In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

Local regulation of antral follicle development and ovulation in monovulatory species

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.(AU)

Retrospectiva dos principais biomarcadores envolvidos no desenvolvimento folicular e oocitário em mamíferos / Retrospective of the main biomarkers involved in follicular and oocyte development in mammals

A análise da expressão das diversas proteínas relacionadas a foliculogênese e oogênese in vivo e in vitro, através da proteômica, tem demonstrado possíveis biomarcadores celulares. A descoberta desses biomarcadores pode ajudar na elucidação de mecanismos complexos como a foliculogênese (entendimento sobre o crescimento folicular e maturação oocitária), no tratamento de doenças relacionadas a infertilidade feminina, bem como, no aprimoramento de biotécnicas reprodutivas como a manipulação de oócitos inclusos em folículos pré-antrais. Desta forma, a presente revisão abordou as principais proteínas relatadas nos mais novos trabalhos referentes aos estudos proteômicos visando melhores esclarecimentos referentes ao desenvolvimento folicular e oocitário in vivo e in vitro. A escolha dos artigos foi realizada com base nas descobertas mais atuais e do impacto que determinadas proteínas poderiam trazer para melhorar o entendimento da foliculogênese e oogênese nas diferentes espécies de animais mamíferos.(AU)

The analysis of the expression of several proteins related to folliculogenesis and oogenesis in vivo and in vitro, through proteomics, has demonstrated possible cellular biomarkers. The discovery of these biomarkers may help in the elucidation of complex mechanisms such as folliculogenesis (understanding of follicular growth and oocyte maturation), in the treatment of diseases related to female infertility, as well as in the improvement of reproductive biotechniques such as the manipulation of oocytes enclosed in preantral follicles. Thus, the present review addressed the main proteins reported in the newest works related to proteomic studies aiming at better clarifications regarding follicular and oocyte development in vivo and in vitro. The choice of articles was based on the most current discoveries and the impact that certain proteins could bring to improve the understanding of folliculogenesis and oogenesis in different species of mammalian animals.(AU)

Influência das espécies reativas de oxigênio durante o cultivo in vitro de oócitos e folículos ovarianos de mamíferos domésticos / Influence of reactive oxygen species during in vitro culture of ovarian oocytes and follicles of domestic mammals

Sistemas de cultivo in vitro de folículos pré-antrais e de complexos cumulus-oócitos (CCOs) se tornaram uma poderosa ferramenta capaz de subsidiar diversas biotécnicas reprodutivas, bem como possibilitar estudos do efeito de substâncias sobre a dinâmica folicular. No entanto, esses sistemas podem resultar em estresse oxidativo e na diminuição da proteção antioxidante das células, distúrbio associado a altas taxas de morte celular durante o cultivo in vitro. Para contornar esses efeitos adversos, a adição de substâncias antioxidantes aos meios de cultivo tem sido proposta. Abrangendo diferentes classes e mecanismos de ação, compostos antioxidantes têm por função interferir no processo de oxidação para inibir ou retardar o dano oxidativo causado pelos radicais livres às biomoléculas. Além de antioxidantes que têm sido rotineiramente utilizados com esse propósito, recentemente, substâncias alternativas de origem natural como extratos vegetais e óleos essenciais têm ganhado destaque. Dessa forma, diante da influência do estresse oxidativo e da importância do uso de antioxidantes no cultivo celular, a presente revisão objetiva abordar os principais mecanismos de síntese e atuação dos radicais livres bem como o papel dos antioxidantes em protocolos de cultivo in vitro de folículos ovarianos e de CCOs de animais domésticos.(AU)

