular growth and morphology, estradiol production, oocyte nuclear maturation, and relative expression of key genes related to steroidogenesis (CYP19A1, CYP17, and insulin receptor) and basement membrane remodeling (MMP-9 and TIMP-2). In both treatments, a decrease was observed in the percentage of morphologically intact follicles with a concomitant increase in the rates of extruded and degenerated follicles (P < 0.05). The GDF-9 treatment showed higher rates of extruded follicles only on day 6 of culture (P < 0.05). Follicle diameter increased progressively throughout the culture period (P < 0.05) with similar diameters between treatments at all culture times (P > 0.05). Growth and differentiation factor 9 increased the daily growth rate from the first to the second third of culture, with higher values (P < 0.05) than control in the second third. Oocyte maturation rate as well as estradiol levels and relative mRNA expression for CYP19A1, CYP17, MMP-9, TIMP-2, and insulin receptor genes were similar between treatments (P > 0.05). This study shows for the first time that GDF-9 added to a culture medium increased the follicle growth rate of goat early antral follicles cultured in vitro.

Resumo Este estudo teve como objetivo investigar o efeito do GDF-9 durante o cultivo in vitro de folículos antrais iniciais caprinos isolados. Os folículos antrais iniciais isolados e selecionados foram cultivados individualmente por 18 dias, e os seguintes tratamentos foram testados: α MEM+ (tratamento controle) ou α-MEM+ suplementado com 200 ng/mL de GDF-9 (tratamento GDF-9). Os seguintes parâmetros foram avaliados: crescimento e morfologia folicular, produção de estradiol, maturação nuclear do oócito e expressão relativa de genes-chave relacionados a esteroidogênese (CYP19A1, CYP17 e receptor de insulina) e remodelamento da membrana basal (MMP-9 e TIMP-2). Em ambos os tratamentos, observou-se diminuição na porcentagem de folículos morfologicamente intactos com aumento concomitante nas taxas de folículos extrusos e degenerados (P < 0,05). O tratamento GDF-9 apresentou maiores taxas de folículos extrusos apenas no 6º dia de cultivo (P < 0,05). O diâmetro do folículo aumentou progressivamente ao longo do período de cultivo (P < 0,05) com diâmetros semelhantes entre os tratamentos em todos os tempos de cultivo (P > 0,05). O GDF-9 aumentou a taxa de crescimento diário do primeiro para o segundo terço de cultivo, sendo maior (P < 0,05) que o controle no segundo terço. A taxa de maturação oocitária assim como os níveis de estradiol e a expressão relativa de RNAm para os genes CYP19A1, CYP17, MMP-9, TIMP-2 e receptor de insulina foram similares entre os tratamentos (P > 0,05). Em conclusão, este estudo mostra pela primeira vez que GDF-9 adicionado a um meio de cultivo aumentou a taxa de crescimento de folículos antrais iniciais caprinos cultivados in vitro.

Resumo Os compostos bioativos naturais obtidos de microrganismos têm despertado especial interesse da indústria nos últimos anos. Esta atenção ocorre em um momento em que o esgotamento de recursos naturais é pronunciado, e a aquisição de novos insumos e produtos bioativos de origem vegetal representa um desafio para as próximas gerações. Neste sentido, a prospecção para a produção e uso em larga escala dos pigmentos bacterianos tem representado uma importante estratégia para o desenvolvimento de novos produtos. Uma grande variedade de propriedades foi atribuída a estas substâncias, entre elas, o potencial terapêutico contra doenças importantes, como o câncer. Existe um consenso de que os protocolos quimioterápicos disponíveis são conhecidos por afetarem negativamente a fertilidade de pacientes com câncer. Grande parte dos efeitos deletérios da quimioterapia está relacionado à citotoxicidade das drogas usadas para este fim, que além das células cancerosas, afetam as células normais. Nesse sentido, as propriedades naturais atribuídas aos pigmentos bacterianos associadas à baixa citotoxicidade e relevante seletividade, os qualificaram como potenciais drogas anticâncer. No entanto, pouco se tem de informação a respeito da toxicidade reprodutiva destes novos e promissores compostos. Dessa forma, a presente revisão tem o objetivo de abordar os principais pigmentos bacterianos, suas utilizações potenciais como drogas anticâncer, bem como os seus possíveis efeitos tóxicos, sobretudo, sobre a gônada feminina.

