Blastocyst Cell Number Research Articles

Extracellular vesicles from human Fallopian tubal fluid benefit embryo development in vitro.

Do extracellular vesicles (EVs) from human Fallopian tubes exert an influence on early embryo development in vitro? Human Fallopian tube EVs carrying miRNAs increase murine embryo viability in vitro. Oviductal EVs (oEVs) are recently identified key players in embryo-oviduct interactions that contribute to successful pregnancy in vivo. Their absence in current in vitro systems may partly explain the suboptimal embryo development observed; therefore, further knowledge is needed about their impact on early embryos. The oEVs were isolated from the luminal fluid of human Fallopian tubes using ultracentrifugation. We cocultured oEVs with murine two-cell embryos until the blastocyst stage. The study was conducted between August 2021 and July 2022. A total of 23 premenopausal women were recruited for Fallopian-tubes collection, and the oEVs were isolated. The micro RNA (miRNA) contents were detected using high-throughput sequencing and their target genes and effects were analyzed. After in vitro culture with or without oEVs, the blastocyst and hatching rates were recorded. Furthermore, for the blastocysts formed, we assessed the total cell number, inner cell mass proportion, reactive oxygen species (ROS) level, number of apoptotic cells, and mRNA expression levels of genes involved in development. EVs were successfully isolated from the human Fallopian tubal fluid and the concentrations were evaluated. A total of 79 known miRNAs were identified from eight samples that had been sequenced, all involved in various biological processes. The blastocyst rate, hatching rate, as well as total cell number of blastocysts were significantly increased in the oEVs-treated groups (P < 0.05 versus untreated), while the proportion of inner cell mass showed no significant difference between groups. ROS levels and apoptotic cell proportions were decreased in the oEVs-treated groups (P < 0.05 versus untreated). The genes, Actr3 (actin-related protein 3), Eomes (eomesodermin), and Wnt3a (Wnt family member 3A) were upregulated in blastocysts in the oEVs-treated group. Data are available from Gene Expression Omnibus: Accession number: GSE225122. The Fallopian tubes in the current study were collected from patients with uterine fibroids (the reason they underwent hysterectomy), and this pathological condition may affect the characteristics of EVs in luminal fluid. Also, owing to restrictions for ethical reasons, an in vitro co-culture system using murine embryos was used instead of human embryos, and the findings may not be transferable. Deciphering miRNA contents in human oEVs and providing new evidence that oEVs benefit embryo development in vitro will not only increase our knowledge on embryo-oviduct communication but also potentially improve ART outcomes. This study was supported by the National Key Research and Development Project of China (2021YFC2700603). No competing interests are declared.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 μL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

Leonurine improves bovine oocyte maturation and subsequent embryonic development by reducing oxidative stress and improving mitochondrial function

It is acknowledged that excessive reactive oxygen species (ROS) level attributes greatly to the compromised developmental potential of oocytes matured in vitro. Although agents were applied to alleviate ROS levels, results were varied because of the distinct antioxidative activity and cell toxicity. Leonurine (LEO), extracted from the natural Chinese herb motherwort, is considered to be a potent free radical scavenger. Yet, it is undetermined whether LEO is benefit for oocyte development during in vitro maturation (IVM). In the present study, the effect of LEO on the quality of bovine oocyte as well as the underlying mechanism was investigated. We found that maturation rate (P < 0.01), subsequent blastocyst formation rate (P < 0.05), and the total blastocyst cell number (P < 0.05) after parthenogenetic activation were significantly increased in the group treated with 20 μM LEO. Moreover, a dramatic decline in ROS (P < 0.01), decreased lipid content (P < 0.01), elevated MMP level (P < 0.05), increased ATP content (P < 0.05), and reduced mitochondrial temperature (P < 0.01) were observed in oocytes treated with LEO. Furthermore, the expression level of anti-apoptotic protein BCL2 was significantly higher in LEO treated oocytes (P < 0.01), and the ratio of BAX/BCL2 was obvious decreased (P < 0.01). Finally, we found that LC3B intensity was significantly reduced (P < 0.05) while the rate of EdU positive nuclei was markedly increased (P < 0.05) in embryos derived from LEO-treated oocytes. Our results demonstrate that LEO exhibits a potent protective role in the acquisition of oocyte development capacity against oxidative stress during IVM, and provides a new solution for optimizing the in vitro culture system of bovine embryos.

