Heat shock interferes with the amino acid metabolism of bovine cumulus-oocyte complexes in vitro: a multistep analysis.

By affecting the ovarian pool of follicles and their enclosed oocytes, heat stress has an impact on dairy cow fertility. This study aimed to determine how heat shock (HS) during in vitro maturation affected the ability of the bovine cumulus-oocyte complexes (COCs) to develop, as well as their metabolism of amino acids (AAs). In this study, COCs were in vitro matured for 23 h at 38.5 °C (control; n = 322), 39.5 °C (mild HS (MHS); n = 290), or 40.5 °C (severe HS (SHS); n = 245). In comparison to the control group, the MHS and SHS groups significantly decreased the percentage of metaphase-II oocytes, as well as cumulus cell expansion and viability. The SHS decreased the rates of cleavage and blastocyst formation in comparison to the control and MHS. Compared to the control and MHS-COCs, the SHS-COCs produced significantly more phenylalanine, threonine, valine, arginine, alanine, glutamic acid, and citrulline while depleting less leucine, glutamine, and serine. Data showed that SHS-COCs had the highest appearance and turnover of all AAs and essential AAs. Heat shock was positively correlated with the appearance of glutamic acid, glutamine, isoleucine, alanine, serine, valine, phenylalanine, and asparagine. Network analysis identified the relationship between HS and alanine or glutamic acid, as well as the relationship between blastocyst and cleavage rates and ornithine. The findings imply that SHS may have an impact on the quality and metabolism of AAs in COCs. Moreover, the use of a multistep analysis could simply identify the AAs most closely linked to HS and the developmental competence of bovine COCs.

Ferulago angulata extract alleviates testicular toxicity in male mice exposed to diazinon and lead.

In this study, we investigated the protective effects of Ferulago angulata extract (FAE) against the reproductive toxicants Diazinon (DZN) and Lead (Pb) in mice. These pollutants are known to induce oxidative stress (OS), while FAE acts as a natural antioxidant. Adult male NMRI mice were exposed to DZN, Pb, and DZN+Pb, with or without FAE treatment for six weeks. We evaluated OS markers, testicular histology, and expression of mRNA related to enzymatic antioxidants. Exposure to DZN and Pb led to increased levels of thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) in the testes, along with a decrease in the total antioxidant capacity (TAC). Furthermore, the mRNA expression of antioxidant enzymes such as superoxide dismutase 1 (SOD1) and glutathione peroxidase 4 (GPX4) was altered. However, when FAE was administered concurrently, it restored the biochemical parameters to normal levels, reduced the toxic effects of DZN and Pb, and provided protection against testicular histopathological injury. These findings suggest that FAE has the potential to serve as a protective agent against oxidative damage caused by contaminants in reproductive organs, specifically in the testes.

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.

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.

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.

Vitamin D and calcium, together and separately, play roles in female reproductive performance.

Vitamin D (VD) deficiency reduces the chances of successful fertilization; however, it remains to be validated whether this effect is dependent or not on calcium. To address this question, we generated several situation using a mouse model in which VD content was either increased or decreased in a normo or hypocalcemia context. After the measurement of serum 25-hydroxyvitamin D2, calcium and phosphorus levels, an analysis was carried out in terms of oocytes maturation as well as reproductive performance. VD overdose, despite the fact that it resulted in an increased number of mature oocytes, reduced developmental competence and offspring survival. VD deficiency (VDD), on the contrary, reduced the number and percentage of mature oocytes, blastocyst rate, as well as fertility rate and offspring survival. Hypo-calcemia when VD levels were normal, had a similar effect than VDD. The effects of VDD were reversed by a diet that corrected calcium level. Therefore, both VD overdose (in a context of normal calcium level) VD deficiency as well as hypo-calcemia have an effect on female reproductive function. In conclusion, although closely related, VD and calcium act in part independently of each other in defining the "optimum" for female reproductive performance.

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.

Effect of xylazine, detomidine, medetomidine and dexmedetomidine during laparoscopic SCNT embryo transfer on pregnancy rate and some physiological variables in goats.