In vitro culture systems for preantral follicles and cumulus-oocyte complexes (COCs) have become a powerful tool capable of subsidizing several reproductive biotechniques, as well as enabling studies of the effect of substances on follicular dynamics. However, these systems can favor oxidative stress and decrease the antioxidant protection of cells, a disorder associated with high rates of cell death during in vitro culture. To overcome these adverse effects, the addition of antioxidant substances to the culture media has been proposed. Covering different classes and mechanisms of action, these antioxidant compounds have the function of interfering in the oxidation process to inhibit or delay the oxidative damage caused by free radicals to biomolecules. In addition to antioxidants that have been commonly used for this purpose, recently, alternative substances of natural origin such as plant extracts and essential oils have gained prominence. Thus, given the influence of oxidative stress and the importance of using antioxidants in cell culture, this review aims to address the main mechanisms of synthesis and action of free radicals as well as the role of antioxidants in in vitro protocols for ovarian follicles and COCs in domestic animals.(AU)

Cultivo in vitro de folículos pré-antrais bovinos: revisão, desafios, conquistas e perspectivas futuras / In vitro culture of bovine preantral follicles: review, challenges, achievements and future perspectives

A reprodução animal incide diretamente na eficiência e rentabilidade econômica de um rebanho, sendo considerada um dos fatores de maior importância para os sistemas de produção. Consequentemente, o desenvolvimento e a utilização de biotecnologia são indispensáveis para o aumento da eficiência reprodutiva, do melhoramento genético, da produção de alimentos e geração de riquezas. Nas últimas décadas várias foram as biotécnicas associadas à reprodução animal que apresentaram um grande potencial para a geração de genética, especialmente, ao que se referem aos avanços da reprodução assistida. Estes progressos possibilitaram a utilização de folículos pré-antrais como fonte de oócitos para biotecnologias reprodutivas. Deste modo, a técnica de cultivo in vitro proporciona a retomada do desenvolvimento dos folículos presente na reserva ovariana pela ativação in vitro forçada simulando a ocorrência de distúrbios na foliculogênesse. Até então, apenas a especie murina obteve descendentes vivos através dos folículos primordiais cultivados in vitro. Infelizmente, não há relatos de sucesso em outras espécies, tornando o desenvolvimento de sistemas ideias de cultivo in vitro um desafio. Portanto, o objetivo desta revisão é descrever e discutir os principais avanços da situação atual da pesquisa com folículos pré-antrais inclusos em folículos ovarianos.(AU)

Animal reproduction directly affects the efficiency and economic profitability of a herd, being considered one of the most important factors for production systems. Consequently, the development and use of biotechnology are indispensable for increasing reproductive efficiency, genetic improvement, food production and wealth generation. In the last few decades, biotechnologies associated with animal reproduction have shown great potential for the generation of genetics, especially with regard to advances in assisted reproduction. These advances have enabled the use of pre-antral follicles as a source of oocytes for reproductive biotechnologies. Thus, the in vitro culture technique provides the resumption of the development of follicles present in the ovarian reserve by forced in vitro activation, simulating the occurrence of disturbances in folliculogenesis. Until then, only the murine species obtained living descendants through primordial follicles cultivated in vitro. Unfortunately, there are no reports of success in other species, making the development of ideal in vitro culture systems a challenge.Therefore, the purpose of this review is to describe and discuss the main advances in the current research situation with preantral follicles included in ovarian follicles.(AU)

Cultivo in vitro de folículos pré-antrais bovinos: revisão, desafios, conquistas e perspectivas futuras / In vitro culture of bovine preantral follicles: review, challenges, achievements and future perspectives

A reprodução animal incide diretamente na eficiência e rentabilidade econômica de um rebanho, sendo considerada um dos fatores de maior importância para os sistemas de produção. Consequentemente, o desenvolvimento e a utilização de biotecnologia são indispensáveis para o aumento da eficiência reprodutiva, do melhoramento genético, da produção de alimentos e geração de riquezas. Nas últimas décadas várias foram as biotécnicas associadas à reprodução animal que apresentaram um grande potencial para a geração de genética, especialmente, ao que se referem aos avanços da reprodução assistida. Estes progressos possibilitaram a utilização de folículos pré-antrais como fonte de oócitos para biotecnologias reprodutivas. Deste modo, a técnica de cultivo in vitro proporciona a retomada do desenvolvimento dos folículos presente na reserva ovariana pela ativação in vitro forçada simulando a ocorrência de distúrbios na foliculogênesse. Até então, apenas a especie murina obteve descendentes vivos através dos folículos primordiais cultivados in vitro. Infelizmente, não há relatos de sucesso em outras espécies, tornando o desenvolvimento de sistemas ideias de cultivo in vitro um desafio. Portanto, o objetivo desta revisão é descrever e discutir os principais avanços da situação atual da pesquisa com folículos pré-antrais inclusos em folículos ovarianos.

