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The aim of this study was to investigate the effects of modulating reactive oxygen species (ROS) in vitrified bovine in vitro produced (IVP) embryos. In experiment I we compared ROS production in fresh and vitrified-warmed blastocysts. In experiment II we evaluated the effects of antioxidant supplementation (100 μM of 2-mercaptoethanol; BME; 0 h to 2 h during warming) on ROS levels in vitrified-warmed blastocysts, and in experiment III we compared the development of fresh and vitrified-warmed blastocysts in the presence (BME) or absence (Control) of antioxidant (100 μM BME; 0 h to 48 h during warming). Higher ROS production (Fresh: 68.48 ± 7.92 vs Vitrified: 123.53 ± 13.15; P<0.05) and lower cell number was observed in vitrified compared to fresh embryos (Fresh: 123.01 ± 5.67 vs Vitrified: 103.04 ± 4.25; P<0.05). Antioxidant supplementation reduced ROS levels (Vitrified: 38.24 ± 1.27 vs. Vitrified/BME: 33.54 ± 1.08; P<0.05) and increased cell number in treated embryos (Vitrified: 100.65 ± 3.98 vs . Vitrified/BME: 112.95 ± 3.72; P<0.05). No differences were observed in the re-expansion rates of vitrified embryos cultured in the absence and presence of BME at 0, 2, and 4 h after warming (P>0.05). The embryo hatching rate did not differ (P>0.05) among embryos from the fresh, vitrified and vitrified/BME groups. However, the total cell numbers were higher (P<0.05) in vitrified embryos supplemented with BME (143.02 ± 6.97) than in vitrified embryos without BME (113.25 ± 5.09) but similar (P>0.05) to that observed in fresh embryos cultured with (150.54 ± 8.99) and without BME (142.71 ± 13.60). It was concluded that the vitrification and warming processes increased ROS levels in blastocysts and its attenuation with BME antioxidant improved embryonic quality., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

Zinc oxide nanoparticles (ZnO-NPs) have garnered significant attention in biological applications due to their known antioxidant properties. However, their potential impact on assisted reproduction techniques remains largely unexplored, particularly in the context of oocyte quality maintenance within in vitro culture systems, where free radicals can exert detrimental effects. This study investigated the effects of incorporating ZnO-NPs to in vitro maturation (IVM) media on the developmental, cryosurvival, and metabolic profiles of bovine embryos. Three concentrations of ZnO-NPs (0, 1.0, and 1.5 μg/mL) were evaluated. We observed, for the first time, that the inclusion of ZnO-NPs at a concentration of 1.0 μg/mL led to a significant increase in the number of embryonic cells (p < 0.05) accompanied by a reduction in reactive oxygen species production (p < 0.05). Notably, ZnO-NPs did not alter embryonic development, cryosurvival rates, or mitochondrial viability. These findings suggested that ZnO-NPs has antioxidant properties and are compatible with bovine oocytes. Consequently, they may serve as promising supplements to the IVM media, potentially enhancing the efficiency of assisted reproduction techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower ( P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower ( P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased ( P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower ( P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

Oocyte in vitro maturation (IVM) is a key procedure for the in vitro production (IVP) of bovine embryos; however, the efficiency of this step is limited by the intrinsic developmental competence of oocytes. This study aimed to investigate possible epigenetic changes resulting from using of histone deacetylase (HDAC) inhibitors in the maturation of bovine oocytes and subsequent embryo development. Initially, we investigated the meiotic progression of bovine oocytes matured in vitro in the presence of trichostatin A (TSA). We then evaluated histone H3k9 acetylation levels in oocytes exposed to different TSA concentrations, and the relative expression of genes linked to oocyte competence. Finally, we studied pre-implantation embryonic development by analyzing the cleavage, blastocysts, and hatching rates. Acetylation levels of H3k9 increased (p < 0.05) when oocytes were exposed to 50 nM or 100 nM TSA during IVM, but there were no significant changes in the relative expression of the evaluated genes p34cdc2, cyclin B1, MAPK, GDF9, G6PDH, and HSP70. We found that 5 nM TSA promoted the attenuation of meiotic progression and positively affected pre-implantation embryo development in bovine species, allowing a 10% increase in the blastocyst rate. We concluded that TSA treatment during IVM was efficient in promoting changes in H3k9 acetylation levels from 50 nM and promoted attenuated meiotic progression in bovine oocytes at all concentrations evaluated, with a positive impact on pre-implantation development when used at low concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

While microRNAs (miRNAs) are a class of non-coding RNAs important for embryo development, the relationship between them and heat stress during oocyte maturation has not yet been established. This study investigated the effect of heat shock during in vitro maturation (IVM) on the abundance of bta-miR-20a, -27b, -103, -21-5p, -19b, -1246 miRNAs and DROSHA and DICER1 mRNAs, previously reported for being involved in oocyte maturation, response to heat stress and miRNA biogenesis. Oocytes were exposed for 12h to heat shock during IVM, fertilized in vitro and the presumptive zygotes cultured for eight days. The relative quantification of miRNAs and mRNAs was performed by real-time PCR in vitro-matured oocytes and 8-cell stage embryos. Progression of meiosis, embryonic development and apoptotic indices was also evaluated. Heat shock compromised (p < .05) oocyte nuclear maturation, cleavage and embryo development, with a higher (p < .05) embryonic apoptotic index than the control group. The abundance of bta-miR-19b increased (p < .05) whereas the abundance of DROSHA transcripts decreased (p < .05) in embryos derived from heat-shocked oocytes. In conclusion, heat shock during IVM influences the abundance of bta-miR-19b and DROSHA in pre-implantation embryos, indicating a persistent effect of heat shock that can be associated with impaired embryo development., (© 2021 Wiley-VCH GmbH.)

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study aimed to evaluate chromium supplementation on productive, reproductive, and metabolic parameters at lactating Girolando cows subjected to heat stress conditions in a climatic chamber. Thirty-six lactating Girolando cows were subjected to two sequential trials. In trial 1 (thermoneutral environment), the effect of chromium supplementation was evaluated (0 vs. 0.50 mg/kg of dry matter). In trial 2, the cows were fed the same diets, but they were divided into three environmental conditions: heat stress conditions in climatic chamber, fed ad libitum (HS); thermoneutral environment, fed ad libitum (TN); and thermoneutral environment, pair-fed (PF). In thermoneutral conditions, chromium supplementation did not affect productive or metabolic parameters, although supplemented cows had lower viability of oocytes (65.11 ± 0.08% vs. 76.86 ± 0.08%). During heat stress, chromium supplementation lowered plasma glucose levels (61.17 ± 1.90 vs. 67.11 ± 1.90 mg/dL), and increased the insulin:glucose ratio (0.39 ± 0.04 vs. 0.27 ± 0.04). Cows fed the control diet in the HS group had higher vaginal temperature values (39.40 ± 0.10 °C) than the cows in the TN group and PF group (38.89 ± 0.10 °C and 38.85 ± 0.11 °C, respectively). However, supplemented cows heat-stressed maintained the same vaginal temperature as cows in thermoneutral conditions. In conclusion, chromium supplementation improved glucose metabolism and prevented body temperature increases under heat stress conditions.

Objective: In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors., Methods: A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo -matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR., Results: Oocytes in the IN VITRO-OPU group had lower ( p <0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups ( p <0.05)., Conclusion: In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts., (Copyright © 2017 Elsevier Inc. All rights reserved.)