BACKGROUND: The present study was conducted to determine if using α2-adrenergic agonists results in decreased stress levels (lower cortisol levels) in goats used for laparoscopic embryo [somatic cell nuclear transfer (SCNT)] transfer; and there is an effect on pregnancy rate when stress levels are lessened. Sixty healthy does aged 24 ± 4 months and weighing 30 ± 3 kg were used in experimental, prospective, randomized and blinded study. In this study, embryos were obtained by the Somatic Cell Nuclear Transfer (SCNT) method. Animals were randomly assigned to five groups: control (normal saline); xylazine (100 µg kg- 1); detomidine (50 µg kg- 1); medetomidine (20 µg kg- 1); and dexmedetomidine (5 µg kg- 1). Embryo transfer (through laparoscopic technique) began at 15 min and continued till 45 min post-treatment. Heart rate (HR), respiratory rate (RR), rectal temperature (RT), and ruminal motility were performed before (baseline) and after drug administration. Pregnancy detection was performed 38 days after embryo transfer. RESULTS: Compared to control, HR, RR and ruminal motility were significantly lower in α2-adrenergic agonists groups at 5-90, 15-60, and 5-120 min, respectively. Serum cortisol values significantly increased from baseline in the control group 45 min after drug administration (p = 0.001). At time points 45 and 60 min, serum cortisol concentration was significantly lower in α2-adrenergic agonists groups compared with the control. The pregnancy rate in control group (n = 4/12, 33.3%) was significantly lower than xylazine (n = 9/12, 75%; p = 0.041), detomidine (n = 10/12, 83.3%; p = 0.013), medetomidine (n = 9/12, 75%; p = 0.041) and dexmedetomidine (n = 10/12, 83.3%; p = 0.013); but no significant differences were observed among different α2-adrenergic agonists groups. CONCLUSION: Alph2-adrenergic agonists were effective on increasing the pregnancy rate of recipient goats receiving cloned embryos. No significant differences were detected among different α2-adrenergic agonists.

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.

Effect of Collagen Hydrolysate and Fish Oil on High-Sensitivity C-Reactive Protein and Glucose Homeostasis in Patients with severe Burn; a Randomized Clinical Trial.

INTRODUCTION: Collagen and omega-3 fatty acids (FAs) are suggested to have anti-inflammatory, anti-oxidant, and insulin-sensitizing properties. The aim of this study was to investigate the effect of collagen hydrolysate and omega-3 FAs on inflammation and insulin resistance in patients with major burns. METHODS: In this double-blind randomized clinical trial, 66 patients with 20-45% burns were assigned to either of the three groups of collagen (40 gr/d), collagen (40 gr/d) plus fish oil (10 ml/d), or control. High-sensitivity C-reactive protein (hs-CRP), fasting blood glucose (FBG) and insulin concentrations, and homeostatic model assessment for insulin resistance (HOMA-IR) were assessed at baseline, as well as end of weeks two and three. RESULTS: Based on post-hoc analyses, hs-CRP levels were significantly lower in the collagen (p=0.026) and collagen+omega-3 (p=0.044) groups compared to the control group, at week three. However, pre- to post- (week three) changes of hs-CRP were significantly higher only in the collagen+omega-3 group compared to the control group (173.2 vs. 103.7 mg/l, p=0.024). After three weeks of the intervention, insulin (11.3 and 11.9 vs. 22.8 µIU/ml) and HOMA-IR (2.9 and 2.8 vs. 7.9) values seemed to be clinically, but not statistically, lower in both intervention groups compared to the control group. Pre- to post- (week three) values of FBG decreased significantly in the collagen (p=0.002) and collagen+omega-3 (p=0.036) groups. Insulin (p=0.008) and HOMA-IR (p=0.001) decreased significantly only in the collagen+omega-3 group at week three compared to the baseline. CONCLUSIONS: Supplementation with collagen hydrolysate and omega-3 FAs can improve hs-CRP concentration and probably insulin resistance in patients with severe burns. Omega-3 FAs had additional effects on modulating inflammation. Larger clinical trials are needed to confirm the current findings especially in terms of glucose homeostasis.

Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

Ferulago angulata extract improves the quality of buck spermatozoa post-thaw and counteracts the harmful effects of diazinon and lead.

