In Vitro Maturation Medium Research Articles

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall invitro maturation efficiency and early embryonic stages.

In vitro maturation of bovine oocytes: the role of ROS, oxidative stress, and the impact of natural antioxidant supplementation

According to available data in the literature, Brazil has one of the largest cattle herds in the world and is one of the leading embryo producers, with a clear predominance of embryos produced in vitro. These data influence the ongoing pursuit of optimizing the results obtained in IVEP (in vitro embryo production). In this technique, in vitro maturation (IVM) is such a crucial stage that it justifies studies involving components that, when added to the media, can lead to improved cleavage rates and blastocyst development. It is well known that oocyte quality is influenced by oxidative stress, and in this context, the inclusion of antioxidants in the IVM medium may enhance these outcomes. Thus, the present study aimed to review oocyte maturation, the impact of oxidative stress, and the addition of antioxidants, such as Moringa oleifera, to the IVM media.

Impact of N-acetyl-L-cysteine on spindle morphology and reactive oxygen species in vitrified/warmed invitro matured bovine oocytes.

Low developmental potential of vitrified invitro matured (IVM) bovine oocytes is frequently attributed to high levels of reactive oxygen species (ROS) and abnormal spindle assembly. This study aimed to evaluate the efficacy of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, added to IVM medium in reducing ROS and preserving spindle configuration of vitrified/warmed IVM bovine oocytes. Oocytes collected from abattoir ovaries were either cultured in IVM medium or in IVM medium supplemented with 1 mM NAC for the initial 8 h of IVM. Half of the oocytes of each group were vitrified/warmed, and spindle morphology and ROS production were assessed at 24 h of IVM. Results indicated that fresh oocytes IVM with NAC improved spindle configuration, with significantly lower ROS levels compared to the control group. Vitrification resulted in lower percentages of bovine oocytes reaching the metaphase II stage but similar ROS levels to non-vitrified oocytes, regardless of NAC supplementation. However, the supplementation of NAC during maturation had no effect on spindle or chromosome configuration of vitrified oocytes. These findings emphasize NAC's potential in enhancing the quality of IVM bovine oocytes but its addition at 1 mM for 8 h to IVM medium did not decrease levels of ROS nor improve spindle assembly after vitrification.

Ovarian support cell in vitro maturation (OSC-IVM) results in healthy murine live births with no evidence of reprotoxicology in a multigenerational study

Research questionDoes application of human stem cell-derived ovarian support cells (OSCs) for in vitro maturation (IVM) have a safe reproductive toxicity profile? DesignA total of 70 B6/CBA 6–8-week-old stimulated female mice were used to collect immature oocytes (n=2,025) at the germinal vesicle stage. Oocytes were retrieved denuded (n=930) or with cumulus cells (COCs, n=1,095). These were randomly assigned to traditional IVM media (MediCult-IVMTM, Origio) or the same IVM media (MediCult-IVMTM, Origio) further supplemented with human OSCs (FertiloTM, Gameto Inc.). Metaphase II (M2) oocytes were inseminated by intracytoplasmic sperm injection and cultured to blastocyst stage. Vitrified blastocysts were transferred to pseudopregnant females, and F1 offspring were evaluated for various health and developmental metrics. Select F1 mice were outbred to produce the F2 generation, assessing live births, sex ratio, morphology, behavior, and health. ResultsIn mice OSC-IVM generally led to a decreased maturation rate compared to Traditional-IVM (p=0.0101). Subsequent embryo culture yielded similar cleavage rates between the four groups (p=0.7547). However, there was a significant distinction in the blastocyst formation with OSC-IVM showing higher blastocyst formation rates for both denuded oocytes (p=0.0408) and COCs (p=0.0063). Live births, sex ratio, development, and behavior in F1 and F2 generations revealed no adverse effects or significant differences, confirming the absence of long-term toxicity. ConclusionsThis study demonstrates the safety of the OSC-IVM system, with no adverse effects on embryo development or offspring health and fertility across generations.

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

True empty follicle syndrome is a subtype of oocyte maturation abnormalities.

