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).

La técnica de transferencia de huso meiótico desde la perspectiva legal en la República Argentina / THE MEIOTIC SPINDLE TRANSFER TECHNIQUE FROM THE LEGAL PERSPECTIVE IN THE ARGENTINE REPUBLIC

La transferencia de huso permite evitar enfermedades de herencia mitocondria. El art. 57 del Código Civil y Comercial, que utiliza una redacción amplia para no quedar obsoleto, apuntaría la prohibición a la manipulación de embriones en busca de mejoras determinadas, pero no a aquellas prácticas que tienen un fin terapéutico. Sin embargo, hay que repensar los límites de la prohibición y la razonabilidad de este tratamiento.

Spindle transfer makes it possible to avoid diseases of mitochondrial inheritance. The art. 57 of the Civil and Commercial Code, which uses a broad wording so as not to become obsolete, would point the prohibition to the manipulation of embryos in search of certain improvements, but not to those practices that have a therapeutic purpose. However, it is necessary to rethink the limits of the prohibition and the reasonableness of this treatment.

The effect of melatonin on in vitro maturation fertilization and early embryo development of mouse oocytes and expression of HMGB1 gene in blastocysts

Antioxidants are commonly used for maturation, fertilization and early development of embryos. Melatonin as an antioxidant have been recently proven to be useful for the assisted reproductive technology. In the present study, we evaluated the roles of melatonin in the in vitro maturation, fertilization, development and also the gene expression of high mobility group box-1 (HMGB1) in the blastocysts. The immature oocytes of BDF1 mice were transferred to the media containing different doses of melatonin (10-6, 10-9, 10-12 M). The blastocysts that developed under in vitro fertilization from each group were stained to determine the cell number of embryos and analyzed to determine the expression level of HMGB1 by real-time PCR. The most effective doses of melatonin for maturation of oocytes were 10-6 and 10-12M (P<0.05). Fertilization rate, early development and the cell number of blastocysts were significantly higher in the group that treated with 10-12 M of melatonin comparing to the other groups. The HMGB1 expression decreased in groups that treated with 10-6M and 10-9M of melatonin and increased in the group that treated with 10-12 M of melatonin, but did not show a significant difference (p˃0.05). From the results, it may be concluded that the melatonin could be effective when the embryos undergo maturation, fertilization and early developmental processes. The HMGB1 expression, as a marker of early development in mice embryos, increased in the groups that treated with low doses of melatonin

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Maturação in vitro de oócitos de cutias (Dasyprocta prymnolopha, Wagler 1831) seguida de fertilização in vitro e ativação partenogenética / In vitro maturation of agoutis (Dasyprocta prymnolopha) oocytes followed by in vitro fertilization and parthenogenetic activation

O objetivo foi avaliar protocolos de maturação in vitro (MIV) para oócitos de cutias, seguida de fertilização in vitro (FIV) e ativação partenogenética (AP). Os oócitos imaturos (CCOs) foram obtidos por fatiamento do ovário, após OSH, e submetidos a três grupos: MAT - 16 (16 horas de maturação), MAT - 20 (20 horas de maturação) e MAT - 24 (24 horas de maturação), em incubadora de cultivo a 38,8°C, com atmosfera de 5% de CO2 e 95% de umidade relativa. A maturação foi analisada pela presença do primeiro corpúsculo polar. Em seguida, os CCOs maduros foram submetidos à FIV, com período de coincubação dos CCOs e dos espermatozoides de 15h, a 38,8ºC e 5% de CO2, e AP com ionomicina. Os grupos de MIV foram analisados utilizando-se o teste qui-quadrado e, nos experimentos de FIV e AP, foram analisadas a taxa de clivagem e a proporção de desenvolvimento embrionário. A análise estatística foi realizada utilizando-se o programa SAS. Houve diferença significativa entre os grupos de maturação, tendo os grupos MAT - 20 e MAT - 24 apresentado maior porcentagem de oócitos maturados in vitro. As taxas de clivagem e de desenvolvimento embrionário foram de 8,6% e 2,9%, respectivamente, na FIV, e de 63,6% e 15,1%, na AP. Entretanto, nos dois casos, o embrião não passou do estágio de mórula.(AU)