Abstract Natural bioactive compounds obtained from microorganisms, have awakened particular interest in the industry nowadays. This attention comes when natural resources depletion is pronounced, and the acquisition of both new plant origin resources and bioactive products, represents a challenge for the next generations. In this sense, prospecting for large-scale production and use of bacterial pigments is a necessary strategy for the development of novel products. A wide variety of properties have been attributed to these substances and, among them, their therapeutic potential against important diseases, such as cancer. There is consensus that available chemotherapy protocols are known to detrimentally affect cancer patients fertility. Hence, considerable part of the deleterious effects of chemotherapy is related to the drugs cytotoxicity, which, in addition to cancer cells, also affect normal cells. Therefore, the intrinsic properties of bacterial pigments associated with low cytotoxicity and relevant cell selectivity, certified them as potential anticancer drugs. However, little information is available about reproductive toxicity of these new and promising compounds. Thus, the present review aims to address the main bacterial pigments, their potential uses as anticancer drugs and their possible toxic effects, especially on the female gonad.

Abstract This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation – Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 μM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

ABSTRACT Most anticancer drugs like doxorubicin (DXR) have low specificity that results in undesirable effects especially when it comes to collateral effects on reproduction. Plants are excellent sources when searching for new drugs. Auxemma oncocalyx (A. oncocalyx) and its main component Oncocalyxone A (onco A) have anti-tumoral activity and are less toxic than DXR in reproductive parameters. However, there are no studies on the action of these drugs regarding the porcine in vitro oocyte competence and embryo development. The aim of this study was to evaluate the effect of A. oncocalyx and onco A exposure during in vitro maturation (IVM) of oocytes (Experiment 1) or in vitro embryo culture (IVC) (Experiment 2) on the oocyte developmental competence. For experiment 1, COCs were distributed in IVM medium alone (control) or supplemented with DXR (0.3 (g/mL), A. oncocalyx (1.2 (g/mL) and onco A (1 (g/mL). Then, oocytes were submitted to in vitro fertilization (IVF) and in vitro embryo culture. For experiment 2, zygotes were cultured with DXR, A. oncocalyx and onco A for 7 days. Viability, maturation, fertilization and embryo developmental parameters were evaluated in both experiments. In experiment 1; DXR, A. oncocalyx and onco A reduced (P<0.05) oocyte viability and IVM efficiency. Onco A increased (P<0.05) the meiotic resumption. After IVF, all drugs reduced (P<0.05) viability, IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR decreased the percentage of blastocyst. In experiment 2; all drugs reduced (P<0.05) the percentage of penetration, but only DXR and onco A decreased (P<0.05) IVF efficiency. DXR and A. oncocalyx decreased (P<0.05) the percentage of cleaved embryo, but had no effect on blastocyst formation. In conclusion, the addition of DXR during IVM or IVC negatively affected the IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR only during IVM was more detrimental to oocyte viability and blastocyst formation than A. oncocalyx and onco A.

