Recreating the Follicular Environment: A Customized Approach for In Vitro Culture of Bovine Oocytes Based on the Origin and Differentiation State.

The mammalian ovary is a large source of oocytes organized into follicles at various stages of folliculogenesis. However, only a limited number of them can be used for in vitro embryo production (IVEP), while most have yet to complete growth and development to attain full meiotic and embryonic developmental competence. While the in vitro growth of primordial follicles in the ovarian cortex has the potential to produce mature oocytes, it is still at an experimental stage. The population of early antral follicles (EAFs), instead, may represent a reserve of oocytes close to completing the growth phase, which might be more easily exploited in vitro and could increase the number of female gametes dedicated to IVEP.Here we present in vitro culture strategies that have been developed utilizing physiological parameters to support the specific needs of oocytes at distinct stages of differentiation, in order to expand the source of female gametes for IVEP by maximizing the attainment of fertilizable oocytes. Furthermore, these culture systems provide powerful tools to dissect the molecular processes that direct the final differentiation of the mammalian oocyte.

Physiological parameters related to oocyte nuclear differentiation for the improvement of IVM/IVF outcomes in women and cattle.

In vitro maturation (IVM) has been applied in numerous different contexts and strategies in humans and animals, but in both cases it represents a challenge still far from being overcome. Despite the large dataset produced over the last two decades on the mechanisms that govern antral follicular development and oocyte metabolism and differentiation, IVM outcomes are still unsatisfactory. This review specifically focuses on data concerning the potential consequences of using supraphysiological levels of FSH during IVM, as well as on the regulation of oocyte chromatin dynamics and its utility as a potential marker of oocyte developmental competence. Taken together, the data revisited herein indicate that a significant improvement in IVM efficacy may be provided by the integration of pre-OPU patient-specific protocols preparing the oocyte population for IVM and more physiological culture systems mimicking more precisely the follicular environment that would be experienced by the recovered oocytes until completion of metaphase II.

Synchronization of germinal vesicle maturity improves efficacy of in vitro embryo production in Holstein cows.

Germinal vesicle oocytes obtained by ovum pick-up (OPU) on a random day are heterogeneous in terms of chromatin maturity, and those with an intermediate degree of chromatin compaction present higher developmental competence. We previously developed a synchronization protocol combining follicle aspiration and FSH treatment capable of increasing the percentage of oocytes with intermediate chromatin compaction (classified as GV2 oocytes; within progressive stages of chromatin compaction ranging from GV0 to GV3) at the time of OPU. In this study, we tested the capacity of a similar protocol to synchronize oocyte chromatin maturity before OPU, as well as to improve the efficacy of in vitro embryo production (IVP) in Holstein cows. In the first experiment, eight non-lactating Holstein cows were subjected to the D5/4FSH, during which all follicles larger than 2 mm were aspirated and a progesterone intravaginal device was inserted on a random day (day 0). Subsequently, four IM injections of FSH (Folltropin; 40/40/20/20 mg) were administered 12h apart on days 2 and 3, and removal of the progesterone device and OPU were performed on day 5. Of the oocytes recovered by OPU, 83.2% were at the GV2 stage. In a second experiment, eighteen non-lactating Holstein cows (Synchro group) were subjected to the D5/4FSH protocol followed by IVM/IVF, and embryo production was compared with that of other seventeen cows submitted to OPU on a random day followed by IVM/IVF (Control group). Blastocyst rate in relation to total oocytes recovered was higher in the Synchro group (37.9%) compared to the Control group (21%; P < 0.05). The percentage of good quality blastocysts morphologically selected for freezing and later transfer in relation to the total number of oocytes recovered tended to be higher in the Synchro group (27.68%) compared to the Control group (14.34%; P = 0.1). These data suggest that synchronization protocols increasing the percentage of GV2 oocytes in the population subjected to IVM/IVF can improve the efficacy of embryo in vitro production in cattle.

Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds.

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.