To review the outcomes of in vitro maturation (IVM) and in vitro fertilization (IVF) in women with empty follicle syndrome (EFS). The study evaluated the genetic underpinnings of EFS by analyzing mutations. This retrospective case series involving 17 women with EFS over at least 2 IVF cycles was conducted. The study also employed whole-exome sequencing to analyze the genetic mutations. The treatment approaches included letrozole-primed IVM, follicle-stimulating hormone (FSH)-human chorionic gonadotrophin (hCG)-primed IVM, and conventional IVF. The average female age was 31.5±4.6 years, and the duration of infertility was 7.3±3.5 years. Four patients underwent IVF. IVM oocyte collections yielded oocytes in 12 of 13 subjects. Of these, 75% (9/12) yielded MII oocytes after 48 h of IVM media incubation. Six subjects had fertilized embryos, resulting in a 40.9% intracytoplasmic sperm injection (ICSI) fertilization rate (9 embryos/22 MII oocytes). Genetic analysis revealed mutations in seven patients. This study demonstrated the partial efficacy of letrozole-primed IVM plus growth hormone and FSH-hCG primed IVM protocols. No pregnancies or live births were recorded after IVM. One ongoing pregnancy post-IVF and one spontaneous live birth were observed. Inter-cycle variabilities were observed in women with oocyte maturation abnormalities (OMAs). Almost all patients with EFS had oocytes collected during IVM following IVF. These oocytes have limited potential for maturation, fertilization, and live birth, as demonstrated by the low rates observed after IVM culture and ICSI. These conditions are observed in OMAs due to defects in the oocyte machinery. The proposed flowchart provides a comprehensive classification approach for various forms of EFS.

Antioxidant effects and compatibility of zinc oxide nanoparticles during in vitro maturation of bovine oocytes and subsequent embryo development

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.

Impact of the near-physiological temperature on the in vitro maturation of bovine oocytes: A comparative proteomic approach

In vivo, the temperature inside preovulatory follicles of cows is approximately 1 °C lower than rectal temperature. However, standard bovine oocyte in vitro maturation (IVM) protocols use 38.5 °C based on rectal temperature. This study evaluated the effect of reducing IVM temperature to 37.5 °C on the proteomic profile of oocytes compared to the routine 38.5 °C. Nuclear maturation rate and cumulus cell (CC) expansion (30 COCs per group, 21 replicates) were assessed by observing the first polar body and using a subjective scoring method (0–4). Total nitrite concentrations in the culture medium were measured using the Griess method. Differential proteomics was performed using LC-MS/MS on pooled oocyte samples (500 matured oocytes per group, three replicates), followed by gene ontology enrichment, protein-protein interaction, and putative miRNA target analyses. No significant differences were observed between the groups in nuclear maturation, CC expansion, or nitrite concentration (P > 0.05). A total of 806 proteins were identified, with 7 up-regulated and 12 down-regulated in the treatment group compared to the control. Additionally, 12 proteins were unique to the control group, and 8 were unique to the treatment group. IVM at 37.5 °C resulted in the upregulation of proteins involved in protein folding and GTP binding, and the downregulation of enzymes with oxidoreductase activity and proteins involved in cytoskeletal fiber formation. Furthermore, 43 bovine miRNAs potentially regulating these genes (DES, HMOX2, KRT75, FARSA, IDH2, CARHSP1) were identified. We conclude that IVM of bovine oocytes at 37.5 °C induces significant proteomic changes without impacting nuclear maturation, cumulus cell expansion, or nitrite concentration in the IVM medium.

