ABSTRACT
Metadata analysis of public microarray datasets using bioinformatics tools has been successfully used in several biomedical fields in the search for biomarkers. In reproductive science, there is an urgent need for the establishment of oocyte quality biomarkers that could be used in the clinical environment to increase the chances of successful outcomes in treatment cycles. Adaptive cellular processes observed in cumulus oophorus cells reflect the conditions of the follicular microenvironment and may thus bring relevant information of oocyte's conditions. Here we analyzed human cumulus cells gene expression datasets in search of predictors of oocyte quality, a strategy which uncovered several cellular processes positively and negatively associated with embryo development and pregnancy potential. Secondly, the expression levels of genes that were present in the majority of processes observed were validated in house with clinical samples. Our data confirmed the association of the selected biomarkers with blastocyst formation and pregnancy potential rates, independently of patients' clinical characteristics such as diagnosis, age, BMI, and stimulation protocol applied. This study shows that bioinformatic analysis of cellular processes can be successfully used to elucidate possible oocyte quality biomarkers. Our data reinforces the need to consider clinical characteristics of patients when selecting relevant biomarkers to be used in the clinical environment and suggests a combination of positive (PTGS2) and negative (CYPB1) quality biomarkers as a robust strategy for a complementary oocyte selection tool, potentially increasing assisted reproduction success rates. Also, GPX4 expression as pregnancy potential biomarker is indicated here as a possibility for further investigations.
Subject(s)
Cumulus Cells , Oocytes , Pregnancy , Female , Humans , Cumulus Cells/metabolism , Oocytes/metabolism , Biomarkers/metabolism , Embryonic Development/genetics , Cyclooxygenase 2/metabolismABSTRACT
PURPOSE: To study whether the cumulus cell antioxidant system varies accordingly to patients clinical characteristics' as age, infertility diagnosis, BMI, and stimulation protocol applied and if the antioxidant profile of cumulus cells could be used as a predictor of embryo development. METHODS: A prospective study including 383 human cumulus samples provided by 191 female patients undergoing intracytoplasmic sperm injection during in vitro fertilization treatments from a local in vitro fertilization center and processed in university laboratories. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) enzyme activity levels and reduced glutathione (GSH) levels were measured in cumulus oophorus cells individually collected from each aspirated cumulus-oocyte complex, and the results of each sample were compared considering the oocytes outcome after ICSI and patients clinical characteristics. A total of 223 other human cumulus samples from previous studies were submitted to a gene expression meta-analysis. RESULTS: The antioxidant system changes dramatically depending on patients' age, infertility diagnosis, stimulation protocol applied, and oocyte quality. SOD activity in cumulus cells revealed to be predictive of top-quality blastocysts for young patients with male factor infertility (P < 0.05), while GST levels were shown to be extremely influenced by infertility cause (P < 0.0001) and stimulation protocol applied (P < 0.05), but nonetheless, it can be used as a complementary tool for top-quality blastocyst prediction in patients submitted to intracytoplasmic sperm injection technique (ICSI) by male factor infertility (P < 0.05). CONCLUSION: Through a simple and non-invasive analysis, the evaluation of redox enzymes in cumulus cells could be used to predict embryo development, in a personalized matter in specific patient groups, indicating top-quality oocytes and improving success rates in in vitro fertilization treatments. TRIAL REGISTRATION: The trial was registered at UFRGS Research Ethics Committee and Plataforma Brasil under approval number 68081017.2.0000.5347 in June 6, 2019.
Subject(s)
Cumulus Cells , Infertility, Male , Antioxidants/metabolism , Cumulus Cells/physiology , Embryonic Development/genetics , Female , Fertilization in Vitro , Humans , Infertility, Male/metabolism , Male , Oocytes/metabolism , Prospective Studies , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
Abstract Background: The brilliant cresyl blue (BCB) staining is a non-invasive test to select the best-suited oocytes for embryonic development. This makes it a useful tool to select best-quality oocytes at the times of the year when there is forage restriction. Objective: To evaluate the effect of seasonality on the nuclear maturation and quality of oocytes selected by the BCB test. Methods: The cumulus-oophorus complexes (COCs) were obtained in summer and winter of 2010 and 2011. Selected COCs were maintained for 90 min at 38.5 °C in a CO2 incubator, in TCM 199 medium containing 10% fetal bovine serum and antibiotics, and supplemented with 26 µM brilliant cresyl blue. Afterwards, they were divided according to the ooplasm staining (BCB+ -blue; BCB− -unstained). Subsequently, COCs were matured for 22 h. Nuclear maturation was evaluated at 22 h of culture. Results: The proportion of BCB− oocytes was higher in the winter of 2010, but there was no increase in this group in the winter of 2011. The percentage of oocytes that reached metaphase II was higher in control and BCB+ groups in relation to oocytes from BCB− group. Conclusion: The season of the year influences the percentage of oocytes best suited for embryonic production in situations in which oocyte donors receive pasture-based feeding, since the method was effective in determining the effect of seasonality on the competence of bovine oocytes to reach nuclear maturation.
