Resumo em Inglês:

Abstract In the bovine dairy sector, the pursuit of rapid genetic advancement has prompted the adoption of increasingly younger parental figures for both males and females. While physiological limitations and access to gametes impose certain restrictions, the impact of age on gamete quality remains crucial yet poorly understood. We propose that the age effect encompasses environmental factors, which include the metabolic state of the parents and the conditions surrounding gametes and embryos within the reproductive tracts of both sexes. Emerging evidence indicates that this environment significantly influences not only the functionality of gametes and early embryos but also the future phenotype of the offspring. Recent research utilizing transcriptomic and epigenetic molecular analyses in bovine models has demonstrated that the age of both females and males gamete donors, can alter gene expression and programming within the embryo in a similar way that metabolic post partum conditions can. This embryo adaptation to parent’s age is similarly noted in variations related to different culture conditions and the in vitro fertilization (IVF) process. A common outcome from these circumstances is the development of embryos operating in “economy” mode, where translation, cell division, and ATP production are diminished, seemingly as an anticipated adaptation to environmental conditions. Furthermore, new epidemiological studies have shown that these alterations can lead to distinct phenotypes, particularly in animals conceived through IVF, underscoring the long-term consequences that may unfold later in their lives.

Resumo em Inglês:

Abstract In vitro embryo production (IVEP) offers an alternative approach for fertility preservation, genetic improvement, and reproductive research. However, in vivo, the female reproductive tract constitutes a dynamic microenvironment that undergoes critical changes crucial to support oocyte maturation, fertilization and embryo development. During IVEP, the absence of maternal-gamete and later maternal-embryo cross-talk can compromises both fertility and embryo development as well as quality. Extracellular vesicles (EVs) derived from the maternal reproductive tract, such as those from follicular fluid, oviductal fluid and uterine fluid, have attracted increasing attention due to their ability to carry bioactive biomolecules and partially restore this bidirectional communication when supplemented during IVEP. Moreover, EVs hold the potential to serve as indicator of the physiological or pathological state of reproductive structures as well as serving as real-time biomarkers. In addition, several studies suggest that EVs offer multiples advantages over conventional synthetics carries, opening new frontiers for modern drug or nucleotide delivery systems. Therefore, this review aims to provide a comprehensive overview of EVs derived from female reproductive tract, exploring their potential applications and challenges in enhancing IVEP outcomes and fertility treatments.

Resumo em Inglês:

Abstract Embryo-maternal communication is a critical process that influences early embryonic development, implantation success, and pregnancy outcomes across mammalian species. This review examines the diverse in vitro systems developed to study this complex dialogue, highlighting their applications, advantages, and limitations. We explore conventional approaches such as two-dimensional (2D) cell cultures, which despite their simplicity, face challenges in replicating the three-dimensional (3D) architecture and cellular functions present in vivo. The review progresses through increasingly sophisticated models, including fluid co-culture systems that incorporate bioactive molecules, explant cultures that maintain tissue architecture, air-liquid interface systems that promote epithelial polarization and differentiation, 3D organoid systems that recapitulate complex structural organization, and organ-on-a-chip platforms that recreate mechanical forces and dynamic conditions. Special attention is given to the emerging role of extracellular vesicles (EVs) as mediators of embryo-maternal communication, transporting crucial molecular signals between the embryo and reproductive tract. By comparing these systems across species and developmental stages, we provide a comprehensive framework for selecting appropriate models based on specific research questions. The integration of these in vitro approaches with advanced analytical techniques offers promising avenues for understanding embryo-maternal cross-talk, potentially leading to improved assisted reproductive technologies and strategies to mitigate early pregnancy loss. As technology advances, the continued refinement of these systems will further illuminate the intricate molecular and cellular mechanisms underlying successful embryo development and implantation.