RESUMEN La mayoría de los medicamentos contra el cáncer como la doxorrubicina (DXR) tienen una baja especificidad que produce efectos indeseables, especialmente cuando se trata de efectos colaterales en la reproducción. Las plantas son excelentes fuentes cuando se buscan nuevos medicamentos. Auxemma oncocalyx (A. oncocalyx) y su componente principal Oncocalyxone A (onco A) tienen actividad antitumoral y son menos tóxicos que DXR en los parámetros reproductivos. Sin embargo, no hay estudios sobre la acción de estos medicamentos con respecto a la competencia de los ovocitos in vitro porcina y el desarrollo embrionario. El objetivo de este estudio fue evaluar el efecto de la exposición a A. oncocalyx y onco A durante la maduración in vitro (IVM) de los ovocitos (Experimento 1) o el cultivo de embriones in vitro (IVC) (Experimento 2) en la competencia del desarrollo de los ovocitos. Para el experimento 1, los COC se distribuyeron en medio IVM solo (control) o suplementado con DXR (0.3 µg / mL), A. oncocalyx (1.2 µg / mL) y onco A (1 µg / mL). Luego, los ovocitos se sometieron a fertilización in vitro (FIV) y cultivo embrionario in vitro. Para el experimento 2, los cigotos se cultivaron con DXR, A. oncocalyx y onco A durante 7 días. Se evaluaron los parámetros de viabilidad, maduración, fertilización y desarrollo embrionario en ambos experimentos. En el experimento 1; DXR, A. oncocalyx y onco A redujeron la viabilidad de los ovocitos (P <0.05) y la eficiencia de IVM. Onco A aumentó (P <0.05) la reanudación meiótica. Después de la FIV, todos los medicamentos redujeron la viabilidad (P <0.05), la eficacia de la FIV y el porcentaje de embriones clivados, sin embargo, solo DXR disminuyó el porcentaje de blastocistos. En el experimento 2; todos los medicamentos redujeron (P <0.05) el porcentaje de penetración, pero solo DXR y onco A disminuyeron (P <0.05) la eficacia de la FIV. DXR y A. oncocalyx disminuyeron (P <0.05) el porcentaje de embriones clivados, pero no tuvieron efecto sobre la formación de blastocistos. En conclusión, la adición de DXR durante IVM o IVC afectó negativamente la eficiencia de IVF y la tasa de clivaje. Además, la exposición de los COCs a DXR solo durante la IVM fue más perjudicial para la viabilidad de los ovocitos y la formación de blastocistos que A. oncocalyx y onco A.

Abstract The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

Abstract This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

The aim of the present study was to determine the role of GDF-9 and/or FSH on the growth and mRNA expression for FSH-R, GDF-9, and BMPs in goat secondary follicles after culture in vitro. Goat secondary follicles (~200µm) were isolated and cultured for six days in minimum essential medium (MEM) supplemented with GDF-9 (200 ng/mL), FSH (50 ng/mL) or both. At the beginning and end of culture, the follicular diameter was evaluated and compared. The levels of mRNA for GDF-9, FSH-R and BMPs -2, -4, -6, -7 and -15 in cultured follicles were quantified by real time PCR. The results showed that a significant increase of follicle diameter after six days when compared to day 0, but the presence of GDF-9 and FSH did not influence the follicular growth in comparison with those cultured in MEM. Real time PCR showed that GDF-9 down-regulated the levels of mRNA for BMPs -2 and -15, while FSH either alone or in combination with GDF-9 did not affect the expression of GDF-9, FSH-R and BMPs. In conclusion, GDF-9 reduced the expression of BMP-2 and -15 in caprine preantral follicles after their culture, but FSH either alone or in association with GDF-9 did not control the expression of GDF-9, FSH-R and BMPs.

O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivência e a ultra-estrutura de folículos primordiais caprinos.

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

O presente trabalho foi conduzido de modo a se verificar o efeito de diferentes concentrações da BMP-7 no desenvolvimento in vitro de folículos pré-antrais caprinos. Fragmentos de tecido cortical ovariano caprino foram cultivados por 1 ou 7 dias em Minimum Essential Medium (MEM+) suplementado com diferentes concentrações de BMP-7 (1, 10, 50 ou 100ng/ml). Os fragmentos não cultivados ou aqueles cultivados por 1 ou 7 dias foram processados para histologia clássica e microscopia eletrônica de transmissão (TEM), sendo avaliados parâmetros morfológicos indicativos de viabilidade, ativação e crescimento. Os resultados mostraram que o percentual de folículos morfologicamente normais diminuiu significativamente em todos os tratamentos quando comparados ao controle, exceto na concentração de 1ng/ml por 1 dia de cultivo. Já no D7 todos os tratamentos reduziram significativamente os percentuais de folículos morfologicamente normais. Utilizando 10ng/ml de BMP-7 foi observado um aumento significativo no diâmetro folicular quando comparados os diferentes períodos de cultivo. Não houve influência das demais concentrações de BMP-7 quando avaliados além do diâmetro folicular o diâmetro oocitário. A análise por TEM confirmou a integridade ultra-estrutural nos folículos após 7 dias de cultivo com 1ng/ml de BMP-7 . Em conclusão, o BMP-7 em baixas concentrações pode melhorar a sobrevivência e o crescimento durante o cultivo in vitro de folículos pré-antrais caprinos.