Resumen Antecedentes: La tinción con azul cresil brillante (BCB) es un método no invasivo para seleccionar ovocitos aptos para el desarrollo embrionario. Por tanto, es una herramienta útil para selecionar los ovocitos de mejor calidad en temporadas de restricción de forraje. Objetivo: Evaluar el efecto de la estacionalidad sobre la maduración nuclear y calidad de los ovocitos seleccionados por el test BCB. Métodos: Los complejos cumulus-oophorus (CCOs) fueron obtenidos durante el verano y el invierno de 2010 y 2011. Los CCOs seleccionados se mantuvieron durante 90 min a 38,5 oC en una incubadora de CO2 en un medio TCM 199 con 10% de suero fetal bovino y antibióticos, suplementado con 26 µM de azul cresil brillante. Luego se separaron según el color del citoplasma (BCB+ -azul y BCB− -incoloro). Posteriormente, los CCOs se maduraron durante 22 h. La evaluación de la maduración nuclear se realizó a las 22 h de cultivo. Resultados: La proporción de ovocitos BCB− fue mayor en el invierno de 2010, pero no hubo un aumento de ese grupo en el invierno de 2011. El porcentaje de ovocitos que alcanzaron la etapa de metafase II fue mayor en el grupo control y BCB+ con respecto al grupo BCB−. Conclusión: La estación del año influye en el porcentaje de ovocitos más aptos para la producción de embriones en situaciones donde las donadoras de ovocitos reciben alimentación a base de pastos, ya que este método fue eficaz para determinar el efecto de la estacionalidad en la competencia de ovocitos bovinos en alcanzar la maduración nuclear.
Resumo Antecedentes: O método do azul cresil brilhante (BCB) não é invasivo e seleciona ovócitos mais aptos ao desenvolvimento embrionário. Portanto é ferramenta útil para selecionar ovócitos de melhor qualidade em épocas do ano que ocorre restrição de pastagem. Objetivo: Avaliar o efeito da sazonalidade sobre a maturação nuclear e a qualidade dos ovócitos selecionados pelo teste BCB. Métodos: Os complexos cumulus oophorus (CCOs) foram obtidos no verão e inverno de 2010 e 2011. Os CCOs selecionados foram mantidos por 90 min, a 38,5 °C, em incubadora de CO2, em meio TCM 199 contendo 10% de soro fetal bovino e antibióticos, e suplementado com 26 µM de azul cresil brilhante. Em seguida, estes foram divididos de acordo com a coloração do citoplasma (BCB+ -azuis e BCB− -incolores). Então os CCOs foram maturados durante 22 h. A avaliação da maturação nuclear foi realizada às 22 h de cultivo. Resultados: A proporção dos ovócitos BCB− foi maior no inverno de 2010, mas não houve aumento desse grupo no inverno de 2011. O percentual de ovócitos que atingiu o estágio de metáfase II foi maior no controle e no grupo BCB+ em relação ao grupo BCB−. Conclusão: A estação do ano influencia o percentual de ovócitos mais aptos a produção de embriões, em situações onde as doadoras de ovócitos recebem alimentação baseada em pastagens, já que o método se mostrou efetivo para determinação do efeito da sazonalidade sobre a competência de ovócitos bovinos em atingirem a maturação nuclear.
ABSTRACT
A produção in vitro de embriões suínos tem alcançado resultados insatisfatórios: ovócitos maturados in vivo produzem uma porcentagem maior de embriões em relação aos maturados in vitro. O sucesso da maturação in vitro está diretamente relacionado com a competência ovocitária. Somente ovócitos competentes são capazes de serem fecundados e terem desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada por vários parâmetros. Recentemente têm sido utilizados como parâmetro os estudos da expressão de genes associados com a competência. O presente trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER e ZAR1 em ovócitos imaturos de diferentes classes morfológicas, sendo elas: 1, 2, 3 e 4, com a finalidade de proporcionar importantes marcadores moleculares relacionados com a capacidade ovocitária. O RNA total dos ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando-se modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3; somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de RT-qPCR foi eficiente para detecção desses transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária, são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento, e implicação de outros genes envolvidos com a competência ovocitária.(AU)
The in vitro production of pig embryos has achieved unsatisfactory results; in vivo matured oocytes produce a higher percentage of embryos compared to in vitro maturation. The success of in vitro maturation is directly related to oocyte competence. Only competent oocytes are capable of being fertilized and have normal embryonic development. The oocyte competence can be assessed using several parameters. Recently these parameters have been used for gene expression studies associated with competence. This work aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 as endogenous control and the constitutive gene GAPDH in immature oocytes of different morphological classes which are: 1, 2, 3 and 4, in order to provide significant molecular markers linked to the ability of development. Oocytes Total RNA was extracted and used as a template for synthesis of the first cDNA strand. The results of gene expression were analyzed using a mixed model, considering the dependent gene expression data and independent ovocitary variable classes. The genes BMP15, RYBP ZAR1 and showed similar ovocitary expression in classes 1, 2 and 3 differ only in category 4 in their expression (P<0.05). The MATER gene was similarly expressed in all ovocitary classes studied (P>0.05). The RTQ-PCR technique was effective for detection of these transcripts in oocytes from different classes. However, for better understanding of the involvement of these transcripts in the acquisition of oocyte competence more studies are needed to evaluate different morphological classes of oocytes at different stages of development and the implication of other genes involved in oocyte competence.(AU)
Subject(s)
Animals , Swine/embryology , Gene Expression , In Vitro Oocyte Maturation Techniques/statistics & numerical data , In Vitro Oocyte Maturation Techniques/veterinary , Embryonic Development , Oocytes , Fertilization in Vitro/veterinary , Cytoplasmic StructuresABSTRACT
A produção in vitro de embriões suínos tem alcançado resultados insatisfatórios: ovócitos maturados in vivo produzem uma porcentagem maior de embriões em relação aos maturados in vitro. O sucesso da maturação in vitro está diretamente relacionado com a competência ovocitária. Somente ovócitos competentes são capazes de serem fecundados e terem desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada por vários parâmetros. Recentemente têm sido utilizados como parâmetro os estudos da expressão de genes associados com a competência. O presente trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER e ZAR1 em ovócitos imaturos de diferentes classes morfológicas, sendo elas: 1, 2, 3 e 4, com a finalidade de proporcionar importantes marcadores moleculares relacionados com a capacidade ovocitária. O RNA total dos ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando-se modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3; somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de RT-qPCR foi eficiente para detecção desses transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária, são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento, e implicação de outros genes envolvidos com a competência ovocitária.
