Resemblance in physical activity levels: The Portuguese sibling study on growth, fitness, lifestyle, and health.

OBJECTIVES: To investigate the relationships of biological, behavioral, familial, and environmental characteristics with siblings´ physical activity (PA) levels as well as the intrapair resemblance in PA. METHODS: The sample comprises 834 (390 females) biological siblings [brother-brother (BB), sister-sister (SS), brother-sister (BS)] aged 9 to 20 years. Total PA index (TPAI) was estimated by questionnaire. Information on potential behavioral, familial, and environmental correlates was obtained by self-report; body mass index (BMI), biological maturation, and physical fitness were measured. Multilevel models were used to analyze siblings´ clustered data, and sibling resemblance was estimated with the intraclass correlation (ρ). RESULTS: On average, younger sibs, those more physically fit, and those with more parental support had greater TPAI. Further, BB pairs had higher TPAI levels than SS or BS pairs, but also had greater within-pair variance. When adjusted for all covariates, SS pairs demonstrated greater resemblance in TPAI (ρ = 0.53, 95%CI = 0.38-0.68) than BS (ρ = 0.26, 95%CI = 0.14-0.43) or BB pairs (ρ = 0.18, 95%CI = 0.06-0.44). CONCLUSIONS: Age, physical fitness, and parental support were the best predictors of TPAI levels. A moderate level of resemblance in TPAI was observed in SS pairs, while lower resemblance was found for BS and BB pairs. These findings may be due to differences in the roles of shared genetic factors, familial, and environmental characteristics across different sibling types.

Correlates of children's compliance with moderate-to-vigorous physical activity recommendations: a multilevel analysis.

This study aimed to investigate the association between individual and school characteristics associated with the number of school days children comply with moderate-to-vigorous physical activity (MVPA) recommendations. Sample comprises 612 Portuguese children, aged 9-11 years, from 23 schools. Time spent in MVPA was measured by accelerometry, while individual-level correlates were obtained by anthropometry and questionnaires. School-level variables were collected by questionnaire, and accelerometer wear time and season were also considered. Maximum likelihood estimates of model parameters were obtained via a multilevel analysis with children as level-1, and school as level-2. Children who spent more time in sedentary activities and girls were less likely to comply with MVPA/daily. More mature children and those who use active transportation to school were more likely to attain the PA recommendation. Furthermore, greater accelerometer wear time and spring season increased the chance to achieve the recommended MVPA. In terms of school-level correlates, a greater number of available facilities was negatively associated with children MVPA compliance. Given the set of variables, our results showed that individual characteristics seem to be more relevant for children's compliance rates with PA/day than school context variables, which should be taken into account in the implementation of school policies and practices.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue.

OBJECTIVE: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.

Immunolocalization of the Anti-Müllerian Hormone (AMH) in Caprine Follicles and the Effects of AMH on In Vitro Culture of Caprine Pre-antral Follicles Enclosed in Ovarian Tissue.

The aims of this study were to evaluate the localization, by immunohistochemistry, of the anti-Müllerian hormone (AMH) in goat ovaries and to investigate its effects on the in vitro survival and development of caprine pre-antral follicles enclosed in fragments of ovarian tissue. Pre-antral follicles were cultured in vitro for 1 or 7 days in α-MEM(+) in the absence or presence of kit ligand (KL; 50 ng/ml, positive control) or AMH (50 or 150 ng/ml). The results showed that AMH was localized in oocytes and granulosa cells from the primordial follicle to antral follicle stages. Addition of AMH maintained the percentage of developing follicles, similar to that in the uncultured control; however, the percentage of developing follicles was significantly lower than that in the cultured control and KL. Nonetheless, addition of AMH to the culture medium did not affect survival rates and follicular growth. In conclusion, this study demonstrated that the expression of AMH varies according to the compartment and stage of follicular development. Furthermore, AMH inhibits the activation of caprine primordial follicles.

Modeling the dynamics of BMI changes during adolescence. The Oporto Growth, Health and Performance Study.

