Exploring the impact of heat stress on oocyte maturation and embryo development in dairy cattle using a culture medium supplemented with vitamins E, C, and coenzyme Q10.

Heat stress is a significant factor affecting the fertility of dairy cattle due to the generation of free radicals. In assisted reproductive techniques, the inclusion of protective antioxidants becomes crucial to mitigate potential cellular damage. This study aimed to explore the impact of supplementing vitamins E, C, and coenzyme Q10 into the oocyte culture medium, with the goal of ameliorating the adverse effects of heat stress on oocyte maturation and embryo development in dairy cattle. A group of fifty Holstein dairy cows were synchronized, and their oocytes were harvested using the ovum pick-up method. High-quality oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF) procedures, utilizing a culture medium containing, no supplements (Group 1), 100 µM of vitamins E (Group 2) and C (Group 3), along with 50 µM of coenzyme Q10 (Group 4). The ensuing zygotes were cultured, and the ensuing embryos were evaluated for blastocyst formation by the seventh day. An analysis of the blastocysts' inner cell mass (ICM) and trophectoderm (TE) cells was also conducted. The findings revealed that the group receiving supplementation of vitamin E and coenzyme Q10 exhibited significantly higher maturation and cleavage rates in comparison to both the control and the vitamin C groups. Furthermore, the count of ICM, TE, and blastocyst cells was notably elevated in the vitamin E supplemented group when compared to the control group. In summary, the effectiveness of vitamin E in enhancing IVM, IVF, and embryo development under conditions of heat stress surpassed that of vitamin C and coenzyme Q10.

Effects of ovary storage temperature and embryo vitrification on somatic cell nuclear transfer outcomes in goats.

Improving the genetic potential of farm animals is one of the primary aims in the field of assisted reproduction. In this regard, somatic cell nuclear transfer (SCNT) can be used to produce a large number of embryos from genetically elite animals. The aims of the present study were to assess the effects of: (1) ovary storage conditions on preimplantation development of recovered oocytes and the freezability of the derived blastocysts; and (2) vitrification of goat SCNT-derived blastocysts on postimplantation development. Goat oocytes were recovered from ovaries and stored under warm (25°C-27°C) or cold (11°C-12°C) conditions before being used to produce SCNT embryos. There were no differences in oocytes recovered from ovaries kept under cold versus warm storage conditions in terms of cleavage (mean (±s.d.) 95.68±1.67% vs 95.91±2.93% respectively) and blastocyst formation (10.69±1.17% vs 10.94±0.9% respectively) rates. The re-expansion rate of vitrified blastocysts was significantly lower for cold- than warm-stored ovaries (66.3±8.7% vs 90±11% respectively). To assess the effects of vitrification on postimplantation development, blastocysts from cold-stored ovaries only were transferred from fresh and vitrified-warmed groups. The pregnancy rate was comparable between the fresh and vitrified-warmed groups (41.65% and 45.45% respectively). In addition, established pregnancy in Day 28-38 and full-term pregnancy rates were similar between the two groups. In conclusion, this study shows similar invitro preimplantation developmental potential of warm- and cold-stored ovaries. This study introduces the vitrification technique as an appropriate approach to preserve embryos produced by SCNT for transfer to recipient goats at a suitable time.

Supplementation of maturation medium with CoQ10 enhances developmental competence of ovine oocytes through improvement of mitochondrial function.

In vitro maturation (IVM) can impair the balance between antioxidant capacity and oxidative stress, and jeopardize embryo development by increasing oxidative stress, reducing energy metabolism, and causing improper meiotic segregation. Balancing the energy production and reduction of oxidative stress can be achieved by supplementation with coenzyme Q10 (CoQ10), an electron transporter in the mitochondrial inner membrane. To improve the in vitro production of ovine embryos, we studied the effect of CoQ10 supplementation during the maturation of sheep oocytes. A minimum of 100 cumulus-oocyte complexes (COCs) were matured in the presence of 15, 30, or 50 µM CoQ10 in three to five replicates; next, in vitro fertilization and culture in a subset of oocytes were done. Our data revealed that compared to control oocytes or other concentrations of CoQ10, supplementation with 30 µM CoQ10 resulted in a significant increase in blastocyst formation and hatching rates, improved the distribution, relative mass and potential membrane of mitochondria, decreased the levels of reactive oxygen species and glutathione and lessened the percentage of oocytes with misaligned chromosomes after spindle assembly. The relative expression levels of apoptosis markers CASPASE3 and BAX were significantly reduced in CoQ10-treated oocytes and cumulus cells whereas the relative expression level of GDF9, an oocyte-specific growth factor, significantly increased. In conclusion, supplementation with CoQ10 improves the quality of COCs and the subsequent developmental competence of the embryo.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Vitrification at Day3 stage appears not to affect the methylation status of H19/IGF2 differentially methylated region of in vitro produced human blastocysts.

