Efeito do cilostazol na produção in vitro de ovinos / Effect of cilostazol on in vitro production of sheep / Efecto del cilostazol en la producción in vitro de ovejas

O objetivo deste estudo foi avaliar o efeito da inclusão de cilostazol no meio de maturação in vitro de oócitos sobre produção in vitro de embriões ovinos. Para isso, foram realizadas colheitas de oócitos oriundos de ovários obtidos em abatedouro por meio do método de aspiração folicular com bomba de vácuo. Os oócitos foram divididos em quatro grupos de maturação: grupo CON, onde os complexos cumulus oócitos foram imersos em TCM-199, suplementado com 500 UI de penicilina, 0,5 mg de estreptomicina, 1,25 µg de anfotericina, 0,2 mM de piruvato de sódio, 10% (v/v) de soro fetal bovino (SFB), 10 ng/mL de fator de crescimento epidérmico (EGF), 10 ug/m de FSH, 10 µg/mL de LH, 10 ug/mL de estradiol e 100 µM de cisteamina; e nos grupos CILO0,3; CILO1 e CILO10, os oócitos foram maturados no meio do grupo CON, mas sem a adição de cisteamina e suplementado com as concentrações de 0,3; 1 e 10 µM, respectivamente. Após 24h, os oócitos foram avaliados quanto a presença ou não de células do cumulus e quanto ao grau de expansão e destinados à fecundação in vitro, em meio FIV, juntamente com espermatozoides. Após a FIV, os presumíveis zigotos seguiram para o cultivo in vitro. Foram avaliadas clivagens no dia 2, sendo dia 0 o dia do início do CIV. Os resultados foram expressos em porcentagem e as variáveis de expansão das células do cumulus e número de estruturas clivadas foram comparadas por meio do teste qui-quadrado do software Epi Info (Epi Info 7.2.5, Atlanta, GA, EUA, 2021). Os resultados foram considerados significativos quando P<0,05. Em relação à expansão das células do cumulus, todos os grupos apresentaram 100% de expansão. Não houve diferenças significativas quanto ao grau de expansão das células do cumulus entre os grupos suplementados com cilostazol e cisteamina (P>0,05), assim como não houve diferenças significativas entre as taxas de clivagem entre os grupos suplementados com cilostazol e cisteamina (P > 0,05).

The objective of this study was to evaluate the effect of including cilostazol in the in vitro maturation medium of oocytes on the in vitro production of sheep embryos. Oocytes were collected from ovaries obtained from a slaughterhouse by follicular aspiration with a vacum pump. The oocytes were divided into four maturation groups: the CON group, where the cumulus-oocyte complexes were immersed in TCM-199 supplemented with 500 IU of penicillin, 0.5 mg of streptomycin, 1.25 µg of amphotericin, 0.2 mM of sodium pyruvate, 10% (v/v) fetal bovine serum (FBS), 10 ng/mL of epidermal growth factor (EGF), 10 µg/mL of FSH, 10 µg/mL of LH, 10 µg/mL of estradiol, and 100 µM of cysteamine; and in the CILO0.3, CILO1, and CILO10 groups, the oocytes were matured in the CON group medium without the addition of cysteamine and supplemented with concentrations of 0.3, 1, and 10 µM of cilostazol, respectively. After 24 hours, the oocytes were evaluated for the presence or absence of cumulus cells and the degree of expansion and then subjected to in vitro fertilization (IVF) with sperm in FIV medium. After IVF, the presumptive zygotes were cultured in vitro. Cleavage was evaluated on day 2, with day 0 being the start of IVF. Results were expressed as a percentage, and variables such as cumulus cell expansion and the number of cleaved structures were compared using the chi-square test in the Epi Info software (Epi Info 7.2.5, Atlanta, GA, USA, 2021). Results were considered significant when P < 0.05. All groups showed 100% cumulus cell expansion, and there were no significant differences in cumulus cell expansion degree between the cilostazol- and cysteamine-supplemented groups (P > 0.05), as well as no significant differences in cleavage rates between the cilostazol- and cysteamine-supplemented groups (P > 0.05).