Animal reproduction directly affects the efficiency and economic profitability of a herd, being considered one of the most important factors for production systems. Consequently, the development and use of biotechnology are indispensable for increasing reproductive efficiency, genetic improvement, food production and wealth generation. In the last few decades, biotechnologies associated with animal reproduction have shown great potential for the generation of genetics, especially with regard to advances in assisted reproduction. These advances have enabled the use of pre-antral follicles as a source of oocytes for reproductive biotechnologies. Thus, the in vitro culture technique provides the resumption of the development of follicles present in the ovarian reserve by forced in vitro activation, simulating the occurrence of disturbances in folliculogenesis. Until then, only the murine species obtained living descendants through primordial follicles cultivated in vitro. Unfortunately, there are no reports of success in other species, making the development of ideal in vitro culture systems a challenge.Therefore, the purpose of this review is to describe and discuss the main advances in the current research situation with preantral follicles included in ovarian follicles.

Oogênese e Foliculogênese em Bovinos / Oogenesis and Folliculogenesis in Cattle

Os ovários desempenham papéis relevantes para o sistema reprodutivo, no qual a função exócrina (ou gametogênica) realiza a maturação e liberação do oócito para a fecundação e a função endócrina (ou esteroidogênica) afeta a síntese, secreção de hormônios e fatores de crescimento. Além disso, existe uma interação entre os fatores endócrinos, autócrinos e parácrinos, atuando em associação com o processo de desenvolvimento folicular e oocitário durante a vida reprodutiva da fêmea. O córtex, porção mais externa do ovário, representa a região funcional onde se localizam os folículos ovarianos e as estruturas lúteas, em diferentes estágios de desenvolvimento e/ou atresia. A região medular localiza-se mais internamente, na maioria das espécies, e sua principal função é nutrir e sustentar o ovário. Apesar da numerosa população folicular presente nos ovários, estabelecida durante a vida fetal, quase todos os folículos encontrados no pool de reserva ovariana, ou seja, 99,9%, não atingem a ovulação. Esses folículos passam por um processo de morte celular conhecido como atresia folicular, tornando o ovário um órgão de baixa produtividade. A elucidação dos mecanismos que regulam a foliculogênese, incluindo o processo de atresia, é importante para o melhor aproveitamento dos folículos na melhoria da eficiência reprodutiva de animais de produção.

The ovaries play roles relevant to the reproductive system, in which the exocrine (or gametogenic) function carries out the maturation and release of the oocyte for fertilization and the endocrine (or steroidogenic) function effects the synthesis and secretion of hormones and growth factors. In addition, there is an interaction between endocrine, autocrine and paracrine factors, acting in association with the follicular and oocyte development process during the female's reproductive life. The cortex, the outermost portion of the ovary, represents the functional region where the ovarian follicles and luteal structures are located, at different stages of development and/or atresia. The medullary region is located more internally, in most species, and its main function is to nourish and support the ovary. Despite the numerous follicular population present in the ovaries, established during fetal life, almost all follicles found in the ovarian reserve pool, i.e., 99.9%, do not reach ovulation. These follicles undergo a process of cell death known as follicular atresia, making the ovary a low-productivity organ. The elucidation of the mechanisms that regulate folliculogenesis, including the atresia process, is important for the better use of follicles in improving the reproductive efficiency of production animals.

