Profiling and Functional Analysis of long non-coding RNAs in yak healthy and atretic follicles

Yak is the livestock on which people live in plateau areas, but its fecundity is low. Follicular development plays a decisive role in yak reproductive performance. As an important regulatory factor, the expression of long non-coding RNA (lncRNAs) in yak follicular development and its regulatory mechanism remains unclear. To explore the differentially expressed lncRNAs between healthy and atretic follicular in yaks. We used RNA-seq to construct lncRNA, miRNA, and mRNA expression profiles in yak atretic and healthy follicles, and the RNA sequence results were identified by qPCR. In addition, the correlation of lncRNA and targeted mRNA was also analyzed by Starbase software. Moreover, lncRNA/miRNA/mRNA networks were constructed by Cytoscape software, and the network was verified by dual-luciferase analysis. A total of 682 novel lncRNAs, 259 bta-miRNAs, and 1704 mRNAs were identified as differentially expressed between healthy and atretic follicles. Among them, 135 mRNAs were positively correlated with lncRNA expression and 97 were negatively correlated, which may be involved in the yak follicular development. In addition, pathway enrichment analysis of differentially expressed lncRNA host genes by Kyoto Genome Encyclopedia (KEGG) showed that host genes were mainly involved in hormone secretion, granulosa cell apoptosis, and follicular development. In conclusion, we identified a series of novel lncRNAs, constructed the lncRNA ceRNA regulatory network, and provided comprehensive resources for exploring the role of lncRNAs in yak ovarian follicular development.(AU)

Copper deficiency in dairy goats and kids / Deficiência de cobre em cabras leiteiras e cabritos

The clinical, pathological and reproductive aspects of an outbreak of copper deficiency in dairy goats and kids from the semiarid region of Pernambuco, Brazil are described. Ten adult dairy goats with clinical signs of deficiency and four kids presenting enzootic ataxia born from copper deficient does were separated from the herd, and examined. In the dairy goats, the average serum concentration of copper was 6.1±2.8mmol/L and iron was 39.5±8.2mmol/L. In kids, the average serum concentration of copper was 3.8±0.9mmol/L and iron was 38.5±4.1mmol/L. Clinical signs in dairy goats consisted of pale mucous membranes, anemia, emaciation, diarrhea, achromotrichia, brittle hair and alopecia. The main reproductive alterations consisted of prolonged anestrus, embryonic resorption and high indices of retained placenta. The kids born from copper deficient dairy goats were weak, and presented neonatal or late ataxia until 70 days of life. Six dairy goats and four kids were necropsied. Most ovaries examined were small, firm and did not present viable follicles on their surface. Microscopically, there was reduction of viable follicles in addition to disorganization of follicular and stromal structures, with marked follicular atresia. Microscopically, changes in kids with enzootic ataxia consisted of neuronal chromatolysis and axonal degeneration, mainly in neurons of the spinal cord. In this study, the source of high iron was not identified, but it is known that outbreaks of copper deficiency can occur due to excess iron intake, mainly when adequate mineral supplementation is not provided for the goat herds.

Descrevem-se os aspectos clínicos, patológicos e reprodutivos de um surto de deficiência de cobre em cabras leiteiras e em cabritos da região semiárida de Pernambuco, Brasil. Dez cabras leiteiras adultas com sinais clínicos de deficiência de cobre e quatro cabritos com ataxia enzoótica nascidos dessas cabras foram separados do rebanho e examinados. Nas cabras leiteiras, a concentração média de cobre sérico foi de 6,1±2,8mmol/L e a concentração média de ferro sérico foi de 39,5±8,2mmol/L. Nos cabritos, a concentração média de cobre sérico foi de 3,8±0,9mmol/L e a concentração média de ferro sérico foi de 38,5±4,1mmol/L. Os sinais clínicos nas cabras consistiram em mucosas pálidas, anemia, diarreia, acromotriquia, pelos opacos e quebradiços e alopecia. As principais alterações reprodutivas consistiram em anestro prolongado, reabsorção embrionária e aumento da taxa de retenção de placenta. Os cabritos nascidos vivos dessas cabras nasceram fracos, com ataxia neonatal ou desenvolveram ataxia tardia em até 70 dias de vida. Seis cabras leiteiras e quatro cabritos foram necropsiados. Nas cabras, a maioria dos ovários examinados macroscopicamente eram pequenos, firmes e não apresentavam folículos viáveis em sua superfície. Microscopicamente, houve redução de folículos viáveis, além de desorganização das estruturas foliculares, com marcada atresia folicular. Macroscopicamente não foram observadas alterações nos cabritos com ataxia enzoótica. As lesões histológicas observadas nesses cabritos consistiram em cromatólise neuronal e degeneração axonal, principalmente em neurônios da medula espinhal. Neste estudo a fonte dos altos teores de ferro no soro não foi identificada, mas sabe-se que surtos de deficiência de cobre podem ocorrer devido ao excesso de ingestão de ferro, principalmente quando não é fornecida suplementação mineral adequada para os rebanhos caprinos.