El objetivo de este estudio fue evaluar el efecto de la inclusión de cilostazol en el medio de maduración in vitro de ovocitos sobre la producción in vitro de embriones ovinos. Para ello, se realizaron recolecciones de ovocitos provenientes de ovarios obtenidos en un matadero mediante el método de aspiración folicular con bomba de vacío. Los ovocitos se dividieron em cuatro grupos de maduración: grupo CON, donde los complejos cúmulus ovocitos se sumergieron en TCM-199, suplementado con 500 UI de penicilina, 0,5 mg de estreptomicina, 1,25 ug de anfotericina, 0,2 mM de piruvato de sodio, 10% (v/v) de suero fetal bovino (SFB), 10 ng/mL de factor de crecimiento epidérmico (EGF), 10 ug/m de FSH, 10 µg/mL de LH, 10 µg/mL de estradiol y 100 µM de cisteamina; y en los grupos CILO0,3; CILO1 y CILO10, los ovocitos se maduraron en el medio del grupo CON, pero sin la adición de cisteamina y suplementado con las concentraciones de 0,3; 1 y 10 µM, respectivamente. Después de 24 horas, los ovocitos se evaluaron en cuanto a la presencia o no de células del cúmulus y em cuanto al grado de expansión y se destinaron a la fecundación in vitro, en medio FIV, junto con espermatozoides. Después de la FIV, los presuntos cigotos siguieron para el cultivo in vitro. Se evaluaron las clivajes en el día 2, siendo el día 0 el día del início del CIV. Los resultados se expresaron en porcentaje y las variables de expansión de las células del cúmulos y número de estructuras clivadas se compararon mediante la prueba del chi-cuadrado del software Epi Info (Epi Info 7.2.5, Atlanta, GA, EE. UU., 2021). Los resultados se consideraron significativos cuando P < 0,05. En relación a la expansión de las células del cúmulus, todos los grupos presentaron el 100% de expansión. No hubo diferencias significativas en cuanto al grado de expansión de las células del cúmulus entre los grupos suplementados con cilostazol y cisteamina (P > 0.05), así como no hubo diferencias significativas entre las tasas de clivaje entre los grupos suplementados con cilostazol y cisteamina (P>0,05).

Influence of Media Supplementation with Alpha Tocopherol and/or Epigallocatechin Gallate on in vitro Maturation and Subsequent Fertilization of Bovine Oocytes

The present study was planned to determine the effect of Epigallocatechin Gallate (EGCG), Alpha tocopherol and their combination as an antioxidant in TCM-199 media for in vitro maturation (IVM) and in vitro fertilization (IVF) in bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated from the ovaries derived from slaughter house and in vitro cultured in three groups using TCM-199 supplemented with EGCG @10 μM, Alpha tocopherol @100 μM, and Combination (EGCG @10 μM plus Alpha tocopherol @100 μM). Oocytes of a control group were matured in TCM-199 medium without any treatment. After IVM, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 15–18 h. Compared to no addition, the presence of EGCG @10 μM in medium during IVM significantly (p<0.05) increased the proportion of maturation and fertilization rate. Combination produced significantly higher percentage of in vitro matured bovine oocytes compared to the alpha tocopherol @100 μM alone. The results suggest that EGCG @10 μM in IVM medium had better effect than Alpha tocopherol alone and Combination on in vitro maturation and subsequent fertilization of bovine oocytes.

Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines / Efeito de rolipram na maturação in vitro, expressão gênica e desenvolvimento embrionário em bovinos

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.(AU)

O objetivo deste trabalho foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos, expressão gênica e embriões produzidos in vitro. Os ovários bovinos foram coletados no matadouro. Os COCs foram selecionados e divididos em cinco grupos: controle 0 tempo; controle: MIV por 24 horas; tratamentos rolipram com bloqueio MIV por 24 horas em meio de maturação contendo 100, 150 e 200µM. Após 24 horas, todos os grupos foram recolocados em MIV por mais 24 horas. Subsequentemente COCs foram submetidos ao mesmo sistema MIV e fertilizados, sendo avaliada a taxa de clivagem e de blastocisto, além da expressão dos seguintes genes: Mater, BMP15 e Bax. Nenhuma diferença foi observada na expressão gênica. Dos oócitos avaliados logo após a aspiração folicular, 79,0% estavam em GV, GVBD, MI, enquanto 13,40% estavam em MII, e 7,60% em D/NI. A diferença significativa foi observada em diferentes concentrações (T100, T200 e T150µM) em oócitos que atingiram a fase MII em comparação aos tratamentos de controle (P=0,3). Diferenças foram observadas nas taxas de clivagem (P<0,5) entre T150 e T200 quando comparadas com as taxas do grupo C/24. Uma grande diferença foi observada na taxa de blastocisto (P<0,1) entre os tratamentos em relação ao grupo controle.(AU)

