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.

Smart seq2 revealed distinct molecular responses during in vitro porcine oocyte maturation before or after the addition of mogroside V.

Oocyte maturation involves both nuclear and cytoplasmic maturation. Mogroside V (MV) has been shown to enhance nuclear maturation, mitochondrial content, and developmental potential of porcine oocyte during in vitro maturation (IVM). However, the impact of MV on cytoplasmic maturation and its underlying mechanisms are not understood. This study aimed to assess the effect of MV on cytoplasmic maturation. Germinal vesicle (GV) oocytes treated with MV exhibited a noticeable increase in cortical granules (CGs) formation. Additionally, MV enhanced the expression of NNAT and improved glucose uptake in mature oocytes. Further insights were gained through Smart-seq2 analysis of RNA isolated from 100 oocytes. A total of 11,274 and 11,185 transcripts were identified in oocytes treated with and without MV, respectively. Among quantified genes, 438 differentially expressed genes (DEGs) were identified for further analysis. Gene Ontology (GO) enrichment analysis indicated that these DEGs were primarily involved in DNA repair regulation, cellular response to DNA damage, intracellular components, and organelles. Furthermore, the DEGs were significantly enriched in three KEGG pathways: fatty acid synthesis, pyruvate metabolism, and WNT signalling. To validate the results, lipid droplets (LD) and triglyceride (TG) were examined. MV led to an increase in the accumulation of LD and TG production in mature oocytes. These findings suggest that MV enhances cytoplasmic maturation by promoting lipid droplet synthesis. Overall, this study provides valuable insights into the mechanisms through which MV improves oocyte quality during IVM. The results have significant implications for research in livestock reproduction and offer guidance for future studies in this field.

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.

Acetic acid affects porcine oocyte metabolism and improves oocyte developmental ability.

Improvement in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism.

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.

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.

Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway.

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.

How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy.

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

Effect of low ethanol concentration in maturation medium on developmental ability, mitochondria, and gene expression profile in mouse oocytes.

Ethanol affects pre-conceptional oocyte quality in women. In this study, we examined the effect of low ethanol concentrations on mouse oocytes. Oocytes were collected from the ovaries of 9-10 week old mice and allowed to mature in vitro in the presence of low concentrations of ethanol (0.1% and 0.2% v/v) for 24 h. Treatment of oocytes with ethanol (0.2%) during maturation decreased the mitochondrial DNA content and membrane potential compared to that in untreated ones, whereas the ATP content did not differ between the groups. Both 0.1% and 0.2% ethanol reduced the lipid content in the oocytes. In addition, immunostaining revealed that oocytes cultured in maturation medium containing ethanol (0.2%) had reduced levels of global DNA methylation and DNMT3A compared with untreated oocytes, and decreased rate of blastocyst development with low mitochondrial protein levels (TOMM40) in embryo. RNA-sequencing of the ethanol-treated (0.2%) and untreated oocytes revealed that mitochondria were a major target of ethanol. In conclusion, treatment of oocytes with low concentration of ethanol reduces the developmental rate to the blastocyst stage, with a lower total cell number and global DNA methylation. In addition, ethanol affected mitochondrial function and mitochondria-related gene expression.

Agarose-based 3D culture improved the developmental competence of oocyte-granulosa complex isolated from porcine preantral follicle.

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established. The present study indicated that the use of a modified medium with Ascorbic Acid (50 µM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3-0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac-H3-K9) and methylation of histone H3 at lysine 4 (Me-H3-K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.

Treatment of mares with the non-steroidal anti-inflammatory drug (NSAID) phenylbutazone transiently affects in vitro maturation of equine oocytes and blastocyst development after Intracytoplasmic Sperm Injection (ICSI).

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.

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.

C-Phycocyanin improves the quality of goat oocytes after in vitro maturation and vitrification.

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 µg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 µg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10-4). Meanwhile, supplementation with 30 µg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10-4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 µg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10-3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro.

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

Physiological high temperatures alter the amino acid metabolism of bovine early antral follicles.

Heat stress reduces the developmental competence of bovine oocytes during the growth phase; however, the detailed mechanisms remain unclear. Amino acids play various critical roles in follicular development, including protein synthesis and as energy sources. We performed in vitro growth (IVG) culture of oocyte-cumulus-granulosa complexes (OCGCs) to assess the amino acid metabolism of small follicles at high temperatures. We isolated OCGCs from early antral follicles (0.5-1.0 mm) and subjected them to IVG culture for 12 days. OCGCs in the heat shock group were cultured under a temperature cycle of (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, and 39.5°C: 9 h) to reproduce the body temperature of lactating cows under a hot environment. OCGCs in the control group were cultured at a constant temperature of 38.5°C for 24 h. Of the surviving OCGCs, those showing similar morphology and size between the groups were selected for amino acid analysis. We analyzed the free amino acids and their metabolites in the culture medium and calculated the depletion or appearance of molecular species. The depletion of three essential amino acids (isoleucine, leucine, and valine), two non-essential amino acids (aspartic acid and glycine), and ornithine was higher in the heat shock group (P < 0.05). Alanine depletion was lower in the heat shock group (P < 0.05). We concluded that heat exposure alters the amino acid metabolism of OCGCs isolated from early antral follicles, which might be involved with the diminished developmental potential of oocytes during summer.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes.

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.

The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and somatic cloned embryo development.

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial ß-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm.

Oocyte competence and gene expression in parthenogenetic produced embryos from repeat breeder and normally fertile buffaloes (Bubalus bubalis) raised in sub-humid tropical climate.

The reproductive management of the buffalo species still faces several unresolved problems, which directly affect the productivity of the herd, one of them being the presence of repeat breeder females. Given this scenario, this study aimed to verify the developmental competence of oocytes obtained from repeat breeder females and submitted to parthenogenetic activation. In addition, embryo gene expression was compared to normally fertile females. Murrah buffaloes were divided into two groups: repeat breeder (RB, n = 8) and normally fertile or control (CR, n = 7). Cumulus-oocyte complexes (COCs) were aspirated by transvaginal ovum pick-up from estrus synchronized females. The COCs were submitted to IVM for 24 h, and subsequently, the oocytes were activated using ionomycin, followed by 6-DMAP. Afterwards, the presumptive parthenotes were cultured for six or seven days in a microenvironment of 5 % CO2, 5 % O2, and 90 % N2 at 38.5 °C. The expression of OCT4, GLUT1, BCL2 and TFAM genes from blastocysts was evaluated. The overall COCs recovery rate was 70.9 % (190/268). The maturation (57.8 vs 71.1), cleavage (45.2 vs 62.2) and blastocyst (30.1 vs 45.9) rates did not differ (P > 0.05) between RB and CR females, respectively. Similarly, no significant difference (P > 0.05) was observed for the expression of studied genes in both RB and CR females. In conclusion, oocytes obtained from RB were as developmentally competent as those collected from CR females, with similar energy metabolism and in vitro development capacity. Thus, the low fertility rate of repeat breeder buffaloes, when compared to normal cyclic females, must be due to subsequent events to the blastocyst stage.