The objective was to evaluate IVM protocols for agouti oocytes, followed by in vitro fertilization (IVF) and parthenogenetic activation (PA). The immature oocytes (CCOs) were obtained by slicing the ovary after OSH and submitted to three groups: MAT - 16 (16 hours maturation), MAT - 20 (20 hours maturation) and MAT - (24 hours maturation), in a culture incubator at 38.8°C, with an atmosphere of 5% CO2 and 95% relative humidity. The maturation was analyzed by the presence of the first polar corpuscle. Then, mature CCOs were submitted to IVF, with co-incubation period of CCOs and spermatozoa from 15h to 38.8°C and 5% of CO2, and PA with inomycin. The IVM groups were analyzed using the chi-square test and in the FIV and PA experiment the rate of cleavage and the rate of embryonic development were analyzed. Statistical analysis was performed using the SAS program. There was a significant difference between the maturation groups, and the MAT - 20 and MAT - 24 groups showed a higher percentage of matured oocytes in vitro. The rates of cleavage and embryonic development were 8.6% and 2.9%, respectively in FIV and 63.6% and 15.1% in PA. However, in both cases the embryo did not pass beyond the morula stage.(AU)

Efeito da somatotropina bovina recombinante (rbST) na produção in vivo e na maturação in vitro de oócitos bovinos da raça Sindi / Effect of recombinant bovine somatotropin (rbST) on the in vivo production and in vitro maturation of bovine oocytes of the Sindi breed

A fim de avaliar o efeito de diferentes doses da rbST sobre a dinâmica folicular, a produção e a maturação in vitro de oócitos, 20 vacas Sindi, divididas em três grupos, receberam um dispositivo de progesterona intravaginal, estradiol e PGF2α, além de 2mL de solução salina (grupo controle), 250 (grupo rbST 250) ou 500mg de rbST (grupo rbST 500). Cinco dias depois, realizou-se a ovum pick up, e os complexos cumulus-oócitos (CCOs) recuperados foram selecionados, classificados e maturados in vitro. Os dados de contagem foram comparados pelo procedimento glht (General Linear Hypothesis Test), e os dados em porcentagem foram submetidos ao qui-quadrado, no programa estatístico R, onde as diferenças foram consideradas significativas quando P<0,05. Não houve diferença (P>0,05) entre os grupos quanto à quantidade de folículos e à taxa de maturação. Os grupos rbST 250 e rbST 500 foram significativamente superiores (P<0,05) ao grupo controle em relação ao número de folículos grandes (0,42±0,20 vs. 0). O grupo rbST 500 apresentou maior (P<0,05) porcentagem de oócitos viáveis (91,52%) do que os grupos controle (67,85%) e rbST 250 (53,33%). A rbST aumenta o número de folículos grandes, e 500mg de rbST aumentam a porcentagem de oócitos viáveis em vacas Sindi.(AU)

In order to evaluate the effect of different doses of rbST on the follicular dynamics, production, and in vitro maturation of oocytes, 20 Sindhi cows were divided into three groups, receiving an intravaginal progesterone device, estradiol and PGF2α, and 2mL of solution saline (Control Group), 250 (rbST 250 Group) or 500mg rbST (rbST 500 Group). Five days later, the ovum pick up was performed, and the cumulus-oocyte (CCO) complexes recovered were selected, classified, and matured in vitro. The counting data were compared by the glht (General Linear Hypothesis Test) procedure, and the percentage data were submitted to Qui- square, in the statistical program R, where differences were considered significant when P< 0.05. There was no difference (P> 0.05) between the groups regarding follicle quantity and maturation rate. The rbST 250 and rbST 500 groups were significantly higher (P< 0.05) than the Control group in relation to the number of large follicles (0.42±0.20 versus 0). The rbST 500 group presented higher (P< 0.05) percentage of viable oocytes (91.52%) than the Control (67.85%) and rbST 250 (53.33%) groups. rbST increases the number of large follicles and 500mg rbST increases the percentage of viable oocytes in Sindhi cows.(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)