Evaluación clínica de meningioma intraventricular: reporte de caso paciente pediátrico y paciente adulto / Clinical evaluation of intraventricular meningioma: case report of adult and paediatric patient / Avaliação clínica de meningioma intraventricular: relato de caso e paciente adulto e paciente pediátrico

Los Meningiomas intraventriculares (IVM) representan el 0,5-5% de todos los meningiomas, de los cuales aproximadamente un 80% se localizan en el trígono del ventrículo lateral. Se originan en los ventrículos del tope células aracnoideas contenidos en el plexo coroideo constituyen el grupo tumoral intracraneal frecuente en adultos. En la mayor parte de los casos, se trata de tumores histológicamente benignos, de crecimiento lento. Los meningiomas primarios intraventriculares suponen entre el 0,5 y el 3% de los meningiomas intracraneales. No son comunes en población pediátrica ya que comprenden menos del 3% de los tumores cerebrales pediátricos y sólo 1.5 a 1.8% de todas las neoplasias intracraneales. Los tumores pediátricos también muestran una asociación con neurofibromatosis tipo 2 y la exposición anterior a radiación. Se presenta el contraste de dos casos de meningioma intraventriculares en paciente pediátrico y adulto. La edad y el sitio de la lesión en paciente pediátrico es poco común. La escisión de la lesión y tratamiento pronto permitió la cesación de los síntomas en los 2 pacientes.

The intraventricular meningioma (IVM) account for 0.5-5% of all meningiomas, of which approximately 80% are located in the trigone of the lateral ventricle. They originate in the ventricles of content arachnoid cap cells in the choroid plexus are the group common intracranial tumor in adults. In most cases, tumors are histologically benign, slow-growing. Primary intraventricular meningiomas account for between 0.5 and 3% of intracranial meningiomas. They are uncommon in the pediatric population and comprising less than 3% of pediatric brain tumors and only 1.5 to 1.8% of all intracranial neoplasms. Pediatric tumors also show an association with neurofibromatosis type 2 and previous exposure to radiation. We present two contrasting cases of intraventricular meningioma in pediatric and adult patients. Age and site of injury in pediatric patients is rare. The excision of the lesion and prompt treatment allowed the cessation of symptoms in 2 patients.

A conta intraventricular meningiomas (IVM) em 0,5-5% de todos os meningiomas, dos quais cerca de 80% estão situados na trigone do ventrículo lateral. Eles se originam nos ventrículos de células cap aracnóide conteúdo no plexo coróide são o grupo tumor intracraniano comum em adultos. Na maioria dos casos, os tumores são histologicamente benigno, de crescimento lento. Meningiomas intraventriculares primários representam entre 0,5 e 3% dos meningiomas intracranianos. Eles são raras na população pediátrica e compreendendo menos de 3% dos tumores cerebrais pediátricos e apenas 1,5 a 1,8% de todos os tumores intracranianos. Tumores pediátricos também mostram uma associação coma neurofibromatose tipo 2 e exposição prévia à radiação. Apresentamos dois casos contrastantes de meningioma intraventricular em pacientes pediátricos e adultos. Idade e local da lesão em pacientes pediátricos é raro. A excisão do tratamento da lesão ea tempo permitido a cessação dos sintomas em dois pacientes.

Maturation timing and fetal bovine serum concentration for developmental potential of sheep oocytes in vitro.