Resumo em Inglês:

Abstract Embryo development is a complex process that requires several physiological and molecular events to happen harmoniously, and all of this begins with the interaction of the oocyte and sperm. The ability of an oocyte to become a healthy blastocyst is the result of several critical events that are determinants for the successful development of the embryo. Among these events are the sperm's ability to interact with and penetrate the oocyte, carry out syngamy, the developmental competence of the oocyte to support mitotic divisions, and the proper activation of the molecular machinery to regulate the embryo's developmental competence during the early stages of embryonic development. Some of these events originate from either the paternal or maternal side. The focus of this review is to explore the contributions of the paternal side to reproduction in general, with greater emphasis on early embryo development. A deeper understanding of these paternal factors and their influence on embryo development and overall fertility will support the development of new strategies for selecting sires to improve reproductive efficiency in cattle.

Resumo em Inglês:

Abstract Understanding the molecular mechanisms regulating the development of bovine pre-implantation embryos and formation of embryo-like structures (blastoids) is essential to uncover the causes of infertility and develop promising novel assisted reproductive technologies (ARTs). This review presents an updated view of functional genome characterization of bovine pre-implantation development. The use of genomic phenotyping and candidate gene perturbation approaches to uncover molecular factors governing bovine early embryonic development are discussed. This review also delves into the latest breakthroughs in the development of bovine blastoids and highlights key molecular signaling for the creation of bovine blastoids.

Resumo em Inglês:

Abstract The productivity in livestock systems is related to the reproductive efficiency of herds. Over the years, strategies have been developed to improve the reproductive rates of female cattle. Initially, estrus synchronization protocols were developed, however, difficulties related to prolonged postpartum anestrus and estrus observation resulted in low conception rates in these programs. Subsequently, hormonal associations were used to synchronize ovulation and inseminate female cattle at a predetermined time, eliminating the need for estrus observation and improving the fertility rates of cows in postpartum anestrus. Several adjustments were made to improve the response to a timed-artificial insemination (TAI) protocol in different production systems and animal categories. Finally, the development of recombinant drugs and nanotechnology may optimize production systems. Thus, the objective of this review is to detail the research carried out over the years related to the evolution of TAI protocols.

Resumo em Inglês:

Abstract The ability to develop oocytes from the earliest follicular stages through maturation and fertilization in vitro would revolutionize fertility preservation in human medicine and animal breeding. Instead, current assisted reproductive technologies rely only on a limited portion of the female gamete reserve, corresponding to the antral population, while the preantral follicle reserve remains unexploited, mainly due to a lack of knowledge regarding the mechanisms that guide preantral follicle differentiation and folliculogenesis in vitro. This review highlights the efforts made thus far and suggests an approach to studying the mechanisms and ovarian environment to enhance preantral follicle culture systems.

Resumo em Inglês:

Abstract The increase in free fatty acid (FFA) levels in the circulation and follicular fluid in response to the negative energy balance of dairy cows has received significant attention during the last decades. However, until recently the potential effect of FFA on the periovulatory steroid environment has been overlooked. The well-orchestrated luteinizing hormone (LH) peak induces a steroid shift in the periovulatory follicle, from Estradiol-17β (E2) dominance around the LH peak towards progesterone (P4) dominance around ovulation, and is a prerequisite for optimal cytoplasmic and nuclear maturation in the oocyte and oocyte developmental competence. Recent insights in literature demonstrate a link between saturated and mono-unsaturated FFAs and the expression of gonadotrophin receptors, follicle stimulating hormone (FSH)R and LHR, including steroid related enzymes and E2 synthesis by in vitro granulosa cells. The current review will focus on the potential role of mono-unsaturated oleic acid, the most abundant FFA in follicular fluid, on steroidogenesis and its potential effect on the cumulus-oocyte-complex (COC) during final maturation. The data of this review suggest the potential for a regulatory interlinked system, which includes the oocyte secreted factor FGF10 and oleic acid, that modulates the steroidogenic switch from E2 to P4 in the periovulatory follicle, via actions that involve the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in support of the delicate and well-orchestrated dialogue between the oocyte and cumulus cells during final maturation of COCs.