Síndrome dos ovários policísticos: particularidades no manejo da infertilidade / Polycystic ovary syndrome: particularities in the management of infertility

A síndrome dos ovários policísticos (SOP) é responsável por cerca de 80% dos casos de infertilidade anovulatória. Não há na literatura evidências suficientes para a definição do tratamento ideal da infertilidade na SOP, mas repete-se que deve ser iniciado por mudanças no estilo de vida, e frequentemente envolve a indução farmacológica da ovulação e, em casos selecionados, as técnicas de reprodução assistida e o drilling ovariano laparoscópico. Este texto pretende reunir informações atuais sobre o manejo da infertilidade em mulheres com SOP e, dessa forma, permitir ao ginecologista a escolha da melhor abordagem, de forma Individualizada e baseada nas melhores evidências disponíveis.(AU)

Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1) / Comprometimento na taxa de maturação nuclear em ovócitos de vacas naturalmente infectadas pelo herpesvírus bovino 1 (BoHV-1)

Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P<0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.(AU)

Herpesvírus bovino 1 (BoHV-1) é um importante patógeno bovino, responsável por causar doenças respiratórias e falhas reprodutivas. A presença do BoHV-1 em sistema de produção in vitro de embriões afeta a fertilização, a maturação e o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a capacidade de desenvolvimento de ovócitos oriundos de vacas infectadas naturalmente sem histórico reprodutivo. Além disso, este estudo investigou a presença do DNA viral em Complexos Cumulus Ooforus (COCs). Os tratamentos foram definidos a partir do título de anticorpos detectados pelos ensaios de soroneutralização, sendo estabelecidos três grupos: animais soronegativos (título menor do que 2), título baixo (2 a 8) e animais com título maior ou igual a 16. Os COCs foram obtidos de 15 doadoras durante 22 sessões de aspiração folicular guiada por ultrassom. A extração do DNA foi realizada em um pool de COCs de todas as aspirações de uma mesma doadora e no sangue total para a realização das reações de Nested-PCR. Para avaliação da taxa de maturação nuclear, foram selecionados para o cultivo in vitro somente os COCs com camada compacta de células do cumulus, zona pelúcida íntegra e citoplasma homogêneo. Após 24 horas de cultivo, os ovócitos foram fixados e corados em lâmina para a avaliação do estádio do ciclo celular meiótico. Os ovócitos que apresentaram configuração cromossômica em metáfase II foram considerados como tendo alcançado a maturação nuclear. Verificou-se comprometimento na taxa de maturação nuclear ovocitária (P<0.05) nos animais de título maior ou igual a 16 (50%). No entanto, não houve influência do título viral na taxa de maturação de ovócitos bovinos em animais que apresentaram titulação baixa (62,2%) quando comparados com o grupo controle (76,7%). O DNA viral não foi identificado nas amostras de sangue, mas foi detectado no pool de COCs de três doadoras soropositivas. Diante dos resultados encontrados conclui-se que vacas infectadas naturalmente pelo vírus BoHV-1 apresentam comprometimento na taxa de maturação nuclear, dependendo do grau de titulação de anticorpos contra o vírus. Ademais, o DNA viral pode estar presente em COCs contrariando a hipótese de que animais sorologicamente positivos e sem histórico de sintomatologia clínica oferecem risco negligível de transmissão do BoHV-1 por COCs.(AU)

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times / Maturação in vitro de oócitos de cateto (Pecari tajacu) após diferentes períodos de incubação

Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.(AU)

