Morphological aspects and germ cells of male reproductive tract of river stingray, Potamotrygon amandae

The family Potamotrygonidae are the only species of stingrays restricted to fresh water and located exclusively in South America. The objective of this research was to analyze the morphological aspects and germ cells of the male reproductive tract of Potamotrygon amandae. The samples were fixed in 10% formalin, and then dehydrated in an ascending ethanol series (70 to 100%). To carry out light microscopy analyses, they were embedded in paraffin, cut and stained; as for scanning electron microscopy analyses, the samples were dried, glued in metallic bases and metalized. The gross morphology consisted of the following paired organs: testis, epididymis, deferent duct, Leydig gland, seminal vesicle, clasper, and the clasper gland. Microscopically, several stages of spermatogenesis were observed in the testis, occurring in spherical follicles, similar to other stingrays. The epididymis was formed by one duct subdivided in various tubules. The deferent ducts were continuous with the epididymis, and the lumen was full of spermatozoa. The Leydig glands consisted of glandular units with eosinophilic content in the lumen of some, and the deferent ducts ran parallel to the ventral portion. The seminal vesicles possessed numerous compartments to store the sperm, with a wall similar to a hive, and the lumen was full of spermatozoa. Alcian Blue (AB) and Periodic Schiff-Acid (PAS) performed in the Leydig Gland, deferens ducts and seminal vesicle was positive only in the connective tissue, the cilia were PAS+ and the nuclei stained weakly for AB. The clasper gland was composed of unit glands and was covered with striated muscle externally. It stained very well with Periodic Schiff-Acid. The morphological aspects of the male reproductive tract of Potamotrygon amandae were similar to other stingrays.(AU)

Criopreservação e cultivo de tecido testicular em mamíferos - estado da arte, desafios e perspectivas / Cryopreservation and culture of testicular tissue in mammals - state of the art, challenges and perspectives

O tecido testicular contém células germinativas, que possuem potencial para se desenvolver em espermatozoides viáveis por meio do cultivo in vitro ou xenotransplante, sendo uma alternativa interessante a ser utilizada na formação de biobancos. Esta revisão compila as atualizações, desafios e perspectivas relacionadas às técnicas de criopreservação e cultivo de tecido testicular como estratégia para a conservação de espécies mamíferas. O tecido testicular pode ser obtido tanto de indivíduos adultos como pré púberes, seja após orquiectomia ou até mesmo após a sua morte. O tecido fragmentado pode ser criopreservado por congelação lenta ou rápida e por métodos de vitrificação. Os crioprotetores são indispensáveis durante a criopreservação e podem variar o tipo e concentração de acordo com a espécie. Com os avanços da criopreservação deste material, espermatozoides podem ser obtidos por transplante de fragmentos testiculares ou células germinativas isoladas em camundongos imunodeficientes. No entanto, a obtenção de espermatozoides no cultivo in vitro ainda é um desafio.(AU)

The testicular tissue contains germ cells, which have the potential to develop into viable spermatozoa through in vitro culture or xenotransplantation, being an interesting alternative to be used in the formation of biobanks. This review compiles updates, challenges and perspectives related to cryopreservation techniques and testicular tissue culture as a strategy for the conservation of mammalian species. Testicular tissue can be obtained from both adult and pre-pubertal individuals, either after orchiectomy or even after their death. Fragmented tissue can be cryopreserved by slow or fast freezing and by vitrification methods. Cryoprotectants are indispensable during cryopreservation and may vary in type and concentration according to the species. With advances in cryopreservation of this material, spermatozoa can be obtained by transplanting testicular fragments or isolated germ cells into immunodeficient mice. However, obtaining spermatozoa in in vitro culture is still a challenge.(AU)

Efeitos imediatos, tardios e transgeracionais do estresse térmico em gametas / Immediate, late and transgenerational effects of heat stress on gametes