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

O presente trabalho investigou a taxa de degeneração de folículos primordiais caprinos in situ após conservação em PBS ou TCM 199, em diferentes temperaturas e tempos de incubação. Para cada animal, o par ovariano foi dividido em 19 fragmentos. Um fragmento foi escolhido aleatoriamente e imediatamente fixado (controle). Os 18 fragmentos ovarianos restantes foram aleatoriamente distribuídos em tubos contendo PBS ou TCM 199 e conservados a 4, 20 or 39ºC por 4, 12 or 24h. A conservação de fragmentos ovarianos em PBS ou TCM 199 a 20ºC por 12 e 24h ou a 39ºC, em todos os tempos de incubação testados, aumentou significativamente a percentagem de folículos primordiais degenerados (P<0,05). Ao contrário, em ambos os meios testados, a taxa de degeneração de folículos primordiais conservados a 4ºC em todos os tempos testados e a 20ºC por 4h foi similar ao controle (P>0,05). Em conclusão, este estudo mostrou que o PBS foi tão eficiente quanto o TCM 199 na conservação de folículos primordiais caprinos in situ, sendo os melhores resultados observados na temperatura de 4ºC.

The present work has investigated the degeneration rate of goat primordial follicles in situ after preservation in PBS or TCM 199 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 PBS or TCM 199 and stored at 4º, 20º or 39ºC for 4, 12 or 24h. The storage of ovarian fragments in PBS or TCM 199 at 20ºC for 12h and 24h or at 39ºC, in all incubation times tested, increased significantly the percentage of degenerated primordial follicles (P<0.05). In contrast, for both media tested the degeneration rate of primordial follicles preserved at 4ºC for up to 24h and at 20ºC for 4h was similar to control values (P>0.05). In conclusion, this study shows that PBS was as efficient as TCM 199 in the preservation of goat primordial follicles in situ, being the best results observed at 4ºC.

O presente estudo investigou a eficiência da solução salina e solução à base de água de coco na preservação de folículos pré-antrais inclusos em tecido ovariano, em diferentes temperaturas e diferentes tempos de incubação. No abatedouro, o par ovariano foi dividido em 19 fragmentos; um fragmento ovariano foi imediatamente fixado para histologia clássica (controle-tempo zero). Os outros 18 fragmentos ovarianos foram conservados em ambas as soluções a 4ºC, 20ºC ou 39ºC por 4 h, 12 h ou 24 h. A análise histológica mostrou que a conservação de fragmentos ovarianos em ambas as soluções a 4ºC por até 24 h mantém a percentagem de folículos pré-antrais normais similar aos valores do controle. Ao contrário, a conservação a 20°C ou 39ºC, em ambas as soluções, reduziu significativamente a percentagem de folículos pré-antrais normais comparado aos valores do controle, exceto em solução salina a 20ºC por 4 h ou em solução à base de água de coco a 20ºC por 4 h e 12 h. Em conclusão, esse estudo mostrou que ambas as soluções podem ser usadas com igual eficiência para conservar folículos pré-antrais caprinos a 4°C, independente do tempo de incubação. No entanto, para conservar folículos pré-antrais caprinos a altas temperaturas, a solução à base de água de coco é recomendada.

The present study investigated the efficiency of saline solution and coconut water solution in the preservation of goat preantral follicles enclosed in ovarian tissue, at different temperatures and for different incubation periods. At the slaughterhouse, the ovarian pair was divided into 19 fragments; one ovarian fragment was immediately fixed for histology (control-time zero). The other 18 ovarian fragments were preserved in both solutions at 4ºC, 20ºC or 39ºC for 4 h, 12 h or 24 h. The histological analysis showed that the storage of ovarian fragments in both solutions at 4ºC for up to 24 h kept the percentage of normal preantral follicles similar to the control values. In contrast, preservation at 20°C or 39ºC, in either solution, reduced significantly the percentage of normal preantral follicles compared to the control values, except in saline solution at 20ºC for 4 h or in coconut water solution at 20ºC for 4 h and 12 h. In conclusion, this study shows that both solutions can be used with the same efficiency to preserve goat preantral follicles at 4°C, irrespective of the incubation time. However, to preserve goat preantral follicles at higher temperatures, coconut water solution is recommended.