Resumo em Inglês:

Abstract The acquisition of oocyte competence in cattle, encompassing both cytoplasmic and nuclear maturation, is essential for successful fertilization and embryonic development. This competence is progressively achieved during the latter stages of the oocyte growth phase and completed within the dominant follicle (DF). The unique hormonal and immunological environment of the DF during oestrous supports oocyte “capacitation,” a process involving organelle reorganization, mRNA storage and meiosis resumption, which fully prepares the oocyte for fertilization. These changes differentiate oocytes from the DF from those of subordinate follicles, explaining why only oocytes from the DF mature and ovulate successfully. Despite advances in assisted reproductive technologies like in vitro maturation (IVM) and in vitro fertilization (IVF), developmental outcomes remain inferior compared to in vivo matured oocytes, largely due to incomplete or altered oocyte maturation in vitro. Blastocyst rates after IVM/IVF are substantially lower (~35%) than those from in vivo matured oocytes (58-78%). The heterogeneity of oocytes retrieved from antral follicles and the lack of exposure to the natural follicular environment during IVM are key factors limiting developmental competence. Here we describe the molecular changes in bovine oocytes from DFs, collected at 24 and 2 h before ovulation without ovarian stimulation, using single-cell RNA sequencing and bisulfite sequencing to assess gene expression and DNA methylation dynamics. Results revealed significant shifts in transcripts related to oxidative phosphorylation, highlighting the crucial role of energy metabolism during oocyte capacitation. DNA methylation changes were subtle but indicated a more dynamic and less stable epigenome in fully-grown oocytes than previously assumed. Overall, understanding the gene expression and epigenetic landscape during oocyte maturation in the DF offers valuable insights into improving oocyte quality and ART outcomes in cattle. Optimizing the maturation environment to better mimic natural follicular conditions could enhance reproductive efficiency in bovine production systems.

Resumo em Inglês:

Abstract In vitro embryo production (IVEP) has become a cornerstone of genetic advancement in cattle, yet its efficiency remains suboptimal and highly variable. This review synthesizes current knowledge on donor selection and management strategies aimed at optimizing IVEP outcomes. Central to IVEP success is the quantity and developmental competence of oocytes, which is influenced by both intrinsic donor characteristics and extrinsic management interventions. Ovarian superstimulation using follicle-stimulating hormone (FSH) has emerged as a key strategy to enhance oocyte yield and quality, with evidence supporting dose-dependent improvements in embryo development and yield. Protocol refinements—including timing, duration, and delivery mode of FSH— can further influence IVEP efficacy. Donor-specific factors such as age, pregnancy status, and size of the ovarian reserve, assessed via antral follicle count (AFC) or anti-Müllerian hormone (AMH) concentrations, significantly affect oocyte competence and/or embryo yield. Additionally, newly developed genomic traits and selection indexes, offer predictive value for donor performance and enable integration of IVEP-specific traits into breeding programs. High AMH donors consistently outperform low AMH counterparts, and emerging evidence suggests that tailoring superstimulation protocols to AMH phenotype can further enhance IVEP outcomes. The integration of physiological and genomic data provides the opportunity for developing targeted, phenotype/genotype-driven superstimulation protocols to maximize IVEP efficiency in a cost-effective and biologically sound manner.

Resumo em Inglês:

Abstract Assisted reproductive technologies, particularly sex-sorted semen and in vitro embryo production (IVP) can contribute to accelerating genetic gain in both dairy breeds and beef breeds suitable for mating with dairy cows by increasing the number of offspring produced from genetically elite dams. Use of sexed semen has rapidly increased in recent years, accelerating herd genetic gain through selection of the best genetic merit dams to breed replacements, allowing non-replacement dams to be bred to beef sires or to act as recipients of beef embryos to improve calf marketability. IVP offers significant advantages over traditional multiple ovulation embryo transfer (MOET) including increased flexibility in sire usage allowing multiple pregnancies from elite dam-sire combinations to be generated, the ability to produce more embryos per unit time per genetically elite female, the ability to use oocytes from prepubertal females and the more efficient use of rare or high-cost semen straws. Despite these benefits, significant challenges relating to pregnancy loss after embryo transfer, particularly after cryopreservation of IVP embryos, and issues relating to peri- and postnatal health and development of IVP offspring remain to be resolved and hamper the more widespread application of the technology. Improving our understanding of the underlying physiological and molecular mechanisms that regulate early embryo development, embryo-endometrial interactions and lead to successful pregnancy establishment is necessary to understand and elucidate the causes of pregnancy loss and provide a basis for new strategies to improve pregnancy outcomes and reproductive efficiency.