The in vitro production of pig embryos has achieved unsatisfactory results; in vivo matured oocytes produce a higher percentage of embryos compared to in vitro maturation. The success of in vitro maturation is directly related to oocyte competence. Only competent oocytes are capable of being fertilized and have normal embryonic development. The oocyte competence can be assessed using several parameters. Recently these parameters have been used for gene expression studies associated with competence. This work aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 as endogenous control and the constitutive gene GAPDH in immature oocytes of different morphological classes which are: 1, 2, 3 and 4, in order to provide significant molecular markers linked to the ability of development. Oocytes Total RNA was extracted and used as a template for synthesis of the first cDNA strand. The results of gene expression were analyzed using a mixed model, considering the dependent gene expression data and independent ovocitary variable classes. The genes BMP15, RYBP ZAR1 and showed similar ovocitary expression in classes 1, 2 and 3 differ only in category 4 in their expression (P<0.05). The MATER gene was similarly expressed in all ovocitary classes studied (P>0.05). The RTQ-PCR technique was effective for detection of these transcripts in oocytes from different classes. However, for better understanding of the involvement of these transcripts in the acquisition of oocyte competence more studies are needed to evaluate different morphological classes of oocytes at different stages of development and the implication of other genes involved in oocyte competence.
Subject(s)
Animals , Embryonic Development , Gene Expression , Swine/embryology , In Vitro Oocyte Maturation Techniques/statistics & numerical data , In Vitro Oocyte Maturation Techniques/veterinary , Cytoplasmic Structures , Fertilization in Vitro/veterinary , OocytesABSTRACT
In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ») or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Students t-test, logistic regression and test of proportions, and differences were considered significant when P<0.05. There was no difference between recovery rates of groups G-A (66.5%; 330/496) and G-B (65.5%; 359/548). In the G-A group, grade II oocytes were related to higher recovery rates (46.9%; 145/330) than grade I (23.6%; 72/330), grade III (20.6%; 59/330) and denuded oocytes (8.5%; 24/330; P<0.05).(AU)
Na obtenção de oócitos para a espécie equina, aspectos físicos são de alta significância para o sucesso da técnica, como a pressão de vácuo e o calibre de agulha utilizado, além da forma como é realizada a raspagem da parede folicular. O objetivo deste estudo foi investigar o índice de recuperação de oócitos equinos aspirados pela técnica de raspagem da parede folicular, testando calibres distintos de agulhas descartáveis, assim como avaliação morfológica dos complexos cumulus oophorus (CCOs). Foram utilizados ovários de éguas (n=447), obtidos em abatedouro local, transportados ao laboratório em recipiente térmico (20 ºC) e submetidos à dissecação da túnica albugínea e tecidos conectores. Os folículos aspirados obtinham diâmetro entre 10 mm a 25 mm, e para tanto utilizou-se a agulha 30x8 (21G 1 ») ou 40x12 (18G 1 ½), formando respectivamente, o grupo A (G-A) e grupo B (G-B). No G-A e G-B aspiraram-se 480 e 548 foliculos, respectivamente. Sob lupa estéreo-microscópica avaliou-se os oócitos quanto à qualidade do ooplasma e características das células do cumulus oophorus (grau I, II, III e desnudo). Para análise estatística utilizou-se teste t Student, regressão logistica e teste de proporções, e as diferenças foram consideradas significativas quando P 0,05. A taxa de recuperação entre G-A e G-B não apresentou diferença; 66,5% (330/496) e 65,5% (359/548), respectivamente. Houve diferençaquanto à qualidade dos oócitos no G-A, no qual os oócitos de grau II obtiveram melhor taxa (46,9%; 145/330; P<0,05) frente ao grau I (23,6%; 72/330), grau III (20,6%; 59/330) e desnudo (8,5%; 24/330). Entretanto, no G-B não houve diferença estatística quanto aos graus de qualidade do CCOs recuperados, grau I (23,4%; 77/359), grau II (43,2%; 145/359), grau III (22,5%; 73/359) e desnudo (11,1%; 32/359).(AU)
Subject(s)
Animals , Female , Horses/anatomy & histology , Horses/physiology , Oocyte Retrieval/veterinaryABSTRACT
In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ») or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Students t-test, logistic regression and test of proportions, and differences were considered significant when P<0.05. There was no difference between recovery rates of groups G-A (66.5%; 330/496) and G-B (65.5%; 359/548). In the G-A group, grade II oocytes were related to higher recovery rates (46.9%; 145/330) than grade I (23.6%; 72/330), grade III (20.6%; 59/330) and denuded oocytes (8.5%; 24/330; P<0.05).
