UNIVERSIDADE DE SÃO PAULO FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS GIULIANA DE AVILA FERRONATO Produção in vitro de embriões bovinos

FERRONATO, G. A. In vitro production of bovine embryos in different 3D culture systems: effects on nuclear morphology, methylation pattern, gene transcription and extracellular vesicles. 2022. 97 f. Dissertação (Mestrado) – Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2022. The in vitro embryo production (IVP) is a technique widely used to improve the reproductive parameters in bovines and humans. However, the IVP still have some limitations, such as lower blastocysts and pregnancies rates in comparison with the in vivo produced embryos. Moreover, the embryos produced by IVP harbor epigenetic alterations which can affect the conception rates and the health of the offspring. IVP embryos are cultured in petri dishes, that are ~6 x more rigid than the in vivo environment, causing different external stimuli and generating different intracellular responses in the embryos. An alternative to circumvent this and create a more physiological culture environment is to use the 3D cultures, which consists in the creation of a microenvironment where the cells can interact themselves and with the culture medium. These culture systems can be produced using several methodologies and materials. The Liquid Marbles (LM) are a type of 3D culture that consists in the creation of an environment trapping a small droplet of culture medium using a hydrophobic substance. An alternative material used is the alginate, that possess the capacity to generate a stable hydrogel after the addition of a bivalent ion. On this basis, our hypothesis was that the 3D systems produced using LM or alginate hydrogels can benefit the in vitro maturation and in vitro culture of bovine oocytes and embryos, respectively. To this end, in the experiment 1 (chapter 2), the in vitro maturation was carried out in LM. We observed that the nuclear maturation rate was similar between the LM and control group. However, the cumulus cells expansion and the expression of genes involved in the maturation processes were decreased in the LM group. The culture of presumptive zygotes was also carried out in LM. As a result, on the day 7 was observed lower blastocysts rate, decrease in the expression of the miRNA bta-miR-615 and increased in the global levels of DNA methylation and hydroxymethylation in the LM blastocysts compared with the controls. In the experiment 2 (chapter 3), the 3D system using alginate hydrogels were used to culture the embryos. In this experiment, the conventional culture was termed 2D; the embryo culture on top of an overlay of alginate termed 2.5D; and the culture with the embryos encapsulated in an alginate hydrogel termed 3D. After the culture for 7 days in each system, the blastocysts rates were examined. We observed a decrease in the blastocysts rates in the embryos cultured in the 2.5 and 3 D systems compared to the control. Regarding the miRNas expression, the miRNAs miR-1245 and miR-1260b were decreased in the 2.5D group compared with the other groups. Moreover, the miR-541 presented lower expression in the 3D group when compared with the other groups. The global levels of DNA methylation and hydroxymethylation increased only in the embryos from the 2.5D group. In addition, the transcriptome from embryos from the 2.5D and 3D differed from the control group, and are more similar between them. Also, several genes critical for embryo development, pluripotency, placentation were decreased in the embryos cultured in the alginate hydrogel. Altogether, these results suggest that the 3D culture systems used in our experiments impaired the embryo development. However, more studies are necessary to fully understand the effects caused by these molecules on embryos. The IVP technique has constantly evolved in the past 40 years, and always a new approach was attempted a lot of drawbacks had to be surpassed. In the future, we envision that the embryos will be cultured in more physiological systems and they will be more similar to the in vivo ones.