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1.
Reprod Sci ; 27(8): 1602-1608, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32436196

RESUMO

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher (P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased (P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

2.
Anim Reprod Sci ; 215: 106310, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32216933

RESUMO

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

3.
Reprod Domest Anim ; 55(1): 105-109, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31661715

RESUMO

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

4.
Reprod Sci ; : 1933719119831783, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808260

RESUMO

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher ( P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased ( P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

5.
Anim Reprod Sci ; 196: 120-129, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30049427

RESUMO

The present study aimed to investigate a concentration-response curve of human recombinant FSH (hrFSH) for in vitro culture of isolated preantral and early antral follicles of goats. Isolated follicles were cultured for 18 days using the following treatments: basic culture medium (control); or control medium supplemented with 10, 50, and 100 mIU/mL of hrFSH. At the end of the culture, cumulus-oocyte complexes were recovered and subjected to in vitro maturation. The following endpoints were evaluated: follicle morphology, growth rate and antrum formation, oocyte viability and meiotic stage, and estradiol production, as well as relative expression of FSH receptor (FSHR), and steroidogenic enzyme (3ß-HSD, CYP17, and CYP19A1) genes. In antral follicles, the FSH addition at 50 mIU/mL increased follicular diameter and growth rate, percentage of fully developed oocytes, and oocyte diameter (P < 0.05), and tended to increase the percentage of MII oocytes when compared to the control (P = 0.07). With preantral follicles, FSH addition at 100 mIU/mL increased relative abundance of mRNA for CYP19A1 when compared to the control (P < 0.05). At the same FSH concentrations of 100 and 50 mIU/mL, there was a greater relatively abundance of mRNA for 3ß-HSD and CYP17 in preantral than in antral follicles (P < 0.05). For preantral and antral follicle comparisons when the same treatments were imposed, there were greater concentrations of estradiol for antral follicles (P < 0.05). In conclusion, hrFSH enhanced in a concentration-dependent manner the in vitro development of caprine antral follicles; however, there was no positive effect in the culture of preantral follicles.


Assuntos
Hormônio Foliculoestimulante Humano/farmacologia , Cabras , Folículo Ovariano/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante , Humanos , Oócitos , Folículo Ovariano/fisiologia
6.
PLoS One ; 13(6): e0198108, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29897931

RESUMO

Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) and region (dorsal and ventral), (iii) distance from the geometric center, and (iv) follicular class. Ovaries from young and old mares (n = 8) were harvested in a slaughterhouse and submitted to histological processing for further evaluation. For data analyses, a novel methodology was developed according to the geometric center of each histological section for a precise determination of preantral follicle distribution. Results indicated that (i) equine preantral follicles are clustered and located near to the ovarian geometric center, and that aging induced their dispersion through the ovarian cortex; (ii) the distance from the geometric center was shorter for developing follicles than primordial; and (iii) secondary follicles were more distant from the geometric center but closer to the ovulation fossa. In conclusion, the spatial distribution of preantral follicles was successfully determined in the equine ovary and was affected by age, region, and portion.


Assuntos
Cavalos , Folículo Ovariano/citologia , Reserva Ovariana/fisiologia , Ovário/citologia , Fatores Etários , Animais , Contagem de Células , Feminino , Técnicas Histológicas , Cavalos/fisiologia , Ovulação/fisiologia
7.
Biopreserv Biobank ; 16(4): 258-269, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29957024

RESUMO

AIM: The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. METHODS: This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 µM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. RESULTS: The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 µM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 µM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). CONCLUSIONS: Although 100 µM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.


Assuntos
Catalase/farmacologia , Criopreservação/métodos , Ovário/citologia , Ovário/metabolismo , Ácido Tióctico/farmacologia , Vitrificação , Animais , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ovinos
8.
Theriogenology ; 107: 219-225, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29179058

RESUMO

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50-62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm2. The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 µm2, and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density.


Assuntos
Cabras/cirurgia , Laparoscopia/veterinária , Ovário/patologia , Coleta de Tecidos e Órgãos/veterinária , Animais , Biópsia , Feminino , Laparoscopia/métodos , Coleta de Tecidos e Órgãos/métodos
9.
Reprod Fertil Dev ; 30(2): 359-370, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28768567

RESUMO

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.