The success of in vitro embryo production (IVEP) in animals has improved over time, employing a variety of culture media. Here, we assessed the maturation timing and developmental potential of sheep oocytes in vitro at different concentrations of fetal bovine serum (FBS). Cumulus oocyte complexes (COCs) were aspirated from follicles (2-6 mm) of sheep ovaries collected from local slaughter house. COCs were randomly divided into two groups and matured at 38.5°C, 5% CO2 for 24 h (Group I) and 27 h (Group II). Oocytes cultured for 27 h showed significantly (P <0.05) more maturation than those cultured for 24 h (82 vs. 76%) followed by more cleavage (35 vs. 30%), morula (53 vs. 39%) and blastocyst (17 vs. 11%) percentage. In the second experiment, oocytes were randomly divided into two groups and matured with 10% FBS (Group I) and 20% FBS (Group II) for 27 h supplemented with pyruvate, glutamine, LH, FSH and estradiol. After maturation, oocytes were fertilized by fresh semen for 18 h. Presumptive zygotes in both the groups were again divided into two groups and cultured in 10 and 20% FBS during post fertilization period, respectively. Different FBS concentration in maturation medium did not influence maturation percentage (82 vs. 79%) significantly. Out of culture groups, presumptive zygotes matured in 20% FBS and cultured in 20% FBS during post fertilization period showed significant increase in cleavage percentage (44 vs. 39, 35 and 27%) as compared to other groups but subsequent development to morula (55 vs. 53, 43 and 40%) and blastocyst (20 vs. 17, 16 and 15%) percentage were more in the group matured in 10% FBS and cultured in 20% FBS during post fertilization period.

Técnica de fatiamento do ovário para obtenção de oócitos em cutias (Dasyprocta prymnolopha) / Ovarian slicing technique to obtain oocytes from agoutis (Dasyprocta prymnolopha)

As biotécnicas da reprodução são importantes ferramentas para conservação de espécies domésticas e silvestres, pois permitem a recuperação e uso futuro de material reprodutivo. O objetivo deste estudo foi avaliar parâmetros morfológicos de CCOs de cutias, obtidos pela técnica de fatiamento do ovário para utilização em protocolos de maturação e fecundação na produção in vitro de embriões. Foram utilizadas dezessete cutias fêmeas do NEPAS, CCA- UFPI, com idade e peso médios de 3,9 anos e 2,16Kg, respectivamente, que foram submetidas à ovariossalpingohisterectomia. Os ovários após dissecados e pesados em balança de precisão, foram fatiados individualmente. Procedeu-se a busca e seleção dos CCOs em estereomicroscópio, os quais foram identificados e quantificados por cada ovário, além de classificados quanto a sua morfologia segundo a quantidade de camadas de células do cumulus e ao citoplasma em quatro graus. Verificou-se que a técnica de fatiamento do ovário possibilitou a obtenção de CCOs de cutias, com recuperação de grande quantidade e de variados graus de qualidade. Não houve correlações entre a idade dos animais e o peso dos ovários; a idade e o número de CCOs obtidos; entre o peso das cutias e o peso dos ovários e o número de CCOs obtidos.

The reproductive biotechnologies are important tools for conservation of domestic and wild animal species, because they allow the recovery and future use of reproductive material. The aim of this study was to evaluate morphological parameters of COCs of agoutis obtained by slicing the ovary for using them in maturation and fertilization protocols in vitro production of embryos. Seventeen female agoutis NEPAS, CCA-UFPI, age and weight average of 3.9 years and 2.16 kg, respectively, were underwent ovariossalpingohisterectomy. The ovaries after dissected and weighed on a precision balance were sliced individually. We proceeded the search and selection of COCs in stereomicroscope, which were identified and quantified by each ovary, and classified as to their morphology, by the quantity of layers of cumulus cells and the cytoplasm into four degrees. It has been found that the technique of slicing ovarian possible to obtain agouti COCs with recovery of loads and varying degrees of quality. There was no correlation between age and weight of animal ovaries, age and the number of COCs obtained; between the weight of agoutis and weight of ovaries and the number of COCs obtained.

The effect of COH and IVM on mitochondrial membrane potentials and cytoskeleton / 实用医学杂志

Objective To investigate the effect of different maturation methods on mitochondrial functions of oocytes and the possible mechanism. To explore novel ideas for developing assisted reproductive technology (ART). Methods Female mice were used as models and randomly allocated into three groups, COH, IVM and NC control. Oocytes maturated with different methods which were all simulated with those treatments in human IVF cycle. Immunofluorescence were used to measure the mitochondrial membrane potentials and analyze the cy-toskeleton. Results The mitochondrial membrane potential in the COH group was significantly lower than that in NC group and IVM group (P < 0.05). The proportion of normal cytoskeleton including spindle structure and chromosome configuration in the COH group and IVM group were significantly lower than that in the NC group (PCOH < 0.01, PIVM < 0.05). Conclusions Both COH and IVM can affect mitochondrial functions.

Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 µL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn’t affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn’t have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes’ meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39degrees C under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean +/- SD) per prepubertal and pubertal goats were 5.2 +/- 0.6 and 6.8 +/- 0.6, and 77.3 +/- 0.1% and 80.5 +/- 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 +/- 0.8 vs. 2.6 +/- 0.2, p 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 +/- 1.7 vs. 32.2 +/- 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.

Evaluación de la capacidad de desarrollo in vitro de ovocitos bovinos provenientes de vacas con predominancia fenotípica Bos taurus y Bos indicus / Evaluation of in vitro development capacity of bovine oocytes obtained from predominantly Bos taurus and Bos indicus cows

El objetivo de este estudio fue evaluar la capacidad de desarrollo in vitro de ovocitos bovinos de vacas mestizas B. taurus y B. indicus. Los ovocitos fueron recuperados de ovarios de hembras bovinas provenientes de un matadero comercial. Para la obtención de los complejos cumulus-ovocitos (CCO) se realizó la técnica de slicing, seleccionando los ovocitos que tenían al menos una capa de células del cumulus y un citoplasma homogéneo. Los ovocitos seleccionados fueron madurados y fecundados in vitro (MIV-FIV). Se utilizó semen de un toro Brahman puro (B. indicus). Para la evaluación de la MIV y FIV todos los ovocitos se fijaron por al menos 24 h a 4°C en solución metanol-ácido acético (3:1) y teñidos con aceto-orceína al 1,1%. La tasa de maduración de ovocitos de vacas con predominancia fenotípica B. indicus fue del 66,17% mientras que las vacas con predominancia fenotípica B. taurus alcanzaron un 50,94% (P>0,05). En cuanto a la tasa de fecundación se obtuvo un 14,28 y 35,72% de ovocitos penetrados normalmente y anormales, respectivamente, para el grupo de ovocitos con predominancia fenotípica B. indicus. Mientras que para vacas con predominancia fenotípica B. taurus, un 10,22% correspondió a ovocitos penetrados normales y 19,31% de ovocitos penetrados anormales, sin encontrar diferencias estadísticamente significativas en ambos grupos. Los presentes resultados, tanto para la progresión meiótica como para las tasas de fecundación, indican que los ovocitos de vacas mestizas con predominancia fenotípica B. indicus son más competentes en las primeras etapas de desarrollo in vitro que los ovocitos de vacas mestizas con predominancia fenotípica B. taurus.

The aim of this study was to evaluate the in vitro development capacity of bovine oocytes from crossbred B. taurus and B. indicus cows. Oocytes from bovine cows were collected from commercial slaughterhouse. The cumulus-oocyte complex (COC) ovaries were obtained by Slicing technique, selecting those oocytes that had 2 to 3 layers of cumulus cells and homogeneous cytoplasm. After selection oocytes proceed with maturation (IVM) and fertilization in vitro (IVF). It used semen from a pure Brahman bull (B. indicus). For the assessment of IVM as for IVF oocytes were fixed for about 24 hours at 4°C in methanol-acetic acid (3:1) solution and stained with 1.1% aceto-orcein. The maturation rate of oocytes from cows with B. indicus phenotypic predominance was 66.17%, whereas cows with B. taurus phenotypic predominance 50.94% (P>0.05). Fertilization rate obtained in B. indicus phenotypic predominance group was 14.28% of oocytes normal penetrated and abnormal penetrated 35.72%, for cows with a phenotypic predominance B. taurus oocytes normal penetrated were 10.22% and 19.31% of abnormal oocytes penetrated. In conclusion, the present results indicate that oocytes from cows with phenotypic predominance B. indicus are more competent in the early stages of development in vitro than oocytes from cows with phenotypic predominance B. taurus.

Efecto de la suplementación con L-cisteína en la maduración in vitro de ovocitos bovinos / L-cysteine supplementation effect on the in vitro maturation of bovine oocytes