Qualitative and quantitative aspects of atresia during mammalian folliculogenesis / Aspectos qualitativos e quantitativos da atresia durante a foliculogênese em mamíferos

Durante a foliculogênese ovariana, cerca de 99,9% dos folículos morrem pelo processo de atresia folicular. O processo de atresia pode ocorrer pelas vias degenerativa ou apoptótica. Este processo compromete todos os estágios de desenvolvimento folicular, sendo os folículos antrais os mais afetados. Por outro lado, os folículos préantrais são mais resistentes, em função de sua menor taxa metabólica, bem como do número reduzido de células e camadas de células somáticas, células da granulosa e/ou da teca. Embora folículos pré-antrais sejam menos afetados pelo processo de atresia, quando este evento ocorre, pode-se classificá-lo de duas formas, degeneração do tipo I e degeneração do tipo II. Na degeneração do tipo I, o oócito é o compartimento mais comprometido, apresentando núcleo picnótico, embora suas células da granulosa apresentem-se bem organizadas e sem picnose nuclear. Já na degeneração do tipo II, os folículos apresentam oócito retraído e células da granulosa edemaciadas, desorganizadas e sem aderência à membrana basal e ao oócito. É importante destacar que a degeneração do tipo I é mais comum em folículos primordiais e primários, enquanto folículos secundários apresentam mais degeneração do tipo II, ou seja, a medida que os folículos evoluem, a degeneração do tipo II é mais frequente. Considerando todos os aspectos aqui relacionados, essa revisão de literatura abordará aspectos relacionados aos processos de foliculogênese e atresia folicular, bem como as substâncias que induzem a atresia durante a foliculogenese in vitro e in vivo e, ainda, os métodos e parâmetros para análise da atresia em folículos ovarianos. Isto se deve a necessidade de desenvolvimento de protocolos mais eficientes de recuperação dos folículos ovarianos, prevenindo essa grande perda folicular e otimizando as possibilidades de utilização desses materiais biológicos no futuro.

During ovarian foliculogenesis, about 99.9% of follicles die by the follicle atresia process. The atresia process can occur via degeneration or apoptosis. This process compromises all the follicle development stages, with antral follicles being those most affected. On the other hand, the preantral follicles are more residents, since their slow metabolic rate, as well as the reduced number and layers of somatic cells, granulosa and/or thecal cells. Although preantral follicles are less affected by the atresia process, when the event occurs, we can classify it in two ways, type I or type II degenerated follicles. In the type I degeneration the oocyte is the most compromised compartment, showing the picnotic nuclei, although its granulosa cells presented well organized and no picnosis. Already in the type II degeneration, the follicles presented shrunken in the oocyte, and swollen, disorganization and no adhered granulosa cells from the basal membrane and oocyte. It is important to highlight that type I degeneration is the most common in primordial and primary follicles, while in secondary follicles present more type II degeneration, which means that the as follicle evolve, type II degeneration is more frequent. Considering all the aspects related here, this literature review will address aspects related to the folliculogenesis and the process of follicle atresia, as well as substances that induce atresia during in vitro and in vivo folliculogenesis and methods and parameters for analyzing atresia in ovarian follicles. This is due to the need to develop more efficient ovarian follicle recovery protocols, which can prevent this great follicular loss and optimizing the possibilities of use of these biological materials in the future.

Efeito do resveratrol associado a sacarose em protocolo de vitrificação de tecido ovariano de vacas / Effect of resveratrol associated with sacarosis on ovarian tissue vitrification protocol in cows

The objective of this work was to evaluate the effect of the association of the antioxidant resveratrol with sucrose in a vitrification protocol of ovarian tissue in cows, on the morphology of preantral follicles. Ten ovaries of cows were used, collected in local slaughterhouses, fragmented and distributed to the following treatments: fresh control (Co); toxicity (T); (T0) zero toxicity/ only with base vitrification solution (SBV), (TS) toxicity with SBV plus sucrose, (TR) toxicity with SBV plus resveratrol, (TS+R) toxicity with SBV and sucrose plus resveratrol; and for glazing (V); (VS) vitrification with SBV and sucrose, (VR) vitrification with SBV and resveratrol, (VS+R) vitrification with SBV and sucrose plus resveratrol. Preantral follicles were quantified and classified according to morphology into normal and degenerated. The mean percentages between normal and degenerated follicles did not differ (p>0.05) in the following percentages, normal 51.4% and degenerated 48.60%. In the toxicity test there was a difference (p0.05), demonstrating that the vitrification technique is efficient, but the concentration of cryoprotectants used needs to be re-evaluated. Concluding that the natural antioxidant association resveratrol to sucrose in vitrification and rewarming protocols contributes with reservations for the morphological preservation of preantral follicles in cows.