As perdas de produtividade e fertilidade animal associadas ao estresse térmico durante os meses mais quentes do ano é um dos maiores desafios do setor pecuário. Na indústria de leite as perdas econômicas causadas pelo estresse térmico foram estimadas em mais de 1,5 bilhões de dólares por ano. No que tange a reprodução, já foi demonstrado que o estresse térmico exerce múltiplos efeitos deletérios, causando disfunções endócrinas e alterando a sequência orquestrada de eventos importantes para a gametogênese e para o desenvolvimento embrionário inicial. Estudos recentes têm esclarecido o padrão temporal no qual os danos são estabelecidos e carreados dependendo da intensidade do estresse. Enquanto os efeitos imediatos do estresse térmico nos gametas já são bem caracterizados, existem evidências de que alguns danos podem ser carreados de forma tardia e possivelmente entre gerações. Além disso, dados emergentes indicam que o estresse térmico compromete a reprogramação da metilação do DNA que ocorre durante a gametogênese e a programação do desenvolvimento in utero. Dessa forma, esse artigo visa explorar os efeitos imediatos, tardios e transgeracionais do estresse térmico nos gametas.(AU)

The drop on animal productivity and fertility associated with heat stress during the hot months of the year is one of the biggest challenges for the livestock sector. For the dairy industry the economic losses caused by heat stress have been estimated over 1.5 billion dollars per year. It has already been demonstrated that heat stress exerts multiple deleterious effects on reproductive function, causing endocrine dysfunctions as well as changes in the sequence of events required for gametogenesis and early embryonic development. Recent studies have shed a light in the temporal pattern in which heat-induced damage is established and carried forward depending on the intensity of stress. While the immediate effects of heat stress on gametes are well characterized, there is evidence that some damage can be carried over for longer periods and even across generations. Furthermore, emerging data indicate that heat stress compromises DNA methylation reprogramming that occurs during gametogenesis and developmental programming in utero. Thus, this paper aims to explore the immediate, late and transgenerational effects of heat stress on gametes.(AU)

Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

Avances en el manejo de gametos caninos in vitro / Advance in handling canine gametes in vitro

Las características fisiológicas de los gametos caninos que los hace especiales respecto a los de otras especies han dificultado los estudios tendientes a incrementar los porcentajes de éxito en diversas biotecnologías reproductivas. Entre estas características está el inicio de la meiosis de los ovocitos posterior al nacimiento y la ovulación de un gameto inmaduro como ovocito primario, lo que ha complejizado la maduración in vitro de estos ovocitos y, por parte de los espermatozoides, una gran diferencia en la sobrevivencia de los espermatozoides eyaculados versus los congelados-descongelados, que hace imprescindible evaluar la biología reproductiva de estos gametos tendiente a optimizar nuevos protocolos haciendo énfasis previamente en una adecuada evaluación del crecimiento del ovocito in vivo y de la viabilidad espermática en aquellos sometidos a criopreservación(AU)

The physiological characteristics of canine gametes that make them unique compared to those of other species have hindered studies aimed at increasing the success rates in various reproductive biotechnologies. Among these characteristics is the meiosis of the oocytes after birth and the ovulation of an immature gamete as a primary oocyte, which has made the in vitro maturation of these oocytes more complex and, on the part of the spermatozoa, a significant difference in the viability of ejaculated versus frozen-thawed spermatozoa, which makes it essential to evaluate the reproductive biology of these gametes in order to optimize new protocols, previously emphasizing an adequate evaluation of in vivo oocyte growth and sperm viability in those subjected to cryopreservation.(AU)

The role of the oviduct and extracellular vesicles during early embryo development in bovine

The oviduct is an important reproductive structure that connects the ovary to the uterus and takes place to important events such as oocyte final maturation, fertilization and early embryonic development. Thus, gametes and embryo can be directly influenced by the oviductal microenvironment composed by epithelial cells such secretory and ciliated cells and oviductal fluid. The oviduct composition is anatomically dynamic and is under ovarian hormones control. The oviductal fluid provides protection, nourishment and transport to gametes and embryo and allows interaction to oviductal epithelial cells. All these functions together allows the oviduct to provides the ideal environment to the early reproductive events. Extracellular vesicles (EVs) are biological nanoparticles that mediates cell communication and are present at oviductal fluid and plays an important role in gametes/embryo - oviductal cells communication. This review will present the ability of the oviducts based on its dynamic and systemic changes during reproductive events, as well as the contribution of EVs in this process.(AU)