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.

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.

Dermatomiosite canina-símile em cão sem raça definida: relato de caso / Canine dermatomyositis-like disease in a mixed-breed dog: case report

A dermatomiosite canina é uma vasculopatia inflamatória da pele e músculos, com manifestações cutâneas envolvendo a face, orelhas, extremidades da cauda e distais por sobre proeminências ósseas. O envolvimento muscular manifesta-se sob a forma de mioatrofia cefálica, dificuldade de deglutição, redução do reflexo do vômito e marcha atípica. A dermatomiosite canina é classificada em dermatomiosite canina familiar e em forma variante ou dermatomiosite-símile. Neste relato descreve-se um caso de dermatomiosite-símile em cadela de dois anos de idade, sem raça definida, que apresentava necrose isquêmica dos pavilhões auriculares e atrofia dos músculos temporal e masseter. O exame dermo-histopatológico realizado em fragmentos de pele dos pavilhões auriculares, região cefálica e amostras de tecido muscular (temporal e masseter) confirmou a existência de dermatite perivascular e foliculite de interface pobre em células, focos de atrofia folicular e de fibras musculares e moderada fibrose dérmica. Portanto a histopatologia, associada às manifestações clínicas do animal, permitiu o estabelecimento do diagnóstico de dermatomiosite-símile, enfermidade pouco descrita na dermatologia veterinária brasileira.(AU)

Canine dermatomyositis is a skin and muscles inflammatory vasculopathy with cuta-neous manifestations involving face, ears, tail and distal ends over bony prominences. Mus-cle involvement manifests itself in the form of muscles of the head, difficulty in swallowing, reduction of reflex of vomit and atypical gait. Canine dermatomyositis is classified into variant form or dermatomyositis-like. This report describes a case of dermatomyositis-like in a two-year-old femalemixed-breed dog that presented ischemic necrosis of pinna and temporal and masseter muscle atrophy. In histopathological examinations performed with skin fragments of pinna, cephalic region and muscle samples (temporal and masseter), it was detected a perivascular dermatitis and folliculitis with poor cell interface, foci of follicular atrophy and muscle fibers with moderate dermal fibrosis confirming the diagnosis of derma-tomyositis-like, relatively little described in brazilian veterinary dermatology.(AU)

Dermatomiosite canina-símile em cão sem raça definida: relato de caso / Canine dermatomyositis-like disease in a mixed-breed dog: case report

A dermatomiosite canina é uma vasculopatia inflamatória da pele e músculos, com manifestações cutâneas envolvendo a face, orelhas, extremidades da cauda e distais por sobre proeminências ósseas. O envolvimento muscular manifesta-se sob a forma de mioatrofia cefálica, dificuldade de deglutição, redução do reflexo do vômito e marcha atípica. A dermatomiosite canina é classificada em dermatomiosite canina familiar e em forma variante ou dermatomiosite-símile. Neste relato descreve-se um caso de dermatomiosite-símile em cadela de dois anos de idade, sem raça definida, que apresentava necrose isquêmica dos pavilhões auriculares e atrofia dos músculos temporal e masseter. O exame dermo-histopatológico realizado em fragmentos de pele dos pavilhões auriculares, região cefálica e amostras de tecido muscular (temporal e masseter) confirmou a existência de dermatite perivascular e foliculite de interface pobre em células, focos de atrofia folicular e de fibras musculares e moderada fibrose dérmica. Portanto a histopatologia, associada às manifestações clínicas do animal, permitiu o estabelecimento do diagnóstico de dermatomiosite-símile, enfermidade pouco descrita na dermatologia veterinária brasileira.

