Search

Probióticos e prebióticos são classificados como alimentos funcionais e representam importante papel para regulação da microbiota intestinal. Subprodutos da agricultura representam fontes promissoras para produção desses tipos de alimentos, além de representarem uma fonte sustentável. Neste contexto se destaca o caju, um dos principais subprodutos da cajucultura. Diante disso, neste estudo objetivou-se realizar uma revisão de literatura sobre estudos de avaliação do caju na produção de prebióticos e probióticos. Para isto, foi realizada uma revisão de literatura utilizando as bases de dados Periódicos CAPES, PubMed/Medline, Scielo e ScienceDirect com as seguintes palavras-chave: cashew apple Prebiotic e cashew apple probiotic. Foram selecionados trabalhos que utilizaram o caju, bem como destacaram o tipo de espécie utilizadas. Os artigos que não atenderam aos critérios de inclusão não foram incluídos. Após as buscas, se observou que o caju apresenta diversas potencialidades para produção de prebióticos e probióticos a partir da utilização de diferentes tipos de cepas do gênero Lactobacillus, por exemplo, além de conter substâncias de interesse para a saúde, como vitaminas, representando substrato adequado e com baixo custo para produção de oligossacarídeos, quando comparados a outros substratos

INTRODUÇÃO: O Brasil apresenta diversas espécies nativas que apresentam importante papel quanto ao seu uso medicinal, sendo Anacardium occidentale L. uma dessas espécies. Estudos etnofarmacológicos destacam o uso da casca do caule da planta para problemas inflamatórios, antibacteriano, entre outros. Além disso, dados revelam a presença de compostos bioativos que corroboram com seu uso popular. OBJETIVO: Este estudo objetivou realizar uma revisão de literatura acerca dos efeitos da casca do caule de Anacardium occidentale e dos seus constituintes químicos. MÉTODOS: Para isto, foi realizada uma busca nas bases de dados: Periódicos CAPES, PubMed/Medline, Scielo e ScienceDirect, no período de junho-julho de 2022, utilizando descritores DeCS/MeSH: Efeito, Anacardium occidentale, casca de planta, sem considerar o ano de publicação. RESULTADOS: Foram selecionados artigos sobre a casca do caule, em relação aos constituintes químicos e efeitos por meio da avaliação em modelos in sílico, in vitro ou in vivo. Foram selecionados artigos sobre os efeitos antidiarreico, antifúngico, anti-inflamatório e antinociceptivo, antibacteriano, antimutagênico, cicatrizante, citotóxico e hipoglicêmico. Observou-se a presença de diversos compostos fitoquímicos como, compostos fenólicos, ácido anacárdico e taninos. CONCLUSÃO: Portanto, considera-se que a casca de A. occidentale é uma importante fonte de compostos bioativos utilizada etnofarmacológicamente e que tem sido evidenciada em estudos in vitro, in sílico e in vivo.

O presente estudo teve por objetivo promover um levantamento bibliográfico nas bases de dados em torno da seguinte questão norteadora: quais os estudos referentes à utilização da espécie Schinus terebinthifolius Raddi em ensaios antifúngicos do gênero Candida em um período de 10 anos de produtividade científica? Trata-se de uma revisão integrativa da literatura de caráter descritivo, transversal com abordagem quantitativa. Obteve-se um quantitativo total de 26 artigos, contudo, apenas 10 passaram por leitura na íntegra para discussão, LILACS dois artigos, Medline seis e SciELO dois, sendo os demais descartados por não atenderem aos critérios estabelecidos. O estudo indicou pouca produtividade cientifica envolvendo a espécie Schinus terebinthifolius Raddi em relação ao gênero Candida, por mais que os existentes nas bases de dados tenham demostrado um potencial promissor na inibição de crescimento desse patógeno.