Effects of crocin supplementation during in vitro culture of intact and half-destroyed four-cell mouse embryos.

We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 μg/ml of crocin supplementation (Groups 2 and 4). Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

Effect of arachidonic acid on pre- and post-hatching in vitro bovine embryo development.

Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos. Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333μM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100μM AA up to Day 12. Pre-hatching development to blastocyst was completely abrogated at 333μM AA, whereas blastocyst rates and cell numbers were not altered at 100μM AA. Impaired post-hatching development was observed at 100μM AA, whereas no effect on survival rates was noted at 5, 10 and 20μM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20μM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10μM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

Effects of the addition of insulin-transferrin-selenium (ITS) and/or metformin to the in vitro maturation of porcine oocytes on cytoplasmic maturation and embryo development.

One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug. To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development. Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control). ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05). Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS. ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

Schisandrin B protects boar spermatozoa against oxidative damage and increases their fertilization ability during in vitro storage

Oxidative stress due to low temperatures during in vitro preservation reduces boar spermatozoa quality. It has been proven that Schisandrin B (Sch-B) can act against oxidative stress in cells. Therefore, the purpose of this study was to investigate whether the treatment with Sch-B could improve the quality of boar sperm during storage at 17 °C. Semen samples were randomly divided into four groups and added to the Beltsville Thawing Solution containing different concentrations of Sch-B (0, 0.1, 0.5, and 1 mg/L) after collection. Each group was then preserved at 17 °C and the sperm motility, membrane integrity, and acrosome integrity were detected to determine the maximum available concentration of Sch-B for sperm. The optimal concentration was set at 0.1 mg/L and was used in subsequent experiments. Sperms treated with 0 and 0.1 mg/L Sch-B were evaluated for lipid peroxidation (MDA) and fertilization ability through in vitro fertilization. Finally, the quality of blastocysts which were formed by 0 and 0.1 mg/L Sch-B-treated sperm was determined. The results showed that compared with the control, the addition of 0.1 mg/L Sch-B improved boar sperm motility, and the addition of 0.1 and 0.5 mg/L Sch-B improved sperm membrane integrity and acrosome integrity. Treatment with 0.1 mg/L Sch-B reduced the level of MDA and increased the cleavage rate, blastocyst rate, and total cell number of blastocysts compared to the rate and number in the control group. However, no significant difference was observed in the ROS levels of blastocysts between the treatment and the control groups. The expression levels of CAT, SOD2, and Bcl-2 in IVF-blastocysts formed using sperm stored for one day at 17 °C were significantly higher than those in the control blastocysts. On day 4 of storage, CAT and Bcl-2 expression were significantly higher in IVF-blastocysts formed from sperm treated with 0.1 mg/L Sch-B than that in the control blastocysts. The ratio of Bax/Bcl-2 was also significantly higher in IVF-blastocysts formed using Sch-B-treated sperm. Our findings demonstrate that treatment with Sch-B can protect boar sperm from oxidative stress during liquid preservation and can increase the fertilization ability of the sperm.

Interleukin-7 enhances in vitro development and blastocyst quality in porcine parthenogenetic embryos.

Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.

Antioxidants supplementation improves the quality of in vitro produced ovine embryos with amendments in key development gene expressions