During cryopreservation, spermatozoa are exposed to chemical or physical stress that has adverse effects on the quality of mammalian spermatozoa. Recently, much attention has been paid to environmental contaminants (ECs) in livestock, because of their detrimental effects on livestock productivity and fertility. ECs like diazinon (DZN) and lead acetate (LA) are considered ubiquitous and induced oxidative stress, which decreases spermatozoa quality. Since Ferulago angulata extract (FAE) has antioxidant properties, the present study investigated the effect of FAE supplementation in a freezing extender, in the presence or absence of DZN + LA, during cryopreservation, on the quality and fertility ability of buck spermatozoa after thawing. Pooled ejaculates were diluted with a freezing extender and supplemented with FAE (0.002%, w/v) in the presence or absence of DZN (100 µM) + LA (12.5 µM). Post-thaw spermatozoa parameters, ROS production, fertilization ability, and developmental competence of oocytes inseminated with FAE/DZN + LA treated spermatozoa were calculated. The results demonstrated that FAE improves cryopreserved spermatozoa motility, viability, membrane integrity, fertilizability, and developmental competence, and reduced spermatozoa ROS production in the presence or absence of DZN + LA. Besides, FAE significantly restored the adverse effects of DZN + LA exposure during cryopreservation on inner cell mass (ICM) count, trophectoderm (TE) cell count, total cell number (TCN), and the ratio between ICM to TCN. In conclusion, FAE on its own resulted in an improvement in the buck spermatozoa's quality and fertility. Therefore, the addition of FAE, as a natural antioxidant to buck semen extender, can increase spermatozoa cryotolerance and post-thaw resistance even when exposed to ECs.

Effect of rosiglitazone on developmental competence of mouse embryos treated with lipopolysaccharide.

Lipopolysaccharide (LPS) significantly reduces pre- and post-implantation developmental competence of embryos. One of the reason of this effect could be a consequence of TLR4-mediated inflammation. In this study, we assessed the anti-inflammatory effect of peroxisome proliferator activated receptor γ (PPAR γ) agonist, rosiglitazone (RGZ), in LPS-treated mouse embryos. Initially, the optimal doses of LPS, RGZ and GW9662 (a potent and selective PPARγ antagonist) were determined by treating the mouse zygotes up to blastocyst stage and assessment of compaction and blastocyst rates. Quantitative PCR was used to assess the mRNA expression of inflammatory cytokines. Immunostaining was used to study the translocation of PPARγ in blastocysts. Finally, the blastocysts were transferred to surrogate mouse to determine the post-implantation developmental competence. 0.0625 mg/mL of LPS significantly reduced the developmental competency by around 50% compared to control group. 10 µM of RGZ significantly ameliorated the toxic effect of LPS, which was also significantly reversed by 1.25 µM GW9662. Through immunostaining, it was shown that LPS could prevent the translocation of PPARγ to nucleus; and translocation was facilitated by RGZ and this effect was reversed by GW9662. A similar effect was also observed for the mRNA expression of inflammatory cytokines (Il-1ß and Il-6). LPS significantly increased the expression of these cytokines, while RGZ significantly reduced their expression, which was also significantly reversed by GW9662. It was also shown that embryos exposed to LPS had significantly reduced post implantation developmental competence which was considerably improved by treatment with RGZ. In conclusion, these data may have clinical implications for ameliorating the adverse effects of LPS in dairy farming and infertility treatment.

siRNA inhibition and not chemical inhibition of Suv39h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos.

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.

Impact of vitamin D deficiency on mouse sperm structure and function.

BACKGROUND: In rodents and humans, vitamin D deficiency (VDD) is associated with altered sperm structure and function (primarily decreased motility and morphological abnormalities) that are primarily attributed to VDD-induced hypocalcemia. However, it is suspected that VDD has much more drastic effects on mammalian spermatozoa. OBJECTIVES: The purpose of this study was to illustrate that VDD, depending on its severity and duration, can alter sperm nuclear integrity and can also lead to the loss of spermatozoa's ability to support embryonic development. MATERIALS AND METHODS: A mouse model of induced VDD combining the action of a vitamin D-deficient diet, UV exposure limitation, and paricalcitol injections; a vitamin D2 analog that catabolizes endogenous vitamin D by increasing the expression of CYP24A, a member of the cytochrome P450 family, has been used to create different grades of VDD. RESULTS: We show that the most significant sperm defect recorded concerns the integrity of the paternal nucleus, which is both decondensed and fragmented in moderate-to-severe VDD situations. Consistent with the known consequences of fertilization with DNA-damaged spermatozoa, we show that paternal VDD decreases the ability of spermatozoa to optimally support fertilization and embryonic development. DISCUSSION AND CONCLUSION: Given the worldwide high prevalence of VDD in humans, and although obtained in an animal model, the data presented here suggest that subfertile/infertile males may benefit from VDD testing and that attempts to correct serum vitamin D levels could be considered prior to conception, either naturally or through ART.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development.