Ovarian development of Xiphopenaeus kroyeri (Crustacea: Penaeidae) from Espírito Santo, southeastern Brazil / Desenvolvimento ovariano de Xiphopenaeus kroyeri (Crustacea: Penaeidae) no Espírito Santo, sudeste do Brasil

This study aimed to describe and characterize the stages of gonadal development of females of Xiphopenaeus kroyeri caught by artisanal fishers in Espírito Santo state, southeastern region of Brazil. All females (n= 1,831) were subjected to macroscopic and microscopic morphological analysis (n= 333) of the ovaries. From the morphology, coloration and degree of turgidity of the fresh ovary, macroscopic analysis determined five stages of gonadal development. The macroscopic analysis showed difficulties in differentiating the immature and spawning stages due to the similarity between the colors of the ovaries, which confirms the need to perform the macroscopic and histological analysis simultaneously for fisheries management studies. Microscopic observations allowed us to analyze the following six stages of cell development: oogonia, previtellogenic oocytes, primary vitellogenic oocytes, secondary vitellogenic oocytes, mature oocytes and atretic oocytes. From this, five stages of gonadal development were defined, i.e., immature, early development, advanced development, mature and spawned. The presence of peripheral bodies was not observed in this species. These results help to clarify and better understand the reproductive and population aspects of the Atlantic Seabob, which are fundamental for the establishment of management and conservation measures of this resource.

Este estudo teve como objetivo descrever e caracterizar os estágios de desenvolvimento gonadal de fêmeas de Xiphopenaeus kroyeri capturado por pescadores artesanais no Estado do Espírito Santo, região sudeste (Região sudeste = Rio de Janeiro, São Paulo, Minas Gerais e Espírito Santo) do Brasil. Todas as fêmeas (n= 1.831) foram submetidas à análise morfológica macroscópica e microscópica (n= 333) dos ovários. A partir da morfologia, coloração e grau de turgidez do ovário fresco, a análise macroscópica determinou cinco estágios de desenvolvimento gonadal. A análise macroscópica mostrou dificuldades em diferenciar os estágios imaturo e reprodutivo devido à semelhança entre as cores dos ovários, o que confirma a necessidade de realizar a análise macroscópica e histológica simultaneamente para estudos de manejo pesqueiro. As observações microscópicas permitiram analisar os seguintes seis estágios de desenvolvimento celular: oogônias, ovócitos prévitelogênicos, ovócitos em vitelogênese primária, ovócitos em vitelogênese secundária, ovócitos maduros e ovócitos atrésicos. A partir disso, foram definidos cinco estágios de desenvolvimento gonadal, ou seja, imaturo, desenvolvimento inicial, desenvolvimento avançado, maduro e desovado. A presença de corpos periféricos não foi observada nesta espécie. Estes resultados ajudam a esclarecer e compreender melhor os aspetos reprodutivos e populacionais do camarão do Atlântico, que são fundamentais para o estabelecimento de medidas de gestão e conservação deste recurso.

Avanços na andrologia de animais selvagens / Advances in wild animal andrology

As tecnologias de reprodução assistida (TRA) são de fundamental importância para a conexão de indivíduos em diferentes localidades, facilitando assim o intercâmbio genético e favorecendo a variabilidade genética de uma espécie. Por esta razão, as TRAS podem ser ferramentas importantes para a conservação de espécies ameaçadas de extinção. Apesar dos esforços nas últimas décadas, o avanço no desenvolvimento de tais tecnologias está aquém à urgência de reverter processos de baixa variabilidade genética em algumas espécies. A necessidade de refinamento das técnicas para as particularidades fisiológicas e comportamentais de cada espécie, somada à raridade de acesso aos animais são os principais fatores relacionados as dificuldades em se avançar com as TRAS. As técnicas mais recentes desenvolvidas para a recuperação de espermatozoides em animais selvagens são a colheita farmacológica, com uso de alfa-2-agonistas e a criopreservação / vitrificação testicular com posterior cultivo. Pouco de avançou, no entanto, em relação aos métodos de criopreservação, prevalecendo associação clássica de TRIS-gema-glicerol. Discutimos, então os métodos usados para acesso ao gameta masculino em espécies selvagens e suas aplicações na conservação animal.(AU)