One of the most widely used assisted reproductive technology (ART) is vitrification. The aim of this study is to evaluate DNA methylation of H19/IGF2 differentially methylation region (DMR) in in vitro produced human blastocysts derived from non-vitrified and vitrified day3 embryos. Day3 embryos derived from ICSI cycles from fertile couples referring for family balancing program were either biopsied or vitrified/warmed and subsequently biopsied. Following biopsy, embryos were cultured to day 5. Day5 blastocysts with desired sex were transferred or vitrified for future use. Blastocysts with un-desired sex were donated for research. The assessment of the embryos was performed in two non-vitrified and vitrified groups. Methylation level of H19/IGF2 DMR was analysed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. Results showed that the overall methylated CpGs percentages of this region in the vitrified and non-vitrified groups were 35.3% ± 3.6 and 38.27 ± 4.1%, respectively. The difference between the two groups was not significant. Vitrification of day3 embryo appears to have no adverse effect on DNA methylation status of H19/IGF2 DMR of embryos cultured in vitro to blastocyst stage. These data may have implications for performing frozen embryo cycles transfer instead of fresh embryo transfer cycles, owing to the naturally synchronized uterus and subsequently improved endometrial receptivity in frozen embryo transfer instead of imbalanced hormonal milieu in fresh embryo transfer cycles.

Contrasting effects of G1.2/G2.2 and SOF1/SOF2 embryo culture media on pre- and post-implantation development of non-transgenic and transgenic cloned goat embryos.

This study compared the efficiency of two embryo culture media (SOF1/SOF2 and G1.2/G2.2) for pre- and post-implantation development of somatic cell nuclear transfer goat embryos derived from non-transgenic and transgenic (for htPA and hrcfIX genes) fibroblasts. Despite similar cleavage rates, G1.2/G2.2 supported significantly higher blastocyst development than SOF1/SOF2 (30-35% versus 21%; P < 0.05), irrespective of cell transgenesis. However, following embryo transfer, pregnancy outcomes (establishment, full-term development and live birth) were all significantly higher (P < 0.05) for embryos developed in SOF1/SOF2 versus G1.2/G2.2. Gene expression profiling of 17 developmentally important genes revealed that: (i) SOX2, FOXD3, IFNT, FZD, FGFR4, ERK1, GCN5, PCAF, BMPR1, SMAD5, ALK4, CDC25 and LIFR were significantly induced in blastocysts developed in SOF1/SOF2 but not G1.2/G2.2; (ii) OCT4, CTNNB and CDX2 were similarly expressed in both groups; and (iii) AKT was significantly higher in G1.2/G2.2 than SOF1/SOF2 (P < 0.05). Following IVF, although blastocyst development in G1.2/G2.2 was significantly higher than SOF1/SOF2 counterparts, the majority of assessed genes were similarly expressed in blastocysts developed in both groups. It was concluded that the long-term programming effects of embryo culture medium and/or embryo production method may irreversibly affect post-implantation development of cloned embryos through defined molecular pathways.

The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst.

Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers.

Advanced Glycation End-Products of Follicular Fluid are Associated with Embryo Morphokinetic Parameters and ART Outcomes.

Advanced glycation end products (AGEs) can disrupt antioxidant system and steroidogenesis, resulting in detrimental effects on assisted reproductive technology (ART) outcomes. This study aimed to investigate the association of AGEs in follicular fluid (FF) with morphokinetic parameters of embryos and ART outcomes. Fifty women undergoing ART treatment were studied. AGEs, glucose, 25(OH) vitamin D, malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were evaluated in FF. The expression of 3ßHSD, CYP11A1, and CYP19A1 genes were analyzed in granulosa cells (GCs) by qRT-PCR technique. Morphokinetic parameters were evaluated using time-lapse technology. The FF level of AGEs was reversely associated with CAT, SOD, and GPX activities, and total and mature oocytes number, blastocyst formation rate, and high-grade embryos number, while it showed positive correlations with the FF MDA levels, the expression of steroidogenesis genes, number of immature oocytes, morphokinetic parameters, and number of low-grade embryos. Furthermore, the level of vitamin D in FF had an inverse association with AGEs and positive correlations with ART outcomes and morphokinetic parameters. Comparison between the those with positive and negative biochemical pregnancy showed no significant differences in terms of FF factors and just the expression of 3ßHSD, CYP11A1, and CYP19A1 genes were higher in pregnant women (p < 0.05). AGEs could delay blastomere division and lead to an increase in the number of low-quality embryos, while vitamin D have an adverse effect on AGEs and a protective function against AGEs negative effects.