The present study assessed the effects of supplementation of different antioxidants on oocyte maturation, embryo production, reactive oxygen species (ROS) production and expression of key developmental genes. In this study, using ovine as an animal model, we tested the hypothesis that antioxidant supplementation enhanced the developmental competence of oocytes. Ovine oocytes aspirated from local abattoir-derived ovaries were subjected to IVM with different concentrations of antioxidants [(Melatonin, Ascorbic acid (Vit C), alpha-tocopherol (Vit E), Sodium selenite (SS)]. Oocytes matured without any antioxidant supplementation were used as controls. The oocytes were assessed for maturation rates and ROS levels. Further, embryo production rates in terms of cleavage, blastocysts and total cell numbers were evaluated after performing in vitro fertilization. Real-Time PCR analysis was used to evaluate the expression of stress related gene (SOD-1), growth related (GDF-9, BMP-15), and apoptosis-related genes (BCL-2 and BAX). We observed that maturation rates were significantly higher in alpha-tocopherol (100 μM; 92.4%) groups followed by melatonin (30 μM; 89.1%) group. However, blastocyst rates in ascorbic acid (100 μM; 19.5%), melatonin (30 μM; 18.4%), alpha-tocopherol (100 μM; 18.2%), and sodium selenite (20 μM; 16.9%) groups were significantly higher (P 0.05) than that observed in the control groups. Total cell numbers in blastocysts in the melatonin, ascorbic acid and alpha-tocopherol groups were significantly higher than those observed in sodium selenite and control groups. ROS production was reduced in groups treated with melatonin (30 μM), vitamin C (100 μM), sodium selenite (20 μM) and α-tocopherol (200 μM) compared with that observed in the control group. Supplementation of antioxidants caused the alterations in mRNA expression of growth, stress, and apoptosis related gene expression in matured oocytes. The results recommend that antioxidants alpha-tocopherol (200 μM), sodium selenite (40 μM), melatonin (30 μM) and ascorbic acid (100 μM) during IVM reduced the oxidative stress by decreasing ROS levels in oocytes, thus improving embryo quantity and quality.

Using Cumulus Cell Biopsy as a Non-Invasive Tool to Access the Quality of Bovine Oocytes: How Informative Are They?

The present study aimed to determine whether cumulus cells (CC) biopsy, acquired before or after in vitro maturation (IVM), presents similar gene expression pattern and if would compromises oocyte quality. First, immature cumulus oocyte complexes (COCs) were distributed: (1) maturated in groups (control); (2) individually maturated, but not biopsied; (3) subjected to CC biopsy before maturation and individually matured; (4) individually matured and submitted to CC biopsy after maturation; (5) individually matured and CC biopsied before and after maturation. Secondly, candidate genes, described as potential markers of COCs quality, were quantified by RT-qPCR in CCs before and after IVM. After in vitro fertilization (IVF), zygotes were tracked and sorted regarding their developmental potential: fully developed to embryo, cleaved and arrested, and not-cleaved. The COC's biopsy negatively affects embryo development (p &lt; 0.05), blastocyst cell number (p &lt; 0.05), and apoptotic cell ratio (p &lt; 0.05), both before and after IVM. The PTGS2, LUM, ALCAM, FSHR, PGR, SERPINE2, HAS2, and PDRX3 genes were differentially expressed (p &lt; 0.05) on matured CCs. Only PGR gene (p = 0.04) was under-expressed on matured CCs on Not-Cleaved group. The SERPINE2 gene was overexpressed (p = 0.01) in the Cleaved group on immature CCs. In summary, none of the selected gene studies can accurately predict COC's fate after fertilization.

Inhibition of METTL5 improves preimplantation development of mouse somatic cell nuclear transfer embryos.

Several factors affect the reprogramming efficiency of nuclear transfer embryos. This study shows that inhibiting 18S rRNA m6A methyltransferase METTL5 during nuclear transfer can improve the developmental rate of nuclear transfer embryos. N6-methyladenosine (m6A) is one of the most important epigenetic modifications in eukaryotic RNAs, which regulates development and diseases. It is identified by several proteins. Methyltransferase-like 5 (METTL5), an enzyme that methylates 18S rRNA m6A, controls the translation of proteins and regulates pluripotency in embryonic stem cells. However, the functions of METTL5 in embryonic development have not been explored. Here, we found that Mettl5 was upregulated in somatic cell nuclear transfer (SCNT) embryos compared with normal fertilized embryos. Therefore, we hypothesized that METTL5 knockdown during the early stage of SCNT would improve the developmental rate of SCNT embryos. Notably, injection of Mettl5 siRNA (si-Mettl5) into enucleated oocytes during nuclear transfer increased the rate of development and the number of cells in blastocysts. Moreover, inhibition of METTL5 reduced the activity of phosphorylated ribosomal protein S6, decreased the levels of the repressive histone modification H3K27me3 and increased the expression of activating histone modifications H3K27ac and H3K4me3 and mRNA levels of some 2-cell-specific genes. These results expand our understanding of the role of METTL5 in early embryonic development and provide a novel idea for improving the efficiency of nuclear transfer cloning.