OBJECTIVES: The aims of this study were twofold: (i) to model changes in body mass index (BMI) of 10-18-year-old adolescents, and (ii) to investigate the effects of total physical activity (TPA), physical fitness (PF), sleep duration and fruit/vegetable consumption in BMI trajectories across time. METHODS: Data were obtained from the Oporto Growth, Health and Performance Study and comprised 6894 adolescents (3418 girls) divided into four age cohorts (10, 12, 14 and 16 years) measured annually for 3 years. BMI was computed using the standard formula (kg m(-2)); TPA was estimated with the Baecke questionnaire; PF measures included 1-mile run/walk, 50 yard dash (50YD), standing long jump (SLJ), handgrip strength (HGr) and agility shuttle run. Longitudinal changes in BMI were analyzed using the multilevel modeling approach. RESULTS: The average BMI at age of peak of height velocity was 20.7±0.07 kg m(-2) for girls (P<0.001) and 20.58±0.06 kg m(-2) for boys (P<0.001). The annual increment in BMI was 1.36±0.04 kg m(-2), P<0.001 and 1.23±0.03 kg m(-2), P<0.001 for girls and boys, respectively. PF were related to BMI trajectories in both sexes (Girls: ß1mile=0.12±0.02, P<0.001; ßSLJ=-0.01±0.00, P<0.001; ß50YD=0.28±0.05, P<0.001; ßHGr=-8.91±0.54, P<0.001; Boys: ß1mile=0.18±0.02, P<0.001; ßSLJ=-0.01±0.00, P<0.001; ß50YD=0.26±0.04, P<0.001; and ßHGr=-8.15±0.45, P<0.001). TPA only showed significant, but positive, association with girls' BMI trajectories (ß=0.10±0.03, P=0.001). After adjusting for the covariates, sleep duration and fruit/vegetable intake did not show any significant association with BMI trajectories either sex. CONCLUSIONS: BMI increased linearly with age in both gender. PF levels are negatively associated with BMI across time in both boys and girls. Therefore, promotion of PF in the adolescent years seems to be effective in the early prevention of obesity.

In vitro development of secondary follicles from pre-pubertal and adult goats cultured in two-dimensional or three-dimensional systems.

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

Effect of sequential medium with fibroblast growth factor-10 and follicle stimulating hormone on in vitro development of goat preantral follicles.

A sequential medium with fibroblast growth factor-10 (FGF-10) and follicle stimulating hormone (FSH) was evaluated on the survival, ultrastructure, activation and growth rate of caprine preantral follicles submitted to long-term culture, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with FGF-10 and/or FSH added sequentially on different days of culture. Ovarian fragments were cultured during the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+) (cultured control), FSH/FSH, FSH/FGF-10, FSH/FSH+FGF-10, FGF-10/FGF-10, FGF-10/FSH, FGF-10/FSH+FGF-10, FSH+FGF-10/FSH+FGF-10, FSH+FGF-10/FSH and FSH+FGF-10/FGF-10. Follicle morphology, viability and ultrastructure were analyzed. The FSH/FGF-10 treatment showed a higher (P<0.05) percentage of normal follicles compared to all other treatments. In addition, follicles from the FSH/FGF-10 treatment maintained ultrastructural integrity after the culture period. After 16 days of culture, the FSH/FGF-10 and FSH/FSH treatments showed a higher percentage of activation compared to the cultured control (α-MEM(+)/α-MEM(+)). Moreover, the FSH/FGF-10 treatment promoted greater follicular and oocyte diameters compared to the fresh control. In conclusion, this study showed that a sequential medium with FSH followed by FGF-10 (FSH/FGF-10 and FSH/FSH) maintains follicular viability and ultrastructure and promotes transition from the primordial to primary stage (activation) and growth in goat preantral follicles cultured in vitro.

Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro / Esfingosina 1-fosfato promove ativação de folículos pré-antrais de caprinos in vitro

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability...

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular...

Is the mouse follicle culture a good model for the goat with respect to the development of preantral follicles in vitro?

The present study evaluated the efficiency of using 2 culture media developed for mice and for goats in the in vitro preantral follicle culture of each species. Murine and caprine secondary follicles were cultured in vitro with human recombinant follicle-stimulating hormone (murine medium) or with bovine recombinant follicle-stimulating hormone in association with growth hormone (caprine medium). The results showed that murine follicles cultured in caprine medium had lower (P < 0.05) rates of follicular survival and growth, whereas for caprine follicles, these variables were not affected by the type of medium used (P > 0.05). After in vitro maturation, a higher (P < 0.05) number of oocytes that resumed meiosis were observed in the murine medium for both species. In contrast, only in the caprine species estradiol production was significantly superior when the caprine medium was used. Higher progesterone production was observed in the presence of the murine medium only for murine follicles (P < 0.05). In conclusion, murine and caprine preantral follicles cultured under the same in vitro culture medium conditions respond differently; caprine oocytes grown in vitro in the presence of the murine medium show the greatest developmental competence among the tested combinations. Therefore, under the present experimental conditions, the mouse follicle culture has proved be a good model for the development of new culture media for caprine preantral follicles.