O presente trabalho investigou a eficiência da solução salina 0,9% e tampão fosfato salina (PBS) na conservação de folículos pré-antrais caprinos in situ a diferentes temperaturas e tempos de incubação. O par ovariano de cada animal foi dividido em 19 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado (controle). Os outros 18 fragmentos foram distribuídos aleatoriamente em tubos contendo solução salina 0,9% ou PBS a 4, 20 ou 39 °C por 4, 12 ou 24 h. Um total de 5.921 folículos pré-antrais foram analisados. A qualidade dos folículos pré-antrais foi avaliada através de histologia clássica. A incubação de fragmentos ovarianos em solução salina 0,9% ou PBS a 4 ºC não reduziu significativamente a percentagem de folículos morfologicamente normais quando comparados com o controle, exceto após a conservação em solução salina 0,9% por 24 h. A incubação de fragmentos ovarianos a 20 ou 39°C reduziu a percentagem de folículos pré-antrais normais quando comparados com o controle, exceto após conservação em PBS a 20°C por 4 h. Em conclusão, este estudo mostrou pela primeira vez que folículos pré-antrais caprinos podem ser conservados in situ com sucesso a 4 ºC em solução salina 0,9% por 12 h e em PBS por 24 h, e a 20 ºC em PBS por 4 h.

The present work investigated the efficiency of 0.9% saline solution and Phosphate Buffered Saline (PBS) in the preservation of goat preantral follicles in situ at different temperatures and incubation times. The ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was taken randomly and fixed (control). The other 18 fragments were randomly distributed in tubes containing 0.9% saline solution or PBS at 4, 20 or 39 ºC for 4, 12 or 24 h. A total of 5,921 preantral follicles were examined. The quality of preantral follicles was evaluated by classical histology. The storage of ovarian fragments in 0.9% saline solution or PBS at 4 ºC did not reduce significantly the percentage of morphologically normal follicles when compared with the control, except after preservation in 0.9% saline solution for 24 h. The storage of ovarian fragments at 20 or 39°C reduced the percentage of normal preantral follicles when compared to the control, except after preservation in PBS at 20°C for 4 h. In conclusion, this study showed for the first time that goat preantral follicles can be stored in situ successfully at 4 ºC in 0.9% saline solution for 12 h and in PBS for 24 h, and at 20 ºC in PBS for 4 h.

O presente trabalho descreve os efeitos do estágio reprodutivo (animais impúberes, adultos não-gestantes e gestantes), posição anatômica do ovário (direito e esquerdo) presença de corpo lúteo funcional, peso do ovário e número de folículos antrais superficiais (FAS) sobre o número de folículos pré-antrais (FPA) isolados mecanicamente. Os FPA isolados foram divididos em 3 classes a saber: primordiais, primários e secundários. O número médio (± EP) de FPA isolados por ovário foi de 1.324 ± 193; 866 + 170 e 779 ± 139, respectivamente para ovários oriundos de animais impúberes, não-gestantes e gestantes, sendo observada diferença significativa somente entre animais impúberes e gestantes. A posição anatômica do ovário (exceto para os animais não-gestantes) e a presença ou ausência de corpo lúteo não exerceram nenhum efeito sobre o número e distribuição dos FPA isolados. De um modo geral, o número de folículos primários e secundários foi negativamente correlacionado com o peso ovariano. Já o número de folículos primordiais e secundários foi, respectivamente, positiva e negativamente correlacionado com o número de FAS. Analisando as categorias de animais isoladamente, observou-se que, nos animais impúberes, o número de folículos primordiais foi positivamente correlacionado com o número de FAZ, enquanto que o número de folículos primários e secundários foi negativamente correlacionado com o número de FAS. No tocante à categoria de animais gestantes, o número de folículos primários foi negativamente correlacionado com o peso ovariano. Dependendo da categoria de ovários estudada, foram observadas associações significativas entre o número de folículos primordiais, primários e secundários.