Objetivo: o presente artigo objetivou promover uma discussão a respeito do paradoxo da teoria contrapondo a prática da saúde do homem na assistência de enfermagem na atualidade, com base no arsenal científico. Metodologia: trata-se de um artigo teórico-reflexivo, com abordagem metodológica de revisão da literatura, que se utilizou como foco norteador a implementação da Política Nacional de Atenção Integral à Saúde do Homem e os assuntos pautados sobre sua aplicabilidade dentro do contexto da Assistência de Enfermagem. Resultados: obteve-se um quantitativo de 34 artigos, dos quais apenas 26 se enquadravam nos critérios pré-estabelecidos. Quanto à exclusão, 4 não estavam disponíveis na íntegra, 2 tratavam-se de teses de doutorado e 2 não respondiam ao tópico norteador. Já no que se refere às bases de dados, 16 estavam contidos na LILACS, 3 na Medline e 7 na SciELO. Considerações finais: compreende-se assim que, o estudo conseguiu estabelecer uma relação próxima entre a teoria da Política de Atenção Integral à Saúde do Homem e a realidade prática por meio de uma reflexão, de modo a apontar as falhas e desafios que ainda precisam ser enfrentados pelo corpo gestor e equipe profissional, para atender essa parcela tão distante de seus direitos perante cidadania.

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.

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 - P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

Background:Auxemma oncocalyx and its main component oncocalyxone A (onco A) have a high level of antioxidant and antitumor activity, but there are no studies on the action of both of these drugs regarding folliculogenesis. Material and Methods: Caprine ovarian tissue fragments were fixed (non-cultured control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented with dimethyl sulfoxide (DMSO; 20% v/v), bone morphogenetic protein 15 (BMP-15; 100 ng/ml), doxorubicin (DXR; 0.3 g/ml), or different concentrations of A. oncocalyx (1.2, 12, or 34 g/ml) or onco A (1, 10, or 30 g/ml). We analyzed for follicular morphology and growth, apoptosis (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay), and cell proliferation (silver staining of argyrophilic nucleolus organizer regions (AgNOR) and test for proliferating cell nuclear antigen (PCNA)). Results:A. oncocalyx and onco A (in a concentration-dependent manner) and DXR decreased (P < 0.05) the number of morphologically normal follicles, with no effect (P > 0.05) on follicular growth. A. oncocalyx reduced (P < 0.05) the percentage of normal follicles compared to onco A, whereas DXR, A. oncocalyx 1.2 g/ml, and onco A 1 g/ml increased (P < 0.05) the percentage of TUNEL-positive follicles. DXR decreased (P < 0.05) the number of nucleolus organizer regions. Conclusion:A. oncocalyx and onco A affected the in vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 g/ml) has a less harmful effect than DXR on goat preantral follicle survival.

SummaryThe objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding–refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

This study aimed to demonstrate the expression of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles in order to investigate the effects of GH on the survival and development of preantral follicles. The ovaries were processed for the isolation of follicles to study GH-R mRNA expression or to localization of GH-R by immunohistochemical analysis. Pieces of ovarian cortex were cultured for 7 days in minimum essential medium(+) (MEM(+)) in the presence or absence of GH at different concentrations (1, 10, 50, 100, and 200 ng/mL). High expression levels of GH-R mRNA were observed in granulosa/theca cells from large antral follicles. However, preantral follicles do not express mRNA for GH-R. Immunohistochemistry demonstrated that the GH-R protein was expressed in the oocytes/granulosa cells of antral follicles, but any protein expression was observed in preantral follicles. The highest (P 0.05) rate of normal follicles and intermediate follicles was observed after 7 days in MEM(+) plus 10 ng/mL GH (70%). In conclusion, GH-R mRNA and protein are expressed in caprine antral follicles, but not in preantral follicles. Moreover, GH maintains the survival of goat preantral follicles and promotes the development of primordial follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM+) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM+ alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM+ alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