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.

Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro / Fator de crescimento transformante-β (TGF-β) mantém a ultraestrutura folicular e estimula o crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano cultivado in vitro

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture...

O objetivo desse estudo foi investigar se o TGF-β afeta a sobrevivência, ativação e crescimento de folículos primordiais caprinos inclusos no córtex ovariano após o cultivo in vitro. Ovários de cabras foram coletados em abatedouro e fragmentos de tecido ovariano foram cultivados por um e sete dias em meio essencial mínimo alfa (α-MEM+) sozinho ou suplementado com TGF-β (1, 5, 10 ou 50ng/mL). Fragmentos ovarianos não cultivados e cultivados foram processados para análise histológica e ultraestrutural. Os resultados mostraram que, comparado ao controle fresco, houve diminuição no percentual de folículos morfologicamente normais em todos os tratamentos somente após sete dias de cultivo. O TGF-β não afetou a ativação folicular independente da concentração testada, contudo, o diâmetro folicular foi superior (P<0.05) no tratamento com 10ng/mL de TGF-β quando comparado ao controle fresco e aos demais tratamentos. Além disso, essa mesma concentração manteve a ultraestrutura normal dos folículos após sete dias de cultivo. Em conclusão, o TGF-β apresentou efeito adicional no crescimento folicular e na manutenção da integridade ultraestrutural de folículos pré-antrais caprinos inclusos no tecido ovariano quando utilizado na concentração de 10ng/mL durante sete dias de cultivo...

Role of nerve growth factor (NGF) and its receptors in folliculogenesis.

Summary Nerve growth factor (NGF) is a prototype member of the neurotrophins family and has important functions in the maintenance of viability and proliferation of neuronal and non-neuronal cells, such as certain ovarian cells. The present review highlights the role of NGF and its receptors on ovarian follicle development. NGF initiates its multiple actions through binding to two classes of receptors: the high affinity receptor tyrosine kinase A (TrkA) and the low-affinity receptor p75. Different intracytoplasmic signalling pathways may be activated through binding to NGF due to variation in the receptors. The TrkA receptor activates predominantly phosphatidylinositol-3-kinase (PI3K) and mitogenic activated protein kinase (MAPK) to promote cell survival and proliferation. The activation of the phospholipase type Cγ (PLCγ) pathway, which results in the production of diacylglycerol (DAG) and inositol triphosphate (IP3), culminates in the release of calcium from the intracytoplasmic cellular stocks. However, the details of activation through p75 receptor are less well known. Expression of NGF and its receptors is localized in ovarian cells (oocyte, granulosa, theca and interstitial cells) from several species, which suggests that NGF and its receptors may regulate some ovarian functions such as follicular survival or development. Thus, the use of NGF in culture medium for ovarian follicles may be of critical importance for researchers who want to promote follicular development in vitro in the future.

High insulin concentrations promote the in vitro growth and viability of canine preantral follicles.

To determine whether the effects of different concentrations of insulin on the development of canine preantral follicles in vitro were associated or not with FSH, secondary follicles were isolated and cultured. In Experiment 1, follicles were cultured in the following media: modified minimum essential medium (CtrlMEM) alone; CtrlMEM plus 5 ng mL⁻¹ insulin (Ins5ng); CtrlMEM plus 10 ng mL⁻¹ insulin (Ins10ng); and CtrlMEM plus 10 µg mL⁻¹ insulin. In Experiment 2, follicles were cultured in the same media but in the presence of sequential FSH (i.e. CtrlFSH, Ins5ngF, Ins10ngF and 10µgF, respectively). Increasing concentrations of FSH (100, 500 and 1000 ng mL⁻¹) were added sequentially to the culture medium on Days 0, 6 and 12 of culture. Viability were assessed at the end of culture and follicular diameter and the antrum formation rate at four time points (Days 0, 6, 12 and 18). In Experiment 1, the high insulin concentration significantly increased follicular viability (P<0.05). In contrast, in Experiment 2, viability was not affected by the inclusion of insulin. In addition, viability was significantly better in follicles cultured in CtrlFSH (P<0.05). The diameter of follicles in the high-insulin group in Experiment 1 and high-insulin plus FSH group in Experiment 2 was superior to other groups tested. In experiment 2, the Ins10µg and Ins10µgF groups exhibited significantly higher antrum formation rates than the other groups. In conclusion, in the absence of FSH, high concentrations of insulin have beneficial effects on follicular viability. However, to promote the growth of canine preantral follicles in vitro, it is recommended that a combination of insulin and FSH be added to the medium.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture.