Assuntos
Criopreservação/veterinária , Cabras/embriologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Folículo Ovariano/fisiologia , Ovário/citologia , Animais , Antígenos Nucleares/metabolismo , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Fármacos para a Fertilidade Feminina/farmacologia , Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/farmacologia
10.
Theriogenology ; 86(4): 924-933, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27087533

RESUMO

Improvements in the estimation of male fertility indicators require advances in laboratory tests for sperm assessment. The aims of the present work were (1) to apply a multivariate analysis to examine sperm set of alterations and interactions and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development. Bulls (n = 3) were subjected to scrotal insulation, and ejaculates were collected before (preinsulation = Day 0) and through 56 days (Days 7, 14, 21, 28, 35, 42, 49, and 56) of the experimental period. Sperm head morphometry and chromatin variables were assessed by a computational image analysis, and IVF was performed. Scrotal heat stress induced alterations in all evaluated sperm head features, as well as cleavage and blastocyst rates. A principal component analysis revealed three main components (factors) that represented almost 89% of the cumulative variance. In addition, an association of factor scores with cleavage (factor 1) and blastocyst (factor 3) rates was observed. In conclusion, several sperm traits were simultaneously altered as a result of a thermal insult. These sperm traits likely play specific roles in IVF and embryonic development.


Assuntos
Bovinos/fisiologia , Desenvolvimento Embrionário/fisiologia , Fertilização In Vitro/veterinária , Escroto/fisiologia , Espermatozoides/fisiologia , Animais , Criopreservação/veterinária , Temperatura Alta , Masculino , Preservação do Sêmen/veterinária
11.
PLoS One ; 11(2): e0149693, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26900687

RESUMO

Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles.


Assuntos
Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Ovário/citologia , Ovário/fisiologia , Animais , Biópsia , Contagem de Células , Feminino , Cavalos , Células Estromais/citologia
12.
Anim Reprod Sci ; 146(3-4): 117-25, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24674823

RESUMO

This study was designed to evaluate the influence of heat stress (HS) on the metabolic profile of serum and follicular fluid (FF), ovarian follicle development, and oocyte quality of Girolando dairy cows. Oocytes, blood, and FF (follicles ≥9mm) samples were obtained at 30, 45, 60, 75, and 90 days postpartum in the summer and winter seasons. During transvaginal follicular aspiration, rectal temperature (RT), body condition score (BCS), number of ovarian follicles, and quality of oocytes were recorded. The ambient air temperature (AT) and relative humidity (RH) were also recorded to calculate the temperature humidity index (THI). Glucose, total cholesterol (TC), triglycerides (TG), urea, sodium (Na), potassium (K), and calcium (Ca) concentrations were determined using serum and FF samples. The RT, THI, and BCS loss were greater (P<0.01) in the summer; however, glucose, Na, and K serum concentrations decreased in the same season (P<0.05). Degenerated oocytes were positively associated (P<0.05) with THI (r=0.14) and AT (r=0.13), and negatively associated with glucose (r=-0.12) and K (r=-0.11) serum concentrations. HS induces metabolic changes, which compromise the number of ovarian follicles and the follicular environment, thus resulting in morphologically damaged oocytes.


Assuntos
Bovinos/fisiologia , Líquido Folicular/química , Oócitos/fisiologia , Ovário/fisiologia , Período Pós-Parto/fisiologia , Animais , Indústria de Laticínios , Feminino , Temperatura Alta/efeitos adversos , Lactação/fisiologia , Gravidez , Estações do Ano , Estresse Fisiológico , Fatores de Tempo
13.
Reprod Fertil Dev ; 26(6): 866-74, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23815864

RESUMO

This study was designed to monitor the biochemical profiles of serum and follicular fluid (FF) of postpartum dairy cows during the summer (n=30) and winter (n=30). Blood and FF (follicles ≥ 9 mm) were obtained from Girolando cows at 30, 45, 60, 75 and 90 days postpartum. The samples were collected and analysed to determine glucose, total cholesterol (TC), triglyceride (TG), urea, sodium (Na), potassium (K) and calcium (Ca) levels. Throughout the study, the following clinical variables were measured: rectal temperature (RT), respiratory rate (RR) and body condition score (BCS). In addition, the temperature humidity index (THI) was calculated for each season. During the summer season, THI was higher, BCS decreased, there was an increase in RT, and glucose, urea, Na and K serum levels were decreased (P<0.05). The levels of TC, TG, urea, K and Ca in follicular fluid increased (P<0.05). Positive correlations (P<0.05) were observed between the serum and FF levels for glucose (r=0.29), TC (r=0.24) and Ca (r=0.30). Therefore, the biochemical profile of serum and FF of dairy cows under summer heat-stress conditions demonstrates marked changes that may impair fertility during lactation.


Assuntos
Bovinos , Líquido Folicular/metabolismo , Metaboloma , Período Pós-Parto/metabolismo , Estações do Ano , Soro/metabolismo , Animais , Glicemia/metabolismo , Bovinos/metabolismo , Indústria de Laticínios , Feminino , Líquido Folicular/química , Resposta ao Choque Térmico , Lipídeos/sangue , Período Pós-Parto/sangue , Soro/química
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