The aim of the present study was to investigate the influence of two insulin concentrations (10ng/mL and 10μg/mL) combined or in the absence of BMP15 and/or GDF9, on the in vitro survival and development of preantral follicles of goat ovarian tissue. Ovarian slices from the same goat ovary pair were randomly assigned to a non-cultured control treatment or to be in vitro cultured for 1 or 7days in α-MEM containing 10ng/mL (Low) or 10μg/mL (High) of insulin in the absence or presence of BMP15 and/or GDF9. With the low insulin treatment, there was a greater percentage of normal follicles than with the high insulin treatment. The addition of BMP15 alone or in association with GDF9 to the medium containing low insulin resulted in a lesser percentage of normal follicles (P

Auxemma oncocalyx and its main component oncocalyxone A (onco A) have a high level of antioxidant and antitumor activity, but there are no studies on the action of both of these drugs regarding folliculogenesis.Caprine ovarian tissue fragments were fixed (non-cultured control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented with dimethyl sulfoxide (DMSO; 20% v/v), bone morphogenetic protein 15 (BMP-15; 100 ng/ml), doxorubicin (DXR; 0.3 g/ml), or different concentrations of A. oncocalyx (1.2, 12, or 34 g/ml) or onco A (1, 10, or 30 g/ml). We analyzed for follicular morphology and growth, apoptosis (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay), and cell proliferation (silver staining of argyrophilic nucleolus organizer regions (AgNOR) and test for proliferating cell nuclear antigen (PCNA)).A. oncocalyx and onco A (in a concentration-dependent manner) and DXR decreased (P0.05) the number of morphologically normal follicles, with no effect (P0.05) on follicular growth. A. oncocalyx reduced (P0.05) the percentage of normal follicles compared to onco A, whereas DXR, A. oncocalyx 1.2 g/ml, and onco A 1 g/ml increased (P0.05) the percentage of TUNEL-positive follicles. DXR decreased (P0.05) the number of nucleolus organizer regions.A. oncocalyx and onco A affected the in vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 g/ml) has a less harmful effect than DXR on goat preantral follicle survival.

We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a significant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a significant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies confirmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles.

This study evaluated the levels of c-kit mRNA in goat follicles and the effects of kit ligand (KL) on the in vitro development of cultured preantral follicles. Preantral follicles isolated from goat ovarian cortex were cultured for 18 days in α-MEM + supplemented with KL (0, 50 or 100 ng/mL) in the absence or presence of follicle stimulating hormone (FSH). Real-time PCR showed that c-kit mRNA was higher in primordial and primary follicles than in secondary stage. Regarding the culture, KL addition in the absence of FSH improved the follicular survival, antrum formation, oocyte growth and meiotic resumption. KL-positive effects were not observed in the presence of FSH. In conclusion, c-kit mRNAs are detected in all follicular categories. KL promotes the survival and antral cavity formation of caprine preantral follicles after in vitro culture, as well as the growth and meiotic resumption of their oocytes in the absence of FSH.

Contents We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 μm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.

The majority of thousands of oocytes in the ovaries are small, non-growing and reside in preantral follicles. The development of a culture system for preantral follicle may be very useful for understanding the complex mechanism in folliculogenesis at early stages of development as wells as could offer a significant way for the propagation of livestock, including goats. This paper describes the results of a number of studies aimed to evaluate the effects of several substances on in vitro culture of caprine preantral follicles highlighting the many advances, limitations and prospects.

The aim of this study was to evaluate the effects of a dynamic medium containing kit ligand (KL) and follicle-stimulating hormone (FSH) on the in vitro culture of caprine preantral follicles for 16 days. Ovarian fragments were cultured in α-MEM+ containing or not containing KL (50 ng/ml) and/or FSH (50 ng/ml) added during the first (days 0–8) and/or second half (days 8–16) of the culture period. Noncultured (control) and cultured fragments were processed for histological and ultrastructural evaluation. After 1 day of culture, only the treatments performed with KL or FSH maintained a percentage of normal follicles similar to that of the control. After 16 days, all treatments using KL until day 8 (KL/KL, KL/FSH, and KL/FSH+KL) and only FSH during the entire culture period (FSH/FSH) showed higher rates of follicular survival compared to α-MEM+ alone. After 1 and 8 days, the treatments initially cultured with KL increased the percentage of follicular activation in comparison to α-MEM+ alone and other treatments. The highest follicular diameter after 16 days was observed in follicles cultured with KL until day 8 followed by FSH (KL/FSH). Furthermore, this treatment promoted, as early as after 1 day of culture, an increase in oocyte growth compared to α-MEM+ alone. Ultrastructural analysis confirmed the integrity of follicles cultured in KL/FSH after 16 days. In conclusion, a dynamic medium containing KL and FSH maintained follicular integrity and promoted follicular activation and growth during the long-term in vitro culture of caprine preantral follicles.