Na obtenção de oócitos para a espécie equina, aspectos físicos são de alta significância para o sucesso da técnica, como a pressão de vácuo e o calibre de agulha utilizado, além da forma como é realizada a raspagem da parede folicular. O objetivo deste estudo foi investigar o índice de recuperação de oócitos equinos aspirados pela técnica de raspagem da parede folicular, testando calibres distintos de agulhas descartáveis, assim como avaliação morfológica dos complexos cumulus oophorus (CCOs). Foram utilizados ovários de éguas (n=447), obtidos em abatedouro local, transportados ao laboratório em recipiente térmico (20 ºC) e submetidos à dissecação da túnica albugínea e tecidos conectores. Os folículos aspirados obtinham diâmetro entre 10 mm a 25 mm, e para tanto utilizou-se a agulha 30x8 (21G 1 ») ou 40x12 (18G 1 ½), formando respectivamente, o grupo A (G-A) e grupo B (G-B). No G-A e G-B aspiraram-se 480 e 548 foliculos, respectivamente. Sob lupa estéreo-microscópica avaliou-se os oócitos quanto à qualidade do ooplasma e características das células do cumulus oophorus (grau I, II, III e desnudo). Para análise estatística utilizou-se teste t Student, regressão logistica e teste de proporções, e as diferenças foram consideradas significativas quando P 0,05. A taxa de recuperação entre G-A e G-B não apresentou diferença; 66,5% (330/496) e 65,5% (359/548), respectivamente. Houve diferençaquanto à qualidade dos oócitos no G-A, no qual os oócitos de grau II obtiveram melhor taxa (46,9%; 145/330; P<0,05) frente ao grau I (23,6%; 72/330), grau III (20,6%; 59/330) e desnudo (8,5%; 24/330). Entretanto, no G-B não houve diferença estatística quanto aos graus de qualidade do CCOs recuperados, grau I (23,4%; 77/359), grau II (43,2%; 145/359), grau III (22,5%; 73/359) e desnudo (11,1%; 32/359).
Subject(s)
Female , Animals , Horses/anatomy & histology , Horses/physiology , Oocyte Retrieval/veterinaryABSTRACT
Background: The goat rearing is practiced mainly in the Northeast region with semiarid climate, since climate conditionsand vegetation are extremely favorable to the species, but it has low productivity due to the low level of technology employed. Management alternatives aimed at maximizing productivity at low cost are essential tools for the reversal of thisframework. Aiming to focus the onset of estrus for females at low cost is common to use the male effect with the breedingseason, representing a practical, inexpensive and efficient methodology for the producer Thus, this study is to evaluate theeffect of different durations of mating season associated with the male effect on reproductive performance of multiparousAlpine goats during the dry and rainy seasons.Materials, Methods & Results: The survey was conducted in the municipality of Serra Talhada-PE from October 2011 toMay 2013. A total of 160 multiparous females aged between 22 and 74 months and four breeders aged between 24 and 38months were used. In the first step, we tested the mating season durations 25 (MS-25) and 45 days (MS-45). In the secondstep, the mating seasons were 25 (MS-25) and 15 days (MS-15) in different climate periods, each experimental groupconsisting of 20 females and one breeder that has been away from females for 15 days before starting the season in orderto stimulate the male effect and thus synchronizing the estrus. The lots were observed at 6:00 and 16:00 pm by qualifiedpersonnel to identify the mated females and after 45 days of the last mating, an ultrasound examination was performed todetect the matrices that had positive pregnancy, assessing the data by the Chi-square test at 5% significance. The estrusresults obtained during the first stage of...
Subject(s)
Male , Female , Animals , Apoptosis , Goats , Estrus , Ovarian Follicle , In Situ Nick-End Labeling/veterinaryABSTRACT
Background: The goat rearing is practiced mainly in the Northeast region with semiarid climate, since climate conditionsand vegetation are extremely favorable to the species, but it has low productivity due to the low level of technology employed. Management alternatives aimed at maximizing productivity at low cost are essential tools for the reversal of thisframework. Aiming to focus the onset of estrus for females at low cost is common to use the male effect with the breedingseason, representing a practical, inexpensive and efficient methodology for the producer Thus, this study is to evaluate theeffect of different durations of mating season associated with the male effect on reproductive performance of multiparousAlpine goats during the dry and rainy seasons.Materials, Methods & Results: The survey was conducted in the municipality of Serra Talhada-PE from October 2011 toMay 2013. A total of 160 multiparous females aged between 22 and 74 months and four breeders aged between 24 and 38months were used. In the first step, we tested the mating season durations 25 (MS-25) and 45 days (MS-45). In the secondstep, the mating seasons were 25 (MS-25) and 15 days (MS-15) in different climate periods, each experimental groupconsisting of 20 females and one breeder that has been away from females for 15 days before starting the season in orderto stimulate the male effect and thus synchronizing the estrus. The lots were observed at 6:00 and 16:00 pm by qualifiedpersonnel to identify the mated females and after 45 days of the last mating, an ultrasound examination was performed todetect the matrices that had positive pregnancy, assessing the data by the Chi-square test at 5% significance. The estrusresults obtained during the first stage of...(AU)
Subject(s)
Animals , Male , Female , Ovarian Follicle , Apoptosis , Goats , Estrus , In Situ Nick-End Labeling/veterinaryABSTRACT