Assisted reproduction technologies (ART) are of fundamental importance for connecting individuals in different locations, thus facilitating genetic exchange and favoring the genetic variability of a species. For this reason, TRAS can be important tools for the conservation of endangered species. Despite efforts in recent decades, the advance in the development of such technologies is short of the urgency of reversing processes of low genetic variability in some species. The need to refine the techniques for the physiological and behavioral particularities of each species, added to the rarity of access to animals, are the main factors related to the difficulties in advancing with TRAS. The most recent techniques developed for sperm collection in wild animals are pharmacological collection, with the use of alpha-2-agonists and testicular cryopreservation / vitrification with subsequent cultivation. Little progress has been made, however, in relation to cryopreservation methods, prevailing the classic association of TRIS-yolk-glycerol. We therefore discuss the methods used to access the male gamete in wild species and their applications in animal conservation.(AU)

Effect of environmental contamination on female and male gametes – A lesson from bovines

Endocrine-disrupting compounds (EDCs) and foodborne contaminants are environmental pollutants that are considered reproductive toxicants due to their deleterious effects on female and male gametes. Among the EDCs, the phthalate plasticizers are of growing concern. In-vivo and in-vitro models indicate that the oocyte is highly sensitive to phthalates. This review summarizes the effects of di(2-ethylhexyl) phthalate and its major metabolite mono(2-ethyhexyl) phthalate (MEHP) on the oocyte. MEHP reduces the proportion of oocytes that fertilize, cleave and develop to the blastocyst stage. This is associated with negative effects on meiotic progression, and disruption of cortical granules, endoplasmic reticulum and mitochondrial reorganization. MEHP alters mitochondrial membrane polarity, increases reactive oxygen species levels and induces alterations in genes associated with oxidative phosphorylation. A carryover effect from the oocyte to the blastocyst is manifested by alterations in the transcriptomic profile of blastocysts developed from MEHP-treated oocytes. Among foodborne contaminants, the pesticide atrazine (ATZ) and the mycotoxin aflatoxin B1 (AFB1) are of high concern. The potential hazards associated with exposure of spermatozoa to these contaminants and their carryover effect to the blastocyst are described. AFB1 and ATZ reduce spermatozoa's viability, as reflected by a high proportion of cells with damaged plasma membrane; induce acrosome reaction, expressed as damage to the acrosomal membrane; and interfere with mitochondrial function, characterized by hyperpolarization of the membrane. ATZ and AFB1-treated spermatozoa show a high proportion of cells with fragmented DNA. Exposure of spermatozoa to AFB1 and ATZ reduces fertilization and cleavage rates, but not that of blastocyst formation. However, fertilization with AFB1- or ATZ-treated spermatozoa impairs transcript expression in the formed blastocysts, implying a carryover effect. Taken together, the review indicates the risk of exposing farm animals to environmental contaminants, and their deleterious effects on female and male gametes and the developing embryo.

Effect of environmental contamination on female and male gametes A lesson from bovines

