Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing in vitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing in vitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

Transvaginal aspiration, oocyte maturation and production of blastocysts and a pregnancy via ICSI in an endangered breed: Baudet du Poitou donkey.

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey.

Dynamics of transcription is affected by oxygen tension and developmental speed during in vitro production of bovine embryos.

This study examines the impact of oxygen tension and embryo kinetics on gene transcription dynamics in pathways crucial for embryonic preimplantation development, including lipid metabolism, carbohydrate transport and metabolism, mitochondrial function, stress response, apoptosis and transcription regulation. Bovine embryos were generated in vitro and allocated into two groups based on oxygen tension (20% or 5%) at 18 h post insemination (hpi). At 40 hpi, embryos were categorized into Fast (≥4 cells) or Slow (2 cells) groups, resulting in four experimental groups: FCL20, FCL5, SCL20 and SCL5. Embryo collection also occurred at 72 hpi (16-cell stage; groups FMO20, FMO5, SMO20 and SMO5) and at 168 hpi (expanded blastocyst (BL) stage; groups FBL20, FBL5, SBL20 and SBL5). Pools of three embryos per group were analysed in four replicates using inventoried TaqMan assays specific for Bos taurus, targeting 93 genes. Gene expression patterns were analysed using the K-means algorithm, revealing three main clusters: genes with low relative abundance at the cleavage (CL) and 16-cell morula (MO) stages but increased at the BL stage (cluster 1); genes with higher abundances at CL but decreasing at MO and BL (cluster 2); and genes with low levels at CL, higher levels at MO and decreased levels at BL (cluster 3). Within each cluster, genes related to epigenetic mechanisms, cell differentiation events and glucose metabolism were particularly influenced by differences in developmental kinetics and oxygen tension. Fast-developing embryos, particularly those cultured under low oxygen tension, exhibited transcript dynamics more closely resembling that reported in vivo-produced embryos.

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 µM CGA treatment during the in vitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes.

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Influence of glucose and oxygen tension on the trophectoderm and the inner cell mass of in vitro produced bovine embryos.

The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O2) also interferes with TE formation. The roles of GLC and O2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O2 (low [5%O2] or high [20%O2]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD + or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O2 (p = 0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to + GLC (p = 0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O2 condition compared to the other three conditions. No effects of GLC, O2, or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p = 0.007) and TFAP2C (p = 0.038) were increased in -GLC 20%O2 compared to + GLC 20%O2. SOX2 was decreased in +GLC 20%O2 compared to + GLC 5%O2 (p = 0.027) or compared to -GLC 20%O2 (p = 0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression.

MCT1-mediated transport of valeric acid promotes porcine preimplantation embryo development by improving mitochondrial function and inhibiting the autophagic AMPK-ULK1 pathway.

Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.

FGF18 impairs blastocyst viability, DNA double-strand breaks and maternal recognition of pregnancy genes.

Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.

Paraspeckles / CARM1 mediates the regulation of OEVs on cell differentiation during in vitro embryonic development of yak.

Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of blastocysts, the low ratio of the number of inner cell mass cells to the number of trophoblastic cells, and the high apoptosis rate of blastocysts, resulting in low embryo implantation rate. Therefore, optimizing in vitro culture conditions has become a key technology to im-prove the quality of preimplantation embryos. Oviduct Epithelial cells exosomes (OEVs) can be absorbed and internalized by embryos to improve the blastocyst rate and blastocyst quality of embryos in vitro. As a special nuclear structure, Paraspeckles are involved in the fate determination of mammalian early embryonic mammalian cells. However, the regulation of embryonic cell differentiation by OEVs remains unknown. We aimed to investigate the effects of OEVs on paraspeckle formation and cell fate determination in yak in vitro fertilization (IVF) of em-bryos. To simulate the in vivo oviduct environment after ovulation, we used follicular fluid exosomes (FEVs) to stimulate yak oviduct epithelial cells and collect OEVs. OEVs were added to the yak IVF embryo culture system. Paraspeckle formation, cell differentiation, and blastocyst quality in yak embryos were determined. Our results show that, development of yak embryos is unique compared to other bovine species, and OEVs can be used as a supplement to the in vitro culture system of yak embryos to improve embryonic development and blas-tocyst quality. And also Paraspeckles/CARM1 mediated the regulation of OEVs on cell differentiation during in vitro yak embryo production. These results provide new insights into the study of yak embryonic development and the role of OEVs in embryonic development.

The presence of Leptospira spp. in the follicular fluid of naturally infected cows affects the overall efficiency of the in vitro embryo production technique.