Objetivou-se com este estudo determinar a influência das estações seca e chuvosa na maturação de oócitos e produção in vitro (PIV) de embriões na espécie caprina. Os ovários das cabras nas estações seca (outubro a março) e chuvosa (abril a setembro) foram colhidos em abatedouro e transportados ao Laboratório de Biotécnicas da Reprodução da UFRPE. Os complexos cumulus oophorus (CCOs) foram colhidos pela técnica de "slicing" dos folículos entre 2 a 6 mm de diâmetro e selecionados com base na classificação morfológica. Foram realizadas 12 repetições, nas quais os CCOs foram submetidos à maturação, fertilização e cultivo in vitro dos embriões. A média e desvio padrão da taxa de clivagem foi determinada no dia 3 (D-3) e dos embriões que se desenvolveram aos estádios de 8-16 células, mórula e blastocisto foi determinada nos dias 4 (D-4), 5 (D-5) e 8 (D-8) após a fecundação, respectivamente. A quantidade de blastômeros foi determinada com o corante DAPI e os blastômeros positivos para apoptose por meio do teste de TUNEL. A produção de embriões no D-3 e mórulas foram inferiores ao obtido no período chuvoso (P < 0,05). Não apresentaram diferenças (P > 0,05) quanto às fases de maturação, fertilização, cultivo no D-4 e blastocisto. Os embriões produzidos na estação seca apresentaram maior incidência de apoptose (P < 0,05). Nas condições descritas neste estudo, os resultados permitem concluir que as fases iniciais do desenvolvimento embrionário sofrem maior impacto negativo durante a estação seca em protocolos de PIV na espécie caprina.(AU)
This study aimed to determine the influence of dry and rainy seasons on oocyte maturation and in vitro production (IVP) of embryos in goats. The ovaries of does in dry (October to March) and rainy season (April-September) were collected at a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing of the follicles from 2 to 6 mm in diameter, and selected based on morphologic classification. We used 12 replicates, in which the COCs were submitted to maturation, fertilization and in vitro culture. The cleavage rate was determined on day 3 (D-3) and embryos that developed to 8-16 cells (D-4), morulae (D-5) and blastocyst (D-8) stages after fertilization. The number of blastomeres was assessed with DAPI staining, and the determination of apoptotic blastomeres was performed by TUNEL assay. In the dry season, D-3 embryos and morulae development was lower than that obtained in the rainy season (P<0.05). However, no differences (P>0.05) were observed on oocyte maturation, fertilization, D-4 embryo yield and blastocyst development. Embryos produced during the dry season had a higher incidence of apoptosis on D- 3 and at the morulae stage (P<0.05). Under the conditions described in this study, the results suggest that the early stages of embryonic development suffer greater negative impact during the dry season in IVP protocols in goats.(AU)
Subject(s)
Animals , Female , Ruminants/embryology , Dry Season , Rainy Season , Tropical Climate/adverse effects , In Vitro Oocyte Maturation Techniques/veterinaryABSTRACT
Objetivou-se com este estudo determinar a influência das estações seca e chuvosa na maturação de oócitos e produção in vitro (PIV) de embriões na espécie caprina. Os ovários das cabras nas estações seca (outubro a março) e chuvosa (abril a setembro) foram colhidos em abatedouro e transportados ao Laboratório de Biotécnicas da Reprodução da UFRPE. Os complexos cumulus oophorus (CCOs) foram colhidos pela técnica de "slicing" dos folículos entre 2 a 6 mm de diâmetro e selecionados com base na classificação morfológica. Foram realizadas 12 repetições, nas quais os CCOs foram submetidos à maturação, fertilização e cultivo in vitro dos embriões. A média e desvio padrão da taxa de clivagem foi determinada no dia 3 (D-3) e dos embriões que se desenvolveram aos estádios de 8-16 células, mórula e blastocisto foi determinada nos dias 4 (D-4), 5 (D-5) e 8 (D-8) após a fecundação, respectivamente. A quantidade de blastômeros foi determinada com o corante DAPI e os blastômeros positivos para apoptose por meio do teste de TUNEL. A produção de embriões no D-3 e mórulas foram inferiores ao obtido no período chuvoso (P 0,05) quanto às fases de maturação, fertilização, cultivo no D-4 e blastocisto. Os embriões produzidos na estação seca apresentaram maior incidência de apoptose (P < 0,05). Nas condições descritas neste estudo, os resultados permitem concluir que as fases iniciais do desenvolvimento embrionário sofrem maior impacto negativo durante a estação seca em protocolos de PIV na espécie caprina.
This study aimed to determine the influence of dry and rainy seasons on oocyte maturation and in vitro production (IVP) of embryos in goats. The ovaries of does in dry (October to March) and rainy season (April-September) were collected at a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing of the follicles from 2 to 6 mm in diameter, and selected based on morphologic classification. We used 12 replicates, in which the COCs were submitted to maturation, fertilization and in vitro culture. The cleavage rate was determined on day 3 (D-3) and embryos that developed to 8-16 cells (D-4), morulae (D-5) and blastocyst (D-8) stages after fertilization. The number of blastomeres was assessed with DAPI staining, and the determination of apoptotic blastomeres was performed by TUNEL assay. In the dry season, D-3 embryos and morulae development was lower than that obtained in the rainy season (P0.05) were observed on oocyte maturation, fertilization, D-4 embryo yield and blastocyst development. Embryos produced during the dry season had a higher incidence of apoptosis on D- 3 and at the morulae stage (P<0.05). Under the conditions described in this study, the results suggest that the early stages of embryonic development suffer greater negative impact during the dry season in IVP protocols in goats.