A maturação in vitro (MIV) oocitária é a primeira etapa das tecnologias reprodutivas in vitro que permite que oócitos maturados sejam gerados ex vivo e depois usados para a produção de embriões. Nesse sentido, o estabelecimento do ambiente de cultivo, como o período de incubação de oócitos, é essencial para o sucesso da MIV. Portanto, o estudo foi realizado para investigar a relação entre o potencial meiótico e os períodos de MIV de oócitos derivados de catetos, mamíferos silvestres de grande interesse comercial e ecológico. Para tanto, os ovários foram coletados de fêmeas derivadas de cativeiro e transportados ao laboratório dentro de 1 h após o abate. Os oócitos derivados de folículos (3-6mm de diâmetro) foram recuperados por aspiração e fatiados. Oócitos de boa qualidade (citoplasma uniformemente granulado com pelo menos uma camada circundante de células cumulus) foram selecionados e submetidos ao cultivo em TCM 199 suplementado com 10µg/mL de FSH, 10% de SFB e 100μM de cisteamina a 38,5°C, 5% de CO2 e umidade máxima por 24 e 48 h. Após o período de incubação, o estado nuclear, a presença do primeiro corpúsculo polar e a expansão das células do cumulus dos oócitos foi avaliada. Os dados obtidos foram analisados pelo teste exato de Fisher (P<0,05). Um total de quatro sessões (2-3 fêmeas por sessão) foi realizado, resultando em dezoito ovários aspirados e fatiados com características morfológicas normais. Uma taxa de recuperação oocitária de aproximadamente 83,1% (59/71) foi obtida com 3,3 oócitos/ovário e 2,3 oócitos viáveis/ovário. Após diferentes períodos de incubação, diferenças (P<0,05) foram observadas entre 24 e 48 h para a expansão das células cumulus (38,1% vs. 100%), presença de primeiro corpúsculo polar (52,4% vs. 90,5%) e estado nuclear na segunda metáfase (19,0% vs. 76,2%), respectivamente. Em conclusão, 48 h é o período adequado para a maturação in vitro de oócitos derivados de catetos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico observado. Estudos adicionais devem ser conduzidos para melhorar a qualidade do ambiente de cultivo oocitário, como a composição de meio, objetivando obter oócitos maturados viáveis para outras biotecnologias in vitro.(AU)

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 μg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 μg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Developmental competence of in vitro-matured human oocytes obtained from pregnant and non-pregnant women / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. METHODS: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. RESULTS: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p < 0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. CONCLUSION: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.

Influence of epidermal growth factor (EGF) supplementation at different times of in vitro maturation of canine oocytes / Influência da suplementação do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos

The aim of this study was to evaluate the influence of epidermal growth factor (EGF) on in vitro maturation of canine oocytes at different times of the process. Ovaries were collected from 55 bitches considered healthy and aseptically isolated, immersed in physiological solution (0.9% NaCl) and transported under refrigeration. Grade 1 cumulus-oocyte complexes (COCs) were selected and divided into two groups: control group (CG) and treatment group (TG). In CG 698 grade I COCs were placed in 4-well plates containing TCM-199 medium supplemented with 25 mM HEPES, 100 IU/mL penicillin, 100 mg/mL streptomycin, 26 mM sodium bicarbonate, 1.5 mM sodium pyruvate, 2.9 mM sodium lactate pentahydrate, 0.6 mM cysteine, 0.03 IU/mL hCG, 0.5 µg/mL FSH, 20 µg/mL estrogen at 38.5ºC in a humidified atmosphere of 5% CO2 in times of 24 h, 48 h, and 72 h. In TG 547 COCs received the same maturation medium plus 10 ηg/mL EGF. Logistic regression models (SAS, 2011) were constructed in order to estimate the chances of oocytes being observed at nuclear maturation stages in different culture times (24 h, 48 h, and 72 h). Based on the results found EGF-supplemented medium showed 2.56 times more chances of having an oocyte at metaphase I (M-I) than medium without EGF (p < 0.0001). The results of this study demonstrated that the time of 72 h showed 5.88 times more chances of having an oocyte at metaphase II (M-II) compared to time of 24 h (p = 0.0001) and 7.69 times more chance than time of 48 h (p = 0.0001). The chances of finding an oocyte at M-II were also 9.09 times higher in medium supplemented with EGF than in medium without EGF (p = 0.0001). Thus, these results demonstrated the essential importance of EGF at different moments of oocyte maturation, being a key component for the acquisition of meiotic competence in bitches, increasing the M-I and M-II rates.(AU)