utilizada como herramienta para el estudio de diversos aspectos relacionados con la maduración de ovocitos, la fecundación y el desarrollo temprano del embrión en condiciones in vitro. La L-cisteína es un sustrato externo requerido para la síntesis del glutation en la maduración de ovocitos bovinos. Con el objetivo de evaluar el efecto de la suplementación con L-cisteína sobre la maduración in vitro de ovocitos bovinos, se llevó a cabo un experimento con ovarios obtenidos de vacas mestizas beneficiadas en una sala de matanza local. Se utilizaron tres tratamientos con diferentes concentraciones T1: 0 mM; T2: 0,1 mM y T3: 1,0 mM. Aproximadamente 400 Complejos Cumulus Ovocito (COCs) por tratamiento se maduraron en pozos de 500 µL del medio de maduración (TCM-199), distribuyéndose 50 COC por pozo en una incubadora a 5% de CO2, a 38,5°C y con humedad saturada. Los datos se analizaron por medio de un análisis de varianza (ANAVAR). No se observaron diferencias significativas entre los tratamientos en el porcentaje de ovocitos que llegaron a Metafase II (MII, T1: 72,77%, T2: 67,47% y T3: 70,43%). Los resultados indican que la suplementación con L-cisteína durante la maduración no ejerció un efecto sobre el porcentaje de ovocitos bovinos que alcanzaron el estado de MII, sin embargo se deben realizar otras investigaciones para determinar el efecto ulterior que tiene la L-cisteína adicionada en la maduración sobre la fecundación y el desarrollo embrionario in vitro.

In vitro fertilization (IVF) is now a routine technique in a number of biotechnology research laboratories worldwide. IVF is used as a tool for the study of various aspects related to oocytes maturation, fertilization and early embryo development. L-cysteine constitutes an external substrate required for synthesis of glutathione in the maturation of cattle oocytes. In order to study the effect of L-cysteine supplementation on in vitro maturation of cattle oocytes, ovaries were obtained from crossbred cows culled at a local slaughterhouse. Three different in vitro maturation treatments were utilized. Each treatment had a different concentrations of L-cysteine: T1: 0 mM, T2: 0.1 mM and T3: 1.0 mM. Approximately 400 Cumulus Oocyte Complexes (COCs) were matured in wells of 500 ìL of maturation medium (TCM-199), with 5% CO2 at 38.5°C and saturated relative humidity. Data were analyzed using analysis of variance (ANOVA). There were no significant differences between treatments in term of oocytes that arrived to Metafase II (MII, T1: 72.77%, T2: 67.47% and T3: 70.43%). Results indicate that supplementation with L-cysteine during maturation did not possitively affect the percentage of bovine oocytes that reached the M II state. Nonetheless further research should be carried out to determine the effect of L-cysteine supplementation during maturation, on fertilization and on embryonic development in vitro.

Influência do fator de crescimento semelhante à insulina I (IGF-I) adicionado ao meio fluido sintético de tuba uterina (SOF) sobre amaturação in vitro de oócitos caninos (Canis familiaris) / The effect of the synthetic oviductal fluid medium (SOF) supplementedwith insulin-like growth factor-I (IGF-I) on in vitro maturation of canineoocytes (Canis familiaris)

O conhecimento da regulação do crescimento folicular e da maturação oocitária é de grande importânciano desenvolvimento e aperfeiçoamento de novas biotecnologias como a fecundação in vitro e atransferência nuclear. Considerando-se a necessidade de elucidação dos mecanismos básicos envolvidosna maturação oocitária na espécie canina, a presente pesquisa foi desenvolvida com o objetivo deavaliar o efeito do fator de crescimento semelhante à insulina-I (IGF-I), adicionado ao meio fluido sintéticode tuba uterina (SOF), sobre a maturação de oócitos caninos. Foram utilizadas 37 cadelas submetidas àovariohisterectomia, eletivas e terapêuticas, como doadoras dos complexos cumulus oócitos grau 1(n=875) que foram alocados em três grupos: M0 (coloração no momento da colheita), Controle (72 h nomeio SOF) e Experimental (72h no meio SOF + 100 ng de IGF-I). Após 72 horas de maturação dos oócitoso estádio de maturação nuclear foi avaliado, por meio de coloração com Hoechst 33342. Os maioresíndices de recuperação das estruturas foram obtidos daquelas doadoras com raças definidas, fêmeasjovens, nulíparas e em estro. Não houve diferenças estatísticas entre os dados analisados quanto àmaturação nuclear entre os grupos controle (SOF) e experimental (IGF-I).