Resumo em Inglês:

Abstract As cattle have not been traditionally considered a model species and the molecular details of germ cell development don’t directly inform production practices, the specification of primordial germ cells in the bovine embryo has remained understudied and poorly understood. Recent work by our laboratory builds on previous investigations to establish the molecular profile of primordial germ cells (PGC) at the critical moment when they are being specified in the embryo during the gastrulation stage. Combining advanced immunolocalization, confocal imaging and single-cell RNA sequencing, we identified PGC in the bovine embryo approximately on day 16 of development by co-expression of the core transcription factors OCT4, SOX17, PRDM1, and TFAP2C as demonstrated for several other species in which the embryo develops a bilaminar disc at the onset of gastrulation. Soon after specification, between days 20 and 22 of embryo development, early migratory PGC repress transcripts responsible for the establishment of somatic lineages. Notably, these cells do not seem highly proliferative during the early migratory stage, another aspect of early germ cells that is conserved in cows and other species such as pigs. Advancing the study of germ cell specification and development during bovine embryonic development, particularly at stages when human embryos are unavailable for investigation, places cows as an additional domestic species capable of providing crucial information about events that are paramount for fertility. As the field of in vitro gametogenesis continues to rapidly evolve, the study of bovine PGC and fetal germ cell development will provide invaluable information to facilitate the development and advancement of future assisted reproduction technologies for the improvement of agricultural animals and human reproduction.

Resumo em Inglês:

Abstract The collection of early embryonic stages from the donor animal and their in vitro development through to transfer back into a recipient animal has gained enormous importance both in understanding embryo physiology and in its application for breeding purposes. Whereas not so long ago the focus was on embryo retrieval after superovulation from the donor animal, oocyte collection by follicular puncture followed by in vitro production (IVP) is now the main source of bovine embryos. However, as recent years of intensive research have shown, it appears to be very difficult to reproduce the extreme complex in vivo processes under laboratory conditions. Consequently, the quality/developmental capacity of embryos available for cryopreservation/storage/transport and transfer still lags behind that of embryos derived directly from animals. Embryo collection in bovine MOET programs is limited to the success of the animal's hormone treatment, embryo collection itself, and transfer on Day 7. IVP largely bypasses these developmental steps in the animal and focuses primarily on the presence of healthy follicular waves. It uses follicular puncture (ovum pick-up: OPU) to obtain immature oocytes, which undergo a three-stage in vitro process to produce embryos that are transferred to the uterus on Day 7. However, it is now known that important processes take place in the oviduct that have a lasting effect on the further development of the embryo. In animals, however, the development of embryos in the oviduct has not yet received sufficient attention. This review will present some highlights of the use of early embryonic stages from the oviduct in different species, but the scientific work mentioned is also largely based on the recent presentation at the AETE 2023 conference in Heraklion, Greece.

