In vitro embryo production from early antral follicles of goats fed with a whole full-fat linseed based diet.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300-400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus-oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

Metabolic stress and reproductive features in post-partum goats supplemented for a long period with detoxified castor meal as the source of dietary nitrogen / Estresse metabólico e características reprodutivas em cabras pós-parto suplementadas por longo período com mamona desintoxicada como fonte de nitrogênio na dieta

This study evaluated the effect of detoxified castor meal on the reproductive performance, metabolic stress, milk production, and kid development in peripartum goats. The diet of the animals were with (DCM, n= 20) or without (WDCM, n= 21) detoxified castor meal during the entire gestation and until weaning, 60 days post-birth. No differences were observed in the gestation period, litter size, rate of multiple births, and mortality between the two groups. The postpartum plasma concentrations of progesterone remained below 1ng/mL in all animals, thus, confirming the absence of active corpora lutea. The thickness of sternum adipose tissue and loin area, levels of urea and cholesterol, milk production, and daily weight gain in the kids were low in the DCM group when compared to those in the WDCM group (P< 0.05). To conclude, the use of detoxified castor meal in peripartum goats resulted in lower level of performance in the kids because of reductions in the amount of milk received from their mothers during lactation. In addition, the diet containing detoxified castor meals was not efficient in recovering from the loss of stored body reserves able to initiate the recovery of the cyclic activity of the goats.(AU)

Este estudo avaliou o efeito da torta de mamona desintoxicada na reprodução, no estresse metabólico, na produção de leite e no desenvolvimento de cabritos no periparto de cabras. Um grupo foi alimentado com torta de mamona (DCM, n=20), e o outro (WDCM, n=21) não recebeu tal suplemento , durante a gestação até o desmame, 60 dias pós-parto. Não foram observadas diferenças significativas no período de gestação, no número de cabritos, na taxa de partos múltiplos e na mortalidade entre os dois grupos. Em todos os animais, a concentração plasmática de progesterona ficou abaixo de 1ng/mL, confirmando a ausência de atividade lútea. A espessura da gordura subcutânea do esterno e da área de olho-de-lombo, a concentração de ureia e colesterol, a produção de leite e o ganho de peso dos cabritos foram menores no grupo DCM (P<0,05). Conclui-se que o uso de torta de mamona desintoxicada no periparto de cabra resultou em cabritos mais leves devido à redução na produção de leite das matrizes e as cabras não retornaram ao cio, pois não recuperaram a massa corporal.(AU)

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats.

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.

Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

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.

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.

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...

SANASA Capivari II - the first full-scale municipal membrane bioreactor in Latin America.

The macro region of Campinas (Brazil) is rapidly evolving with new housing developments and industries, creating the challenge of finding new ways to treat wastewater to a quality that can be reused in order to overcome water scarcity problems. To address this challenge, SANASA (a publicly owned water and wastewater concessionaire from Campinas) has recently constructed the 'EPAR (Water Reuse Production Plant) Capivari II' using the GE ZeeWeed 500D(®) ultrafiltration membrane system. This is the first large-scale membrane bioreactor (MBR) system in Latin America with biological tertiary treatment capability (nitrogen and phosphorus removal), being able to treat an average flow of 182 L/s in its first phase of construction. The filtration system is composed of three membrane trains with more than 36,000 m(2) of total membrane filtration area. The membrane bioreactor (MBR) plant was commissioned in April 2012 and the permeate quality has exceeded expectations. Chemical oxygen demand (COD) removal rates are around and above 97% on a consistent basis, with biochemical oxygen demand (BOD5) and NH3 (ammonia) concentrations at very low levels, and turbidity lower than 0.3 nephelometric turbidity unit (NTU). Treated effluent is sent to a water reuse accumulation tank (from where will be distributed as reuse water), and the excess is discharged into the Capivari River.

Alginate hydrogel matrix stiffness influences the in vitro development of caprine preantral follicles.

This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption.

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...

The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles.

The effects of varying concentrations of EGF were evaluated in terms of in vitro follicular development and the mRNA expression levels of EGF, EGF-R, FSH-R and P450 aromatase. After 6 days, the addition of 50 ng/mL of EGF to the culture medium increased the antrum formation rates in comparison to cultured control and after 18 days of culture produced oocytes with higher rates of meiosis resumption when compared to the other treatments (P<0.05). The daily follicular growth rates in presence of EGF (50 or 100) were increased in comparison to the cultured control (P<0.05). Treatment with EGF 50 stimulated the expression of EGF mRNA but reduced EGF-R mRNA expression and estradiol secretion as compared to the cultured control (P<0.05). After 18 days of culture, the mRNA levels for FSH-R and P450 aromatase were greater than those of the non-cultured controls (P<0.05). In conclusion, the effects of EGF treatment on the mRNA levels for EGF, EGF-R, FSH-R, and P450 aromatase varied according to the stage of follicle development.

A simple model to assess nitrogen and phosphorus contamination in ungauged surface drainage networks: application to the Massaciuccoli Lake Catchment, Italy.