Supplementation of sperm cryopreservation media with H2S donors enhances sperm quality, reduces oxidative stress, and improves in vitro fertilization outcomes.

Cryopreservation of sperm can cause oxidative stress and damage, leading to decreased different functional parameters and fertilization potential. In this study, we evaluated two types of H2S donors: NaHS, a fast-releasing donor, and GYY4137, a slow-releasing donor during cryopreservation of goat sperm. Initially, we determined that 1.5 and 3 µM NaHS, and 15 and 30 µM GYY4137 are optimal concentrations that improved different sperm functional parameters including motility, viability, membrane integrity, lipid peroxidation, and ROS production during incubation at 38.5 °C for 90 min. We subsequently evaluated the impact of the optimal concentration of NaHS and GYY4137 supplementation on various functional parameters following thawing during cryopreservation. Our data revealed that supplementation of extender improved different parameters including post-thaw sperm motility, viability, membrane integrity, and reduced DNA damage compared to the frozen-thawed control group. The supplementation also restored the redox state, decreased lipid peroxidation, and improved mitochondrial membrane potential in the thawed sperm. Finally, we found that supplementation of the extender with NaHS and GYY4137 enhanced IVF outcomes in terms of blastocyst rate and quality of blastocysts. Our results suggest that both donors can be applied for cryopreservation as antioxidants to improve sperm quality and IVF outcomes of frozen-thawed goat sperm.

Omega 6/Omega 3 Ratio Is High in Individuals with Increased Sperm DNA fragmentation.

An imbalance between omega-6 and omega-3 fatty acids in sperm has been linked with lipid peroxidation and DNA damage in sperm, indicating a possible correlation to fertility potential. This cross-sectional study involved 56 infertile men (aged 25-45), and assessed the relationship between the omega-6 to omega-3 fatty acid ratio in sperm and seminal plasma with sperm DNA fragmentation. Individuals were categorized based on high or low levels of sperm DNA fragmentation according to two tests (TUNEL and SCSA assay less or greater than 10 and 30%, respectively), and their fatty acid composition, as well as sperm functional tests, were analyzed. Results showed that men with high DNA fragmentation exhibited higher percentages of total saturated, monounsaturated, and omega-6 to omega-3 fatty acid ratios in both sperm (P < 0.001) and seminal plasma (P < 0.001) compared to men with low DNA fragmentation. The percentage of sperm lipid peroxidation, and residual histone (P < 0.05) were higher, while the percentage of sperm motility (P < 0.001) was lower in the former compared to the latter group. Moreover, Pearson's correlation revealed positive associations between the omega-6 to omega-3 fatty acid ratio with sperm lipid peroxidation, DNA fragmentation, and residual histones in both sperm and seminal plasma. Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better sperm quality compared to men with a lower omega-3 fatty acid.

Follicular fluid advanced glycation end products in assisted reproduction: A systematic review.

Follicular fluid (FF) advanced glycation end products (AGEs) have been associated with low oocyte quality and number, low fertilization rate, impaired embryonic development and low pregnancy rate. These findings are especially relevant in women undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), ie, assisted reproductive technology (ART). A systematic literature search was conducted to examine various AGEs including pentosidine, carboxymethyl-lysine (CML), methylglyoxal 5-hydro-5-methylimidazolones (MG-H1), toxic AGE (TAGE), and soluble receptor for AGE (sRAGE) with ART outcomes. Studies showed that total AGEs and sRAGE in FF were associated with the ovarian response, follicle number, retrieved oocyte number, mature (MII) oocyte number, fertilization rate, embryo number, embryo quality, and successful pregnancy. Although FF AGEs could be considered predictive biomarkers, population heterogeneity and differences in ovulation induction protocols make the findings less clear. This review highlights important role of AGEs in ART and necessity of evaluating AGEs in serum vs with FF to better predict ART outcomes.