Avanços na tecnologia do ovário artificial em ruminantes / Advances in artificial ovary technology in ruminants

O cultivo in vitro de folículos (CIVF), também conhecido como Ovário Artificial, representa uma excelente ferramenta para aumentar nossa compreensão a respeito do controle da foliculogênese inicial, que é crucial para otimizar o uso futuro de um grande número de oócitos imaturos inclusos em folículos pré-antrais (FPs) (principalreserva ovariana) em técnicas de reprodução assistida em humanos, bem como em outras espécies de mamíferos, incluindo os ruminantes. Até o momento, os melhores resultados de CIVF foram relatados em camundongos com a produção de crias vivas a partir de folículos primordiais cultivados in vitro. No entanto, em outras espécies como os ruminantes, esses resultados têm se limitado à produção de um número variável de oócitos maturos e baixas porcentagens de embriões após o cultivo in vitro de folículos pré-antrais secundários tardios isolados de ovários de cabras, búfalas e ovelhas. A presente revisão apresenta e discute os principais achados, limitações e perspectivas da foliculogênese in vitro em ruminantes com foco nas espécies bovinas, caprinas e ovinas.

The in vitro follicle culture (IVFC), also known as artificial ovary, represents an outstanding tool to increase our understanding of the control of early folliculogenesis which is crucial to optimize the future use of a large, number of immature oocytes enclosed in preantral follicles (PFs) (main ovarian reserve) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. To date, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated late secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.

Avanços na tecnologia do ovário artificial em ruminantes / Advances in artificial ovary technology in ruminants

O cultivo in vitro de folículos (CIVF), também conhecido como Ovário Artificial, representa uma excelente ferramenta para aumentar nossa compreensão a respeito do controle da foliculogênese inicial, que é crucial para otimizar o uso futuro de um grande número de oócitos imaturos inclusos em folículos pré-antrais (FPs) (principalreserva ovariana) em técnicas de reprodução assistida em humanos, bem como em outras espécies de mamíferos, incluindo os ruminantes. Até o momento, os melhores resultados de CIVF foram relatados em camundongos com a produção de crias vivas a partir de folículos primordiais cultivados in vitro. No entanto, em outras espécies como os ruminantes, esses resultados têm se limitado à produção de um número variável de oócitos maturos e baixas porcentagens de embriões após o cultivo in vitro de folículos pré-antrais secundários tardios isolados de ovários de cabras, búfalas e ovelhas. A presente revisão apresenta e discute os principais achados, limitações e perspectivas da foliculogênese in vitro em ruminantes com foco nas espécies bovinas, caprinas e ovinas.(AU)

The in vitro follicle culture (IVFC), also known as artificial ovary, represents an outstanding tool to increase our understanding of the control of early folliculogenesis which is crucial to optimize the future use of a large, number of immature oocytes enclosed in preantral follicles (PFs) (main ovarian reserve) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. To date, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated late secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.(AU)

Medium and time conservation affect follicular morphology and superoxide dismutase enzyme activity of bovine preantral follicles during storage at 4ºC / Meio e tempo de conservação afetam a morfologia folicular e atividade da enzima superóxido dismutase de folículos pré-antrais bovinos durante a conservação a 4 ºC

