RESUMEN
The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.
O objetivo do presente trabalho foi associar o método de swim-up modificado à centrifugação em gradiente de densidade para a separação de espermatozoides portadores do cromossomo X. A viabilidade e a integridade espermática foram avaliadas pelo método de coloração Azul de Tripan e Giemsa. O controle de qualidade dos espermatozoides centrifugados foi realizado por meio da produção in vitro de embriões bovinos. Os resultados foram validados pela técnica de PCR para verificar a proporção sexual dos embriões produzidos in vitro, com o uso de sequências Y especificas presente no DNA genômico de machos bovinos. Após determinar o sexo genético dos embriões produzidos in vitro, os resultados não mostraram diferença (P<0,05) no desvio da proporção do sexo quando comparou o grupo controle (45,2% de fêmeas) com os outros processos de seleção de espermatozoides (60,6% de fêmeas) (P<0,05). Os métodos de seleção de espermatozoides são capazes de selecionar espermatozoides portadores do cromossomo X sem comprometer a fertilidade, medida pelas taxas de clivagem e blastocisto de 70% e 26%, respectivamente, e foram considerados métodos de relevância para serem introduzidos nos programas de produção in vitro de embriões bovinos.
RESUMEN
In the last years, enormous progress has been made in the analysis of gene transcription at the blastocyst stage. The study of gene expression at this early stage of development is challenging because of the very small amount of starting material, which limits the use of traditional mRNA analysis approaches such as Northern blot. Another problem is the difficulty for data normalization, particularly the identification of the best housekeeping gene with the lowest fluctuation under different developmental conditions. Moreover, the transcriptional analysis of embryo biopsies or individual embryos needs to take into consideration that the blastocyst is a transitional stage of development, which is composed of three different types of cells (trophoblast, epiblast and primitive ectoderm) with different patterns of gene expression, and that there are large differences between male and female blastocysts. In this review, we analyse the different specific and sensitive tools available to compare mRNA expression levels of specific genes at the blastocyst stage, and how the protocol and the analytical method used can influence the results dramatically. Finally, we describe future research challenges to identify candidate genes related to developmental competence of bovine blastocysts, not only in terms of pregnancy rates but also in relation to adverse long-term consequences in the adult animal.