O objetivo deste estudo foi avaliar a influência do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos. Os ovários foram coletados de 55 cadelas consideradas sadias e isolados assepticamente, imersos em solução fisiológica e transportados refrigerados. Os complexos cumulus-oócito (COCs) grau 1 foram selecionados e divididos em dois grupos, denominados grupo controle (GC) e grupo tratamento (GT). No GC, 698 COCs grau I foram cultivados em placas de quatro poços contendo meio TCM-199 suplementado com 25 mM de HEPES, 100 UI/mL de penicilina, 100 mg/mL de estreptomicina, 26 mM de bicarbonato de sódio, 1,5 mM de piruvato de sódio, 2,9 mM de lactato de sódio penta hidratado, 0,6 mM de cisteína, 0,03 UI/mL de hCG, 0,5 µg/mL de FSH, 20 µg/mL de estrógeno em estufa úmida a 38ºC, 5% de CO2 nos períodos de 24h, 48 h e 72 h . Já no GT, 547 COCs receberam o mesmo meio de maturação acrescido de 10 ηg/mL do EGF. Modelos de regressão logística foram elaborados para estimar as chances do oócito ser observado nos estágios de maturação nuclear em diferentes tempos de cultivo. Com base nos resultados encontrados, o meio suplementado com EGF demonstrou 2,56 vezes mais chances de ter um oócito no estágio de metáfase I (M-I) do que o meio sem EGF (p < 0,0001). Os resultados desse estudo demonstraram também que o tempo de 72 h mostrou 5,88 vezes mais chances de ter um oócito no estágio de metáfase II (M-II) do que o tempo de 2 h (p = 0,0001) e 7,69 vezes mais chance do que o tempo de 48h (p = 0,0001). As chances de se encontrar um oócito em M-II também foram 9,09 vezes maiores no meio suplementado com EGF do que no meio sem EGF (p = 0,0001). Dessa forma, estes resultados demonstraram a importância essencial do EGF em diferentes momentos da maturação oocitária, sendo componente chave para a aquisição da competência meiótica nas cadelas, aumentando os índices de M-I e M-II.(AU)

Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes

Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein [PAWP], phospholipase Czeta [PLCzeta], and truncated form of the kit receptor [TR-KIT]] as candidates of oocyte activation with fertilization rate and early embryonic development

Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection [ICSI] candidates and analyzed according to World Health Organization criteria [2010]. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation [DGC] and the second part was prepared for assessment of sperm morphology [Papanicolaou staining], DNA fragmentation [transferase dUTP nick end labeling [TUNEL]], and three Sperm-borne oocyte-activating factor [s] [SOAFs]-PLCzeta, PAWP, and TR-KIT

Results: Significant positive correlations existed between the percentages of PLCzeta, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCzeta and PAWP. We did not find a relationship between percentages of PLCzeta, PAWP, and TR-KIT with embryo quality and pregnancy rate [P>0.05]. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality

Conclusion: Oocyte activation was associated with the studied sperm factors [PAWP, PLCzeta, and TR-KIT]. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCzeta, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future

Low-dose urinary human chorionic gonadotropin is effective for oocyte maturation in vitro fertilization/ intracytoplasmic sperm injection cycles independent of body mass index

Background: Currently, there is no agreement on the optimal urinary derived human chorionic gonadotropin [u-hCG] dose requirement for initiating final oocyte maturation prior to oocyte collection in in vitro fertilization [IVF], but doses that range from 2500-15000 IU have been used. We intended to determine whether low dose u-hCG was effective for oocyte maturation in IVF/intracytoplasmic sperm injection [ICSI] cycles independent of body mass index [BMI]