Modeling is a common practice to evaluate factors affecting water quality in environmental systems impaired by point and nonpoint losses of N and P. Nevertheless, in situations with inadequate information, such as ungauged basins, a balance between model complexity and data availability is necessary. In this paper, we applied a simplified analytical model to an artificially drained floodplain in central-western Italy to evaluate the importance of different nutrient sources and in-stream retention processes and to identify critical source areas. We first considered only a set of chemical concentrations in water measured from February through May 2008 and from November 2008 through February 2009. We then broadened available data to include water discharge and hydraulic-head measurements to construct a hydrogeological model using MODFLOW-2000 and to evaluate the reliability of the simplified method. The simplified model provided acceptable estimates of discharge (ranging from 0.03-0.75 m s) and diffuse nutrient inputs from water table discharge and in-stream retention phenomena. Estimates of PO-P and total P retention (ranging from 1.0 to 0.6 µg m s and from 1.18 to 0.95 µg m s for PO-P and total P, respectively) were consistent with the range of variability in literature data. In contrast, the higher temporal variability of nitrate concentrations decreased model accuracy, suggesting the need for more intensive monitoring. The model also separated the dynamics of different reaches of the drainage network and identified zones considered critical source areas and buffer zones where pollutant transport is reduced.

Dynamic medium containing kit ligand and follicle-stimulating hormone promotes follicular survival, activation, and growth during long-term in vitro culture of caprine preantral follicles.

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.

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue.

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.

Expression of keratinocyte growth factor in goat ovaries and its effects on preantral follicles within cultured ovarian cortex.

The aims of this study were to evaluate the expression of keratinocyte growth factor (KGF) in goat ovaries and to study its effects on preantral follicle survival and development. The ovaries were used for immunohistochemistry or for in vitro culture for 1 or 7 days with KGF (0, 1, 10, 50, 100, 150, or 200 ng/mL). Noncultured (fresh control) and cultured ovarian slices were processed for histological analysis and transmission electron microscopy (TEM). The results showed that after 7 days of in vitro culture, all treatments had a significant reduction in the percentage of normal follicles compared with the fresh control. After 7 days of culture, the highest KGF concentrations (150 and 200 ng/mL) induced a significant reduction in the percentage of normal follicles compared with the tissues cultured in the absence (α-MEM(+) alone) or presence of 1, 10, and 50 ng/mL KGF. Transmission electron microscopy confirmed follicular integrity after 7 days of culture in 1 ng/mL KGF. In addition, compared with the fresh control, the percentage of growing follicles was significantly increased in all treatments after 1 or 7 days of culture. Immunohistochemical analyses showed the expression of KGF in oocytes and granulosa cells in all follicle developmental stages as well as in thecal and stromal cells. In conclusion, this study demonstrated that, at the lowest concentration (1 ng/mL), KGF maintained the ultrastructure of goat preantral follicles cultured in vitro for up to 7 days. Furthermore, the KGF protein was widely distributed in goat ovaries, especially in ovarian 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.

Moment of addition of LH to the culture medium improves in vitro survival and development of secondary goat pre-antral follicles.

The present study investigated the effects of time of addition of luteinizing hormone (LH) to culture medium on the in vitro development of caprine pre-antral follicles. Pre-antral follicles (≥ 150 µm) were isolated from fragments of the goat ovarian cortex and individually cultured for 18 days in the absence (control) or presence of 100 ng/ml LH, added on days 0, 6 or 12 of culture. Follicular development was assessed based on antral cavity formation, increased follicular diameter as well as follicular and fully grown oocyte (>110 µm) viability. The results showed that after 18 days of culture, the percentage of surviving follicles in the control treatment was significantly lower when compared to other treatments (p < 0.05). There were no significant differences in antrum formation, follicular diameter and oocyte viability. The addition of LH at D6 of culture significantly increased the rates of oocytes ≥ 110 µm and the resumption of meiosis (p < 0.05). In contrast, when LH was added at the onset of culture, only germinal vesicle oocytes were obtained. In conclusion, the moment of addition of LH to the culture medium affects the performance of in vitro culture of caprine pre-antral follicles. The addition of LH to the medium from day 6 of culture onward improved the rates of follicular survival, as well as the ability of oocytes to resume meiosis. However, prolonged exposure to LH (addition at the onset of culture onward) showed detrimental effects for the meiotic resumption.

Dynamic medium produces caprine embryo from preantral follicles grown in vitro.

The aim of this study was to develop a dynamic culture medium containing FSH, LH and EGF to promote the in vitro development of oocytes obtained from goat preantral follicles to complete maturation and to improve the capacity of these oocytes for in vitro fertilization (IVF) and embryo production. For experiment I, preantral follicles were cultured for 18 days in medium supplemented with increasing concentrations of FSH (T1 - control) or in control medium added LH alone or in association with EGF: T2 (LH 50 ng/ml), T3 (LH 50 ng/ml + EGF 50 ng/ml), T4 (LH 50 ng/ml + EGF 100 ng/ml), T5 (LH 100 ng/ml), T6 (LH 100 ng/ml + EGF 50 ng/ml) and T7 (LH 100 ng/ml + EGF 100 ng/ml). For experiment II, preantral follicles were cultured only in the culture medium used in T7, and after 18 days, their oocytes underwent in vitro maturation (IVM) followed by IVF. At the end of the culture period, T3, T4 and T7 had a positive influence on the daily follicular growth rate. Oocytes grown in T4 and T7 had a meiosis resumption percentage significantly superior to the other treatments. Two embryos were obtained, in which preantral follicles in medium supplemented with 100 ng/ml LH and 100 ng/ml EGF (T7). In conclusion, our sequential culture system was able to promote the in vitro growth of preantral follicles, promoting their oocyte maturation and caprine embryo production from preantral follicles.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

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 percent), and the highest percent of primary follicles was achieved with IGF-I (57.7 percent). 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.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles.

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.