P-795 Robust and simple in vitro maturation of ovarian tissue oocytes

Abstract Study question Using in-vitro gametogenesis results in mice, can we perform simple, robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? Summary answer In vitro oogenesis in mice reveals 3 phases of oocyte maturation, IVD, IVG, and IVM. In vivo IVD and IVG simplifes a more robust IVM. What is known already For fertility preservation in cancer patients, IVM of GV’s obtained from ovary tissue with live birth has been achieved, but the results are variable, and great effort has gone into trying to make the “right” culture media, and most are given booster shots of gonadotropin with questionable results. It is easy to obtain many GV oocytes from tiny follicles using open cortical dissection. But it still has remained a puzzle how to do robust IVM in the best way. Study design, size, duration A total of 146 females underwent ovary tissue cryopreservation since 1977. The most recent 38 consecutive such patients between 2 and 38 years of age underwent in-vitro maturation of oocytes (IVM) derived from their ovarian tissue. This is a prospective non-randomized pilot study based upon our results with in vitro oogenesis. Participants/materials, setting, methods After ovarian cortex dissection, the spent media is examined for cumulous complexes (CC’s), which were placed in culture with widely varying concentrations of FSH and HCG, and in a variety of different media. Based on now established mechanisms of in vitro oogenesis, we postulated that the specific media for IVM and the concentrations of HCG would not impact results. There was no prior hormone administration. Main results and the role of chance The harvested germinal vesicle oocyte developed into mature metaphase II oocytes with simple culture media just as well as with “IVM media”, and with HCG concentration from 10 mIU/l to 1000 mIU/l. Of the 38 cases, 18 had had previous chemotherapy or were pre-pubertal. None of these had CC’s. Of the 20 who were post-pubertal and had not yet undergone chemotherapy, the number of CC’s averaged 20 per patient. Of a total of 402 GV oocytes, 137 became mature MII’s (34.1%), varying between 20% and 56%. The only case with low maturation rate (9%) occurred when the cumulus was stripped immediately instead of at 24 hours. Thus mature oocytes can robustly and easily be obtained from ovary tissue with simple media and no need for ovarian stimulation. Furthermore, ovarian stimulation and ovulation are not necessary for oocyte maturation. Oocytes that have just exited IVD that have been exposed to less than 7 days of gonadotropin (IVG) will not mature. Those oocytes exposed to more than 11 days of IVG will degenerate. Thus about 35% of oocytes at any given time will have already been properly prepared in vivo for IVM. Limitations, reasons for caution This is an early proof of concept study in humans with only 30 participants. However, it is completely consistent with what we have predicted from in vitro oogenesis in the mouse. Wider implications of the findings Ovary tissue freezing along with robust IVM will at once provide cancer patients with two possibilities for fertility preservation, without having to delay cancer treatment. Four months of in vivo IVD (non-hormonal) followed by 11 days of IVG (FSH) have already prepared oocytes for IVM. Trial registration number not applicable

P-355 Effect of biphasic CAPA-IVM on ovarian tissue oocytes of transgender men

Abstract Study question Can CAPA-IVM (Biphasic in-vitro maturation) with C-type natriuretic peptide (CNP), followed by in vitro maturation (IVM) improve ovarian tissue oocyte (OTO) maturation in transgender patients? Summary answer CAPA-IVM did not show any significant difference in maturation competence and frequency of chromosomal aberrations when compared with standard and in-house IVM. What is known already OTO-IVM is a method of fertility preservation in patients where prior ovarian stimulation is undesired. It has resulted in live births in cancer and polycystic ovarian syndrome (PCOS) patients. CAPA-IVM has further improved oocyte competency in these patients by synchronizing nuclear and cytoplasmic maturity. Similarly, OTO can be collected from transgender men without ovarian stimulation during gender reassignment surgery. While oocytes do survive and mature, OTO-IVM in transgender men is characterized by decreased fertilization rate and severely compromised developmental competency. We investigated whether CAPA-IVM can improve cytoplasmic immaturity for transgender OTO. Study design, size, duration Patients were recruited from July 2022 to July 2023. Both ovaries were collected from 9 transgender patients (age= 20-24 years, mean age=22 years) who underwent gender reassignment surgery after testosterone treatment (mean length= 34 months). All ovaries were collected in cold medium (4oC) and manipulation was performed within 30 minutes of the collection for the retrieval of cumulus oocyte complexes (COCs). Participants/materials, setting, methods Collected COCs were cultured either in in-house IVM and Standard (Medicult) IVM medium for 48 hours or in biphasic CAPA-IVM for 54 hours (24 hours pre-maturation culture and 30 hours IVM). Following maturation, oocytes were analyzed for calcium (Ca2+)-releasing potential and developmental competency after ICSI. In vitro matured MI (metaphase I) oocytes from stimulated IVF patients served as controls. Genetic analysis was performed on subsequent embryos to detect chromosomal abnormalities in all groups. Main results and the role of chance After culturing 330 COCs, the survival rate following maturation was similar in the in-house IVM (79%) compared to Medicult (72%, p = 0.221) and CAPA-IVM (73%, p = 0.251). A similar maturation rate (i.e. 55-57%) was observed in the different groups. Following ICSI, 15/25 (60%) CAPA-IVM, 13/19 (68.5%) Medicult IVM, and 17/28 (61%) in-house IVM oocytes were normally fertilized, which was lower than controls (24/29, 83%). Blastocyst rate was inferior in all OTO groups and significantly lower (p = 0.031) in Medicult-IVM (0/13) compared to controls (7/24). Ca2+-releasing potential of oocytes was determined as a product of amplitude and frequency, in arbitrary units (AU). The average values for CAPA-IVM (1.74AU), Medicult IVM (1.24AU), and in-house IVM (1.54AU) were significantly lower (p = 0.022, p = 0.008 and p = 0.017 respectively) compared to controls (4.76AU). Shallow whole genome sequencing of developed embryos (Day 2- Day 5) showed that 8/13 (61.5%) in CAPA-IVM, 4/8 (50%) in In-house IVM, 1/6 (17%) in Medicult, and 4/12 (33%) in the Control group were chromosomally normal. Limitations, reasons for caution The limited inclusion of patients warrants caution in the interpretation of the results. Moreover, the lack of a better control group might mask the properties of OTO-IVM. Wider implications of the findings Our preliminary results do not demonstrate an improvement in oocyte maturation and embryo development with the implementation of CAPA-IVM. However, future proteomics and transcriptomic studies would shed more light on the advantages of the current IVM systems and decipher the true potential of OTO-IVM from transgender men. Trial registration number not applicable

