Fernanda Fagali Franchi

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull , a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus–oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.

Histone acetylation is an epigenetic modification responsible for changes in chromatin architecture, accessibility, and ultimately gene expression. At the onset of a new life, when the fully differentiated parental genomes fuse together to generate a new totipotent cell, the gametes’ epigenetic program must be erased, and new ones are progressively installed. Together with other epigenetic modifications, histone acetylation participates in the early events of embryogenesis, undergoing dynamic changes that involve several amino acid residues on different histone proteins. By analyzing studies that followed these changes during the preimplantation development in different mammals, we identified critical windows of acetylation/deacetylation in relation to the oocyte-to-zygote transition, the activation of the embryonic genome, and the specification of cell lineages, all crucial events for early embryo development, the establishment of pluripotent embryonic tissue, and ultimately of a multicellular organism. Finally, this survey points out the possibility that while contributing to the necessary plasticity of the embryonic stem cells, the reversibility of histone acetylation/deacetylation patterns renders this mechanism prone to be hijacked by environmental conditions, such as maternal diet or pollutants, leading to the alterations of epigenetic marks that can be potentially transmitted to the daughter cells and up to adulthood.

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.

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.

Lipid metabolism is essential for ensuring oocyte maturation and embryo development. β-Oxidized fatty acids (FA) are a potent source of energy for cells, particularly for bovine somatic follicular cells. Superstimulatory protocols using follicle stimulating hormone (FSH) or FSH combined with equine chorionic gonadotropin (eCG) are capable of stimulating the follicular microenvironment and drive the expression of biomarker genes associated with lipid metabolism in the cumulus-oocyte complex (COC) for better embryo development. In this study, we assesed the effects of FSH and FSH/eCG protocols on the expression of genes related to lipid metabolism in bovine granulosa cells (GCs). Further, we measured triglyceride levels in follicular fluid (FF) obtained from both superstimulatd and non-superstimulated cows (synchronized cows). In summary, superstimulation with gonadotropins maintained the TG levels in bovine FF and ensured GCs mRNA abundance of ACSL1, ACSL3, ACSL6, SCD, ELOVL5, ELOVL6, FASN, FADS2, and SREBP1. We, however, found the abundance of CPTIB mRNA to be lower in GCs obtained from cows subjected to FSH/eCG protocols than synchronized cows. In conclusion, the findings of this study showed that ovarian superstimulation around the preovulatory phase has a mild impact on the lipid metabolism in GCs.

During embryo development, the endoplasmic reticulum (ER) acts as an important site for protein biosynthesis; however, in vitro culture (IVC) can negatively affect ER homeostasis. Therefore, the aim of our study was to evaluate the effects of the supplementation of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, in the IVC of bovine embryos. Two experiments were carried out: Exp. 1: an evaluation of blastocyst rate, hatching kinetics, and gene expression of hatched embryos after being treated with different concentrations of TUDCA (50, 200, or 1000 μM) in the IVC; Exp. 2: an evaluation of the re-expansion, hatching, and gene expression of hatched embryos previously treated with 200 µM of TUDCA at IVC and submitted to vitrification. There was no increase in the blastocyst and hatched blastocyst rates treated with TUDCA in the IVC. However, embryos submitted to vitrification after treatment with 200 µM of TUDCA underwent an increased hatching rate post-warming together with a down-regulation in the expression of ER stress-related genes and the accumulation of lipids. In conclusion, this work showed that the addition of TUDCA during in vitro culture can improve the cryotolerance of the bovine blastocyst through the putative modulation of ER and oxidative stress.

During embryo development, the endoplasmic reticulum (ER) acts as an important site for protein biosynthesis, however, in vitro culture (IVC) can negatively affect ER homeostasis. Thereby, the aim of our study was to evaluate the effects of supplementation of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, in the IVC of bovine embryos. Two experiments were carried out: Exp. 1: evaluation of blastocyst rate, hatching kinetics and gene expression of hatched embryos after being treated with different concentrations of TUDCA (50, 200 or 1,000 μM) in the IVC; Exp. 2: evaluation of the re-expansion, hatching, and gene expression of hatched embryos previously treated with 200 µM of TUDCA at IVC and submitted to vitrification. There was no increase in the blastocyst and hatched blastocyst rates treated with TUDCA in the IVC. However, embryos submitted to vitrification after treatment with 200 µM of TUDCA, increased hatching rate post-warming, together with a down-regulation in the expression of ER stress-related genes and the accumulation of lipids. In conclusion, this work showed that the addition of TUDCA during in vitro culture can improve the cryotolerance of the bovine blastocyst through the putative modulation of ER and oxidative stress.

Insulin-like growth factor-1 (IGF-1) regulates cellular lipid content, whereas pregnancy-associated plasma protein-A (PAPP-A) increases IGF-1 bioavailability. Using in vitro-matured cumulus-oocyte complexes, we aimed to evaluate the impact of PAPP-A on the blastocyst lipid content, embryo cryotolerance and embryonic transcriptional profile. We determined that PAPP-A did not affect the lipid content of oocytes, blastocysts, or blastocyst yield (P > 0.05). However, PAPP-A modulated the embryo transcriptional profiles by downregulating PPARGC1A and AKR1B1, which are related to lipid metabolism; CASP9, a pro-apoptotic gene; and IFN-τ, a marker of embryo quality (P < 0.05). Furthermore, the use of PAPP-A improved blastocyst re-expansion in the first 3 h of culture after vitrification (P < 0.05). Although PAPP-A did not affect the blastocyst lipid content or embryo production, we suggest that embryonic transcriptional modulation could contribute to maintain the balance in embryo lipid metabolism. Furthermore, PAPP-A’s approach seems to control key intracellular pathways that improve post-cryopreservation development of blastocysts.

Insulin‐like growth factor 1 (IGF‐1) activity is established by the regulation of IGF binding protein activity, which blocks IGF‐1 functions, whereas pregnancy‐associated plasma protein‐A (PAPP‐A) improves IGF‐1 bioavailability and facilitates binding to IGF receptors. To further extend our understanding of the effect of exogenous PAPP‐A on bovine embryo production, we added this protein during in vitro maturation of cumulus‐oocyte complexes (COCs); moreover, we assessed its effects on IGF‐1 quantity in the maturation medium, embryonic yield and postwarming survival, blastocyst quality, and transcript abundance. Bovine COCs were matured in a serum‐free medium, either with PAPP‐A supplementation (100 ng/ml) or without (control). The treatment group produced higher IGF‐1 concentrations in the maturation medium; however, showed no difference on cleavage, blastocysts rates, and embryonic survival 3 and 24 hr postcryopreservation. Regarding gene expression, VNN1 was upregulated, whereas AGPAT9, FASN, EGFR, HAS2, and IMPDH1 were downregulated in PAPP‐A treated. PAPP‐A treated, CPT2, DNMT3A, and TFAM were upregulated, whereas ATF4 and IFITM3 were downregulated. We concluded that although the addition of PAPP‐A did not affect embryo yield and blastocyst survival, higher IGF‐1 levels may affect embryo competence through differential expression of genes involved in lipid metabolism, oocyte competence, and mitochondrial function.