Canine dermatomyositis is a skin and muscles inflammatory vasculopathy with cuta-neous manifestations involving face, ears, tail and distal ends over bony prominences. Mus-cle involvement manifests itself in the form of muscles of the head, difficulty in swallowing, reduction of reflex of vomit and atypical gait. Canine dermatomyositis is classified into variant form or dermatomyositis-like. This report describes a case of dermatomyositis-like in a two-year-old femalemixed-breed dog that presented ischemic necrosis of pinna and temporal and masseter muscle atrophy. In histopathological examinations performed with skin fragments of pinna, cephalic region and muscle samples (temporal and masseter), it was detected a perivascular dermatitis and folliculitis with poor cell interface, foci of follicular atrophy and muscle fibers with moderate dermal fibrosis confirming the diagnosis of derma-tomyositis-like, relatively little described in brazilian veterinary dermatology.

Diagnóstico ultrassonográfico de distúrbios reprodutivos em pequenos ruminantes / Ultrasonographic diagnosis of reproductive disorders in small ruminants

A ultrassonografia (USG) é uma técnica de imagem em tempo real e pouco invasiva. Na produção de ovinos e caprinos, a USG vem sendo utilizada como uma ferramenta auxiliar no manejo reprodutivo, assim como no diagnóstico clínico-reprodutivo de doenças ginecológicas, obstétricas e andrológicas. Por meio da USG é possível a realização de exames em série para monitorar a progressão de uma anormalidade, resposta ao tratamento ou a funcionalidade e morfologia do órgão. Assim, por meio da USG são fornecidas informações substanciais ao profissional do campo e da pesquisa auxiliando na definição de prognósticos, tratamentos, descarte zootécnico e, ainda, na aquisição de reprodutores e matrizes sem distúrbios reprodutivos adquiridos ou congênitos.(AU)

Ultrasonography (USG) is a less invasive, real-time imaging modality. In the sheep and goat farming, USG has been used as an auxiliary tool in the reproductive management, as well as in the clinical-reproductive diagnosis of gynecological, obstetric and andrological disorders. Through the USG, it is possible to perform successive evaluations to monitor the progression of an abnormality, response to treatment or organ function and morphology. Therefore, through USG are provide substantial information to the practical and research veterinarian and assists in the definition of prognostics, treatments, culling and on the acquisition of breeding males and females without acquired or congenital reproductive disorders.(AU)

Diagnóstico ultrassonográfico de distúrbios reprodutivos em pequenos ruminantes / Ultrasonographic diagnosis of reproductive disorders in small ruminants

A ultrassonografia (USG) é uma técnica de imagem em tempo real e pouco invasiva. Na produção de ovinos e caprinos, a USG vem sendo utilizada como uma ferramenta auxiliar no manejo reprodutivo, assim como no diagnóstico clínico-reprodutivo de doenças ginecológicas, obstétricas e andrológicas. Por meio da USG é possível a realização de exames em série para monitorar a progressão de uma anormalidade, resposta ao tratamento ou a funcionalidade e morfologia do órgão. Assim, por meio da USG são fornecidas informações substanciais ao profissional do campo e da pesquisa auxiliando na definição de prognósticos, tratamentos, descarte zootécnico e, ainda, na aquisição de reprodutores e matrizes sem distúrbios reprodutivos adquiridos ou congênitos.

Ultrasonography (USG) is a less invasive, real-time imaging modality. In the sheep and goat farming, USG has been used as an auxiliary tool in the reproductive management, as well as in the clinical-reproductive diagnosis of gynecological, obstetric and andrological disorders. Through the USG, it is possible to perform successive evaluations to monitor the progression of an abnormality, response to treatment or organ function and morphology. Therefore, through USG are provide substantial information to the practical and research veterinarian and assists in the definition of prognostics, treatments, culling and on the acquisition of breeding males and females without acquired or congenital reproductive disorders.