ATF7-dependent epigenetic changes induced by high temperature during early porcine embryonic development.

Activating transcription factor 7 (ATF7) is a member of the ATF/cAMP response element (CRE) B superfamily. ATF2, ATF7, and CRE-BPa are present in vertebrates. Drosophila and fission yeast have only one homologue: dATF2 and Atf1, respectively. Under normal conditions, ATF7 promotes heterochromatin formation by recruiting histone H3K9 di- and tri-methyltransferases. Once the situation changes, all members are phosphorylated by the stress-activated kinase P38 in response to various stressors. However, the role of ATF7 in early porcine embryonic development remains unclear. In this study, we found that ATF7 gradually accumulated in the nucleus and then localized on the pericentric heterochromatin after the late 4-cell stage, while being co-localized with heterochromatin protein 1 (HP1). Knockdown of ATF7 resulted in decreases in the blastocyst rate and blastocyst cell number. ATF7 depletion resulted in downregulation of HP1 and histone 3 lysine 9 dimethylation (H3K9me2) expression. These effects were alleviated when P38 activity was inhibited. High temperatures increased the expression level of pP38, while reducing the quality of porcine embryos, and led to ATF7 phosphorylation. The expression level of H3K9me2 and HP1 was decreased and regulated by P38 activity. Stress-induced ATF7-dependent epigenetic changes play important roles in early porcine embryonic development.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

The Improvement of Porcine In Vitro Embryo Development through Regulating Autophagy by miRNA-143 Inhibition.

Simple SummaryThe improvement of in vitro embryo development is an important factor for the advancement of reproductive technology. Recently, studying microRNA related to early embryos is a strategic approach. This research applies miR-143 (mimics and inhibitors) to the porcine parthenogenetically activated embryos. Interesting results are revealed in this study: the miR-143 inhibitor improves the embryo development to the blastocyst. In addition, our research reveals the enhancement of autophagy and ER-phagy associated markers with higher levels of gene expression.In vitro embryo research is an important stage for the advancement of many reproductive technologies in research and agriculture. For this reason, the improvement of in vitro embryo development is a strategic field worthy of investigation. Relatively little is known about miR-143 and its effects on autophagy associated with embryo development and in vitro embryo culture. In this study, we examined the effect of miR-143 (via mimics and inhibitors) on embryonic development threatened by microinjection after parthenogenetic activation. We evaluated rates of cleavage, blastocyst, and total cell number of blastocyst; additionally, we performed LC3 immunofluorescence analysis and mRNA expression analyses of genes associated with autophagy, endoplasmic reticulum (ER)-phagy, ER stress, embryo quality, and apoptosis. The inhibition of miR-143 positively influenced embryo development by increasing the activity of autophagy and ER-phagy and the expression of embryo quality-related genes, while reducing apoptosis. In contrast, treatment with miR-143 mimics increased ER stress-related gene expression and apoptosis, and reduced embryo development. Together, our findings indicate that miR-143 plays a role in the interplay between autophagy, ER-phagy, and embryo quality during early porcine embryo development.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

Leukemia inhibitory factor enhances the development and subsequent blastocysts quality of yak oocytes in vitro.

Leukemia inhibitory factor (LIF) is a multipotent cytokine of the IL-6 family which plays a critical role in the maturation and development of oocytes. This study evaluated the influence of LIF on the maturation and development ability of yak oocytes, and the quality of subsequent blastocysts under in vitro culture settings. Different concentrations of LIF (0, 25, 50, and 100 ng/mL) were added during the in vitro culture of oocytes to detect the maturation rate of oocytes, levels of mitochondria, reactive oxygen species (ROS), actin, and apoptosis in oocytes, mRNA transcription levels of apoptosis and antioxidant-related genes in oocytes, and total cell number and apoptosis levels in subsequent blastocysts. The findings revealed that 50 ng/mL LIF could significantly increase the maturation rate (p < 0.01), levels of mitochondria (p < 0.01) and actin (p < 0.01), and mRNA transcription levels of anti-apoptotic and antioxidant-related genes in yak oocytes. Also, 50 ng/mL LIF could significantly lower the generation of ROS (p < 0.01) and apoptosis levels of oocytes (p < 0.01). In addition, blastocysts formed from 50 ng/mL LIF-treated oocytes showed significantly larger total cell numbers (p < 0.01) and lower apoptosis rates (p < 0.01) than the control group. In conclusion, the addition of LIF during the in vitro maturation of yak oocytes improved the quality and the competence of maturation and development in oocytes, as well as the quality of subsequent blastocysts. The result of this study provided some insights into the role and function of LIF in vitro yak oocytes maturation, as well as provided fundamental knowledge for assisted reproductive technologies in the yak.