Chemical reversion of age-related oocyte dysfunction fails to enhance embryo development in a bovine model of postovulatory aging

PurposeThere are no clinical treatments to prevent/revert age-related alterations associated with oocyte competence decline in the context of advanced maternal age. Those alterations have been attributed to oxidative stress and mitochondrial dysfunction. Our study aimed to test the hypothesis that in vitro maturation (IVM) medium supplementation with antioxidants (resveratrol or phloretin) may revert age-related oocyte competence decline.MethodsBovine immature oocytes were matured in vitro for 23 h (young) and 30 h (aged). Postovulatory aged oocytes (control group) and embryos obtained after fertilization were examined and compared with oocytes supplemented with either 2 μM of resveratrol or 6 μM phloretin (treatment groups) during IVM.ResultsAged oocytes had a significantly lower mitochondrial mass and proportion of mitochondrial clustered pattern, lower ooplasmic volume, higher ROS, lower sirtuin-1 protein level, and a lower blastocyst rate in comparison to young oocytes, indicating that postovulatory oocytes have a lower quality and developmental competence, thus validating our experimental model. Supplementation of IVM medium with antioxidants prevented the generation of ROS and restored the active mitochondrial mass and pattern characteristic of younger oocytes. Moreover, sirtuin-1 protein levels were also restored but only following incubation with resveratrol. Despite these findings, the blastocyst rate of treatment groups was not significantly different from the control group, indicating that resveratrol and phloretin could not restore the oocyte competence of postovulatory aged oocytes.ConclusionResveratrol and phloretin can both revert the age-related oxidative stress and mitochondrial dysfunction during postovulatory aging but were insufficient to enhance embryo developmental rates under our experimental conditions.

Accomplishment of canine cloning through in vitro matured oocytes: a pioneering milestone.

The in vitro maturation (IVM) rate of canine oocytes remains low compared to other mammals due to their unique reproductive characteristics. This study aimed to explore the effect of hormone supplementation during the IVM of canine immature oocytes on nuclear maturation and subsequently assess its potential application in canine somatic cell nuclear transfer (SCNT). Immature oocytes were collected and cultured in an IVM medium supplemented with hormones (follicle-stimulating hormone [FSH] and progesterone [P4]) or without hormones (control) for 24 hours. The maturation rates of oocytes in the hormone-treated group (94.92 ± 3.15%) were significantly higher than those in the control group (61.01 ± 4.23%). Both in vitro and in vivo matured oocytes underwent NT to evaluate their utility, and the fusion rates were higher in the in vitro matured group than those in the vivo matured group, not significant between in vivo and in vitro matured group (73.28% and 82.35%, respectively). As a result, 14 fused embryos from the in vitro matured group were transferred into two surrogates, with one surrogate achieving a successful pregnancy and delivering four puppies. Whereas in the in vivo matured group, 85 fused embryos were transferred to 8 surrogate mothers, leading to three surrogates becoming pregnant and delivering one, four, and two puppies. The pregnancy rates were not significant between both groups (50% and 37.50%), but the number of offspring exhibited a significant difference (28.57% and 8.23%). In conclusion, we achieved a remarkable milestone by successfully producing cloned puppies using in vitro matured oocytes, underscoring the feasibility of canine cloning from in vitro recovered oocytes. It is important to note that this study focused only on immature oocytes after ovulation and only during the estrus stage. Further research targeting other stages of the estrous cycle could potentially enhance canine cloning efficiency.

MicroRNAs associated with the quality of follicular fluids affect oocyte and early embryonic development.

