RESUMO
In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays.
RESUMO
One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.
RESUMO
A produção de búfalos está se expandindo na Amazônia, sendo assim o uso de biotecnologias reprodutivas em programas de melhoramento genético tornou-se uma estratégia para melhorar a produtividade dos rebanhos sem a necessidade de aumentar as áreas de pastagens. A Produção In Vitro de Embriões (PIVE) é uma das biotecnologias disponíveis, a qual é contiuamente aperfeiçoada através da pesquisa de novos componentes para os meios de cultivo e da pesquisa de protocolos espécie específicos que resultem em maior número de embriões de boa qualidade. Contudo, a falta de informações sobre os requerimentos metabólicos de gametas e embriões têm dificultado a consolidação de protocolos de PIVE específicos para búfalos. Neste artigo discutimos as abordagens transcriptômicas que estão ajudando a preencher esta falta de informações, as limitações destas abordagens e os tópicos de pesquisa que ainda precisam ser investigados em búfalos.
Buffalo production is expanding in the Amazon region therefore the use of reproductive biotechnologies in breeding programs became a strategy to improve the herd productivity without a need for increasing pasture areas. In Vitro Production (IVP) is one of the available biotechnologies, which is continuously being improved through the research of novel components of culture media and the research of specie-specific protocols to obtain an increased number of good quality embryos. However, the lack of information about the metabolic requirements of gametes and embryos have delayed the consolidation of buffalos IVP specific protocols. Herein, we discussed the transcriptomic approaches that are helping to fill the lack of information, the limitations of these approaches and the research topics that still needs further investigations in buffaloes.
Assuntos
Animais , Búfalos/genética , Embrião de Mamíferos , Transcriptoma/genética , Técnicas In VitroRESUMO
A produção de búfalos está se expandindo na Amazônia, sendo assim o uso de biotecnologias reprodutivas em programas de melhoramento genético tornou-se uma estratégia para melhorar a produtividade dos rebanhos sem a necessidade de aumentar as áreas de pastagens. A Produção In Vitro de Embriões (PIVE) é uma das biotecnologias disponíveis, a qual é contiuamente aperfeiçoada através da pesquisa de novos componentes para os meios de cultivo e da pesquisa de protocolos espécie específicos que resultem em maior número de embriões de boa qualidade. Contudo, a falta de informações sobre os requerimentos metabólicos de gametas e embriões têm dificultado a consolidação de protocolos de PIVE específicos para búfalos. Neste artigo discutimos as abordagens transcriptômicas que estão ajudando a preencher esta falta de informações, as limitações destas abordagens e os tópicos de pesquisa que ainda precisam ser investigados em búfalos.(AU)
Buffalo production is expanding in the Amazon region therefore the use of reproductive biotechnologies in breeding programs became a strategy to improve the herd productivity without a need for increasing pasture areas. In Vitro Production (IVP) is one of the available biotechnologies, which is continuously being improved through the research of novel components of culture media and the research of specie-specific protocols to obtain an increased number of good quality embryos. However, the lack of information about the metabolic requirements of gametes and embryos have delayed the consolidation of buffalos IVP specific protocols. Herein, we discussed the transcriptomic approaches that are helping to fill the lack of information, the limitations of these approaches and the research topics that still needs further investigations in buffaloes.(AU)