The aim of this study was to verify the influence of three different protocols for medium refreshing on the in vitro culture of isolated caprine preantral follicles. Independently of the protocol, preantral follicles were individually cultured for 18 days, the initial volume of medium was 25 μl, and the interval of medium refreshing was every two days. The protocols tested were: T1 (Control) – refreshing of 15 μl (removal of 15 μl of culture medium followed by the addition of the same volume of fresh medium), maintaining a final volume of 25 μl, T2 – only the addition of 5 μl of fresh medium every two days (the medium volume increases 5 μl for each change up to a final volume of 65 μl at day 18), and T3 – initial removal of 15 μl of medium in the first change, with addition of 20 μl of fresh medium (net increase of 5 μl in the final volume at each change). In the subsequent changes for T3, the amount of medium added in the previous change was removed, followed by the addition of the same volume plus 5 μl fresh medium (as occurred for T2 the final volume at day 18 is also 65 μl). Analyses of survival, diameter and antrum formation, as well as the rate of daily follicular growth were performed every 6 days. At the end of the culture period, normal oocytes ≥110 μm were destined for in vitro maturation (IVM). The results showed that only T2 (addition without removal of medium) maintained follicular survival until the end of the culture period. In day 18, both follicular diameter and the rate of daily growth was similar in T2 and T3 (Removal + Addition of medium), which were both higher than in T1 (Partial change). Moreover, T2 obtained a greater percentage of oocytes >110 μm destined for IVM and was the only treatment that achieved an oocyte in the telophase-I stage. In conclusion, periodic addition of medium is recommended because it is more practical, maintains survival and promotes the development of caprine preantral follicles in vitro.

SummaryThis study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus–oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

In this study, we analysed the effect on morphology and viability of ovine primordial follicles, when ascorbic acid (AA) was added to vitrification and in vitro culture (IVC) media. For morphological analysis, ovarian tissue was vitrified using DMSO or ethylene glycol (EG), to which AA was added or omitted. After warming, the tissue was fixed for histology or 1-day cultured in the presence or absence of AA. Isolated primordial follicles from ovine ovarian tissue vitrified with DMSO or EG, both supplemented with AA were stained with trypan blue for viability analysis, or 5-day cultured with or without AA followed by a viability analysis. In this study, we report on the successful vitrification protocol developed for ovine ovarian tissue using EG. Vitrification using DMSO reduced the percentage of morphological normal primordial follicles, whereas addition of AA to the vitrification and culture media did enhance these results (p < 0.05). However, vitrification in a DMSO + AA medium followed by 5-day IVC resulted in a significant decrease in the follicular viability, independently of the presence of AA in the IVC medium.