Endocrine-disrupting compounds (EDCs) and foodborne contaminants are environmental pollutants that are considered reproductive toxicants due to their deleterious effects on female and male gametes. Among the EDCs, the phthalate plasticizers are of growing concern. In-vivo and in-vitro models indicate that the oocyte is highly sensitive to phthalates. This review summarizes the effects of di(2-ethylhexyl) phthalate and its major metabolite mono(2-ethyhexyl) phthalate (MEHP) on the oocyte. MEHP reduces the proportion of oocytes that fertilize, cleave and develop to the blastocyst stage. This is associated with negative effects on meiotic progression, and disruption of cortical granules, endoplasmic reticulum and mitochondrial reorganization. MEHP alters mitochondrial membrane polarity, increases reactive oxygen species levels and induces alterations in genes associated with oxidative phosphorylation. A carryover effect from the oocyte to the blastocyst is manifested by alterations in the transcriptomic profile of blastocysts developed from MEHP-treated oocytes. Among foodborne contaminants, the pesticide atrazine (ATZ) and the mycotoxin aflatoxin B1 (AFB1) are of high concern. The potential hazards associated with exposure of spermatozoa to these contaminants and their carryover effect to the blastocyst are described. AFB1 and ATZ reduce spermatozoa's viability, as reflected by a high proportion of cells with damaged plasma membrane; induce acrosome reaction, expressed as damage to the acrosomal membrane; and interfere with mitochondrial function, characterized by hyperpolarization of the membrane. ATZ and AFB1-treated spermatozoa show a high proportion of cells with fragmented DNA. Exposure of spermatozoa to AFB1 and ATZ reduces fertilization and cleavage rates, but not that of blastocyst formation. However, fertilization with AFB1- or ATZ-treated spermatozoa impairs transcript expression in the formed blastocysts, implying a carryover effect. Taken together, the review indicates the risk of exposing farm animals to environmental contaminants, and their deleterious effects on female and male gametes and the developing embryo.(AU)

Looking at the big picture: understanding how the oviduct’s dialogue with gametes and the bryo shapes reproductive success

The oviduct is a tubular organ comprising three distinct anatomical regions (the infundibulum, the ampulla and the isthmus) connecting the ovary and the uterus. Oviductal function is regulated by ovarian hormones, gametes, and embryo-derived factors, for optimally facilitating key reproductive events. A crosstalk is established between the oviduct and the gametes and embryo and this dialogue shapes the microenvironment in which gamete transport, fertilization, and early embryonic development occur. This review aims to address each participant in this conversation in a holistic manner by delineating several advances in the field within the greater context of understanding how oviduct-gamete and oviduct-embryo dialogue shape reproductive success and furthermore how this knowledge can be applied in vitro.

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Avaliação in vitro do sêmen congelado de carneiros com diluidor suplementado com miricetina / In vitro evaluation of ram sperm frozen with extender supplemented with myricetin

O objetivo deste estudo foi avaliar o efeito da suplementação do diluidor de congelação de sêmen ovino com o flavonoide miricetina contra os danos ocasionados aos espermatozoides. Oito pools de sêmen, obtidos de quatro reprodutores ovinos, foram congelados com diferentes concentrações de miricetina (0, 1, 10, 100 e 1000nM). Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, ao potencial de membrana mitocondrial, aos níveis de ROS intracelular, à peroxidação lipídica e à estabilidade de membrana. Amostras tratadas com miricetina 10nM apresentaram menor percentual de células rápidas (P≤0,05), quando comparadas ao grupo miricetina 1000nM. Amostras do grupo controle apresentaram maior (P≤0,05) VAP que o grupo 10nM de miricetina, enquanto amostras criopreservadas com miricetina (10, 100 e 1000nM) evidenciaram maior (P<0,05) BCF, quando comparadas ao grupo controle. O grupo tratado com miricetina 1000nM apresentou maior percentual (P<0,05) de células com peroxidação lipídica, quando comparado ao grupo controle. Em conclusão, a suplementação do diluidor de criopreservação de sêmen ovino com 10 e 100nM de miricetina afeta a cinética espermática sem provocar alterações na estrutura geral do gameta, enquanto 1000nM de miricetina provoca mudanças na cinética associadas à danos peroxidativos.(AU)