Growth hormone on ovarian morphology of lambaris (Astyanax bimaculatus) after induced spawning / Hormônio de crescimento sobre a morfologia ovariana de lambaris (Astyanax bimaculatus) após desova induzida

Lambari, Astyanax bimaculatus, is an oviparous, multiple-spawning fish that is reproductively active throughout the year, which makes it promising for cultivation and research. This research histologically evaluates the ovaries of lambari that have undergone artificial spawning induced with pituitary extract (control group), and the effect of growth hormone at a dose of 2 mg/g body weight (treatment group) on the subsequent process of ovarian recovery. Ovaries of fish in both the control and treatments groups were collected at 120 hours after spawning and analyzed using optical microscopy to characterize the average quantities of: follicles in different stages of development, post-ovulatory follicles, follicular atresia and granulocytes. Quantity and morphology of early and advanced primary follicles did not differ between the treatment and control groups; an important and necessary factor for ovarian recovery for subsequent spawning. There was a greater amount of granulocytes in initial atresia in the group treated with growth hormone. These results demonstrated that the administration of growth hormone may potentiate the process of ovarian recovery after induced spawning.(AU)

O Lambari Astyanax bimaculatus é um peixe ovíparo de desova múltipla que é reprodutivamente ativo durante todo o ano, o que o torna promissor para cultivo e pesquisa. Este trabalho avalia histologicamente os ovários de lambaris submetidos à desova artificial, induzida pelo extrato hipofisário (grupo controle) e o efeito do hormônio de crescimento na concentração de 2 g/g de massa corporal (grupo tratamento) no subsequente processo de recuperação ovariana. Os ovários dos peixes dos grupos controle e tratamento foram coletados às 120 horas após a desova e analisados em microscopia óptica para caracterizar as quantidades médias de: folículos em diferentes estágios de desenvolvimento, folículos pós-ovulatórios, atresia folicular e granulócitos. A quantidade e a morfologia dos folículos primários iniciais e avançados não diferiram entre os grupos tratamento e controle; um fator importante e necessário para a recuperação dos ovários para posterior desova. Houve maior quantidade de granulócitos na atresia inicial no grupo tratado com hormônio de crescimento. Esses resultados demonstram que a administração do hormônio do crescimento pode potencializar o processo de recuperação ovariana após a desova induzida.(AU)

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Morfometria e população de folículos pré-antrais de vacas Nelore vazias, gestantes e de fetos nos terços inicial, médio e final da gestação / Morphometry and population of preantral follicles of not pregnant Nelore cows, pregnant and fetus in the initial, medium and final third of pregnancy

The aim of this study was to describe population and morphological characteristics of preantral follicles of not pregnant cows, pregnant cows and fetus. Ten ovaries of non-pregnant Nelore cows, eighteen ovaries of pregnant cows and eighteen ovaries of fetus were used. For pregnant cows, six ovaries from each third (initial, middle and final) were evaluated, acquired from a slaughterhouse. For fetus, the same methodology and proportion of ovaries were used. Ovaries were washed, fixed and embedded in paraffin. They were then sectioned in longitudinal sections and stained by the Hematoxylin-Eosin method. Preantral follicles were classified according to morphology (primordial, primary and secondary) and degree of viability (intact and in initial, moderate and marked atresia). Descriptive and statistical analyzes were performed through KS300 image analysis program and Tukey's test. A greater proportion of primordial follicles were found in all categories. Secondary follicles were not observed in ovaries of fetus and cows in the initial third of pregnancy. All the ovary dimensions were higher in non-pregnant cows and in the final third of cows' pregnancy, and lower in final third of pregnancy fetus. It was concluded that follicle isolation was effective in describing population and morphological characteristics of preantral follicles of cows and fetus.(AU)

Morfometria e população de folículos pré-antrais de vacas Nelore vazias, gestantes e de fetos nos terços inicial, médio e final da gestação / Morphometry and population of preantral follicles of not pregnant Nelore cows, pregnant and fetus in the initial, medium and final third of pregnancy

