Methods to study ovarian function in monovulatory species using the cow as a model

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functionsin vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.

Methods to study ovarian function in monovulatory species using the cow as a model

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functionsin vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.(AU)

Three-dimensional modeling of color Doppler images: a new approach to study follicular vascularization in cattle

Ovarian blood supply is directly related to follicle developmental potential and to oocyte quality, and color Doppler ultrasonography might be a valuable tool to predict in vitro fertilization outcomes. In most studies in large domestic animals, however, the evaluation of follicle blood flow is qualitative (presence or absence of color signal) or dependent on the analysis of a single image. The objective of the present study was to first describe the use of a three-dimensional (3D) modeling of color Doppler images for a quantitative assessment of vascularization in bovine ovarian follicles. Follicular wave emergence was synchronized in Holstein and Gir heifers (n = 20), and follicular dynamics were assessed every 12 h using a color Doppler ultrasound device. The recorded cine-loop of the dominant follicle was decomposed into frames and medical image processing software was used to isolate the Doppler signal, generate the 3D model and calculate the volume of vascularization. In experiment 1, the model was validated by comparing the expected and calculated volumes and was used to predict possible variations in the results of the 2D approach. In experiment 2, vascularization was analyzed during follicular development. In both breeds, the volume of vascularization increased after follicle deviation and was positively correlated (P < 0.05) to follicular diameter (r = 0.65 and 0.54 for Holstein and Gir heifers, respectively). Spatial analysis of the three-dimensional model showed an uneven distribution of vascularization in the follicular wall, with a more intense blood flow being detected in the basal (nearest the ovarian hilus) and lateral regions of the dominant follicles. These results demonstrate the potential of this technique as a new tool for in vivo studies of ovarian physiology in large animals.

Three-dimensional modeling of color Doppler images: a new approach to study follicular vascularization in cattle

Ovarian blood supply is directly related to follicle developmental potential and to oocyte quality, and color Doppler ultrasonography might be a valuable tool to predict in vitro fertilization outcomes. In most studies in large domestic animals, however, the evaluation of follicle blood flow is qualitative (presence or absence of color signal) or dependent on the analysis of a single image. The objective of the present study was to first describe the use of a three-dimensional (3D) modeling of color Doppler images for a quantitative assessment of vascularization in bovine ovarian follicles. Follicular wave emergence was synchronized in Holstein and Gir heifers (n = 20), and follicular dynamics were assessed every 12 h using a color Doppler ultrasound device. The recorded cine-loop of the dominant follicle was decomposed into frames and medical image processing software was used to isolate the Doppler signal, generate the 3D model and calculate the volume of vascularization. In experiment 1, the model was validated by comparing the expected and calculated volumes and was used to predict possible variations in the results of the 2D approach. In experiment 2, vascularization was analyzed during follicular development. In both breeds, the volume of vascularization increased after follicle deviation and was positively correlated (P < 0.05) to follicular diameter (r = 0.65 and 0.54 for Holstein and Gir heifers, respectively). Spatial analysis of the three-dimensional model showed an uneven distribution of vascularization in the follicular wall, with a more intense blood flow being detected in the basal (nearest the ovarian hilus) and lateral regions of the dominant follicles. These results demonstrate the potential of this technique as a new tool for in vivo studies of ovarian physiology in large animals.(AU)

DESENVOLVIMENTO FOLICULAR EM FRAGMENTOS OVARIANOS DE CADELAS (Canis lupus familiares) CULTIVADOS EM MEIOS MEM SUPLEMENTADO COM SORO DE CADELA NO PRÓ ESTRO E CADELA GESTANTE

Avaliou-se o uso de diferentes protocolos de cultivo in vitro de fragmentos ovarianos de cadelas domésticas adultas em anestro, em casuística de programa de controle populacional de animais errantes no município de Viçosa MG. Após a colheita dos ovários, foram obtidos fragmentos da zona parenquimatosa de cada ovário, um dos quais foi incluído fresco como controle, e os demais incubados por 48 horas em atmosfera de 5 % de CO2, 95 % de umidade relativa e temperatura de 39 ºC, em diferentes tratamentos: T1: cultivados em Meio Essencial Mínimo (MEM); T2: cultivados em MEM associado a Soro de Cadela prenhe (MEM + SCpre) e T3: cultivados em MEM associado a Soro de Cadela no pró-estro (MEM + SCpro). Todos os fragmentos foram fixados por imersão em solução de Karnowisky por até 24 horas e processados segundo rotina para confecção de lâminas histológicas. Estas foram avaliadas quanto ao desenvolvimento folicular, sob microscópio de luz, em aumento de 100 vezes. A análise estatística foi feita pelo teste paramétrico de comparação de médias T Student. Todos fragmentos cultivados apresentaram aumento na proporção de folículos em desenvolvimento, assim conclui-se que o meio de cultivo utilizado associado à técnica de cultivo in situ foi suficiente para promover o desenvolvimento folicular em ovários de cadelas, sendo que a suplementação hormonal com SCpro e SCpre não influenciaram na taxa de desenvolvimento.