The relationship between Leptospira infection and reproductive failures, as well as the mechanisms that lead to it, has not yet been fully established. It has been hypothesized that the presence of Leptospira spp. in the follicular fluid (FF) could impair the oocyte developmental competence. Thus, the impact of the presence of Leptospira spp. in the FF on in vitro embryo production (IVEP) outcomes was assessed. Dairy cows (n=244) from different farms were subjected to ovum pick-up for cumulus-oocyte complexes (COCs) collection. After PCR analysis of the FF, cows were retrospectively allocated into either: positive (POS-FF) or negative (NEG-FF) group. Statistical modeling was conducted using the farm, PCR result, and laboratory in which the IVEP was performed as effects. Noteworthy, 26.6% of the animals were positive for Leptospira spp., and 70% of farms had at least one POS-FF cow in the herd. POS-FF cows had a lower number of COCs recovered (22.6 ± 1.2 vs 15.0 ± 2.8, P=0.036), rate of viable COCs (85.6 ± 0.9% vs 78.1 ± 2.8%, P=0.015), number of good-quality COCs (16.0 ± 0.9 vs 9.8 ± 2.1, P=0.026), cleaved embryos (11.9 ± 0.7 vs 7.5 ± 1.5, P=0.032), and blastocysts (7.3 ± 0.4 vs 2.3 ± 0.7, P=0.044) yielded per cow. In conclusion, the presence of Leptospira spp. in the FF of naturally infected cows impaired the amount of COCs recovered, decreasing the overall IVEP efficiency.

Selecting oocyte donors based on anti-Müllerian hormone (AMH) concentrations: A critical analysis of using cutoff values as exclusion criterion for an in vitro embryo production program in Gir cattle.

The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700 pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.

Impact of reducing lipid content during in vitro embryo production: A systematic review and meta-analysis.

There is still no consensus regarding the role of lipid modulators during in vitro embryo production. Thus, we investigated how lipid reducers during the in vitro maturation of oocytes (IVM) or in vitro culture (IVC) of embryos impact their cryotolerance. A literature search was performed using three databases, recovering 43 articles for the systematic review, comprising 75 experiments (13 performed in IVM, 62 in IVC) and testing 13 substances. In 39 % of the experiments, an increase in oocyte and/or embryo survival after cryopreservation was reported, in contrast to 48 % exhibiting no effect, 5 % causing negative effects, and 8 % influencing in a dose-dependent manner. Of the 75 experiments extracted during IVM and IVC, 41 quantified the lipid content. Of those that reduced lipid content (n = 26), 50 % increased cryotolerance, 34 % had no effect, 8 % harmed oocyte/embryo survival, and 8 % had different results depending on the concentration used. Moreover, 28 out of the 43 studies were analyzed under a meta-analytical approach at the IVC stage in cattle. There was an improvement in the cryotolerance of bovine embryos when the lipid content was reduced. Forskolin, l-carnitine, and phenazine ethosulfate positively affected cryotolerance, while conjugated linoleic acid had no effect and impaired embryonic development. Moreover, fetal bovine serum has a positive impact on cryotolerance. SOF and CR1aa IVC media improved cryotolerance, while mSOF showed no effect. In conclusion, lipid modulators did not unanimously improve cryotolerance, especially when used in IVM, but presented positive effects on cryotolerance during IVC when reaching lipid reduction.

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Sperm fertilizing ability in vitro influences bovine blastocyst miRNA content.

MicroRNAs (miRNAs) are small highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through post-transcriptional regulation of gene expression. During development, miRNAs play a key role in driving embryo patterning and morphogenesis in a specific and stage-dependent manner. Here, we investigated whether sperm from bulls with different fertilizing ability in vitro influence blastocyst quality and miRNA content. Results demonstrate that blastocysts obtained using sperm from high fertility sires (H group) display significantly greater cleavage and blastocyst development as well as greater transcript abundance in blastocysts for the developmental competence markers CDX2, KRT8, NANOG, OCT4, PLAC8, PTGS2, SOX17, and SOX2, compared to blastocysts generated using sperm from low fertility sires (L group). In parallel, high throughput deep sequencing and differential expression studies revealed that H blastocysts exhibit a greater miRNA content compared to L blastocysts, with hsa-miR-4755-5p and hsa-miR-548d-3p uniquely detected in the H group, and greater abundance of hsa-miR-1225-3p in the H group. Gene ontology (GO) and KEGG pathway analyses indicated that the 3 differentially expressed miRNAs identified are involved in the regulation of many biological mechanisms with a key role in aspects of early embryo development, including transcriptional regulation, cellular biosynthesis, nucleic acid metabolism, cellular differentiation, apoptosis, cytoskeleton remodeling, cell-to-cell interactions, and endocytosis. Overall, our results indicate that sperm fertilizing ability influences blastocyst developmental ability and miRNA content. In addition, we demonstrate an association between blastocyst quality and miRNA content, thus suggesting the possibility to score miRNA expression as biomarkers for improved routine embryo selection technologies to support assisted reproductive efforts.

Successful equine in vitro embryo production by ICSI - effect of season, mares' age, breed, and phase of the estrous cycle on embryo production.

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Nobiletin as a novel agent to enhance porcine in vitro embryo development and quality.

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3',4' hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 µM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

Meta-analysis to determine efficacy of colony-stimulating factor 2 for improving pregnancy success after embryo transfer in cattle.

Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended.