Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

Betaine-loaded CaCO3 microparticles improve survival of vitrified feline preantral follicles through higher mitochondrial activity and decreased reactive oxygen species.

Ovary fragments from six sexually mature cats were vitrified in the presence or absence of betaine or ascorbic acid, loaded (7.4 or 74µM betaine; 20 or 200µM ascorbic acid) or not (1mM betaine or 0.3mM ascorbic acid) into CaCO3 microparticles, and assessed for follicular morphology, oxidative stress and mitochondrial activity Feline ovarian tissue was successfully preserved after vitrification in the presence of 74µM betaine loaded in CaCO3 microparticles, as confirmed by morphological analysis and the density of preantral follicles and stromal cells, as well as by the increased mitochondrial activity and decreased production of reactive oxygen species.

The subtle balance of insulin and thyroxine on survival and development of in vitro cultured caprine preantral follicles enclosed in ovarian tissue.

Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 µg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 µg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 µg/mL thyroxine (P < 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 µg/mL (P > 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 µg/mL thyroxine (P < 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P < 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P < 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.

Effect of different extracts and fractions of Senecio biafrae (Oliv. &Hiern) J. Moore on in vivo and in vitro parameters of folliculogenesis in experimental animals.

BACKGROUND: Senecio biafrae is a medicinal plant widely used in traditional medicine to cure female infertility. Some effects have been pharmacologically demonstrated on immature female rats but in vivo and in vitro investigations are still necessary for determining its mechanism of action. The aim of the present study was to evaluate the estrogenic and FSH-like effects of the plant extracts and fractions on some fertility parameters in immature female rats and on in vitro survival and growth of swine preantral follicles. METHODS: 21-23 days old female Wistar rats orally received extracts and fractions of S. biafrae at 0, 8 and 64 mg/kg doses over 20 days. The LH, FSH, estradiol and progesterone serum levels were evaluated as well as the ovarian cholesterol, uterus and ovaries masses and proteins. The numbers of follicles at different developmental stages were recorded in ovarian cortexes after histology. Slices of swine ovarian cortexes were cultured along 1 or 7 days in alpha-minimum essential medium (α-MEM) and fixed for morphological analysis of preantral follicles. The fresh control, cultured control (CIV control) and different Senecio biafrae-treated ovarian fragments were analyzed for preantral follicles development. Treatments that showed the best follicle growth in culture were submitted to AgNOR test. The aqueous and MeOH/CH2Cl2 extracts as well as the ethyl acetate and hexane fractions of S. biafrae were submitted to the HPLC for analysis of polyphenolic secondary metabolites. RESULTS: Ovarian and uterine proteins were significantly high (p < 0.01) in animals treated with the two dosages of ethyl acetate and n-butanol fractions. The same result was recorded with uterine proteins in animals treated with the hexane fraction. The FSH level significantly dropped with all ethanolic extract doses and with the 64 mg/kg dosage of the methanol/methylene chloride (MeOH/CH2Cl2) extract while LH was reduced (p < 0.01) in almost all the treated groups. Estradiol level was significantly increased (p < 0.001) in the three groups receiving the extracts, but reduced (p < 0.001) in the three groups receiving the fractions of the plant. The progesterone level increased with almost all the treated groups. Primary and secondary follicles augmented (p < 0.01) in MeOH/CH2Cl2 extract and n-butanol fraction while tertiary follicles increased with the same extract and the ethyl acetate fraction (p < 0.05). Treatments with aqueous and ethanolic extracts as well as ethyl acetate fraction led to a significant increase (p < 0.05) in the number of morphologically normal follicles after 7 days of culture as compared to the CIV control. The number of AgNOR dots per follicle was significantly low (p < 0.05) in all cultured groups as compared to the fresh control, except the ethyl acetate 2.8 ng/ml dosage. The same observation was done with AgNOR dots per cell in the 2.8 ng/ml dosage aqueous extract-treated fragments. The phenolic compounds mainly encountered in the plant, independently of the extract or fraction are apigenin, eugenol and rutin. CONCLUSION: Extracts and fractions of S. biafrae have an important FSH-like effect which induces follicular survival and growth.

Linfoma leucemizado em felino coinfectado com os vírus da imunodeficiência felina e da leucemia felina: relato de caso / Lymphoma in leukemic phase in feline coinfected with feline immunodeficiency virus and feline leukemia virus: case report

O presente trabalho teve por objetivo relatar um caso de linfoma leucemizado em um felino coinfectado com os vírus da imunodeficiência felina (FIV) e o da leucemia felina (FeLV). Foram realizados exames de hemograma, contagem de reticulócitos, mielograma, bioquímica, teste de imunocromatografia para FIV e FeLV, imunofluorescência indireta (IFA) para FeLV, radiografia torácica e citologia renal. Esse último exame revelou um linfoma extranodal. Foi determinante para a conclusão diagnóstica a associação dos sinais clínicos corroborados com a infiltração de elevada quantidade de células linfoblásticas na medula óssea, exibindo critérios citomorfológicos de malignidade, como mitoses atípicas, relacionadas à presença de corpúsculos linfoglandulares e material hematopoiético inter-relacionado. O linfoma é uma neoplasia relativamente comum em felinos, entretanto, a apresentação leucemizada é rara, podendo representar um desafio diagnóstico clínico, o que torna fundamental a inclusão da citologia medular na prática clínica dessa espécie.(AU)

The present study aimed to report a case of lymphoma in leukemic phase in feline coinfected with feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Blood counts, reticulocyte counts, bone marrow avaluation, biochemistry, immunochromatography assay for FIV and FeLV, indirect immunofluorescence (IFA) for FeLV, thoracic radiography and renal citology were performed. This last examination revealed extranodal lymphoma. The association of the clinical signs with the infiltration of a high number of lymphoblastic cells in the bone marrow with cytomorphological criteria of malignancy, atypical mitoses, lymphoglandular corpuscles and hematopoietic material were determinant for the diagnostic conclusion. Lymphoma is a relatively common neoplasm in felines, however the leukemic phase is rare and may represent a clinical diagnostic challenge, making it essential to include bone marrow cytology in the clinical practice of this species.(AU)

Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence.

The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 µL-drop) or individually (1 COC/10 µL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 µm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 µm/day (fast-growing) and that reached at least 600 µm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 µm, follicle diameter of at least 600 µm, and oocyte diameter ≥120 µm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.

Expectations and limitations of ovarian tissue transplantation.

Constant progress in the diagnosis and treatment of cancer disease has increased the number and prognosis of cancer survivors. However, the toxic effects of chemotherapy and radiotherapy on ovarian function have resulted in premature ovarian failure. Patients are, therefore, still expecting methods to be developed to preserve their fertility successfully. Several potential options are available to preserve fertility in patients who face premature ovarian failure, including immature or mature oocyte and embryo cryopreservation. However, for children or prepubertal women needing immediate chemotherapy, cryopreservation of ovarian tissue is the only alternative. The ultimate aim of this strategy is to implant ovarian tissue into the pelvic cavity (orthotopic site) or in a heterotopic site once oncological treatment is completed and the patient is disease free. Transplantation of ovarian tissue with sufficiently large numbers of follicles could potentially restore endocrine function and allow multiple cycles for conception. However, the success of ovarian tissue transplantation still has multiple challenges, such as the low number of follicles in the graft that may affect their longevity as well as the survival of the tissue during ex vivo processing and subsequent transplantation. Therefore, this review aims to summarize the achievements of ovary grafting and the potential techniques that have been developed to improve ovarian graft survival.

Caprine ovarian follicle requirements differ between preantral and early antral stages after IVC in medium supplemented with GH and VEGF alone or in combination.

The aim of the present study was to evaluate the effect of growth hormone (GH) and vascular endothelial growth factor (VEGF) added alone, sequentially or in combination, in the presence of insulin at physiological concentration (10 ng/mL) on the IVC of two different follicular categories: preantral (experiment 1; Exp.1) and early antral (experiment 2; Exp.2). Isolated follicles were individually cultured for 24 (Exp.1) and 18 days (Exp.2) in the following treatments: αMEM+ (Control), or Control medium supplemented with 50 ng/mL GH (GH), 100 ng/mL VEGF (VEGF), the combination of both (GH + VEGF), GH during the first 12 days and VEGF from Day 12 until the end of the culture (GH/VEGF) and vice versa (VEGF/GH). At the end of the culture, cumulus-oocyte complexes from in vitro-grown follicles were recovered and subjected to IVM. The following end points were evaluated: Follicle morphology, growth rates and antrum formation, production of estradiol, progesterone and testosterone, oocyte viability and meiotic stage, as well as relative expression of LHR, Amh, HAS2, PTGS2, CYP17, CYP19A1, and 3ßHSD. A considerable amount of viable fully grown oocytes were recovered after the IVC of early antral follicles in all treatments. Nevertheless, the GH treatment presented the highest percentage of fully grown oocytes (60%), mean oocyte diameter (117.74 ± 2.61 µm), and meiotic resumption (50%). Furthermore, GH treatment produced higher (P < 0.05) rates of metaphase II oocytes than all the other treatments, and similar LHR, Amh, and PTGS2 transcript levels to in vivo. Contrary to early antral follicles, preantral follicles were not affected by medium supplementation. In conclusion, the addition of GH to a culture medium containing physiological concentrations of insulin improves oocyte growth and maturation after the IVC of goat early antral follicles.

Impact of insulin concentration and mode of FSH addition on the in vitro survival and development of isolated bovine preantral follicles.

UNLABELLED: The insulin and FSH are two important substances in the folliculogenesis process. Thus, the hypothesis of this experiment is that insulin concentration and the form of FSH addition affect the in vitro survival, growth, and estradiol production after culture of isolated bovine preantral follicles. The effects of insulin concentration (experiment 1) and the influence of both fixed and sequential concentrations of FSH (experiment 2) on the in vitro survival and development of bovine preantral follicles were investigated in this study by IVC for 18 days. In experiment 1, on Day 18 of culture, the addition of insulin at all concentrations promoted follicular survival rates significantly higher than that of the control, with the 10-ng/mL insulin treatment showing values significantly higher than the other treatments. The addition of 5- and 10-ng/mL insulin promoted higher follicular growth than the control and other treatments. In experiment 2, FSH 100 had a higher percentage of follicular viability compared with the control. FSH 100 produced follicle diameters significantly higher than those of the control and FSH seq. TREATMENT: Estradiol levels in the presence of FSH (fixed concentration) were significantly higher than the other treatments. In conclusion, the association of insulin (10 ng/mL) and fixed concentration FSH (100 ng/mL) provides high rates of survival, growth, and estradiol production in bovine preantral follicles.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Effect of the aqueous extract of Senecio biafrae (Oliv. & Hiern) J. Moore on some fertility parameters in immature female rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Senecio biafrae is a plant from the huge family of Asteraceae used in the African pharmacopoeia for the treatment of many ailments among which is infertility. MATERIAL AND METHODS: The aqueous extract, which was primarily subjected to polyphenol analysis, has been administered to immature female rats for 20 days at 8, 32, 64 and 128 mg/kg of body weight. The day following the treatment, the animals were sacrificed; their serum, ovary and uterus were retained respectively for reproductive hormones, ovarian and uterine proteins, and ovarian cholesterol assays. RESULTS: Light body weight gain variation of treated animals was observed during the experimental period. A significant increase (p ˂ 0.05) in serum estradiol and proteins as well as in uterine weight (p ˂ 0.01) of all Senecio biafrae treated animals was noted. No significant variation was noticed in the ovarian weight and follicle numbers. CONCLUSION: The various biochemical and physiological parameters of fertility were significantly improved with the aqueous extract of Senecio biafrae, thus attesting some of its traditional usage.

Short-term preservation of Pecari tajacu ovarian preantral follicles using phosphate buffered saline (PBS) or powdered coconut water (ACP(r)) media / Preservação de folículos ovarianos pré-antrais de Pecari tajacu por curtos períodos utilizando meios à base de solução salina fosfatada tamponada (PBS) ou água de coco em pó (ACP(r))