Resumo em Inglês:

Abstract Pregnancy loss (PL) in cattle significantly impacts reproductive efficiency and economic viability of herds. Of particular interest, PL in in vitro embryo production (IVP) systems, represents a major challenge to the success of this technique. Establishment and maintenance of pregnancy is influenced by factors such as fertilization, maternal environment, and embryonic signaling issues. Data on dairy cattle have shown that embryo transfer (ET) may lead to greater initial pregnancy, but greater PL compared to artificial insemination (AI), and the impact of environmental conditions on reproductive outcomes seems to be manageable with proper heat stress mitigation strategies, for example. Data on beef cattle submitted to IVP and ET have shown that recipient cows had greater pregnancy per ET (P/ET) and lower PL compared to recipient heifers, with sex-sorted sperm yielding similar or even greater P/ET than conventional semen. Distinct synchronization protocols for recipients yield different reproductive outcomes, and recipient breed also affects P/ET and PL. Moreover, embryo recipients that express estrus after synchronization, as well as recipients in which better quality embryos are transferred, tend to have greater P/ET and lower PL. These findings highlight the importance of management strategies to improve fertility and reduce PL in embryo recipients.

Resumo em Inglês:

Abstract Reproductive tract inflammatory diseases (RTID) present significant health challenges in domestic animals, impacting welfare, fertility, and productivity. Traditionally, antibiotics have been the primary treatment for these conditions, however, the rise of antimicrobial resistance calls for alternative approaches. The microbiome of the female reproductive tract plays a vital role in maintaining reproductive health, and emerging evidence suggests that microbiome-based therapies, such as ‘natural’ or ‘synthetic’ microbiome transplantation, may offer sustainable solutions for RTID management. This review explores the composition and dynamics of the reproductive microbiome in both healthy and diseased states in cows, mares, sows, dogs, and cats. It also examines current treatments and the potential for microbiome-based interventions to replace or complement antibiotic therapies. Although research on microbiome-based therapies for preventing or treating RTID in domestic animals is virtually non-existent, vaginal and uterine microbiomes transplantation in mice and women show promise but require further investigation to evaluate their efficacy and safety across species with varying reproductive physiologies. Additionally, synthetic microbiome therapies present a controlled and reproducible alternative, though they face challenges in design, engraftment, and regulatory approval. The transition from antibiotic dependence to microbiome-based solutions marks a paradigm shift in veterinary medicine, but successful implementation demands a deeper understanding of host-microbiome interactions, rigorous safety protocols, and species-specific research.

Resumo em Inglês:

Abstract This review provides an up-to-date overview of the roles of the oviduct during the periconception period and underlying mechanisms. The functions of the oviduct before, during, and after fertilization are highlighted, with special focus on the effects of epithelial cell contact and luminal secretions on sperm selection mechanisms and acquisition of fertilization ability. The current knowledge on how the oviduct contributes to support fertilization and embryo development via the overall physical milieu (oxygen tension, fluid current, ciliated epithelial cells) and the role of its secretions is also provided. Altogether, the review underlines the unique role of the oviduct during gamete selection and early embryo development, which so far has not been completely possible to mirror when assisted reproductive technologies (ART) are used. Unveiling the most important functional components of oviductal secretions that contribute to better sperm selection, and boost sperm fertilizing ability and early embryo development, can indeed be useful to improve the outcomes of current in vitro systems used in ART.

Resumo em Inglês:

Abstract Unlocking the developmental potential of oocytes at various stages of folliculogenesis represents a major challenge in reproductive biology and assisted reproductive technologies. While in vitro maturation (IVM) of fully grown oocytes is widely applied, the vast majority of oocytes enclosed within early-stage follicles remain underutilized. This review outlines current advancements in in vitro culture systems designed to support oocyte growth and differentiation, with particular attention to the contributions of the authors. Key developments, mainly encompassing the bovine species, include the use of prematuration strategies to enhance the competence of oocytes retrieved from antral follicles, stepwise in vitro culture protocols for growing oocytes from early antral follicles, and efforts to establish defined systems for preantral follicle culture. Emerging insights into chromatin dynamics, cumulus–oocyte communication, and epigenetic regulation are shaping the design of tailored culture environments. Despite promising progress, significant challenges remain in replicating the complexity of in vivo folliculogenesis, particularly in non-rodent models. Addressing these challenges will be critical to expanding the oocyte pool available for reproductive and biotechnological applications, with broad implications for fertility preservation, livestock breeding, and fundamental research.