The aim of this study was to describe population and morphological characteristics of preantral follicles of not pregnant cows, pregnant cows and fetus. Ten ovaries of non-pregnant Nelore cows, eighteen ovaries of pregnant cows and eighteen ovaries of fetus were used. For pregnant cows, six ovaries from each third (initial, middle and final) were evaluated, acquired from a slaughterhouse. For fetus, the same methodology and proportion of ovaries were used. Ovaries were washed, fixed and embedded in paraffin. They were then sectioned in longitudinal sections and stained by the Hematoxylin-Eosin method. Preantral follicles were classified according to morphology (primordial, primary and secondary) and degree of viability (intact and in initial, moderate and marked atresia). Descriptive and statistical analyzes were performed through KS300 image analysis program and Tukey's test. A greater proportion of primordial follicles were found in all categories. Secondary follicles were not observed in ovaries of fetus and cows in the initial third of pregnancy. All the ovary dimensions were higher in non-pregnant cows and in the final third of cows' pregnancy, and lower in final third of pregnancy fetus. It was concluded that follicle isolation was effective in describing population and morphological characteristics of preantral follicles of cows and fetus.(AU)

Evaluation of buffaloes' follicular dynamics and stress state under different ovulation synchronization protocols

The aims of the present study were to analyze the effect of different hormonal protocols using an implant containing Norgestomet, at the ovulation synchronization in buffaloes and to verify the effect of the stress caused by manipulation of the herd during the experiment. Twenty-four female Murrah breed buffaloes, lactating, multiparous, aged from three to eight years, with a body condition score of 3.5 or higher (0 to 5) and with more than 45 days post partum, were used. These females were randomly distributed into one of the three groups (group 1, group 2 and group 3) with eight subjects in each. On day 0 (day 0), all animals received an ear progesterone implant with 3.0 mg of Norgestomet and an intramuscular (IM) injection with 2.0 mg of estradiol benzoate (EB). The implants were removed after nine days (day 9) and one single dose of PGF2α (0.15 mg d-cloprostenol, IM) was administered to all animals. On the same day, the group ́1 and group 3 buffaloes were treated with 500 UI of eCG (IM). Two days later (day 11), 1000UI of hCG (IM) were administered to the group 1 and group 2 buffaloes. After the implant had been removed, an ultrasound evaluation was performed every 12 h, in order to access the ovarian follicular dynamics, using an Aloka 500 equipment with a 5 MHz transrectal probe. Blood samples were also taken on days 0, 9 and 11 to determine the plasmatic concentrations of cortisol and progesterone. No difference was observed in the ovulation rate (group 1: 62.5%, group 2: 50%, group 3: 75%). However, the size of the preovulatory follicles and the plasmatic concentration of cortisol had (P <0.05). G3 was the most efficient group in promoting the ovulation synchronization, which suggests that this protocol can be used in Fixed Timed Artificial Insemination programs (FTAI) among postpartum, lactating Murrah breed buffaloes’.

Evaluation of buffaloes' follicular dynamics and stress state under different ovulation synchronization protocols

The aims of the present study were to analyze the effect of different hormonal protocols using an implant containing Norgestomet, at the ovulation synchronization in buffaloes and to verify the effect of the stress caused by manipulation of the herd during the experiment. Twenty-four female Murrah breed buffaloes, lactating, multiparous, aged from three to eight years, with a body condition score of 3.5 or higher (0 to 5) and with more than 45 days post partum, were used. These females were randomly distributed into one of the three groups (group 1, group 2 and group 3) with eight subjects in each. On day 0 (day 0), all animals received an ear progesterone implant with 3.0 mg of Norgestomet and an intramuscular (IM) injection with 2.0 mg of estradiol benzoate (EB). The implants were removed after nine days (day 9) and one single dose of PGF2α (0.15 mg d-cloprostenol, IM) was administered to all animals. On the same day, the group ́1 and group 3 buffaloes were treated with 500 UI of eCG (IM). Two days later (day 11), 1000UI of hCG (IM) were administered to the group 1 and group 2 buffaloes. After the implant had been removed, an ultrasound evaluation was performed every 12 h, in order to access the ovarian follicular dynamics, using an Aloka 500 equipment with a 5 MHz transrectal probe. Blood samples were also taken on days 0, 9 and 11 to determine the plasmatic concentrations of cortisol and progesterone. No difference was observed in the ovulation rate (group 1: 62.5%, group 2: 50%, group 3: 75%). However, the size of the preovulatory follicles and the plasmatic concentration of cortisol had (P <0.05). G3 was the most efficient group in promoting the ovulation synchronization, which suggests that this protocol can be used in Fixed Timed Artificial Insemination programs (FTAI) among postpartum, lactating Murrah breed buffaloes.(AU)