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Eight-cell parthenotes originated from in vitro grown sheep 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 follicles >110 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.

Bovine dominant follicular fluid promotes the in vitro development of goat preantral follicles.

The aim of this study was to evaluate the effect of follicular fluid collected from bovine dominant follicles (bFF) on the in vitro development of goat preantral follicles and determine the best time to add this supplement to the culture medium. The preantral follicles were isolated and randomly distributed into four treatments in absence (control) or presence of 10% of bFF added on Days 0 (FF0-18), 6 (FF6-18) or 12 (FF12-18) of culture onwards. After 18 days, follicular development was assessed based on follicular survival, antral cavity formation, increased follicular diameter as well as fully grown oocyte (>110 µm) viability and meiosis resumption. The oocytes from the cultured follicles were in vitro-matured and processed for fluorescence or ultrastructural analysis. The results showed that on Day 18 the treatment FF0-18 had a significantly higher (P<0.05) survival than control and FF12-18, but not FF6-18. The addition of bFF at the beginning of culture (FF0-18 and FF6-18) promoted a high percentage of follicular growth, meiosis resumption and early antrum formation. Moreover, this study described for the first time the ultrastructural analysis of caprine oocytes grown in vitro. This evaluation revealed that in the presence of bFF on (FF0-18) the in vitro-grown oocytes presented normal organelle distribution and well-defined, intact plasma and nuclear membranes. In conclusion, bFF originating from dominant follicles maintain the survival and promote the in vitro growth of goat preantral follicles when added at the beginning of culture.

The effect of LIF in the absence or presence of FSH on the in vitro development of isolated caprine preantral follicles.

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.

How the concentration of insulin affects the development of preantral follicles in goats.

This study investigated the effect of adding different insulin concentrations to the culture medium for goat preantral follicle development in vitro. The ovarian fragments were immediately fixed or cultured for 7 days in MEM with insulin (0, 5, 10 ng/ml and 5 or 10 µg/ml). The results showed that, after 7 days of culture, insulin at 10 ng/ml was the best concentration to preserve follicular viability and ultrastructure, resulting in the highest rates of normal follicles. After 7 days, only treatments with 10 ng/ml and 5 µg/ml of insulin increased follicular activation when compared to other concentrations. Regarding follicular and oocyte growth, the presence of 10 ng/ml of insulin promoted a larger diameter than other treatments. In conclusion, this study shows that addition of 10 ng/ml of insulin to the culture medium improved the survival and stimulated growth of goat preantral follicles.

Steady-state level of bone morphogenetic protein-15 in goat ovaries and its influence on in vitro development and survival of preantral follicles.

This study investigates steady-state level of bone morphogenetic protein-15 (BMP-15) mRNA in caprine follicles, and the effects of BMP-15 on in vitro development of preantral follicles. Ovarian fragments were cultured for one or seven days in Minimal Essential Medium (MEM(+)) with BMP-15 (0, 1, 10, 50, 100 or 200 ng/mL), and further analyzed by histology, transmission electron and fluorescent microscopy. BMP-15 mRNA in secondary follicles was higher than in primordial and primary follicles. After seven days, 10, 50 or 100 ng/mL of BMP-15 maintained the percentage of normal follicles similar to the control (non-cultured), and increased the oocyte and follicle diameters when compared to the control and MEM(+). BMP-15 at 100 ng/mL increased the secondary follicles and maintained their ultrastructural integrity. In conclusion, the BMP-15 mRNAs were detected in all follicular categories. BMP-15 (100 ng/mL) maintained the integrity and promoted the growth of caprine preantral follicles cultured for seven days.

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles.

This 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.