The aim of this study was to evaluate the morphology and superoxide dismutase enzyme (SOD) activity of bovine preantral follicles (PFs) preserved in TCM 199, saline solution or PBS at different conservation periods. Cow ovaries (n=6) were divided into 7 fragments. One small piece of each ovarian fragment was randomly removed to evaluate SOD activity, while the remainder was immediately fixed for morphological evaluation as a control group. The other 6 fragments were randomly distributed in tubes containing TCM 199, saline solution, or PBS and maintained at 4ºC for 6 or 24 h. For histological evaluation, the fragments were fixed in Carnoy and stained with PAS-hematoxylin, following being classified PFs in relation to their follicular morphology in normal or degenerated. Determination of SOD activity was based on the ability to inhibit autoxidation of adrenaline in adrenochrome. Evaluation of follicular morphology showed that follicles preserved in TCM 199 for 6 h did not differ from the control (P > 0.05). In contrast, preservation in saline solution and PBS for 6 or 24 h and TCM 199 for 24 h decreased normal PFs compared to the control (P < 0.05). SOD showed a lower activity in ovarian cortical tissue kept in TCM 199 for 6 h and saline solution for 24 h than in the other groups. Our study shows that incubation using TCM 199 at 4°C for 6 h can be used to efficiently conserve female bovine PFs in situ.(AU)

O objetivo desse estudo foi avaliar a morfologia e atividade da enzima superóxido dismutase (SOD) de folículos pré-antrais bovinos (PFs) preservados em TCM 199, solução salina ou PBS por diferentes períodos de conservação. Ovários de vacas (n=6) foram divididos em 7 fragmentos. Um pequeno pedaço de cada fragmento ovariano foi removido para avaliar a atividade da SOD enquanto que o restante foi imediatamente fixado para avaliação morfológica como grupo controle. Os outros 6 fragmentos foram distribuídos aleatoriamente em tubos contendo TCM 199, solução salina ou PBS e mantidos a 4ºC por 6 ou 24 h. Para avaliação histológica, os fragmentos foram fixados em Carnoy e corados com PAShematoxilina, sendo classificados em seguida os PFs em relação à sua morfologia folicular em normal ou degenerados. A determinação da atividade da SOD foi baseada na capacidade de inibir a autooxidação da adrenalina no adrenocromo. A avaliação da morfologia folicular mostrou que os folículos preservados em TCM199 por 6 h não diferiram do controle (P > 0,05). Em contraste, a preservação em solução salina e PBS por 6 ou 24 h e TCM 199 por 24 h diminuiu os PFs normais em comparação com o controle (P < 0,05). A SOD mostrou uma menor atividade no tecido cortical ovariano mantido em TCM 199 por 6 h e solução salina por 24 h do que nos outros grupos. Nosso estudo mostra que a incubação usando TCM 199 a 4° C por 6 h pode ser usada eficientemente para conservar PFs de fêmeas bovinas in situ.(AU)

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models

Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models

Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.(AU)

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.(AU)

Advances in in vitro folliculogenesis in domestic ruminants

The in vitro follicle culture (IVFC) represents an outstanding tool to enhance our understanding of the control of folliculogenesis and to allow the future use of a large number of immature oocytes enclosed in preantral follicles (PFs) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. So far, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. Live birth has been obtained after the in vitro culture of bovine early antral follicles. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.

Advances in in vitro folliculogenesis in domestic ruminants

The in vitro follicle culture (IVFC) represents an outstanding tool to enhance our understanding of the control of folliculogenesis and to allow the future use of a large number of immature oocytes enclosed in preantral follicles (PFs) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. So far, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. Live birth has been obtained after the in vitro culture of bovine early antral follicles. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.(AU)

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P 0.05) among treatments. Overall, all treatments had lower primordial follicles (P 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

Effect of somatotropin on survival and diameter of bovine preantral follicles / Efeito da somatotropina na sobrevivência e no diâmetro de folículos pré-antrais de bovinos

The aim of this study was to evaluate the effect of Recombinant bovine somatotropin (rbST) on survival and diameter of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovaries were collected from adult cows and fragments of ovarian cortex were immediately fixed (non-cultured control) or cultured in vitro in α-MEM+ alone or containing 10, 50, 100 or 1,000ng/mL rbST. The fragments were processed for Classical Histology and Transmission Electron Microscopy. After one and seven days of culture, the percentage of normal follicles in the non-cultured control was superior (P< 0.05) to the follicles cultured in α-MEM+ alone or with different rbST concentrations. The oocyte and follicular mean diameter did not increase during the culture for one and seven days, both in media containing rbST and in the medium without this hormone. The only medium in which there was no reduction in follicular diameter with the time of culture was the medium without rbST. Ultrastructural damage in PAOF cultured in vitro was found. It is concluded that the use of rbST at different concentrations in in situ culture of bovine preantral follicles has no beneficial effects on survival and growth of bovine PAOF.(AU)