SummaryThe aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

The aim of this study was to investigate the effects of nerve growth factor (NGF) on the in vitro culture of goat preantral follicles. Ovarian cortex fragments were cultured in α-MEM+ supplemented with 0, 1, 10, 50, 100 or 200 ng/ml NGF for 1 or 7 days. Small fragments of noncultured ovarian tissue as well as those cultured for 1 or 7 days were processed for histology and transmission electron microscopy. The results showed that after 1 or 7 days of culture at all concentrations of NGF, except at 1 ng/ml after 1 day of culture, there was a significant reduction in the percentage of normal follicles compared to noncultured tissues. At higher NGF concentrations (100 and 200 ng/ml) after 7 days of culture, there was a significant reduction in the percentage of normal follicles compared to tissues cultured in α-MEM+ alone or at the other concentrations of NGF. It is important to note that ultrastructural and fluorescent analyses confirmed only the integrity of follicles cultured with 1 ng/ml of NGF after 7 days. In contrast to noncultured control tissues, the percentage of developing follicles was significantly increased at all concentrations of NGF after 1 or 7 days of culture. We observed that follicular diameter was greater at 1 and 10 ng/ml NGF after culture for 7 days than at the other concentrations but was similar to follicles cultured in α-MEM+ alone. In conclusion, NGF improved the survival of goat preantral follicles cultured in vitro in a dose-dependent manner.

The aims of this study were to investigate steady-state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT-PCR was used to analyze caprine steady-state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1-3 mm) and large (3-6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM(+)) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT-PCR demonstrated an increase in steady-state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady-state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM(+) after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth.

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 degrees C for 1h (protocol 1) or at 4 degrees C for 24h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P

The roles of gonadotrophins in the regulation of primordial ovarian follicular development remains unclear. The aims of the present study were to investigate the effects of LH alone or in combination with FSH on the survival, activation and growth of in vitro caprine primordial follicles, using histology and transmission electron microscopy (TEM). To this end, samples of the caprine ovarian cortex were cultured for 1 or 7 days (39 °C), in an atmosphere of 5% CO2, in minimum essential medium (MEM—control medium) supplemented with different concentrations of LH (0, 1, 5, 10, 50 or 100 ng/ml—Experiment 1). In Experiment 2, the control medium was supplemented with FSH (50 ng/ml) and the different concentrations of LH (0, 1, 5, 10 or 50 ng/ml also used). Small samples of non-cultured ovarian tissue, as well as those cultured for 1 or 7 days in a specific medium were processed for classical histological evaluation and TEM to evaluate the follicular integrity and to calculate the percentage of normal follicles. Additionally, the effects of FSH on the oocyte and follicle diameters of the cultured follicles were evaluated. Results showed that after 7 days of culture, the presence of LH (10, 50 or 100 ng/ml) in the culture media significantly increases the percentage of developing follicles (P

Investigou-se a eficiência da solução salina 0,9% (SS) e TCM 199 na conservação de folículos pré-antrais (FOPA) bovinos in situ em diferentes temperaturas e tempos de incubação. Cada par ovariano foi dividido em 25 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado imediatamente após a coleta (controle). Os demais foram distribuídos em tubos contendo SS ou TCM 199 a 4, 20 ou 39ºC por 2, 4, 12 ou 24h. A análise histológica mostrou que a conservação a 4ºC em ambas as soluções manteve a porcentagem de FOPA normais similar ao controle. A conservação em SS a 20ºC por 12 ou 24h, TCM 199 a 20ºC por 24h e em ambas as soluções a 39ºC a partir de 2h aumentou (P

Isolated or cortical tissue-enclosed (in situ) sheep early-stage follicles were exposed to 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or unexposed, or frozen/thawed in the presence of these cryoprotectants and then cultured for 5 days in enriched minimal essential medium (MEM) or not cultured. Cultured and uncultured follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. Follicular diameter was measured and the percentages of primordial and developing follicles calculated. Exposure of isolated or in situ follicles to DMSO or EG led to a marked decrease in the percentage of viable follicles. The percentage of viable isolated and in situ follicles further decreased when they were in vitro-cultured for 5 days, EG-exposed follicles generally showing a more damaging effect than DMSO-exposed follicles. Cultured follicles, both isolated and in situ, which were exposed to EG and DMSO, as well as in situ follicles, which had been frozen/thawed in the presence of one of these cryoprotectants, showed similar growth rates as cultured, untreated follicles, while in these groups significantly lower percentages of primordial follicles and higher percentages of more advanced follicular stages were observed. Among the treated groups, the highest percentage (71-75%) of developing follicles was observed after culturing cryoprotectant-exposed isolated follicles. In contrast, when cryopreserved, isolated follicles were cultured, they did not increase in diameter and did not develop into more advanced stages. In conclusion, exposure to or cryopreservation in the presence of EG and DMSO, as well as their further in vitro culture, negatively affected the viability of ovine isolated and in situ early-stage follicles. In vitro growth of early-stage follicles and activation of primordial follicles were better maintained when follicles had been frozen/thawed and cultured in situ.

