Allogenic adipose derived mesenchymal stem cells are effective than antibiotics in treating endometritis.

Endometritis is a uterine inflammatory disease that causes reduced livestock fertility, milk production and lifespan leading to significant economic losses to the dairy industry. Mesenchymal stem cells (MSC) may act as an alternative for inefficacy of antibiotics and rising antibiotic resistance in endometritis. The present study aimed to cure the chronic endometritic buffaloes using allogenic adipose-derived MSCs (AD-MSC). AD-MSCs were isolated from buffalo adipose tissue and characterized by multilineage differentiation as well as MSC-specific markers. The in vivo safety and efficacy were assessed after infusion of AD-MSCs. In safety trial, cells were administered in healthy buffaloes via different routes (IV and IC) followed by examination of clinical and hematological parameters. In efficacy study, AD-MSCs treatments (IV and IC) and antibiotic therapy (ABT) in endometritic buffaloes were comparatively evaluated. AD-MSCs did not induced any immunological reaction in treated buffaloes. PMN count, CRP levels and VDS were significantly (p ≤ 0.05) reduced after AD-MSCs infusions in IV and IC groups and no significant difference was observed in antibiotic group. The IV group was marked with 50% absolute risk reduction in endometritis and 50% live calf births after artificial insemination in comparison with ABT group. Anti-inflammatory cytokines (IL4 and IL10) and anti-microbial peptides (PI3, CATHL4, LCN2 and CST3) expressions were significantly (p ≤ 0.05) upregulated in IV group. The calf delivery rate after the treatments in IV group was higher (50%, 3 calves) than the other groups (IC: 33.3%, 2 calves; ABT: 16.6%, 1 calf). In conclusion, the administration of AD-MSCs through IV route was found to be safe and efficacious for alleviating chronic endometritis in dairy buffaloes.

Stage specific gene expression of folate mediated one-carbon metabolism enzymes and transporters in buffalo oocytes and pre-implantation embryos.

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies.

Allogenic umbilical cord blood-mesenchymal stem cells are more effective than antibiotics in alleviating subclinical mastitis in dairy cows.

Subclinical mastitis is an inflammatory disease that affects the milk production, fertility, and lifespan of animals, leading to significant losses to dairy industry. Antibiotics therapies are resulting in suboptimal benefits in treating subclinical mastitis due to prevalent antibiotic resistance in dairy herds. In a quest to develop alternative therapy, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) and its extracellular vesicles (UCB-MSC-EVs) are used, in the present study, to validate its safety and efficacy as potential therapy for treatment of subclinical mastitis in dairy cows with respect to conventional antibiotic therapy (ABT). We isolated, in vitro cultured, and characterized the UCB-MSCs as well as UCB-MSC-EVs. The repeated infusions of low dose MSCs and EVs were delivered in healthy animals for safety analysis, followed by the same administrations in infected animals for therapeutic efficacy analysis. UCB-MSCs and UCB-MSC-EVs were found to be safe at 2 doses with 7-day gap of 5 × 107 cells/injection and EV equivalent to 500 µg protein in DPBS, respectively. Efficacy trials demonstrated significantly decreased somatic cell count to safe levels in milk samples of UCB-MSCs and UCB-MSC-EVs treated groups compared to antibiotic group. The leucocytes expression of anti-inflammatory cytokines, anti-microbial peptides, and angiogenic genes were significantly upregulated in UCB-MSCs and UCB-MSC-EVs treated groups as compared to antibiotic therapy group. Antibiotic therapy and UCB-MSC-EV groups failed to significantly decrease the gene expression of pro-inflammatory cytokine. In conclusion, the administration of UCB-MSCs and UCB-MSC-EVs through intravenous and local routes was found to be safe and efficacious for alleviating subclinical mastitis in dairy cows.

Production of biologically active recombinant buffalo leukemia inhibitory factor (BuLIF) in Escherichia Coli.

BACKGROUND: Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). RESULTS: The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. CONCLUSIONS: The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos.

Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. Although the oocytes contain endogenous pool of folates for development, the present study investigated the effect of external 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 improves oocyte maturation, the blastocyst production rate, reduces ROS production as well as upregulate the expression of FOLR1 and folate metabolism enzyme, MTR.

Folate receptor-1 is vital for developmental competence of goat embryos.

Folate is essential for DNA synthesis and methylation via one-carbon (C1) metabolism during embryonic development. It is transported into the developing oocytes via folate receptors (FOLR1 and FOLR2) and transporters (RFC1) for utilization during embryo development. However, the role of folate receptors during pre-implantation stages of embryos is not well known. Thus, the present study aimed to investigate the expression of folate transport genes and proteins in mature oocytes and pre-implantation embryos and the effect of FOLR1 knockdown in zygotes on blastocyst outcome. For this, immature goat oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard conditions. A group of zygotes was transfected with esiRNA against FOLR1 and in vitro cultured for blastocyst outcome assessment. The transcripts and proteins for FOLR1, FOLR2 and RFC1 were present in oocytes as well as all the stages of pre-implantation embryos. Immunofluorescence revealed the presence of FOLR1 in the nuclei of embryos but not in the metaphase (matured) oocytes. The knockdown of FOLR1 in embryos was effective and significantly reduced the blastocyst production rate. The present study demonstrates the existence of active folate transport in oocytes and pre-implantation goat embryos. FOLR1 is vital for pre-implantation embryo development and may aid in the progression by functioning as a transcription factor.