Histomorphometric analysis of gonads of green turtles (chelonia mydas) stranded on the coast of Espírito Santo state, Brazil / Análise histomorfométrica das gônadas de tartarugas verdes (Chelonia Mydas) provenientes de encalhe no litoral do Espírito Santo, Brasil

Studies on reproduction in sea turtles are important due to its life cycle, migratory patterns, high juvenile mortality and environmental impacts. This study aimed to analyse histomorphometrically gonads of C. mydas from the coastline of the Espírito Santo State, Brazil. Ovaries and testicles were collected between 2014 and 2015 from stranded animals. The material was fixed in formalin 10%, assessed macroscopically and processed for histomorphometrical evaluation. Gonads from 34 individuals were evaluated, twenty-four females and ten males. Macroscopic sexual identification presented 100% accuracy confirmed by histology. Sexual dimorphism was observed in one individual, which was considered as adult (CCL=1.023 m). Microscopy of female gonads revealed predominant previtellogenic follicles; oocyte diameter ranged between 161µm and 750µm and a positive correlation between ovarian length, largest oocyte and CCL was found. In males, autolysis was verified in five individuals. Viable testicles revealed predominant spermatogonia, primary spermatocytes and Sertoli cells in the seminiferous tubules and, Leydig cells and fibroblasts in the stroma. There was a positive correlation between tubular diameter and CCL and testicle length and CCL. Maturation of stromal tissue and a positive correlation between tubular lumen and CCL were also observed. Gonad development is proportional to individual growth.(AU)

Estudos em reprodução de tartarugas marinhas são importantes devido ao ciclo de vida, ao padrão migratório, à alta mortalidade juvenil e aos impactos ambientais. Objetivou-se analisar histomorfometricamente gônadas de C. mydas no litoral do Espírito Santo. Foram coletados ovários e testículos dessa espécie, entre 2014 e 2015. O material foi fixado em formol a 10% e avaliado macroscopicamente. Em seguida, foi processado para avaliação histomorfométrica. Foram avaliadas gônadas de 34 indivíduos, 24 fêmeas e 10 machos. Verificaram-se 100% de acurácia na identificação sexual à macroscopia, confirmada pela histologia. Observou-se dimorfismo sexual em um macho, que foi considerado adulto (CCC=1,023m). A microscopia dos ovários revelou folículos pré-vitelogênicos, cujos ovócitos apresentaram diâmetro médio entre 161µm e 750µm. Houve correlação positiva entre comprimento ovariano e diâmetro do maior ovócito e CCC. Nos machos, verificou-se autólise em cinco indivíduos. Os testículos viáveis revelaram espermatogônias, espermatócitos primários e células de Sertoli nos túbulos seminíferos, além de células de Leydig e fibroblastos no estroma. Houve correlação positiva entre diâmetro tubular e CCC e comprimento testicular e CCC. Verificou-se maturação do tecido estromal e correlação positiva entre o diâmetro do lúmen tubular e o CCC. Verifica-se que o desenvolvimento das gônadas é proporcional ao crescimento dos indivíduos.(AU)

Histomorphometric analysis of gonads of green turtles (chelonia mydas) stranded on the coast of Espírito Santo state, Brazil / Análise histomorfométrica das gônadas de tartarugas verdes (Chelonia Mydas) provenientes de encalhe no litoral do Espírito Santo, Brasil