O objetivo deste trabalho foi avaliar o efeito da somatotropina recombinante bovina (rbST) sobre a sobrevivência e o diâmetro de folículos ovarianos pré-antrais (FOPA) bovinos cultivados in vitro. Ovários foram coletados de vacas adultas e fragmentos do córtex ovariano foram imediatamente fixados (controle não cultivado) ou cultivados in vitro em α-MEM + sozinho ou contendo 10, 50, 100 ou 1.000ng/mL de rbST. Os fragmentos foram processados para histologia clássica e microscopia eletrônica de transmissão. Após um e sete dias de cultivo, o percentual de folículos normais no controle não cultivado foi superior (P<0,05) aos cultivados em α-MEM + sozinho ou acrescido de diferentes concentrações de rbST. Os diâmetros médios oocitário e folicular não aumentaram durante o cultivo por um e sete dias, tanto nos meios contendo rbST, como no meio sem esse hormônio (α-MEM + ). O único meio em que não houve redução no diâmetro folicular com o tempo de cultivo foi o sem rbST. Verificaram-se ainda danos ultraestruturais em FOPA cultivados in vitro. Conclui-se que o uso de rbST em diferentes concentrações no cultivo in situ de folículos pré-antrais bovinos não tem efeitos benéficos na sobrevivência e no crescimento de FOPA bovinos.(AU)

Effect of somatotropin on survival and diameter of bovine preantral follicles / Efeito da somatotropina na sobrevivência e no diâmetro de folículos pré-antrais de bovinos

The aim of this study was to evaluate the effect of Recombinant bovine somatotropin (rbST) on survival and diameter of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovaries were collected from adult cows and fragments of ovarian cortex were immediately fixed (non-cultured control) or cultured in vitro in α-MEM+ alone or containing 10, 50, 100 or 1,000ng/mL rbST. The fragments were processed for Classical Histology and Transmission Electron Microscopy. After one and seven days of culture, the percentage of normal follicles in the non-cultured control was superior (P< 0.05) to the follicles cultured in α-MEM+ alone or with different rbST concentrations. The oocyte and follicular mean diameter did not increase during the culture for one and seven days, both in media containing rbST and in the medium without this hormone. The only medium in which there was no reduction in follicular diameter with the time of culture was the medium without rbST. Ultrastructural damage in PAOF cultured in vitro was found. It is concluded that the use of rbST at different concentrations in in situ culture of bovine preantral follicles has no beneficial effects on survival and growth of bovine PAOF.(AU)

O objetivo deste trabalho foi avaliar o efeito da somatotropina recombinante bovina (rbST) sobre a sobrevivência e o diâmetro de folículos ovarianos pré-antrais (FOPA) bovinos cultivados in vitro. Ovários foram coletados de vacas adultas e fragmentos do córtex ovariano foram imediatamente fixados (controle não cultivado) ou cultivados in vitro em α-MEM + sozinho ou contendo 10, 50, 100 ou 1.000ng/mL de rbST. Os fragmentos foram processados para histologia clássica e microscopia eletrônica de transmissão. Após um e sete dias de cultivo, o percentual de folículos normais no controle não cultivado foi superior (P<0,05) aos cultivados em α-MEM + sozinho ou acrescido de diferentes concentrações de rbST. Os diâmetros médios oocitário e folicular não aumentaram durante o cultivo por um e sete dias, tanto nos meios contendo rbST, como no meio sem esse hormônio (α-MEM + ). O único meio em que não houve redução no diâmetro folicular com o tempo de cultivo foi o sem rbST. Verificaram-se ainda danos ultraestruturais em FOPA cultivados in vitro. Conclui-se que o uso de rbST em diferentes concentrações no cultivo in situ de folículos pré-antrais bovinos não tem efeitos benéficos na sobrevivência e no crescimento de FOPA bovinos.(AU)