SummaryThe aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM – control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

SummaryThis study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days ofin vitroculture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) afterin vitroculture of ovine ovarian tissue.

SummaryThis study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus–oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

Crude extract of the heartwood of Auxemma oncocalyx (A. oncocalyx) and its main component i.e., Oncocalyxone A (onco A), have elevated antioxidant and anti-tumoral activity, but studies on the action of these drugs regarding folliculogenesis are lacking. The aim of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture of isolated secondary follicles and on the in vitro maturation of oocytes from caprine antral follicles grown in vivo. Isolated secondary follicles were randomly distributed in six groups; the non-cultured control was immediately fixed upon isolation. The remaining follicles were cultured for 7 days in ?-MEM+ alone (control) or supplemented with DMSO, doxorrubicin, A. oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL) and cellular proliferation (PCNA), as well as gene expression of Bcl2 and Bax. Additionally, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for in vitro maturation: control, cultured only in maturation base medium (TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After in vitro maturation, oocyte chromatin configuration and viability were assessed. After 7 days of culture, there was a reduction (P < 0.05) in the percentage of morphologically intact follicles, antrum formation, growth rate and number of PCNA positive granulosa cells in DXR treatment compared to the other treatments. In the DXR treatment a higher percentage (P < 0.05) of TUNEL positive follicles and higher (P < 0.05) relative BAX:BCL2 mRNA ratio’s were observed. After in vitro maturation of the COCs DXR, A. oncocalyx and onco A treatments had a greater (P < 0.05) percentage of abnormal oocytes and a lower (P < 0.05) percentage of viable oocytes as compared with the control group. However, only DXR and onco A treatments increased (P < 0.05) the percentage of alive oocytes with abnormal chromatin configuration. There were no differences in maturation rates between the control group and DXR, A. oncocalyx and onco A treatments. In conclusion, under our culture conditions, A. oncocalyx and onco A do not exhibit a toxic effect on isolated secondary follicles and on maturation rates of COCs recovered from antral follicles, however, these drugs negatively affected the COCs viability. Thus, the use of culture biotechnologies as an in vitro secondary follicle culture and in vitro oocyte maturation toxicity testing are appropriated methods to evaluate the possible effects of drugs in folliculogesis. O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados durante 7 dias em ?-MEM+ sozinho (controle) ou suplementado com DMSO, doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso, os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV, a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na formação de antro, na taxa de crescimento e no número de células PCNA positivas (P < 0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de folículos TUNEL positivos (P < 0,05) e também aumento na taxa de RNAm BAX: BCL2 (P < 0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A, observou-se maior (P < 0,05) percentagem de oócitos anormais e menor de oócitos viáveis quando comparados ao grupo controle (P < 0,05). No entanto, apenas nos tratamentos DXR e onco A aumentou a percentagem de oócitos viáveis com configuração da cromatina anormais (P < 0,05). Não houve diferenças nas taxas de maturação entre o grupo controle e os tratamentos DXR, A. oncocalyx e onco A. De acordo com nossas condições de cultivo, pode-se concluir que a A. oncocalyx e onco A não apresentaram efeitos tóxicos sobre folículos secundários isolados e as taxas de maturação dos CCOs recuperados a partir de folículos antrais. No entanto, estas substâncias afetam negativamente a viabilidade oocitária. Assim, o uso de biotecnologias como o cultivo de folículos secundários in vitro e MIV de oócitos para testes de toxicidade são métodos apropriados para avaliar possíveis efeitos das drogas na foliculogênese.