First deliveries of felines by transcervical transfer of in vitro-cultured embryos

Transcervical transplantation of embryos has been wildly used in several species. However, it has not been successful in feline species because of their unique cervix structure combined with poor in vitro culture systems for embryos. In the present study, a simple device to guide the embryo transfer (ET) tube through the cervix and into the uterus was developed. To supply better blastocysts for ET, the in vitro culture system of feline embryos was also optimized. Results showed that 91.67 ± 4.17% of embryos developed to the blastocyst stage in QAU-4 medium compared with 49.17 ± 6.51% in KSOM medium. The total cell number of blastocysts reached 87.1 ± 22.1 in QAU-4 medium compared with 29.8 ± 2.1 in KSOM. Embryoid body formation was also observed in QAU-4 medium. Finally, by the improved technical scheme, CRX (Cone-Rod Homeobox) gene-edited kittens were successfully bred. To date, this is the first report, to our knowledge, of knockout kittens successfully delivered after transcervical ET with a simple transfer tube.

Can we Make SpermFAST? Improving Sperm Function to Increase Fertilisation Rates after in vitro Fertilisation

Background: Intracytoplasmic sperm injection (ICSI) usage ([Formula: see text]64% of autologous cycles) has increased in Australia as an attempt to avoid low fertilisation and total fertilisation failure after standard in vitro fertilization (IVF). ICSI and to a lesser extent IVF, bypass key sperm maturation (hyperactivation, capacitation/acrosome reaction) events that naturally occur in the female tract and are vital for successful fertilisation. Current commercial sperm preparation media are not fully designed to induce these changes in sperm prior to insemination. Aim: To improve fertilisation rates following IVF by increasing sperm capacitation and hyperactivation between sperm preparation and insemination ultilising a new sperm medium (SpermFAST). Method: Sperm from 12 consenting normospermic men were incubated in either G-IVF+ (Vitrolife) or SpermFAST (UoA/Monash IVF) following a direct swim-up. Measures of capacitation, hyperactivation, sperm binding, acrosome reaction and oxidative DNA damage were assessed. Further, sperm from male CBAF1 mice (N=8) were incubated in either G-IVF+ or SpermFAST prior to IVF insemination. Fertilisation rates, embryo development, blastocyst cell numbers and DNA damage were assessed. Results: Incubation of human sperm in SpermFAST increased tyrosine phosphorylation (15.8% vs 9.5%, P[Formula: see text]0.05), hyperactive motility (38.3% vs 14.9%, P[Formula: see text]0.01), sperm binding (73.1% vs 47.7%, P[Formula: see text]0.01), while having no impact on sperm acrosomal status or oxidative DNA damage levels. Following IVF in the mouse, sperm incubated in SpermFAST increased fertilisation rates (94% vs 88%, P[Formula: see text]0.05), blastocyst total cell (92.2% vs 77.4%, P[Formula: see text]0.05), inner cell mass (14.9% vs 18.9%, P[Formula: see text]0.01) and epiblast cell numbers (3.7% vs 1.6%, P[Formula: see text]0.01), while the proportion of DNA damaged cells decreased in blastocysts (2.3% vs 4.8%, P[Formula: see text]0.001). Conclusion: Sperm function and fertilisation rates are improved when the sperm medium better mimics the environment of the female reproductive tract during natural conception. Improving IVF culture media to better meet the physiological needs of sperm could potentially improve outcomes following IVF.

