Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos

Association between BMP4 gene polymorphism and in vitro embryo production traits in Gyr cows

Background: candidate genes and their polymorphisms have been associated with traits of economic interest in cattle, representing an important strategy for genetic improvement of complex traits. Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGFβ) superfamily, which is involved with several events of embryonic, fetal, and adult development in vertebrates. Objective: the aim of this study was to evaluate the degree of association between polymorphism in the BMP4 gene (guanine for thymine- G>T, SNP rs109778173) and the performance of Gyr oocyte donors, including the rate of Cumulus-oophorus complex obtained in each session of follicular aspiration (OPU), embryo development, and pregnancy rates. Methods: DNA was extracted from hair follicles from 50 oocyte donors and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data from 212 OPU-IVP sessions was collected, and the following traits were associated with BMP4 polymorphism (SNP rs109778173): number and ratio of viable Cumulus-oophorus complexes; number of cleaved embryos at day 4 of culture; number of transferable embryos at day 7 of culture, and pregnancies on days 30 and 60 after embryo transfer. Results: the studied BMP4 polymorphism was significantly associated (pT-, SNP rs109778173) e o desempenho de doadoras de oócitos da raça Gyr, incluindo a taxa de complexos Cumulus-oophorus obtidos em cada sessão de aspiração folicular (OPU), o desenvolvimento embrionário e a taxa de prenhez dos embriões. Métodos: o DNA foi extraído do folículo piloso de 50 vacas Gyr doadoras de oócitos, e a genotipagem foi realizada pela técnica de reação em cadeia da polimerase-polimorfismo no comprimento de fragmentos de restrição (PCR-RFLP). Dados de 212 sessões de OPU-IVP foram coletados, e as características a seguir foram associadas com o polimorfismo no gene BMP4 (SNP rs109778173): número e taxa de complexos Cumulus-oophorus viáveis dos embriões clivados no dia 4 de cultivo, dos embriões transferíveis no dia 7 de cultivo e de prenhezes no dia 30 e 60 após a transferência dos embriões. Resultados: o polimorfismo estudado foi significativamente associado (pT-, SNP rs109778173) y el desempeño de donantes de ovocitos de la raza Gyr, incluyendo la tasa de complejos cummulus-oophorus obtenidos en cada sesión de aspiración folicular (OPU), el desarrollo embrionario y la tasa de preñez. Métodos: el ADN fue extraído del folículo piloso de 50 vacas Gyr donantes de ovocitos, el genotipado fue realizado por la técnica de reacción en cadena de polimerasa-polimorfismos de longitud de fragmentos de restricción (PCR-RFLP). Datos de 212 sesiones de OPU-IVP fueron colectados y las características a seguir fueron asociadas con el polimorfismo en el gen BMP4 (SNP rs109778173): número y tasa de complejos de Cumulus-oophorus viables, de embriones clivados al día 4 de cultivo, de embriones transferibles al día 7 de cultivo, y de preñeces a los días 30 y 60 posterior a la transferencia de los embriones. Resultados: el polimorfismo fue significativamente asociado (p<0,01) con número y tasa de complejos Cumulus-oophorus viables y tasa de preñez al día 30. Conclusión: el genotipo GT (SNP rs109778173) fue asociado a resultados inferiores relacionados con características de OPU-IVP. Este resultado sugiere posibles efectos genéticos del gen BMP4 sobre el desempeño de donadoras de oocitos de la raza Gyr, siendo necesarios estudios del gen BMP4 en relación a las alteraciones en el desarrollo embrionario subsecuentes a la mutación citada

Association between BMP4 gene polymorphism and in vitro embryo production traits in Gyr cows

Background: candidate genes and their polymorphisms have been associated with traits of economic interest in cattle, representing an important strategy for genetic improvement of complex traits. Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGFβ) superfamily, which is involved with several events of embryonic, fetal, and adult development in vertebrates. Objective: the aim of this study was to evaluate the degree of association between polymorphism in the BMP4 gene (guanine for thymine- G>T, SNP rs109778173) and the performance of Gyr oocyte donors, including the rate of Cumulus-oophorus complex obtained in each session of follicular aspiration (OPU), embryo development, and pregnancy rates. Methods: DNA was extracted from hair follicles from 50 oocyte donors and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data from 212 OPU-IVP sessions was collected, and the following traits were associated with BMP4 polymorphism (SNP rs109778173): number and ratio of viable Cumulus-oophorus complexes; number of cleaved embryos at day 4 of culture; number of transferable embryos at day 7 of culture, and pregnancies on days 30 and 60 after embryo transfer. Results: the studied BMP4 polymorphism was significantly associated (p<0.01) with the number and ratio of viable cumulus-oocyte complexes, and the ratio of pregnancies at 30 days. Conclusion: the GT genotype (SNP rs109778173) was associated with inferior performance regarding OPU-IVP traits. This finding suggests possible genetic effects of BMP4 on performance of Gyr oocyte donors. Studies are needed on the BMP4 gene regarding subsequent changes in embryonic development of said mutation