This present study evaluated different protocols of in vitro culture from ovarian fragments of domestic female dog (Canis lupus familiaris) in anestrus. For the study, ovaries from female dogs were used, obtained from the neutering of the stray animal population control of the municipal kennel program/ Veterinary Department - Universidade Federal de Viçosa (UFV). After the ovaries were collected, random fragments of the ovary parenchymal were obtained, one of them were used fresh as control, and the rest were incubated for 48 hours in 5 % of CO2 atmosphere, 95 % of relative humidity and 39 ºC temperature, in different treatments: T1 cultivated in Minimum Essential Medium (MEM); T2 cultivated in MEM associated with pregnant dog serum and T3 cultivated in MEM associated with proestrus dog serum. Those ovaries cultivated in treatments T1, T2 and T3, were fixed by Karnowisky immersion for up to 24 hours, and processed following the normal laboratorial procedures for making histological slides. They were then evaluated under light microscope, using the 100X objective lens. The statistical analysis was made by the Student T`s parametric comparison measures test. All fragments of assessed ovaries showed a proportional increase of developing follicles, concluding that the culture medium used, associated to the in situ culture technique, was enough to promote the follicle development in dogs ovaries, being that the hormonal supplementation with proestrus serum and pregnant serum did not influence the development rate.

EFEITO DA CONCENTRAÇÃO DE INSULINA E DO FSH NO CULTIVO DE FOLÍCULOS OVARIANOS PRÉ-ANTRAIS CAPRINOS CULTIVADOS IN VITRO EM MEIO CONTENDO HORMÔNIO DO CRESCIMENTO

O objetivo deste estudo foi avaliar o efeito de diferentes concentrações de insulina, sozinha ou associada a concentrações fixa ou crescentes de FSH no cultivo in vitro de folículos pré-antrais caprinos isolados. Folículos secundários foram cultivados individualmente durante 18 dias em meio de base contendo 50 ng/mL de GH suplementado com insulina em baixa concentração (INS-LW: 10 ng/mL) ou alta concentração (INS-HG: 10 g/mL) sozinha ou associada ao FSH em concentrações fixa (FSH100: 100 ng/mL) ou crescentes (FSH-SEQ: 100 ng/mL, dias 0 ao 6; 500 ng/mL, dias 6 ao 12; 1000 ng/mL, dias 12 ao 18 ). Após o cultivo, os seguintes pontos foram avaliados: morfologia e taxas de crescimento folicular, formação de antro, produção de estradiol (E2), expressão do mRNA para os genes para FSHR, GHR, INSR, CYP19A1, CYP17, 3ßHSD, bem como as taxas de viabilidade e de maturação oocitária. O tratamento INS-LW mostrou uma taxa mais elevada (P < 0,05) de folículos normais no dia 18 de cultivo. No entanto, aumento global (P < 0,05) no crescimento folicular, diâmetro oocitário e taxa de retomada da meiose foram obtidos utilizando INS-HG+FSH100. Os tratamentos INS-HG e INS-HG+FSH100 apresentaram maiores níveis de produção de estradiol e dos níveis de mRNA para CYP19A1, CYP17, 3ßHSD quando comparado aos tratamentos INS-LW e INS-LW+FSH100. Em conclusão, na presença de GH, o meio básico suplementado com 10 g/mL de insulina e 100 ng/mL de FSH durante todo o período de cultivo, melhorou o crescimento folicular e oocitário, a retomada da meiose oocitária e a produção de E2 a partir de foliculos pré-antrais caprinos isolados cultivados in vitro.

The aim of this study was to evaluate the effect of different insulin concentrations, alone or associated with either a fixed FSH concentration or increasing FSH concentrations on in vitro culture of isolated goat preantral follicles. Secondary follicles were individually cultured for 18 days in a basic medium containing 50 ng/mL GH supplemented with low insulin concentration (INS-LW: 10 ng/mL) or high insulin concentration (INS-HG: 10 g/mL) alone or with a fixed FSH concentration (FSH100: 100 ng/mL) or with increasing FSH concentrations (FSH-SEQ: 100 ng/mL, days 0 to 6; 500 ng/mL, days 6 to 12; 1000 ng/mL days 12 to 18). After culture the following end points were evaluated: follicle morphology and growth rates, antrum formation, , estradiol (E2) production, gene expression for FSHR, GHR, INSR, CYP19A1, CYP17, 3ßHSD as well as oocyte viability and maturation. INS-LW treatment showed a higher (P < 0.05) incidence of normal follicles at day 18 of culture. However, overall higher (P < 0.05) follicular growth, oocyte diameter and meiotic resumption rates were obtained using INS-HG + FSH 100. INS-HG and INS-HG+FSH100 showed higher E2 production and mRNA levels for CYP19A1, CYP17, 3ßHSD when compared to INS-LW and INS-LW+FSH100. In conclusion, in presence of GH, a basic medium supplemented with 10 g/mL insulin and 100 g/mL FSH throughout the culture period, improved follicular and oocyte growth, oocyte meiotic resumption and E2 production from isolated preantral caprine follicles cultured in vitro.