Exposure time and addition of sucrose to the vitrification medium as well as the solid-surface vitrification (SSV) on the morphology of bovine preantral follicles were evaluated. Ovarian tissue was exposed for 1, 5, or 10 min to 4.0 M ethylene glycol with or without the addition of 0.5 M sucrose. Subsequently, the tissue was washed out from cryoprotectants or vitrified by the SSV method. Independently of the presence of sucrose, exposure to vitrification solution for 10 min did reduce the percentages of normal follicles when compared with control. However, the highest rates of normal follicles were attained when tissue was previously exposed to the vitrification solution, with sucrose added or not, for 10 min. Although the SSV is a promising procedure to be applied in ovarian tissue, an optimal vitrification solution for bovine ovarian tissue needs to be developed.

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.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles110 μm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P.05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P.05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.

This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture.

Our aim was to verify the steady-state level of epidermal growth factor (EGF) mRNA in goat follicles at various developmental stages and to investigate the influence of EGF on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify EGF mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of EGF and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for EGF and FSH receptor (FSH-R) was determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. EGF mRNA levels in secondary follicles were significantly higher than those in primordial follicles, whereas in small and large antral follicles, EGF mRNA levels in cumulus–oocyte complexes (COCs) were significantly higher than in granulosa/theca cells. During culture, EGF in the presence or absence of FSH increased the follicular daily growth rate of secondary follicles when compared with that in enriched alpha minimal essential medium. FSH, EGF or both reduced EGF mRNA levels, whereas EGF reduced FSH-R mRNA levels after follicle culture for 6 days. Thus, EGF mRNA levels are higher in secondary follicles than in earlier stages, with both FSH and EGF promoting the growth of goat secondary follicles. EGF and/or FSH reduce EGF mRNA levels, whereas EGF decreases FSH-R mRNA levels, in cultured secondary follicles.

The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

The effect of exposure to cryoprotectant and cryopreservation of goat and sheep ovarian cortical fragments on the morphology of primordial follicles, stromal cell density and follicular development was performed. Goat and sheep ovarian fragments were exposed to 1.0 or 1.5 M ethylene glycol (EG) for 5, 10 or 20 min, followed or not by conventional cryopreservation. Follicular morphology and stromal cell density were evaluated by means of classical histological analysis. In addition, ovarian fragments were cultured for 1 or 7 days after cryopreservation to evaluate follicular development. Both exposure to cryoprotectant and cryopreservation of goat and sheep ovarian tissue did affect the morphology of primordial follicles and stromal cell density, except when goat ovarian tissue was exposed to EG for 5 min. Although exposure time did not influence follicular morphology in both species, increase in the exposure time from 5 to 20 min did reduce goat stromal cell density. Increase in EG concentration from 1.0 to 1.5 M did result in the decrease of the percentage of goat morphologically normal primordial follicles evaluated after exposure only. In vitro culture of frozen–thawed goat and sheep ovarian tissue showed that exposure to 1.0 M, for 10 min, before freezing of goat and sheep ovarian tissue does not impair follicular developmental capacity. In addition, stromal cell density may play a role in follicular survival and development after cryopreservation of ovarian tissue.

We investigated the effects of epidermal growth factor on the survival and growth of caprine preantral follicles. Ovarian fragments were cultured for 1 and 7 days in enriched minimal essential medium with epidermal growth factor (0, 1, 10, 50, 100, or 200 ng/mL). Non-cultured and cultured tissues were processed for histological and ultrastructural studies. Results showed that after 7 days, the epidermal growth factor (1 and 10 ng/mL) maintained the percentage of normal follicles similar to control. An increase in the percentage of primary follicles was observed with 1, 10, and 50 ng/mL of epidermal growth factor compared to enriched minimal essential medium. Ultrastructural studies confirmed follicular integrity after 7 days in epidermal growth factor (1 and 10 ng/mL). In conclusion, the low concentrations of epidermal growth factor maintain caprine follicular viability and promote the transition from primordial to primary follicles.