Studies on reproduction in sea turtles are important due to its life cycle, migratory patterns, high juvenile mortality and environmental impacts. This study aimed to analyse histomorphometrically gonads of C. mydas from the coastline of the Espírito Santo State, Brazil. Ovaries and testicles were collected between 2014 and 2015 from stranded animals. The material was fixed in formalin 10%, assessed macroscopically and processed for histomorphometrical evaluation. Gonads from 34 individuals were evaluated, twenty-four females and ten males. Macroscopic sexual identification presented 100% accuracy confirmed by histology. Sexual dimorphism was observed in one individual, which was considered as adult (CCL=1.023 m). Microscopy of female gonads revealed predominant previtellogenic follicles; oocyte diameter ranged between 161µm and 750µm and a positive correlation between ovarian length, largest oocyte and CCL was found. In males, autolysis was verified in five individuals. Viable testicles revealed predominant spermatogonia, primary spermatocytes and Sertoli cells in the seminiferous tubules and, Leydig cells and fibroblasts in the stroma. There was a positive correlation between tubular diameter and CCL and testicle length and CCL. Maturation of stromal tissue and a positive correlation between tubular lumen and CCL were also observed. Gonad development is proportional to individual growth.(AU)

Estudos em reprodução de tartarugas marinhas são importantes devido ao ciclo de vida, ao padrão migratório, à alta mortalidade juvenil e aos impactos ambientais. Objetivou-se analisar histomorfometricamente gônadas de C. mydas no litoral do Espírito Santo. Foram coletados ovários e testículos dessa espécie, entre 2014 e 2015. O material foi fixado em formol a 10% e avaliado macroscopicamente. Em seguida, foi processado para avaliação histomorfométrica. Foram avaliadas gônadas de 34 indivíduos, 24 fêmeas e 10 machos. Verificaram-se 100% de acurácia na identificação sexual à macroscopia, confirmada pela histologia. Observou-se dimorfismo sexual em um macho, que foi considerado adulto (CCC=1,023m). A microscopia dos ovários revelou folículos pré-vitelogênicos, cujos ovócitos apresentaram diâmetro médio entre 161µm e 750µm. Houve correlação positiva entre comprimento ovariano e diâmetro do maior ovócito e CCC. Nos machos, verificou-se autólise em cinco indivíduos. Os testículos viáveis revelaram espermatogônias, espermatócitos primários e células de Sertoli nos túbulos seminíferos, além de células de Leydig e fibroblastos no estroma. Houve correlação positiva entre diâmetro tubular e CCC e comprimento testicular e CCC. Verificou-se maturação do tecido estromal e correlação positiva entre o diâmetro do lúmen tubular e o CCC. Verifica-se que o desenvolvimento das gônadas é proporcional ao crescimento dos indivíduos.(AU)

Avanços, aplicações, limitações e perspectivas da tecnologia do ovário artificial de ruminantes / Advances, applications, limitations and prospects of the technology of artificial ovary in ruminant

A maioria dos oócitos imaturos nos ovários encontra-se armazenada nos folículos pré-antrais, sendo este a principal reserva ovariana. Entretanto, durante o desenvolvimento folicular uma pequena proporção (0,1%) destes folículos chega ao estágio pré-ovulatório sendo o restante eliminado por atresia. A tecnologia de cultivo folicular in vitro, denominada ovário artificial, representa uma excelente ferramenta para desvendar o controle da foliculogênese nos estágios iniciais de desenvolvimento, bem como poderá assegurar no futuro a utilização de um grande número de oócitos imaturos, previamente crescidos in vitro, em técnicas de reprodução assistida em humanos e em outras espécies, incluindo os ruminantes. Os melhores resultados da tecnologia do ovário artificial foram relatados em camundongos, com a produção de crias vivas a partir de folículos primordiais crescidos in vitro. Entretanto, em outras espécies, incluindo os ruminantes, os resultados têm sido restritos à produção de um pequeno e variável número de oócitos maturos e uma baixa taxa de produção de embriões após o cultivo in vitro de folículos secundários isolados. A presente revisão discute as aplicações, critérios para avaliação de eficiência, estado atual, limitações e perspectivas da tecnologia do ovário artificial em ruminantes, com ênfase nas espécies bovina, caprina e ovina.(AU)