The aïm of this study was to describe the effects of reproductive stages (pre-pubertal, non-pregnant and pregnant adult animals), ovarian anatomic position (left or right), presence of functional corpus luteum, ovarian weight and number of superficial antral follicles on the number of preantral follicles mechanically isolated. The mean number (mean ± SEM) of isolated preantral follicles per ovary was 1,324 ± 193, 866 ±170 and 779 ± 139, respectively for prepubertal, non-pregnant and pregnant adult animals. Anatomic position (except for nonpregnant animals) and the presence or absence of corpus luteum dia nof affect the number and distribution of isolated preantral follicles. In general, the number of primary and secondary follicles was negatively correlated with the ovarian weight. The numbers of primordial and secondary follicles were, respectively, positively and negatively correlated with the number of superficial antral follicles. Analyzing animal categories separately, it was observed that in prepurbetal animals, the number of primordial follicles was positively correlated with the number of superficial antral follicles. However the numbers of primary and secondary follicles were negatively correlated with the number of superficial antral follicles. In the category of pregnant animals, the number of primary follicles was negatively correlated with the ovarian weight. According to ovary category analyzed, were observed significant correlation between the number of primordial, primary and secondary isolated follicles.

Com o objetivo de isolar mecanicamente folículos pré-antrais caprinos, 52 ovários de cabras sem raça definida (SRD) foram coletados em matadouros ou extraídos por ovariectomia. O protocolo utilizado para o isolamento dos folículos, denominado procedimento mecânico direto (PMD), consistiu no corte do tecido ovariano em pequenos fragmentos, utilizando-se um cortador de tecidos. Em seguida, os fragmentos obtidos foram dissociados mecanicamente com pipetas de pasteur (1600mim e 600mim de diâmetro) e a suspensão resultante, contendo os fragmentos ovarianos, foi sucessivamente filtrada em malhas de náilon (500mim e 100mim). A realização deste procedimento permitiu isolar um número médio (± EP) de 1.067 ± 160 folículos por ovário. Com a finalidade de aumentar o número de folículos pré-antrais isolados por ovário, foi utilizado um procedimento mecânico complementar (PMC), que consistiu em submeter os fragmentos retidos nas malhas de 500mim e 100mim às etapas descritas no PMD. A utilização desses fragmentos resultou em um incremento médio de 5,2% no total de folículos isolados por ovário. O número médio (± EP) de folículos isolados por ovário, utilizando-se o procedimento mecânico (PMD + PMC), foi 1.126 + 163. Independentemente do procedimento utilizado, observou-se uma grande variação individual no número de folículos isolados por ovário. Os limites mínimo e máximo desta variação foram 53 e 7.482 folículos, respectivamente. O tamanho dos folículos isolados variou de 15,0mim a 92,7mim. É viável a utilização do procedimento mecânico, descrito pela primeira vez, para o isolamento de folículos ovarianos pré-antrais caprinos.

For mechanical isolation of caprine preantral follicles, ovaries from 52 undefinined breed type goats were collected at a local slaughterhouse or cirurgically extracted. The method used for preantral follicle isolation was a direct mechanical procedure (DMP). Briefly, ovaries were cut into small fragments using a tissue chopper adjusted to 500mum. The ovarian fragments were suspended 80 times with a pasteur pipette and then, the suspension was filtered successively through 500 and 100mum nylon mesh filters. The DMP resulted in a large number of isolated preantral follicles. On average, 1,067 ± 160 (mean number ± SEM) preantral follicles were isolated per ovary. To increase the number of preantral follicles recovered per ovary, the ovarian fragments remaining in the 500 and 100mum nylon mesh filters were transferred to the tissue chopper, and the procedure was repeated (complementary mechanical procedure - CMP). The use of CM P resulted in a mean increment of 5.2% of the total preantral follicles isolated per ovary. The mean number (mean ± SEM) of follicles isolated per ovary using the mechanical procedure (DPM + CPM) was 1,126 + 163. Irrespective of the procedure used, the number of preantral follicles isolated was characterized by extreme individual variation. The limits minimum and maximum of this variation were 53 and 7,482, respectively. The diameter of follicles isolated showed a range from l5.0mum to 92.7 mum. In conclusion, these results showed, for the first time, that preantral follicles can be successfully isolated from caprine ovary by applying a mechanical procedure.