Short-Term Exposure with Long-Term Effects: The Ketone Body, Beta-Hydroxybutyrate, Alters Mouse Embryo Developmental Programming and Reduces Viability

Background: Diet-induced nutritional changes in the maternal reproductive tract stimulate embryonic adaptations to maintain growth and survival, directly impacting child and adult health via developmental programming. This is plausibly facilitated by an interrelationship between metabolism and epigenetic control of gene expression, termed ‘metaboloepigenetics’, resulting in heritable modifications that affect long-term offspring health and viability. The ketogenic diet (KD) is increasingly popular amongst sub-fertile women trying to conceive, and results in elevated maternal ketone levels, including beta-hydroxybutyrate (bOHB). The impact of high bOHB levels on embryonic development, physiology, and developmental programming remains unknown. Aim: To assess how bOHB affects preimplantation mouse embryo development, metaboloepigenetic regulation, and post-transfer viability. Method: Preimplantation mouse embryos were cultured in vitro with or without 2 mM bOHB, representing serum concentrations with KD consumption. Day 5 blastocyst cell number and lineage allocation was assessed via differential nuclear stain, metabolism of bOHB and carbohydrates was assessed by ultramicrofluorescence, and acetylation of histone 3 lysine 9 (H3K9ac) and lysine 27 (H3K27ac) were assessed by immunofluorescence. Day 4 blastocysts were transferred to pseudo-pregnant females for analysis of embryonic day 14.5 placental and fetal development. Results: A reduction in total and trophectoderm cell number (P&lt;0.05) was observed, indicating reduced viability. Blastocysts were shown to consume bOHB in vitro, with uptake increasing with bOHB concentration. Further, bOHB increased glycolytic rate (P&lt;0.01), while epigenetic analyses revealed H3K9ac and H3K27ac were unaffected by bOHB. Of significance, post-transfer implantation rates were reduced (P&lt;0.05), concurrent with smaller placental diameter (P&lt;0.01) and fetal crown-rump length (P&lt;0.05), indicating developmental programming alterations. Conclusion: These data infer that bOHB-stimulated metabolic alterations are a contributing factor to ketone-induced developmental programming, which may incorporate epigenetic components other than H3K9ac or H3K27ac. Therefore, consumption of a KD during conception and pregnancy may have negative impacts on the long-term viability and health of offspring.

Novel Oocyte-Secreted Factors Improve Mouse IVM Outcomes

Background: In vitro maturation (IVM) is a technology designed to obtain mature oocytes following culture of immature cumulus–oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for patients with excessive AFC or those requiring immediate fertility preservation. However, the clinical uptake of IVM has been slow, primarily due to lower embryo yield and live birth rate relative to IVF, therefore improving IVM culture is required. Aim: To assess whether supplementation of IVM culture medium with the novel in-house engineered TGFβ proteins cumulin and super-GDF9 improves subsequent embryo development. Method: Immature mouse COCs were cultured by standard IVM or bi-phasic IVM ± cumulin or super-GDF9. Following IVM, cumulus expansion was scored and COCs were fertilized, and cultured to assess embryo development. Differential staining was performed on day 6 blastocysts following bi-phasic IVM to assess cell allocation. In a separate experiment, hyperspectral imaging of autofluorescence was carried out on oocytes and cumulus cells following standard IVM ± cumulin to assess the molecular composition of these cells. Results: Both cumulin and super-GDF9 in standard IVM significantly increased cumulus expansion (P&lt;0001; n=104-115 COCs) and blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; P=0.006; n=382-406 oocytes). Hyperspectral imaging showed that oocytes (n=115-158) and cumulus cells (n=532-600) exposed to cumulin during IVM had a distinct spectral profile that varied dramatically (P&lt;0.005) from untreated cells, demonstrating that cumulin has a major impact on the molecular composition of these cells, likely contributing to the improved oocyte quality. In bi-phasic IVM, cumulin did not significantly alter embryo yield (n=387-424 oocytes) or blastocyst cell number or allocation (n=84-112 blastocysts). Conclusion: Cumulin did not provide an additional beneficial effect in bi-phasic IVM, however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence suggesting that their effects in human IVM should be investigated.