Most immature oocytes in the ovaries are stored in the preantral follicles, which represent the main ovarian reserve. However, during follicular development, a small proportion (0.1%) of these follicles reaches the preovulatory stage, the rest being eliminated by atresia. The in vitro follicle culture technology, called artificial ovary, represents an excellent tool to understand the control of folliculogenesis in the early stages of development, as well as to ensure in the future the use of a large number of immature oocytes, previously grown in vitro, in assisted reproductive technologies in humans and other species, including ruminants. The best results from artificial ovary technology so far were reported in mice, with the production of live offspring from primordial follicles grown in vitro. However, in other species, including ruminants, the results have been limited to the production of a small and variable number of mature oocytes and a low rate of embryo production after in vitro culture of isolated secondary follicles. The present review discusses the applications, criteria for evaluating efficiency, current status, limitations and perspectives of artificial ovary technology in ruminants, with emphasis on bovine, caprine, and ovine species.(AU)

Avanços, aplicações, limitações e perspectivas da tecnologia do ovário artificial de ruminantes / Advances, applications, limitations and prospects of the technology of artificial ovary in ruminant

A maioria dos oócitos imaturos nos ovários encontra-se armazenada nos folículos pré-antrais, sendo este a principal reserva ovariana. Entretanto, durante o desenvolvimento folicular uma pequena proporção (0,1%) destes folículos chega ao estágio pré-ovulatório sendo o restante eliminado por atresia. A tecnologia de cultivo folicular in vitro, denominada ovário artificial, representa uma excelente ferramenta para desvendar o controle da foliculogênese nos estágios iniciais de desenvolvimento, bem como poderá assegurar no futuro a utilização de um grande número de oócitos imaturos, previamente crescidos in vitro, em técnicas de reprodução assistida em humanos e em outras espécies, incluindo os ruminantes. Os melhores resultados da tecnologia do ovário artificial foram relatados em camundongos, com a produção de crias vivas a partir de folículos primordiais crescidos in vitro. Entretanto, em outras espécies, incluindo os ruminantes, os resultados têm sido restritos à produção de um pequeno e variável número de oócitos maturos e uma baixa taxa de produção de embriões após o cultivo in vitro de folículos secundários isolados. A presente revisão discute as aplicações, critérios para avaliação de eficiência, estado atual, limitações e perspectivas da tecnologia do ovário artificial em ruminantes, com ênfase nas espécies bovina, caprina e ovina.

Most immature oocytes in the ovaries are stored in the preantral follicles, which represent the main ovarian reserve. However, during follicular development, a small proportion (0.1%) of these follicles reaches the preovulatory stage, the rest being eliminated by atresia. The in vitro follicle culture technology, called artificial ovary, represents an excellent tool to understand the control of folliculogenesis in the early stages of development, as well as to ensure in the future the use of a large number of immature oocytes, previously grown in vitro, in assisted reproductive technologies in humans and other species, including ruminants. The best results from artificial ovary technology so far were reported in mice, with the production of live offspring from primordial follicles grown in vitro. However, in other species, including ruminants, the results have been limited to the production of a small and variable number of mature oocytes and a low rate of embryo production after in vitro culture of isolated secondary follicles. The present review discusses the applications, criteria for evaluating efficiency, current status, limitations and perspectives of artificial ovary technology in ruminants, with emphasis on bovine, caprine, and ovine species.

The life and death of the dominant follicle

Much work has been conducted over the years to determine the major factors that control follicle growth, including the role of FSH, LH and IGF1. These factors permit the dominant follicle to grow while subordinate follicles regress. The dominant follicle enters a phase of growth, and then that growth slows as the follicle reaches maximum size. The dominant follicle remains morphologically larger for a few days in the static phase, before starting to regress with the loss of functional dominance. Few studies have addressed the factors that determine follicle fate during the static phase. In this review, we summarize the differences in gene expression between growing and non-growing (static or early regressing) dominant follicles, highlighting areas that require further study. Potential factors that may help survival of the dominant follicle include IGF1, estradiol and BMP4/BMP7, and intrafollicular factors that likely initiate regression and apoptosis include FGF18 and AMH acting through FASLG. It is also very likely that the influence of microRNAs, especially miR-21, play a role in determining the fate of the dominant follicle.(AU)