The aim of this study was to evaluate the effect of the supplementation of ram semen frozen with extender with the flavonoid myricetin against damage to sperm. Eight pools of semen obtained from four ram breeders, were frozen with different concentrations of myicetin (0, 1, 10, 100 and 1000nM). After thawing, the semen was evaluated for spermatic kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, intracellular ROS levels, lipid peroxidation, and membrane stability. Samples treated with 10nM myricetin preserved a lower percentage of rapid cells (P≤0.05) when compared to the 1000nM myricetin group. Samples from the control group presented higher (P≤0.05) VAP than 10nM group of myricetin, while cryopreserved samples with myicetin (10, 100 and 1000nM) showed greater (P<0.05) BCF, when compared to control group. The group treated with 1000nM myricetin had a higher percentage (P<0.05) of cells with lipid peroxidation, when compared to the control group. In conclusion, supplementation of ram semen cryopreservation extender with 10 and 100nM myricetin affects sperm kinetics, without causing changes in the overall structure of the gamete, while 1000nM myricetin causes changes in the kinetics associated with peroxidative damage.(AU)

Avaliação in vitro do sêmen congelado de carneiros com diluidor suplementado com miricetina / In vitro evaluation of ram sperm frozen with extender supplemented with myricetin

O objetivo deste estudo foi avaliar o efeito da suplementação do diluidor de congelação de sêmen ovino com o flavonoide miricetina contra os danos ocasionados aos espermatozoides. Oito pools de sêmen, obtidos de quatro reprodutores ovinos, foram congelados com diferentes concentrações de miricetina (0, 1, 10, 100 e 1000nM). Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, ao potencial de membrana mitocondrial, aos níveis de ROS intracelular, à peroxidação lipídica e à estabilidade de membrana. Amostras tratadas com miricetina 10nM apresentaram menor percentual de células rápidas (P≤0,05), quando comparadas ao grupo miricetina 1000nM. Amostras do grupo controle apresentaram maior (P≤0,05) VAP que o grupo 10nM de miricetina, enquanto amostras criopreservadas com miricetina (10, 100 e 1000nM) evidenciaram maior (P<0,05) BCF, quando comparadas ao grupo controle. O grupo tratado com miricetina 1000nM apresentou maior percentual (P<0,05) de células com peroxidação lipídica, quando comparado ao grupo controle. Em conclusão, a suplementação do diluidor de criopreservação de sêmen ovino com 10 e 100nM de miricetina afeta a cinética espermática sem provocar alterações na estrutura geral do gameta, enquanto 1000nM de miricetina provoca mudanças na cinética associadas à danos peroxidativos.(AU)

The aim of this study was to evaluate the effect of the supplementation of ram semen frozen with extender with the flavonoid myricetin against damage to sperm. Eight pools of semen obtained from four ram breeders, were frozen with different concentrations of myicetin (0, 1, 10, 100 and 1000nM). After thawing, the semen was evaluated for spermatic kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, intracellular ROS levels, lipid peroxidation, and membrane stability. Samples treated with 10nM myricetin preserved a lower percentage of rapid cells (P≤0.05) when compared to the 1000nM myricetin group. Samples from the control group presented higher (P≤0.05) VAP than 10nM group of myricetin, while cryopreserved samples with myicetin (10, 100 and 1000nM) showed greater (P<0.05) BCF, when compared to control group. The group treated with 1000nM myricetin had a higher percentage (P<0.05) of cells with lipid peroxidation, when compared to the control group. In conclusion, supplementation of ram semen cryopreservation extender with 10 and 100nM myricetin affects sperm kinetics, without causing changes in the overall structure of the gamete, while 1000nM myricetin causes changes in the kinetics associated with peroxidative damage.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.(AU)

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.(AU)

Looking at the big picture: understanding how the oviducts dialogue with gametes and the bryo shapes reproductive success

The oviduct is a tubular organ comprising three distinct anatomical regions (the infundibulum, the ampulla and the isthmus) connecting the ovary and the uterus. Oviductal function is regulated by ovarian hormones, gametes, and embryo-derived factors, for optimally facilitating key reproductive events. A crosstalk is established between the oviduct and the gametes and embryo and this dialogue shapes the microenvironment in which gamete transport, fertilization, and early embryonic development occur. This review aims to address each participant in this conversation in a holistic manner by delineating several advances in the field within the greater context of understanding how oviduct-gamete and oviduct-embryo dialogue shape reproductive success and furthermore how this knowledge can be applied in vitro.(AU)

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.