We compare protocols for the short-term preservation of collared peccarie's ovarian preantral follicles (PFs) by using phosphate buffered saline- (PBS) or powdered coconut water- (ACP(r)) based medium. For morphology analysis each pair of ovaries collected from six females was divided into nine fragments. One fragment was destined for morphology analysis (histology and transmission electron microscopy - TEM), constituting the control group and the other fragments were placed in tubes with PBS or ACP(r), packed in 5 L Styrofoam boxes, stored for 4h, 12h, 24h, and 36h, and then analyzed. For viability analysis a pair of ovaries from two additional females was divided into nine fragments; one fragment was immediately destined for viability analysis (Trypan blue test) and the other fragments were stored as previously described, until 24h and then analyzed. After 4h storage in ACP(r) medium, the follicular integrity was similar to control (87.8% vs 94.4%, respectively); however, ultrastructural analyses revealed swollen mitochondria as the first signals of PF degeneration. It was observed that ACP(r) (66.7%) was more efficient than PBS (49.4%) to preserve the morphological integrity after 36h storage (P<0.05); however, no differences were observed on follicular viability (P>0.05). In conclusion, the use of the ACP(r) is recommended for the short-term preservation of Pecari tajacu preantral follicles...

Compararam-se protocolos para a preservação por curtos períodos de folículos ovarianos pré-antrais (PFs) de catetos, utilizando meios à base de solução salina tamponada (PBS) ou água de coco em pó (ACP(r)). Para a análise morfológica, cada par de ovários coletados de seis fêmeas foi dividido em nove fragmentos. Um fragmento foi destinado para a análise da morfologia (histologia e microscopia eletrônica de transmissão - MET), constituindo o grupo controle, e os demais fragmentos foram colocados em tubos contendo PBS ou ACP(r), acondicionados em caixas térmicas de poliestireno expandido de 5L, armazenados durante quatro, 12, 24 e 36 horas, e, então, analisados. Para a análise da viabilidade, pares de ovários de duas fêmeas adicionais foram divididos em nove fragmentos; um deles foi imediatamente destinado à análise da viabilidade (teste com azul de Trypan), os outros fragmentos foram armazenados como descrito previamente até 24h e, então, foram analisados. Após quatro horas de armazenamento em meio ACP(r), a integridade folicular foi similar ao grupo controle (87,8% vs. 94,4%, respectivamente); contudo, a análise ultraestrutural revelou mitocôndrias edemaciadas como os primeiros sinais de degeneração dos PFs. Foi observado que o ACP(r) (66,7%) foi mais eficiente do que o PBS (49.4%) em preservar a integridade morfológica após 36h (p<0,05); entretanto, nenhuma diferença foi observada para a viabilidade folicular (P>0,05). Em conclusão, o uso da ACP(r) é recomendado para a preservação por curtos períodos de folículos pré-antrais de Pecari tajacu...

Effect of morphological integrity, period, and type of culture system on the in vitro development of isolated caprine preantral follicles.

The aims of this study were the following: (1) to define an optimal period for the IVC of isolated caprine preantral follicles, (2) to verify the relationship between follicular morphology (intact, extruded, and degenerate follicles) and estradiol production, and (3) to evaluate the effects of the bidimensional (2D) and three-dimensional (3D) culture systems on the in vitro development of caprine preantral follicles. Three experiments were performed. In experiments 1 and 2, the isolated secondary follicles were cultured for 18, 24, and 30 days or 30, 36, and 42 days, respectively. In experiment 3, the optimal culture period from experiment 2 was used for 2D and 3D culture systems. After culture, the oocytes were submitted to IVM. The morphological integrity, antral cavity formation rates, follicular diameter, presence of healthy, grown oocytes (≥110 µm), rates of resumption of meiosis, and estradiol concentrations were evaluated. In experiment 1, the percentage of oocytes that resumed meiosis was higher in oocytes cultured for 30 days (48.84%) than in oocytes cultured for 18 and 24 days (15% and 20.93%, respectively). In experiment 2, the percentage of oocytes that resumed meiosis was significantly higher in oocytes cultured for 30 and 36 days (47.5% and 50%, respectively) than in oocytes cultured for 42 days (20%). The estradiol concentrations on Day 12 of culture were similar for normal and extruded follicles and higher than those observed in degenerate follicles at the end of the culture period. In conclusion, the 36-day culture period resulted in the highest rates of meiosis resumption. In addition, because the loss of follicular integrity affects the patterns of estradiol production, follicular integrity is a good predictor of follicular quality.

Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture.