Pluripotency and embryonic lineage genes expression in the presence of small molecule inhibitors of FGF, TGFß and GSK3 during pre-implantation development of goat embryos.

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFß inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2-7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

Oct-4 activating compound 1 (OAC1) could improve the quality of somatic cell nuclear transfer embryos in the bovine.

Previous studies described aberrant nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos that is distinctly different from fertilized embryos. This abnormal nuclear reprogramming hampers the proper pre- and/or post-implantation development. It has been demonstrated that SCNT blastocysts aberrantly expressed POU5F1 and POU5F1-related genes. With regard to this, it has been postulated that promoting the expression of POU5F1 in SCNT embryos may enhance reprogramming in SCNT embryos. In this study, we treated either fibroblast donor cells or SCNT embryos with OAC1 as a novel small molecule that has been reported to induce POU5F1 expression. Quantitative results from the MTS assay revealed that lower concentrations of OAC1 (1, 1.5, and 3 µM) are non-toxic after 2, 4, and 6 days, but higher concentrations (6, 8, 10, and 12 µM) are toxic and reduced the proliferation of cells after 6 days. No enhancement in the expression of endogenous POU5F1 was observed when both mouse and bovine fibroblast cells were treated with 1.5 and 3 µM OAC1 for up to 6 consecutive days. Subsequently, we treated either fibroblast as donor cells in the SCNT procedure (BFF-OAC1 group) or SCNT embryos [for 4 days (IVC-OAC1: D4-D7 group) or 7 days (IVC-OAC1: D0-D7 group)] with 1.5 µM OAC1. We observed that neither treatment of fibroblast donor cells nor SCNT embryos improved the cleavage and blastocyst rates. Interestingly, we observed that treatment of SCNT embryos all throughout the in vitro culture (IVC) (IVC-OAC1: D0-D7) with 1.5 µM OAC1 improves the quality of derived blastocyst which was indexed by morphological grading, blastomere allocation, epigenetic marks and mRNA expression of target genes. In conclusion, our results showed that supplementation of IVC medium with 1.5 µM OAC1 (D0-D7) accelerates SCNT reprogramming in bovine species.

Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling.

The specific role of the canonical WNT/ß-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of ß-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of ß-catenin using IWR1. Initially, we observed cytoplasmic expression of ß-CATENIN in 2 and 8-16 cell stage embryos and membranous expression of ß-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of ß-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development.

A 3D printed polylactic acid-Baghdadite nanocomposite scaffold coated with microporous chitosan-VEGF for bone regeneration applications.

Three-dimensional (3D) printing technology has become an advanced approach for fabricating patient-specific scaffolds with complex geometric shapes to replace damaged or diseased tissue. Herein, polylactic acid (PLA)-Baghdadite (Bgh) scaffold were made through the fused deposition modeling (FDM) 3D printing method and subjected to alkaline treatment. Following fabrication, the scaffolds were coated with either chitosan (Cs)-vascular endothelial growth factor (VEGF) or lyophilized Cs-VEGF known as PLA-Bgh/Cs-VEGF and PLA-Bgh/L.(Cs-VEGF), respectively. Based on the results, it was found that the coated scaffolds had higher porosity, compressive strength and elastic modulus than PLA and PLA-Bgh samples. Also, the osteogenic differentiation potential of scaffolds following culture with rat bone marrow-derived mesenchymal stem cells (rMSCs) was evaluated through crystal violet and Alizarin-red staining, alkaline phosphatase (ALP) activity and calcium content assays, osteocalcin measurements, and gene expression analysis. The release of VEGF from the coated scaffolds was assessed and also the angiogenic potential of scaffolds was evaluated. The sum of results presented in the current study strongly suggests that the PLA-Bgh/L.(Cs-VEGF) scaffold can be a proper candidate for bone healing applications.

Platelet rich fibrin containing nanofibrous dressing for wound healing application: Fabrication, characterization and biological evaluations.