Materials and Methods: We retrospectively evaluated a cohort of 295 women who underwent their first IVF/ICSI cycles between January 2003 and December 2010 at the Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI, USA. Treatment cycles were divided into 3 groups based on BMI [kg/m[2]]: <25 [n=136], 25- <30 [n=84], and >/=30 [n=75] women. Patients received 5000, 10000 or 15000 IU u-hCG for final maturation prior to oocyte collection. The primary outcome was clinical pregnancy rates [CPRs] and secondary outcome was live birth rates [LBRs]

Results: Only maternal age negatively impacted [P<0.001] CPR [odds ratio [OR=0.85, confidence interval [CI: 0.79-0.91]] and LBR [OR=0.84, CI: 0.78-0.90]

Conclusion: Administration of lower dose u-hCG was effective for oocyte maturation in IVF and did not affect the CPRs and LBRs irrespective of BMI. Women's BMI need not be taken into consideration in choosing the appropriate dose of u-hCG for final oocyte maturation prior to oocyte collection in IVF. Only maternal age at the time of IVF negatively influenced CPRs and LBRs in this study

Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation / 대한생식의학회지

OBJECTIVE: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca²⁺ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). METHODS: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca²⁺ chelator to investigate the effect of Ca²⁺ oscillations on their maturation. RESULTS: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca²⁺ chelator-treated group. CONCLUSION: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca2+ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca²⁺ oscillations driven by fertilization.

Vitrificación de ovocitos bovinos parcialmente madurados de la raza frisón rojo chileno / Vitrification of partially matured bovine oocytes of the Chilean red friesian breed

Se evaluó la tasa de maduración in vitro post descongelación de ovocitos bovinos, de la raza Frisón Rojo Chileno, parcialmente madurados y vitrificados. Complejos Cúmulus-ovocito fueron obtenidos por aspiración folicular, clasificados morfológicamente y aleatoriamente cultivados in vitro en TCM199 (10 % Suero Fetal Bovino (SFB), 50 mg/mL gentamicina, 0,2 mM piruvato de sodio, 0,08 µg/mL FSH, 1 µg/mL LH y 1 µg/mL estradiol) en los grupos: a) control (n= 137), madurados por 24 h a 38,5 C, 5 % CO2 y 99 % humedad y, b) tratamiento (n= 156), madurados por 6 h, parcialmente denudados, e incubados hasta completar 20 h, para luego ser vitrificados por el método Open Pulled Straws (OPS). Los ovocitos fueron expuestos a la solución de vitrificación uno (SV1) (Buffer Fosfato Salino (PBS), 10 % Etilenglicol (EG), 10% DMSO) por 30 s, posteriormente traspasados a la SV2 (PBS, 20 % EG, 20 % DMSO) por 25 s. Inmediatamente los ovocitos fueron cargados en pajuelas francesas estiradas (OPS) y sumergidos en nitrógeno líquido. Las ovocitos fueron descongelados introduciéndolos en una secuencia de soluciones con concentraciones decrecientes de sucrosa (0,3; 0,15 y 0 M respectivamente). Finalmente, los ovocitos continuaron con la maduración por 4 h adicionales. Posterior al periodo de maduración, los ovocitos de ambos grupos fueron fijados, teñidos y evaluados. Las proporciones de ovocitos en Metafase I (MI), Metafase II (MII) y degenerados fueron comparadas mediante el test de Chi cuadrado. La vitrificación aumentó (p 0,05) el porcentaje de pérdida y de ovocitos dañados en comparación al control. Además, aumentó (p 0,05) la tasa de ovocitos en MI y el número de ovocitos degenerados, y redujo el porcentaje de ovocitos MII, en comparación al control. Por tanto, la vitrificación por el método Open Pulled Straw de ovocitos parcialmente madurados in vitro es una alternativa viable para la conservación de material genético de hembras Frisón Rojo Chileno.

Post thawing in vitro maturation rate was evaluated for partially matured vitrified oocytes from Chilean Red Friesian cattle. Cumulus-Oocytes Complexes were obtained by follicular aspiration, classified by morphology and randomly in vitro matured in TCM199 (10 % Bovine Fetal Serum (BFS), 50 mg/mL gentamicine, 0.2 mM sodium piruvate, 0.08 µg/ml FSH, 1 µg/mL LH and 1 µg/mL estradiol) in the following groups: a) control (n= 137), matured for 24 h at 38.5 C, 5 % CO2 y 99 % humidity, and b) treatment (n= 156), matured for 6 h, partially denuded, and incubated until completion of 20 h. Then, oocytes were vitrified by the Open Pulled Straws (OPS) method. Oocytes were exposed to vitrification solution one (VS1) (Phosphate Buffered Saline (PBS), 10 % Ethylen glycol (EG), 10 % DMSO) for 30 s, then they were exposed to VS2 (PBS, 20% EG, 20% DMSO) for 25 s. Afterwards, oocytes were loaded into open pulled straws and submerged into liquid nitrogen. Oocytes were thawed by exposure to sequential solutions with decreasing concentrations of sucrose (0.3; 0.15 y 0 M respectively). Finally, oocytes continued the in vitro maturation for 4 additional hours. After completion of maturation period oocytes from both groups were fixated, stained and evaluated. The proportion of lost and damaged, MI, MII, and degenerate oocytes were compared between groups by Chi square test. Vitrification procedure increased (p 0.05) the percentage of oocytes lost and damaged when compared to control group. Additionally, vitrification increased (p 0.05) the proportion of MI and degenerated oocytes, and decreased the proportion of MII oocytes. Therefore, vitrification by the OPS method of partially matured bovine oocytes is a reliable alternative for the conservation of germinal cells from Chilean Red Friesian females.

Desempenho de doadoras leiteiras mestiças F1 (Gir x Holandês) no sistema de produção in vitro de embriões / Performance of dairy crossbred F1 donnors (Gir x Holstein) in the production system in vitro embryo

O Brasil atualmente é detentor do terceiro maior rebanho de vacas leiteiras do mundo, composto, em sua maioria, de animais mestiços F1 (Gir X Holandês), os quais são bem adaptados às áreas tropicais. O objetivo do presente estudo foi investigar a eficiência da ovum pick-up (OPU) e da produção in vitro de embriões oriundos de doadoras Girolando com sêmen sexado de touros provenientes de duas raças, Gir e Holandesa. Foram utilizados dados referentes a 232 animais de diversos rebanhos, totalizando 4334 oócitos recuperados. Os oócitos foram classificados, e as estruturas viáveis (GI, GII e GIII) foram utilizadas para produção in vitro de embriões (PIVE). Foi observada uma média de oócitos recuperados para as doadoras F1 de 18,14±1,33. Além disso, notou-se que não houve diferença entre os dois cruzamentos utilizados, considerando média de total de embriões (4,96±0,40 e 6,56±0,76) para o cruzamento F1 X HPB (Holandês preto e branco) e F1 X Gir, respectivamente. Portanto, as doadoras F1 apresentaram potencial como doadoras de oócitos no sistema de produção in vitro de embriões, independentemente da raça do touro cujo sêmen foi utilizado.(AU)

Brazil currently has the third largest herd of dairy cattle in the world, composed mainly by F1 crossbreed animals (Gyr x Holstein), which are well adapted to tropical areas. The aim of this study was to evaluate the efficiency of Girolando donors in OPU and in vitro production of embryos (IVEP) using sexed semen of bulls from two breeds, Gyr and Holstein. We used data from 232 animals coming from different herds and the total of recovered oocytes was 4334. The oocytes were classified according to their viability and the viable ones (GI, GII and GIII) were used for IVP. The average of oocyte recovery from F1 donors was 18.14±1.33. There was no difference on the average of embryos considering the crossing F1 X Holstein (4.96±0.40) and F1 X Gyr (6.56±0.76). In conclusion, F1 donors showed a potential for being oocyte donors in the system of in vitro production of embryos, regardless the breed of the bull.(AU)