Trypan blue staining is not efficient in determining oocyte viability in Colossoma macropomum and Brycon amazonicus / Coloração com azul de tripan não é eficiente na determinação da viabilidade de oócitos em Colossoma macropomum e Brycon amazonicus

Currently, there is no effective technique to evaluate the quality of oocytes in fish farming in apractical and affordable way. The cell membrane integrity test with the vital dye trypan blue (TB)could be an option. In this study, Colossoma macropomum and Brycon amazonicus oocytes were exposed to different TB concentrations seeking to verify a possible relationship between the results of staining tests and reproductive rates. Oocytes were exposed to concentrations of 0.05, 0.04, 0.03, 0.02 and 0.01% TB for 1 minute and subsequently were evaluated under a stereomicroscope. The percentage of unstained (viable) oocytes from each sample was correlated with fertilization and hatching rates using a linear regression (P>0.05). We observed a weak correlation between the results of the staining tests and the fertilization and hatching rates in both species. TB integrity tests were not effective in predicting spawning viability in C. macropomum and B. amazonicus.(AU)

Atualmente não existem técnicas efetivas para avaliar a qualidade de oócitos na piscicultura deforma prática e acessível. O teste de integridade da membrana celular com corante vital azul detripan (AT) surge como uma alternativa. Nesse estudo, oócitos de Colossoma macropomum e Bryconamazonicus foram expostos a diferentes concentrações de AT, buscando verificar uma possível relação entre os resultados dos testes de coloração e as taxas reprodutivas. Oócitos foram expostos a concentrações de 0,05; 0,04; 0,03; 0,02 e 0,01% de AT durante 1 minuto e, posteriormente avaliados em estereomicroscópio. A porcentagem de oócitos não corados (intactos) de cada amostra foicorrelacionada com as taxas de fertilização e eclosão utilizando análise de regressão linear (P>0,05). Fraca correlação entre resultados dos tratamentos e as taxas de fertilização e eclosão nas duas espécies foi observada. Os testes de integridade com AT foram ineficazes em predizer o sucesso da desova em C. macropomum e B. amazonicus(AU)

Trypan blue staining is not efficient in determining oocyte viability in Colossoma macropomum and Brycon amazonicus / Coloração com azul de tripan não é eficiente na determinação da viabilidade de oócitos em Colossoma macropomum e Brycon amazonicus

Currently, there is no effective technique to evaluate the quality of oocytes in fish farming in apractical and affordable way. The cell membrane integrity test with the vital dye trypan blue (TB)could be an option. In this study, Colossoma macropomum and Brycon amazonicus oocytes were exposed to different TB concentrations seeking to verify a possible relationship between the results of staining tests and reproductive rates. Oocytes were exposed to concentrations of 0.05, 0.04, 0.03, 0.02 and 0.01% TB for 1 minute and subsequently were evaluated under a stereomicroscope. The percentage of unstained (viable) oocytes from each sample was correlated with fertilization and hatching rates using a linear regression (P>0.05). We observed a weak correlation between the results of the staining tests and the fertilization and hatching rates in both species. TB integrity tests were not effective in predicting spawning viability in C. macropomum and B. amazonicus.

Atualmente não existem técnicas efetivas para avaliar a qualidade de oócitos na piscicultura deforma prática e acessível. O teste de integridade da membrana celular com corante vital azul detripan (AT) surge como uma alternativa. Nesse estudo, oócitos de Colossoma macropomum e Bryconamazonicus foram expostos a diferentes concentrações de AT, buscando verificar uma possível relação entre os resultados dos testes de coloração e as taxas reprodutivas. Oócitos foram expostos a concentrações de 0,05; 0,04; 0,03; 0,02 e 0,01% de AT durante 1 minuto e, posteriormente avaliados em estereomicroscópio. A porcentagem de oócitos não corados (intactos) de cada amostra foicorrelacionada com as taxas de fertilização e eclosão utilizando análise de regressão linear (P>0,05). Fraca correlação entre resultados dos tratamentos e as taxas de fertilização e eclosão nas duas espécies foi observada. Os testes de integridade com AT foram ineficazes em predizer o sucesso da desova em C. macropomum e B. amazonicus