BIOTECHNIQUES APPLIED TO ANIMAL REPRODUCTION

Coordinator: BENNER GERALDO ALVES

This proposal aims to increase the efficiency of assisted reproduction techniques and involves the performance of 6 large integrated studies using reproductive biotechniques related to the manipulation of immature oocytes, sperm (sptz) and embryos of small ruminants. The studies are: Study 1: In vitro culture system (IVC) for primordial follicles; Study 2: IVC system for secondary and antral follicles; Study 3: Culture media for in vitro maturation of oocytes, fertilization and in vitro production (IVP) of embryos; Study 4: Cryopreservation of preantral follicles (FOPA); Study 5: Cryopreservation of semen; Study 6: Proteomic and epigenetic analysis of sperm, oocytes and embryos produced in vivo and in vitro. Study 1 targets primordial follicles that are in a quiescent state. The strategy of this study is to develop an IVC system that promotes the activation of primordial follicles and their subsequent growth in vitro, aiming at the IVC of secondary follicles. This IVC system can be used as a tool to evaluate the efficiency of ovarian tissue cryopreservation protocols to be developed in Study 4. Study 2 targets two follicular categories, advanced secondary and early antral. The strategy of this study is to develop an IVC system that allows us to understand the requirements of these follicular categories, aiming at the IVC of fully grown and meiotically competent oocytes, as well as embryos. This culture system will be of fundamental importance in the continuation of the in vitro development of the secondary follicles produced by Study 1. Study 3 aims to develop new media for oocyte maturation, IVF and IVC of embryos using anethole as an antioxidant agent, ACP and capacitating proteins (BSPs) and/or with a protective effect of sptz (osteopontin-OPN), aiming to improve the efficiency of conventional IVC of embryos, that is, using COCs recovered from superficial antral follicles, which contain fully grown oocytes. The new culture media developed in this study will be used to improve the efficiency of oocyte maturation and IVC of embryos from in vitro grown oocytes generated in Study 2. Study 4 aims to improve the efficiency of ovarian tissue vitrification protocols for the implementation of germplasm (oocyte) banks and future use in IVC and in vivo (xenotransplantation) protocols. This tissue bank, in addition to preserving animals of genetic merit, would ensure the storage of a large quantity of tissue that could be used in the development of IVF media for the activation and growth of primordial follicles (Study 1). Study 5 targets male gametes. The strategy of this study is to improve semen freezing techniques by testing the ACP extender, added with Aloe vera, as well as anethole and other activating proteins (BSPs) and/or with a sptz-protective effect (OPN). This study aims to provide high-quality frozen semen that could be used to improve IVF of both in vitro-grown oocytes (Study 2) and those grown in vivo (Study 3). Study 6 initially aims to investigate the normal proteomic profile and methylation pattern (epigenetics) in sptz, FOPA, antral, as well as cumulus complex oocytes and embryos produced in vivo and originating from nutritionally controlled animals. In a later stage, a similar investigation (proteomic profile and methylation pattern) will be carried out in the structures (sptz and cryopreserved follicles, follicles cultured in vitro, oocytes matured in vitro and embryos produced in vitro) resulting from the different assisted reproduction techniques used in the project originating from studies 1 to 5. Knowledge of the normal proteomic and methylation profile to be established in Study 6 makes it strategic for the evaluation of the efficiency of the protocols of follicular culture (Studies 1 and 2), cryopreservation of follicles (Study 4) and IVP of embryos, conventional (Study 3) and cryopreservation of semen (Study 5). The integrated action of these studies will allow increasing pregnancy rates using frozen semen; increasing embryo IVP using COCs recovered from antral follicles, as well as implementing an ovarian tissue bank from animals of genetic merit. In the long term, the proposal is based on the exploration of the thousands of immature oocytes included in FOPA with a view to their use in future embryo IVP programs.