Genetic diversity in oocyte donors used in in vitro bovine embryo production programs in Brazil

Abstract Background: Current reproductive management of bovine elite populations involves the use of assisted reproductive technologies (ARTs), aiming to obtain the greatest genetic gain. However, inadequate use of ARTs may lead to loss of genetic diversity in the offspring. Objective: To assess the genetic diversity in elite female cattle populations used in commercial in vitro embryo production. Methods: Using genetic and ecological approaches for the study of populations based on microsatellite markers, we assessed the genetic diversity between and within populations of cows used in commercial in vitro embryo production programs in Brazil. Results: Endogamy within populations varied from zero to 9.1%, while heterozygosity between populations (FST) was <0.05 in the different population interactions. AMOVA showed 1% variation between populations, 8% between individuals and 91% within individuals. The dimensionality reduction method utilized indicated a lack of structure in the populations analyzed, identifying two main clusters in the three populations. Conclusions: Low genetic diversity between cow populations associated with commercial programs of in vitro embryo production in Brazil was evidenced. Variable levels of endogamy within the populations were observed. Approaches of population genetics as well as ecological diversity can be implemented to more thoroughly estimate genetic diversity in livestock populations.Resumo Antecedentes: O atual manejo reprodutivo das populações de elite em bovinos envolve o uso de tecnologias de reprodução assistida (ARTs), visando obter o maior ganho genético. No entanto, o uso inadequado de ARTs pode levar à perda de diversidade genética na prole. Objetivo: Avaliar a diversidade genética em populações de vacas de elite utilizadas na produção comercial de embriões bovinos in vitro. Métodos: Utilizando abordagens da genética e ecologia de populações baseadas em marcadores microssatélites, foi avaliada a diversidade genética entre e dentro das populações de vacas participantes de programas comercias de produção in vitro de embriões. Resultados: A endogamia dentro das populações variou de zero a 9,1%, enquanto a heterozigosidade entre populações (FST) foi <0,05 nas diferentes interações populacionais. AMOVA mostrou variação de 1% entre populações, 8% entre indivíduos e 91% dentro de indivíduos. O método de redução de dimensionalidade utilizado indicou uma falta de estrutura nas populações analisadas, identificando dois clusters principais nas três populações. Conclusões: Baixa diversidade genética entre populações de vacas associadas a programas de produção in vitro de embriões foi evidenciada. Níveis de endogamia variáveis dentro das populações foram observados. Abordagens da genética populacional assim como de diversidade ecológica podem ser implementadas na tentativa de estimar de maneira mais abrangente a diversidade genética em populações animais de interesse pecuário.Resumen Antecedentes: El actual manejo reproductivo en poblaciones de bovinos de élite incluye la utilización de tecnologías de reproducción asistida (ARTs) con el fin de obtener mayor ganancia genética. Sin embargo, el uso inadecuado de las ART puede llevar a la pérdida de diversidad genética en los descendientes. Objetivo: Evaluar la diversidad genética en poblaciones de vacas de élite utilizadas en la producción comercial de embriones bovinos in vitro. Métodos: Utilizando abordajes de la genética y ecología de poblaciones basados en marcadores microsatélites, evaluamos la diversidad genética entre y dentro de poblaciones de vacas participantes de programas comerciales de producción de embriones in vitro en Brasil. Resultados: La endogamia dentro de las poblaciones varió de cero a 9,1%, mientras que la heterocigosidad entre poblaciones (FST) fue <0,05 en las diferentes interacciones de la población. El AMOVA mostró variación del 1% entre poblaciones, 8% entre individuos y 91% dentro de individuos. El método de reducción de dimensionalidad utilizado indicó una falta de estructura en las poblaciones analizadas, identificando dos grupos principales en las tres poblaciones. Conclusiones: Se evidenció una baja diversidad genética entre las poblaciones de vacas asociadas a programas comerciales de producción de embriones in vitro en Brasil. Se evidenciaron niveles variables de endogamia entre las poblaciones. Abordajes de la genética poblacional, así como de diversidad ecológica pueden ser implementados para estimar de manera más amplia la diversidad genética en poblaciones animales de interés pecuario