The aim of this study was to compare the efficiency of different media for the in vitro culturing of fresh and vitrified bovine ovarian tissues. Fragments of the ovarian cortex were subjected to vitrification and histological and viability analyses or were immediately cultured in vitro using the alfa minimum essential medium, McCoy's 5A medium (McCoy), or medium 199 (M199). Samples of different culture media were collected on days 1 (D1) and 5 (D5) for quantification of reactive oxygen species and for hormonal assays. In non-vitrified (i.e., fresh) ovarian tissue cultures, the percentage of morphologically normal follicles was significantly greater than that recorded for the other media (e.g., M199). In the case of previously vitrified tissues, the McCoy medium was significantly superior to the other media in preserving follicular morphology up until the last culture day (i.e., D5), thus maintaining a similar percentage from D1 to D5. Reactive oxygen species levels were higher in D1 vitrified cultured tissues, but there were no differences in the levels among the three media after 5 days. The hormonal assays showed that in the case of previously vitrified tissues, at D5, progesterone levels increased on culture in the M199 medium and estradiol levels increased on culture in the McCoy medium. In conclusion, our results indicate that the use of M199 would be recommended for fresh tissue cultures and of McCoy for vitrified tissue cultures.

Vascular endothelial growth factor-A(165) (VEGF-A(165)) stimulates the in vitro development and oocyte competence of goat preantral follicles.

The aim of this study was to evaluate the effect of vascular endothelial growth factor-A(165) (VEGF-A(165)) on the in vitro development of goat secondary preantral follicles. Preantral follicles (≥150 µm in diameter) were isolated from the ovaries of adult mixed-breed goats and individually cultured for 18 days in αMEM in the absence (control) or presence of VEGF-A(165) at concentrations of 10 ng/ml (VEGF10) and 100 ng/ml (VEGF100). Analyses of follicular survival, diameter, antrum formation and rate of daily growth were performed every 6 days. At the end of the culture period, morphologically normal oocytes (≥110 µm in diameter) were taken for in vitro maturation (IVM). The results demonstrated that all follicles presented oocytes and granulosa cells that were morphologically normal and after labeling with calcein-AM, high rates of oocyte viability were observed in all treatments. The follicular diameter and the growth rate achieved in the presence of VEGF10 were higher than those of the control. Both treatments with VEGF-A(165) showed higher rates of oocyte recovery for IVM when compared with the control. Moreover, only the addition of VEGF-A(165) permitted oocytes grown in vitro to reach metaphase II. Thus, the addition of VEGF-A(165) to the culture medium improves the development of goat preantral follicles cultured in vitro, allowing the production of mature oocytes.

Presence of c-kit mRNA in goat ovaries and improvement of in vitro preantral follicle survival and development with kit ligand.

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.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.

Conservação de folículos pré-antrais bovinos em solução salina 0,9 por cento ou TCM 199 / Preservation of bovine preantral follicles in 0.9 percent saline solution or TCM 199

Investigou-se a eficiência da solução salina 0,9 por cento (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<0,05) a porcentagem de FOPA degenerados comparada à porcentagem de folículos-controle. Em ambas as soluções, independente do tempo de incubação, a porcentagem de folículos normais, após conservação a 39ºC, foi (P<0,05) menor que a obtida com 4 e 20ºC. FOPA bovinos podem ser conservados eficientemente a 4ºC por até 24h em ambas as soluções, e a 20ºC por 4 e 12h em SS e TCM 199, respectivamente.

The efficiency of 0.9 percent saline solution (SS) and TCM 199 on the preservation of bovine preantral follicles (PF) in situ at different temperatures and incubation times was investigated. Each ovarian pair was divided into 25 fragments. One fragment was taken randomly and immediately fixed (control). The other fragments were distributed in tubes containing SS or TCM 199 at 4, 20 or 39ºC for 2, 4, 12 or 24h. The histological analysis showed that the storage at 4ºC in both solutions kept the percentage of normal follicles similar to control values. Preservation in SS at 20ºC for 12 or 24h, TCM 199 at 20ºC for 24h and in both solutions at 39ºC from 2 h onward (P<0.05) increased the percentage of degenerated follicles when compared with control. In both solutions, independent of incubation time, the percentage of normal follicles observed at 39ºC was (P<0.05) lower them those observed at 4 and 20ºC. Bovine PF can be preserved efficiently at 4ºC for up to 24h in both solutions, at 20ºC for 4 and 12h in SS and TCM 199, respectively.