Recently, nanofibrous structures have shown great potential for a wide range of medical applications. The aim of the current study was to evaluate the wound healing process using Polycaprolactone/Keratin/Platelet-rich fibrin (PCL/Kr/PRF) fibrous scaffold fabricated through electrospinning process. A range of techniques were utilized to fully characterize the chemical, physical and biological properties of the resultant structure. Results revealed that by the addition of only 0.5%w/v PRF to PCL/Kr (PCL/Kr/0.5PRF) sample, the fibers diameter decreased from 193.93 ± 64.80 nm to 65.98 ± 14.03 nm, and the stress at break demonstrated a 18.27% increase in comparison to the PCL sample (from 2.90 ± 0.80 MPa to 3.43 ± 0.90 MPa). The PCL/Kr/0.5PRF scaffold showed more antibacterial activity against gram-positive and gram-negative bacteria than PCL/Kr sample. Based on enzyme-linked immunosorbent assays, the PCL/Kr/0.5PRF sample revealed an independent release of VEGF and PDGF for 7 days. Cell viability studies demonstrated non-cytotoxic nature of PRF-containing dressings. Also, chorioallantoic membrane (CAM) assay was performed to evaluate the angiogenic potential of the wound dressings. The in vivo assessments also showed that PCL/Kr/0.5PRF accelerated the wound healing process in terms of collagen deposition and the formation of skin appendages which was comparable to the normal skin. Overall, the data presented in this study greatly suggest that the PCL/Kr/0.5PRF wound dressing could be a suitable candidate for wound healing and skin regeneration.

Sequential IVM by CNP preincubation and cooperating of PGE2 with AREG enhances developmental competence of SCNT reconstructs in goat.

Developmental competence of in vitro matured cumulus oocyte complexes (COCs) in conventional IVM (C.IVM) is lower than in vivo maturated COCs and is related to unsynchronized nuclear and cytoplasmic maturation. To overcome this dearth, COCs can be exposed to granulosa secreted factors in a two-step system. Therefore, in the first experiment, 1000 nM of C-type natriuretic peptide for 8 h was determined (CAPA), as the best time and concentration to retain oocytes in germinal vesicle stage. This condition, also reduces lipid droplets and increases the expression of ATGL and PLIN2 involved in lipolysis and lipogenesis, respectively. In the second experiment, maturation was stimulated with prostaglandin E2 and amphiregulin for 18 h (CAPA-IVM), and their optimal concentrations based on blastocyst formation rates through in vitro fertilization (IVF) were determined as 1 and 600 nM, respectively. In the third experiment, the in vitro and in vivo developmental competency of SCNT embryos in CAPA-IVM group were determined. Despite similar blastocyst formation rates in IVF and SCNT between CAPA-IVM and C.IVM, the quality of blastocysts were quality was higher in CAPA-IVM, which reflected itself, as higher ICM/TE ratio and also expression of NANOG in SCNT blastocysts. Pregnancy rate, live births rate and SCNT efficiency were not significant between CAPA-IVM and C.IVM groups. Therefore, CAPA-IVM can improve the developmental competency of SCNT derived embryos.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

The Impact of Two Embryo Culture Media, Synthetic Oviduct Fluid and Commercial BO, on pre-and post-Implantation Development of Cloned SAANEN Goat Embryos.

BACKGROUND: Somatic cell nuclear transfer (SCNT) is an approach for the propagation of elite animals. In vitro condition, especially the composition of culture media has a profound effect on the developmental competency of in vitro derived e mbryos. There are limited studies evaluating the effect of culture media on SCNT outcomes. To address this gap, we compare the effect of two culture media synthetic oviduct fluid (SOF) vs. commercial bracket-oliphant (BO) on developmental comptenecy. MATERIALS AND METHODS: In this experimental study, embryos derived from in vitro fertilized (IVF) and SCNT were cultured in both BO and SOF media for 7 days. In addition to the assessment of cleavage and blastocyst on day 3, and 7, the quantitative expression of 16 genes in theresultant blastocysts were assessed. The resultant SCNT blastocysts from SOF and BO groups were also transferred to the synchronized recipient for developmental competency to term. RESULTS: The blastocyst rate in the BO medium was significantly higher than that of the SOF medium in the SCNT group (P<0.05). All of the examined genes showed increased expression levels in SCNT blastocyst in both media compared to IVF Blastocyst. In the IVF group, Oct4, Bmpr1, and Gcn5 showed significantly higher expression in the SOF medium compared to the BO medium while Akt, Fgfr4, Sox2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, Fgfr4, Gcn5, Fzd, Ctnnb, Bmpr1, and Fgfr4 showed significantly higher expression in SOF compared to BO derived blastocyst. CONCLUSION: It appears that in SCNT blastocysts, gene regulation is less controlled compared to IVF ones, irrespective of the type of medium. In addition, there are differences regarding certain genes expressions between IVF and SCNT derived blastocysts between SOF and BO, reiterating that culture composition affects developmental competency and gene expression.