Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. The present study investigated the effect of folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat.Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 µM), were in vitro fertilized and cultured. Cumulus expansion markers (Ptx3 and Ptgs2) in cumulus cells were highly upregulated after 50 µM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 µM folic acid during oocyte maturation resulted in signi cantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 µM folic acid in maturation medium increases the blastocyst production rate, improves oocyte maturation, reduces ROS production as well as upregulate the expression of Folr1 and folate metabolism enzyme, Mtr.
Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. The present study investigated the effect of folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 µM), were in vitro fertilized and cultured. Cumulus expansion markers (Ptx3 and Ptgs2) in cumulus cells were highly upregulated after 50 µM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 µM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 µM folic acid in maturation medium increases the blastocyst production rate, improves oocyte maturation, reduces ROS production as well as upregulate the expression of Folr1 and folate metabolism enzyme, Mtr.
The present study was carried out to isolate mesenchymal stem cells (MSCs) from adipose tissue of cattle (Bos indicus), characterise them, and apply them for the treatment of mastitis and metritis in the cow. Cattle MSCs were isolated from adipose tissue near the loin region of cow. Isolated adipose tissue was subjected to enzymatic digestion using 2% collagenase with agitation at regular intervals. The cells obtained after digestion were resuspended in cell culture flasks containing growth enriched medium and cultured under standard culture conditions. Alkaline phosphatase staining was used as one of the parameters to confirm cultured putative MSCs. Bovine Ad-MSCs were further characterised using real time-PCR by amplification of MSC-specific markers: CD73, CD90, and CD105 as positive markers and CD34, CD45, and CD79a as negative markers. Immunocytochemistry showed the presence of CD73, CD90, and CD105 on the cell surface. Three groups-control (C), local (L), and intravenous (IV)-with 6 cows suffering from mastitis were taken in each group and subjected to MSC transplantation through local and intravenous routes. Control group animals were subjected to antibiotic treatment only. Similarly, another three groups were taken with 6 cows in each group suffering from metritis. Post-transplantation wound healing, tissue repair, and reduction in inflammation were monitored for 26 days, at different time intervals; that is, after Days 1, 3, 7, and 15. Blood samples were also collected from animals at the same time intervals for real time-PCR. A similar examination was also done in metritis groups along with the analysis of the reduction in turbidity of cervical fluid at the abovementioned time intervals. Real time-PCR was performed to determine relative expression of genes for proliferative factors, anti-inflammatory cytokines, and antimicrobial peptides on cells isolated from blood collected at different time intervals. Gene expression in the local group of mastitis subjected to MSC injection was significantly higher than that of the IV and control group. The somatic cell count declined in both local and IV groups compared with the control group. Whereas the expression of the same genes in the IV group of metritis was significantly higher than that of the local and control groups of cows. The turbidity of cervical fluid and mucus was reduced in the IV group compared with the local group. In conclusion, we demonstrated the healing potential of MSCs in a cow model via MSC injection. Promising results were obtained in curing mastitis in both local and IV groups, whereas healing in the case of metritis was significantly higher in the IV group compared with both the control and local groups of cows. The study indicates the potential use of MSc for treatment of mastitis and metritis in cattle through wound healing and decreasing microbial infection.
Periconceptional folic acid is known to have a major role in the prevention of neural tube defects, leading to global recommendations for folic acid supplementation before and in early pregnancy. Maternal folate throughout pregnancy may have other roles in offspring health, including neurodevelopment and cognitive performance in childhood. Folate and folic acid (vitamin B9) act as a co-enzyme essential for single carbon metabolism, a network of pathways involved in several biological processes including nucleotide synthesis, DNA repair, and methylation reactions. In general, rapidly growing and multiplying cells require an adequate supply of folate. A primary deficiency of natural folate resulting in an increase of the total homocysteine concentration may be detrimental to the quality of the oocyte, subsequent fertilisation, embryogenesis, implantation, and fetal development. However, to date, folate-methionine metabolism and folate transport have not been studied in developing buffalo embryos. The present study details transcript expression for genes encoding key enzymes in the linked folate-methionine cycles in the ovary tissue, cumulus cells, immature oocytes, IVM oocytes, and pre-implantation embryos and also estimates the folate concentration in follicular fluid (FF) of buffalo. The FF was pooled and collected by aspiration of different sizes of surface follicles (2-8mm diameter). The total number of analysed samples was three, with different dilutions and estimation of folate in FF of buffalo done by chemiluminescence assay. Total RNA was extracted from oocytes, cumulus cells, ovarian tissue, and embryos produced from IVF. RT-PCR was performed to analyse the expression of folate-methionine cycle enzymes and folate transporters. Transcripts for all the enzymes of the folate-methionine cycle (i.e. SHMT, MTR, MTRR, MAT1A, MAT2B, GNMT, AHCY, CBS, and DHFR) and folate transporters (FOLR1, FOLR2) and reduced folate carrier (SLC19A1) were expressed in ovarian tissue, cumulus cells, oocytes, and pre-implantation embryos. Immunocytochemical analysis revealed FOLR2 and SLC19A1 protein expression on the plasma membrane and/or cytoplasm of the oocytes and embryos, and FOLR1 in the nucleus of pre-implantation embryos. The folate concentration in FF was 24ngmL−1. This is the first report to examine the concentration of folate in FF and revealed the identification of transcripts in different samples of buffalo species. The presence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo, therefore indicating that folate from FF is being disseminated through folate receptors within oocytes and embryos to participate in the folate pathway. This study advocates the necessity for examination of the result of folate supplementation throughout invitro embryo production for improving the quality and quantity of transferable blastocysts and subsequently live calf births in buffalo.
During past few years, studies in the field of nanomaterial sciences were more focused on nanocomposites due to their extraordinary and unexplored interesting properties. Our prime focus in this review is to address recent advancements in their manufacturing, processing, characterization of their functional attributes and also to explore the thrust areas of their utilization. In this way, we objectively explored and analyzed different areas such as aerospace, electric and manufacturing, defense and biomedical by addressing the strength and uses of nanocomposites respectively. Along with this, we also explored the area of biomedical sciences specifically their application in tissue engineering using zirconium oxide nanoceramic modified chitosan as an exemplary nanocomposite. Conclusively, this review will provide critical issues in nanocomposites research with promising functional utility to a greater extent.
Objective: To understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study Method(s): Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization Intervention: None Main Outcome Measure(s): Reproductive outcomes, effect on gametes and embryos Result(s): Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 microg/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant (P<0001) increase in blastocyst production at a concentration of 50 microg/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to developing a potential fertility factor in reproduction. Conclusion(s): The purified rec-BSP1 may affect on male fertility and mediated its effect on in vitro embryo production
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