The follicular growth and oocyte maturation knowledge are very important to the development and improvement of new biotechnologies such as in vitro fertilization and somatic cell nuclear transfer. In order to the necessity of clarify the basic mechanisms related to canine oocyte maturation, this investigation focuses on the evaluation of the effect of insulin-like growth factor-i (IGF-I), added to synthetic oviductal fluid medium (SOF) on the in vitro maturation of domestic dog oocytes. Thirty-seven bitches undergoing ovariohysterectomy for castration or due to pathological conditions of the uterus were selected as oocytes’ donors (n=875). The oocytes were allocated in the following groups: M0 (stained in the collection’s time), Control (72h in SOF) and Experimental (72h in SOF plus 100 ng IGF-I). After 72 hours of maturation the oocytes’ nuclear status were assessed by Hoechst 33342 dye. The best results in terms of oocyte harvest were observed in those juvenile donors, females in estrus, nuliparous and pure breeds. No significant differences were observed between treatments control (SOF) or experimental (IGF-I).

The Influence of Sera,Gonadotropins,EGF and Water on In vitro Maturation of Goat Oocytes / 中国兽医学报

In present study,comparisons were carried out to develop and improve in vitro mauration(IVM) systems of goat oocytes. Oocytes were cultured in TCM-199 supplemented with(1)10% sera (either estrous goat serum (EGS) or fetal bovine serum(FBS)) + 20 mg/L luteinizing hormone (LH) + 10 mg/L follicle stimulating hormone (FSH) + 1 mg/L estradiol (E2); (2)10% EGS with different gonadotropins(LH: FSH) at a concentration of 5 mg/L: 0.5 mg/L or at 20 mg/L: 10 mg/L with0. 075 IU/mL human menopausal gonadotropin(HMG),1 mg/L estradiol 17β; (3)10% EGS+ 0. 075 mg/L HMG+10-20 μg/EGF. The culture was also performed by M199 supplemented with 10% EGS+0. 075 mg/L HMG+ 10-20 μg/L EGF into ultra-filtrated water which was derived either from self-producing or from purchased for use. The oocytes were cultured at 38 ℃,5% CO2 in air for 24 h,and the meterphase Ⅱ stage oocytes were examined under dissecting microscope. The results showed that EGS was better than FBS in supporting goat oocyte IVM. An addition of HMG in M199 could improve oocyte maturation and induce a higher percentage of metaphase Ⅱ oocytes compared to gonadotropins. 10-20 μg/L EGF improved goat oocyte maturation but the influence was not significant. Fresh,high quality water was vital for oocyte IVM. In conclusion,under our conditions with IVM ,the best result in maturation of goat oocyte has been M199 supplemented with 10% EGS+0.075 IU/mL HMG+10-20 μg/L EGF and prepared in fresh purified water.

Thickness of cumulus cell layer is a significant factor in meiotic competence of buffalo oocytes

This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with > or == 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 microL drop of TCM-199 supplemented with 10microgram/mL follicle stimulating hormone (FSH), 10microgram/mL luteinizing hormone (LH), 1.5microgram/mL estradiol, 75microgram/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39degrees C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with > or ==3 layers of cumulus cells showed higher maturation rates (p <0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.

Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

In vitro maturation,fertilization and embryo transfer of immature oocytes derived from nature cycles / 重庆医科大学学报

Objective:To investigate the effect of in vitro maturation(IVM),fertilization and embryo transfer of immature oocytes derived from nature cycles in infertility couples.Methods:30 IVF/ICSI-ET cycles were involved in this study.Oocytes retrieval performed after follicle larger than 10 mm and before endogenesis Luteinizing hormone(LH) surge occurrence in nature cycle.Mature oocytes(Metaphase Ⅱ) were inseminated by standard in vitro fertilization(IVF) on the oocytes pick-up day.Immature oocytes(Metaphase I and germinal vesicle stage) were cultured for 24~48 hours and fertilized by intracytoplasmic sperm injection(ICSI) as soon as it developed to Metaphase Ⅱ stage.Embryo transfer was performed 72 h after fertilization.Results:2 of 30 IVM cycles brought no oocytes in this study.113 oocytes were obtained among other 28 oocyte retrieval cycles,74 of which were immature,29 were mature,and 10 were degenerated.60 of 74 immature oocytes developed to MⅡ after IVM(maturation rate 81.08%).6 cases of clinical pregnancy were obtained in the 20 embryo transfer cycles,five of which brought healthy babies(one came from immature oocyte and four came from mature oocyte) and one was abortive.Conclusion:IVM,fertilization and embryo transfer of immature oocytes derived from nature cycles is a feasible treatment for women with infertility.