Subject(s)
Female , Animals , Tropical Climate/adverse effects , Rainy Season , Dry Season , Ruminants/embryology , In Vitro Oocyte Maturation Techniques/veterinaryABSTRACT
Background: The low productivity of Northeast goat herds has been circumvented by the importation of exotic species; however, caution is needed due to the susceptibility of these breeds to the high temperatures found in this region. It is now known that the oocyte and the embryo are the primary targets of the deleterious effects induced by heat stress, causing cellular damage that triggers the cascade of apoptosis. Therefore, the purpose of this study was to evaluate the effect of thermal heat stress during in vitro maturation of oocytes and its effects on embryo production in goats. Materials, Methods & Results: The ovaries were collected in a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing the follicles from 2 to 6 mm in diameter, selected based on morphology and placed in a basic medium for maturation. In 10 replications, the COCs were submitted to the thermal heat stress at 41°C for 0 (thermoneutrality at 39°C), 3, 6, 12, 18, and 24 h of maturation in vitro. The data was evaluated in maturation, fertilization, cleavage (D-3), stage of 8-16 cells (D4), morula (D-5), and blastocyst (D-8) after fertilization and blastocysts positive for apoptosis through the TUNEL test. For statistics, the results were expressed as mean and standard deviation. Also considering the measurements addressed in percentages, a comparison of variances was carried out, F-test for variances to the level of signifi cance 5% (P < 0.05). Then, a t-test to compare averages was conducted, to the significance level of 5%, for equivalent variances or distinct variances, according to what was observed in the F-test for variances. A signifi cant difference (P < 0.05) was observed during all time periods studied for heat stress on maturation, fertilization, D-3, D-4, and D-5. On D-8 no significant difference (P > 0.05) was observed between the periods of 3 vs 6 and 18 vs 24 h, and in the blastocysts positive by the TUNEL test for the periods of 0 vs 3, 3 vs 6, 12 vs 18, and 18 vs 24 h of heat stress. Discussion: When applying a thermal shock that produces damage to the oocyte maturation in vitro, the characteristic membrane, chromatin configuration, and meiotic spindles are changed, and thus, the developmental potential of oocytes after fertilization is compromised. It was observed in this study that there was a gradual reduction in the number of oocytes as the time of exposure to heat shock increased, reflecting directly on each stage of IVP embryos. These stages are most vulnerable during maturation in vivo (ovulation), fertilization, within two days after fertilization, and in the first division of cleavage, as evidenced in this study in vitro after heat stress, reducing the number of blastocysts. This suggests that apoptosis can be induced in pre-implantation of embryos exposed to maternal hyperthermia. Moreover, the degree of apoptosis experienced by IVP embryos generally reflects the severity of thermal shock. In this study, the percentage of cells that were TUNEL positive increased with the prolongation of thermal shock. Induction of apoptosis was time dependent and the number of apoptotic cells increased proportionally after 6, 12, 18, and 24 h of exposure. Under the conditions observed in this study, the results indicate that the time in which the oocyte is exposed to heat stress during maturation in vitro is of great importance for embryonic development and their level of apoptotic cells.
Subject(s)
Animals , Female , Oocytes/radiation effects , Ruminants/embryology , Heat-Shock Response , In Situ Nick-End Labeling/veterinary , In Vitro Oocyte Maturation Techniques/veterinaryABSTRACT
Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be infl uenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and ß-actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantifi ed to obtain the relative mRNA abundance. Part of oocytes submitted to IVM medium supplemented with FBS (G2) or BSA (G3) was stained with Hoechst 33342 to assess the nuclear maturation rate by fluorescence microscopy. The results revealed that relative abundances of HAS (P = 0.000), link protein 1 (P = 0.001), connexin 43 (P = 0.007) and ß-actin (P = 0.011) transcripts differed between COCs submitted to IVM in FBS medium (G2) and COCs not submitted to IVM (G1) or COCs maturated in BSA medium (G3). When COCs submitted to IVM in FBS or BSA media are compared, no statistically significant differences (P > 0.05) were observed in meiosis resumption (86.7% and 91.5%, respectively) or in nuclear maturation rates (56.1% and 58.5%). Discussion: HAS2 is involved in the synthesis of hyaluronic acid (HA) by cumulus cells, and plays an important role in ECM expansion and in oocyte competence development. This protein organization of the ECM, formed by the aggregation of HA and proteoglycans, depends on link protein 1; it is also produced by cumulus cells and is implicated in COC expansion. Connexin 43 belongs to a protein family that establishes gap junctions that play an important role in the cellular communication and coordinated response processes. The role of gap junctions in bovine oocytes during IVM has been associated with maturation rates and cumulus expansion; this expansion of cumulus cells is accompanied by changes in the transmembrane channels formed by connexin 43. The higher mRNA expression of the HAS2, link protein 1, connexin 43 and ß-actin genes in bovine COCs submitted to IVM in FBS medium, in comparison with COCs before IVM or COCs maturated in BSA medium may be associated with FBS constituents, which would act as transcription factors for these genes during ECM expansion. Although the results obtained allow associating the differential expression of transcripts to the presence of FBS in the IVM medium, the data reveal that meiosis resumption and nuclear maturation apparently were not influenced by the protein supplementation regimens in the IVM medium, supplemented either with FBS or BSA.