Oocyte and embryo quality differs significantly among individuals. Follicular fluid (FF) is a solo environment of oocyte maturation and may flux into the oviduct. Supplementation of invitro maturation (IVM) and culture (IVC) medium with extracellular vesicles of FFs supports oocyte maturation and embryonic development. We addressed a hypothesis that miRNA profiles in FFs are crucial background of oocyte maturation and embryonic development. FFs were collected from the ovaries of individual cows, and the FFs were classified into Good or Poor FF based on the developmental rate to the blastocyst stage of enclosed oocytes. miRNAs associated with the Good FFs were explored using small RNA sequencing. In addition, FFs were classified using the concentration of Good-FF-associated miRNAs. These classified FFs or miRNA were added to the IVM or IVC mediums. Supplementation of IVM and IVC medium with Good FF improved embryonic development. Good FFs contained miR-151-3p and miR-425-5p at a high concentration compared with those in Poor FFs. FFs selected by the concentration of miR-151-3p and miR-425-5p improved oocyte maturation and embryonic development. Supplementation of IVM or IVC medium with either miR-151-3p or miR-425-5p improved embryonic development to the blastocyst stage. miRNAs were associated with the Good FFs determined oocyte maturation and embryonic development.

Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Effects of follicular fluid-derived extracellular vesicles improve in vitro maturation and embryonic development of porcine oocytes

To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn’t significantly different G1 and G3 group. G3 group wasn’t significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.

Piperine improves the quality of porcine oocytes by reducing oxidative stress

Oxidative stress caused by light and high temperature arises during in vitro maturation (IVM), resulting in low-quality embryos compared with those obtained in vivo. To overcome this problem, we investigated the influence of piperine (PIP) treatment during maturation of porcine oocytes on subsequent embryo development in vitro. Porcine oocytes were cultured in IVM medium supplemented with 0, 50, 100, 200, or 400 μM PIP. After parthenogenetic activation, the blastocyst (BL) formation was significantly higher and the apoptosis rate was significantly lower using 200 μM PIP-treated oocytes (200 PIP). In the 200 PIP group, the level of reactive oxygen species at the metaphase II stage was decreased, accompanied by an increased level of glutathione and increased expression of antioxidant processes (Nrf2, CAT, HO-1, SOD1, and SOD2). Consistently, chromosome misalignment and aberrant spindle organization were alleviated and phosphorylated p44/42 mitogen-activated protein kinase activity was increased in the 200 PIP group. Expression of development-related (CDX2, NANOG, POU5F1, and SOX2), anti-apoptotic (BCL2L1 and BIRC5), and pro-apoptotic (BAK, FAS, and CASP3) processes was altered in the 200 PIP group. Ultimately, embryo development was improved in the 200 PIP group following somatic cell nuclear transfer. These findings suggest that PIP improves the quality of porcine oocytes by reducing oxidative stress, which inevitably arises via IVM. In-depth mechanistic studies of porcine oocytes will improve the efficiencies of assisted reproductive technologies.

Ellagic acid treatment during in vitro maturation of porcine oocytes improves development competence after parthenogenetic activation and somatic cell nuclear transfer

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 μM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 μM) and in vitro culture (IVC) medium (0, 0.1 μM). Supplementation of IVM medium with 0.1 μM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 μM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 μM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 μg/mL (AN30); 300 μg/mL (AN300) or 2000 μg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 μg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 μg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 μg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 μg/mL is recommended.

Resveratrol Reduces ROS by Increasing GSH in Vitrified Sheep Embryos

The in vitro production and cryopreservation of mammalian embryos generates reactive oxygen species (ROS) due to conditions of the system that can overcome their antioxidant protection. Resveratrol is an antioxidant used in in vitro systems to improve blastocyst rates, but its effect on antioxidant enzymes such as glutathione (GSH) in embryos produced by in vitro fertilization (IVF) after vitrification has not been reported. The objective of this study was to evaluate the effects of resveratrol on the in vitro maturation medium (IVM) of sheep oocytes (Ovis aries) on the levels of ROS and GSH in embryos produced by IVF subjected to vitrification. Resveratrol was added at 0 µM, 0.25 µM, 0.5 µM, and 1 µM during oocyte in vitro maturation (IVM). Matured oocytes were fertilized with thawed ram sperm. Embryos were cultured in sequential media until blastocysts, were then vitrified for 24 h, and, after heating, they were stained with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) to determine the presence of ROS and with Cell Tracker Blue® for the presence of GSH. The quantitative values of ROS and GSH were obtained through the Image J image processor. The results showed that resveratrol increased GSH and decreased ROS production (p &lt; 0.05) in a dose-dependent manner. It is concluded that its use in sheep oocytes during IVM has a beneficial effect on embryos produced by IVF subjected to vitrification by decreasing reactive oxygen species levels and facilitating the generation of embryo antioxidant enzymes like glutathione.