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8-16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

Comparison of Sperm Telomere Length between Two Sperm Selection Procedures: Density Gradient Centrifugation and Zeta Potential.

BACKGROUND: Telomeres are particular sequences of DNA located at the end of the eukaryotic chromosomes that are essential for genome integrity. Telomere length in spermatozoa differs among males, as well as spermatozoa. Also, decreased telomere length in spermatozoa of infertile men is associated with the reduction of fertility potential and embryo quality. Density gradient centrifugation (DGC) and swim-up are useful techniques for separation of spermatozoa with longer telomeres. Also, the selection of sperm based on surface negative electric charge or "Zeta potential", can separate high percentage of spermatozoa with intact chromatin compared to DGC alone, and also the combination of DGC-Zeta can improve clinical outcomes of infertile men candidate for intracytoplasmic sperm injection (ICSI). Therefore, we compared sperm telomere length and DNA fragmentation between two sperm preparation procedures, namely DGC and zeta potential. MATERIALS AND METHODS: In this experimental study, we assessed sperm telomere length and DNA fragmentation by quantitative real-time polymerase chain reaction (PCR) and TUNEL assay methods, respectively. The spermatozoa were obtained from infertile men with normozoospermia between September 2017 and December 2017 and prepared either by DGC or zeta potential methods. Sperm telomere length was expressed as relative and absolute units. RESULTS: Compared with washed semen samples or control, no significant (P>0.05) difference was observed in the mean relative or absolute sperm telomere length when the two methods DGC or zeta potential were compared. However, the mean percentage of DNA fragmentation was significantly (P<0.05) lower in spermatozoa prepared by DGC or zeta potential methods than spermatozoa obtained from control samples. CONCLUSION: This is the first study that compared the effect of DGC and zeta potential as the sperm preparation methods on sperm telomere length. It seems that both methods can select sperm population with high DNA integrity and the same sperm telomeres length.

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.

The attenuation of the toxic effects of LPS on mouse pre-implantation development by alpha-lipoic acid.

The present study was conducted to evaluate the effects of alpha-lipoic acid (ALA) on quantitative and qualitative indices of mouse embryos challenged by lipopolysaccharide (LPS). Having determined the effective concentrations of LPS (1 mg/mL) that could reduce blastocyst formation rate by around 50% and the optimal concentration of ALA (10 µM) that could attenuate the toxic effects of LPS on blastocyst formation, the following indices were defined: inner cell mass and trophectoderm cell numbers, blastocyst mitochondrial distribution, ROS and GSH levels, as well as the relative expression of Tlr-4. Nrf-2 and Tnf-RI/P-60 receptor involved in inflammatory pathways. Finally, embryos derived from the experimental and control groups were transferred to synchronized recipients and their implantation rate and post-implantation capacity were determined. Treatment with LPS resulted in an increase in intracellular ROS level (P ≤ 0.05), and remarkable decreases (P ≤ 0.05) in intracellular GSH content, mitochondrial mass, and blastocyst quality. ALA attenuated all the aforementioned negative effects of LPS. The relative expression levels of Nrf-2 and Tnf-RI/P-60 receptor (P ≤ 0.05) significantly increased in response to LPS, and treatment with ALA significantly reduced the relative expression of Tnf-RI/P-60. ALA also improved the post-implantation developmental capacity of embryos treated with LPS. In conclusion, our findings indicate that the reproductive toxicity of LPS could be overcome by ALA treatment. These effects were mainly due to the improvements made in intracellular antioxidant capacity as well as suppression of some inflammatory elements, especially the main receptor of TNF-α, the Tnf-RI/P-60, involved in induction of apoptosis. These observations have important implications for dairy farming and treatment of infertility.