Subject(s)
Animals , Female , Cattle , Serum Albumin, Bovine , Cattle/genetics , Gene Expression , Cumulus Cells , In Vitro Oocyte Maturation Techniques/veterinary , Real-Time Polymerase Chain Reaction/veterinary , Hyaluronan Synthases/analysisABSTRACT
Background: The low productivity of Northeast goat herds has been circumvented by the importation of exotic species; however, caution is needed due to the susceptibility of these breeds to the high temperatures found in this region. It is now known that the oocyte and the embryo are the primary targets of the deleterious effects induced by heat stress, causing cellular damage that triggers the cascade of apoptosis. Therefore, the purpose of this study was to evaluate the effect of thermal heat stress during in vitro maturation of oocytes and its effects on embryo production in goats. Materials, Methods & Results: The ovaries were collected in a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing the follicles from 2 to 6 mm in diameter, selected based on morphology and placed in a basic medium for maturation. In 10 replications, the COCs were submitted to the thermal heat stress at 41C for 0 (thermoneutrality at 39C), 3, 6, 12, 18, and 24 h of maturation in vitro. The data was evaluated in maturation, fertilization, cleavage (D-3), stage of 8-16 cells (D- 4), morula (D-5), and blastocyst (D-8) after fertilization and blastocysts positive for apoptosis through the TUNEL test. For statistics, the results were expressed as mean and standard deviation. Also con
O rebanho caprino no país se concentra principalmente na região Nordeste com 9,3 milhões de cabeças, destacando-se os estados da Bahia (29,2%) e Pernambuco (20,5%). Apesar de deter praticamente todo o rebanho caprino, o Nordeste necessita de pesquisas e tecnologias para minimizar a relação custo/benefício do sistema de produção, estimulando o desenvolvimento e criando novas possibilidades de agronegócio. A baixa produtividade dos rebanhos locais tem sido contornada através da importação de raças exóticas para o Semiárido nordestino, porém, é preciso cautela devido à susceptibilidade destas raças às temperaturas elevadas encontrada nesta região. [...]
ABSTRACT
Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta
Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta
ABSTRACT
Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta
Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta
ABSTRACT
Background: The low productivity of Northeast goat herds has been circumvented by the importation of exotic species; however, caution is needed due to the susceptibility of these breeds to the high temperatures found in this region. It is now known that the oocyte and the embryo are the primary targets of the deleterious effects induced by heat stress, causing cellular damage that triggers the cascade of apoptosis. Therefore, the purpose of this study was to evaluate the effect of thermal heat stress during in vitro maturation of oocytes and its effects on embryo production in goats. Materials, Methods & Results: The ovaries were collected in a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing the follicles from 2 to 6 mm in diameter, selected based on morphology and placed in a basic medium for maturation. In 10 replications, the COCs were submitted to the thermal heat stress at 41C for 0 (thermoneutrality at 39C), 3, 6, 12, 18, and 24 h of maturation in vitro. The data was evaluated in maturation, fertilization, cleavage (D-3), stage of 8-16 cells (D- 4), morula (D-5), and blastocyst (D-8) after fertilization and blastocysts positive for apoptosis through the TUNEL test. For statistics, the results were expressed as mean and standard deviation. Also con
O rebanho caprino no país se concentra principalmente na região Nordeste com 9,3 milhões de cabeças, destacando-se os estados da Bahia (29,2%) e Pernambuco (20,5%). Apesar de deter praticamente todo o rebanho caprino, o Nordeste necessita de pesquisas e tecnologias para minimizar a relação custo/benefício do sistema de produção, estimulando o desenvolvimento e criando novas possibilidades de agronegócio. A baixa produtividade dos rebanhos locais tem sido contornada através da importação de raças exóticas para o Semiárido nordestino, porém, é preciso cautela devido à susceptibilidade destas raças às temperaturas elevadas encontrada nesta região. [...]
ABSTRACT
This study aimed to determine the in vitro nuclear maturation and cell death by apoptosis in goat oocytes. The ovaries of goats were collected during the dry (October-March) and rainy (April-September) seasons in slaughterhouses and transported to the Laboratory of Reproductive Biotechniques of UFRPE. Twelve repetitions were performed and cumulus oophorus were collected from follicles of 2-6 mm diameter using the technique of "slicing" and selected based on morphology. The experiment contained two groups: Group-1 (not matured), evaluated immediately after collection, and Group-2 (matured in vitro), assessed after 24 hours of maturation in a CO2 incubator, in which 25 oocytes per drop were placed in alkaline maturation (MBM). After collecting the oocytes from G-1 and G-2, the quality was determined by testing the enzyme activity of caspases and DNA fragmentation (TUNEL), using PhiPhiLux-G1D2 reagent, and in group G-2 the quality was only determined after nuclear maturation. During nuclear maturation stages, germinal vesicle, germinal vesicle breakdown and metaphase I and II were evaluated and no significant differences were found (P> 0.05). There was also no significant difference (P> 0.05) in enzymes activity of caspase and DNA fragmentation of oocytes matured and not matured in vitro. Based on the data obtained, it was concluded that rainy and dry seasons have no effect
