Effect of carvacrol antioxidant capacity on oocyte maturation and embryo production in cattle.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

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.

Folliculogenesis-related genes are differently expressed in secondary and tertiary ovarian follicles.

The relative mRNA abundance of 10 genes associated with folliculogenesis was compared between late preantral (secondary) and early antral (tertiary) ovarian follicles in goats. In total, 100 follicles in each category were mechanically isolated. The relative transcript abundance of the mRNAs were determined by qPCR. Data were analyzed using unpaired Student's t-test. Of the 10 tested genes, ABLIM mRNA was not detected in either follicle category, six genes (SLIT3, TYMS, GTPBP1, AKR1C4, PIK3R6, and MAOB) were upregulated in secondary follicles compared with tertiary follicles, and three genes (ARHGEF12, CLEC6A, and CYTL1) showed similar mRNA abundances in both secondary and tertiary follicles. In conclusion, SLIT3, GTPBP1, AKR1C4, and PIK3R6 mRNA abundance was upregulated in secondary follicles (preantral phase) compared with in tertiary follicles (antral phase) in goats.

Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro.

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression.

Long-term in vitro culture (16 days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in α-MEM(+) alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P < 0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81 % and 79 % respectively) than the treatments cultured with FSH or α-MEM(+) alone (68 % and 63 %). At day 16 of culture, treatments with FSH and/or IGF-I had more (P < 0.05) viable follicles (69 %) than α-MEM(+) (38 %). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P < 0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P < 0.05) follicular and oocyte diameters and the percentage of secondary follicles (28 %). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P < 0.05) than α-MEM(+). IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.

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.

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.

Expression levels of mRNA-encoding PDGF receptors in goat ovaries and the influence of PDGF on the in vitro development of caprine pre-antral follicles.

The aims of this study were to investigate the expression levels of mRNA for platelet-derived growth factor (PDGF) receptors (PDGFR-α and -ß) in caprine follicles at different developmental stages and to evaluate the influence of PDGF on the in vitro development of pre-antral follicles. For this, goat primordial, primary and secondary follicles, as well as small (1-3 mm) and large (3-6 mm) antral follicles, were obtained, and PDGFR-α and -ß mRNA levels were quantified by real-time PCR. Furthermore, pre-antral follicles (≥ 200 µm) were isolated from goat ovaries and cultured for 18 days in α- minimum essential medium supplemented with PDGF at 50 or 100 ng/ml, containing or not FSH. Real-time PCR showed highest PDGFR-α mRNA levels in secondary follicles, while PDGFR-ß mRNA levels were highest in primary follicles onwards. Both receptors showed higher mRNA levels in granulosa/theca cells from small and large antral follicles than in their corresponding cumulus-oocyte complexes. In culture, the percentage of antrum formation was significantly higher in 100 ng/ml PDGF compared with the same PDGF concentration associated with FSH. After 18 days, PDGF in both concentrations associated with FSH promoted follicular growth significantly higher than the control. Moreover, the addition of FSH to 50 ng/ml PDGF positively influenced the follicular growth when compared with the same PDGF concentration in the absence of FSH. In conclusion, PDGF is important for early goat folliculogenesis, because the presence of PDGFR-α and -ß mRNA was detected in all follicular categories, and PDGF associated with FSH stimulated the growth of goat pre-antral follicles isolated and cultured in vitro.

Role of EGF on in situ culture of equine preantral follicles and metabolomics profile.

The effects of epidermal growth factor (EGF) concentrations (0, 10, 50, and 100ng/ml) on in vitro culture (IVC) of equine preantral follicles were evaluated using histology, estradiol and reactive oxygen species (ROS) production and metabolomics. After IVC, the percentage of normal follicles was lower (P<0.05) for all treatments when compared to non-cultured control. EGF 50ng/ml treatment had more (P<0.05) normal follicles at Day 7 of culture when compared with EGF 0 and 100ng/ml. EGF 50ng/ml had more (P<0.05) developing follicles than the 0ng/ml and 10ng/ml EGF treatments. Follicular and oocyte diameters were greater (P<0.05) with EGF 50ng/ml than the other cultured treatments, but similar (P>0.05) to the non-cultured control. From Day 1 to Day 7 estradiol production increased (P<0.05) in all EGF treatments. EGF 50ng/ml was the only treatment that maintained ROS production through IVC. Metabolomics profiles of the spent media indicated that eleven ions from variable influence in the projection (VIP) scores were higher represented in the EGF 50ng/ml treatment. In conclusion, EGF 50ng/ml treatment maintained follicle survival and ROS production, and promoted activation of cultured equine preantral follicles enclosed in ovarian tissue.

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-µg/mL insulin and 100-µg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 µm.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 µg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 µg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.

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.

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.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production.

OBJECTIVE: Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS: Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS: GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS: Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.

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.

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.

Presence of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles and improvement of in vitro preantral follicle survival and development with GH.

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