Este estudo teve como objetivo determinar a maturação nuclear in vitro e a morte celular por apoptose em oócitos caprinos. Os ovários de cabras foram coletados nos períodos seco (outubro a março) e chuvoso (abril a setembro), em abatedouros, e transportados ao Laboratório de Biotécnicas da Reprodução da UFRPE. Foram realizadas 12 repetições e os complexos cumulus oophorus foram colhidos de folículos entre 2 a 6 mm de diâmetro pela técnica de slicing e selecionados com base na morfologia. O experimento continha dois grupos: Grupo-1 (não maturados), avaliados logo após a colheita, e Grupo-2 (maturados in vitro), avaliados após a maturação de 24 horas em incubadora de CO2, na qual 25 oócitos foram colocados por gota em meio básico de maturação (MBM). Após a coleta dos oócitos do G-1 e G-2, foi determinada a qualidade, por meio dos testes de atividade das enzimas caspases e o de fragmentação de DNA (TUNEL), com reagente PhiPhiLux-G1D2, sendo que no grupo G-2 a qualidade só foi determinada após maturação nuclear. Avaliou-se durante os estádios de maturação nuclear as fases de vesícula germinativa, rompimento da vesícula germinativa, metáfase I e II, não havendo diferença significativa (P > 0,05). Também não foi encontrada diferença significativa (P > 0,05) na atividade das enzimas caspases e na fragmentação do DNA dos oócitos não maturados e maturados in vitro. Com base nos
ABSTRACT
This study aimed to determine the in vitro nuclear maturation and cell death by apoptosis in goat oocytes. The ovaries of goats were collected during the dry (October-March) and rainy (April-September) seasons in slaughterhouses and transported to the Laboratory of Reproductive Biotechniques of UFRPE. Twelve repetitions were performed and cumulus oophorus were collected from follicles of 2-6 mm diameter using the technique of "slicing" and selected based on morphology. The experiment contained two groups: Group-1 (not matured), evaluated immediately after collection, and Group-2 (matured in vitro), assessed after 24 hours of maturation in a CO2 incubator, in which 25 oocytes per drop were placed in alkaline maturation (MBM). After collecting the oocytes from G-1 and G-2, the quality was determined by testing the enzyme activity of caspases and DNA fragmentation (TUNEL), using PhiPhiLux-G1D2 reagent, and in group G-2 the quality was only determined after nuclear maturation. During nuclear maturation stages, germinal vesicle, germinal vesicle breakdown and metaphase I and II were evaluated and no significant differences were found (P> 0.05). There was also no significant difference (P> 0.05) in enzymes activity of caspase and DNA fragmentation of oocytes matured and not matured in vitro. Based on the data obtained, it was concluded that rainy and dry seasons have no effect
Este estudo teve como objetivo determinar a maturação nuclear in vitro e a morte celular por apoptose em oócitos caprinos. Os ovários de cabras foram coletados nos períodos seco (outubro a março) e chuvoso (abril a setembro), em abatedouros, e transportados ao Laboratório de Biotécnicas da Reprodução da UFRPE. Foram realizadas 12 repetições e os complexos cumulus oophorus foram colhidos de folículos entre 2 a 6 mm de diâmetro pela técnica de slicing e selecionados com base na morfologia. O experimento continha dois grupos: Grupo-1 (não maturados), avaliados logo após a colheita, e Grupo-2 (maturados in vitro), avaliados após a maturação de 24 horas em incubadora de CO2, na qual 25 oócitos foram colocados por gota em meio básico de maturação (MBM). Após a coleta dos oócitos do G-1 e G-2, foi determinada a qualidade, por meio dos testes de atividade das enzimas caspases e o de fragmentação de DNA (TUNEL), com reagente PhiPhiLux-G1D2, sendo que no grupo G-2 a qualidade só foi determinada após maturação nuclear. Avaliou-se durante os estádios de maturação nuclear as fases de vesícula germinativa, rompimento da vesícula germinativa, metáfase I e II, não havendo diferença significativa (P > 0,05). Também não foi encontrada diferença significativa (P > 0,05) na atividade das enzimas caspases e na fragmentação do DNA dos oócitos não maturados e maturados in vitro. Com base nos
ABSTRACT
O objetivo deste trabalho foi avaliar o efeito da temperatura (37 ou 38,5°C) de fecundação in vitro (FIV) e da retirada das células do cumulus oophorus após a FIV ou após doze horas de cultivo embrionário (CIV), nos índices de poliespermia e no desenvolvimento de embriões suínos in vitro. Para a FIV, incubaram-se oócitos e espermatozoides em duas temperaturas (37 ou 38,5ºC). Após as oito horas de FIV, metade dos zigotos de cada grupo teve as células do cumulus oophorus retiradas e foi colocada em meio de cultivo NCSU23. Colocou-se a outra metade apenas em meio de cultivo e ambos os grupos foram mantidos nas mesmas temperaturas da FIV. Após doze horas de CIV, retiraram-se as células do restante dos zigotos e aferiram-se os índices de poliespermia, de todos os grupos. Na segunda etapa do experimento, os zigotos permaneceram em cultivo para avaliação do desenvolvimento embrionário. Não houve efeito da temperatura de FIV e da retirada das células do cumulus oophorus após doze horas de cultivo nos índices de poliespermia e no desenvolvimento embrionário (p<0,05). Concluiu-se que a temperatura de FIV e a presença das células do cumulus oophorus pós-fecundação não interferiram nos índices de poliespermia e no desenvolvimento embrionário.(AU)
Effects of in vitro fertilization (IVF) temperature and cumulus oophorus cells removal after IVF or 12 h of embryo culture (IVC) on polyspermy and embryo development rates were evaluated in swine. Oocytes and spermatozoa were incubated at 37 or 38.5ºC during IVF procedure. Cumulus oophorus cells were removed from 50% of zygotes of each group 8 hours after IVF and all zygotes were cultured with NCSU23 media. Polyspermy rates were assessed after 12 hours of IVC, when cumulus oophorus cells were removed from the rest of zygotes. In a second experiment, embryos remained in culture for the evaluation of embryo development. No effects of IVF temperature or cumulus oophorus cells removal were observed after 12 hours of IVC on polyspermy and embryo development (p<0.05). In conclusion, IVF temperature and the presence of cumulus oophorus cells after IVF do not interfere on polyspermy and embryo development rates.(AU)