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Mastitis and metritis are two inflammatory diseases with high economic impact on dairy industry and farmers are losing revenues. Antibiotic treatment for these disease conditions typically achieve suboptimal outcomes. Moreover, emerging antibiotic resistance in mastitis and metritis worldwide has a severe effect on productivity, fertility, and lifespan of animals. A paradigm shift in treatment strategies is much needed for these animals. Attracting properties of mesenchymal stem cells may act as an alternative strategy for treating these diseases. In addition to their ability to differentiate into multiple types of cells, mesenchymal stem cells can orchestrate immune responses and modulate tissue microenvironments. In the present study, we evaluated the safety and efficacy of adipose tissue–derived mesenchymal stem cells (AT-MSC) for mastitis and metritis. A well-characterised adipose-derived stem cell line with 3–5 passages cells were used for treatment for these diseases. In a safety trial, mastitis- and metritis-infected cattle were administered with 1×106 cells of AT-MSC through different routes (local, IV, IV+local). Animals were clinically evaluated during 15 days of the experimental period and blood samples were collected for hemogram determination. The efficacy was checked under both invivo and invitro conditions. Invitro efficiency of AT-MSC was determined using a transwell plate experiment, in which AT-MSC were co-cultured with bacteria (Escherichia coli and Staphylococcus). Invivo efficiency was evaluated by administration of in 2mL (1×106 cells) of AT-MSC dose; then, somatic cell count (SCC) in milk of mastitic cattle and polymorphonuclear (PMN) cell count in cervical vaginal fluid (CVF) of metritic cattle were determined. Gene expression profiling of antimicrobial (cathelicidin, lipocalin, cystatin) and anti-inflammatory (IL-4, IL-6, IL-10) peptides were quantified in all groups: IV, local, IV+local, and control ATB (antibiotic). Allogenic AT-MSC did not induce any immunological rejection response in treated animals. A significant reduction of bacteria in CVF invitro when co-cultured with AT-MSC was observed. The PMN count was significantly reduced in CVF of the IV+local group. The SCC in milk of mastitic animals was decreased in the local (intramammary) group. Maximum expression of IL-6, IL-10, cathelicidin, lipocalin, and cystatin and angiopoietin genes were observed on Day 3 followed by Day 7 in the local group in case of mastitis. In metritis, expression of IL-6, IL-10, cathelicidin, lipocalin, cystatin, and angiopoietin were observed at Day 3 in the IV+local group. All mastitic and metritic cattle were completely and permanently cured within 30 days after treatment with mesenchymal stem cells. In conclusion, we provide initial evidence for the use of allogenic AT-MSC and their safety and efficacy to treat mastitis and metritis in dairy cattle.

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 (vitamin B9) is the principal component of one-carbon (C1) metabolism in mammalian cells. It acts as a coenzyme in C1 unit transfer during nucleotide and amino acid metabolism. Therefore, it is unquestionably essential for the formation of thymidylate (TMP) for DNA synthesis and methylation, which is in turn crucial for post-implantation embryonic brain and nerve chord development. However, the role of folic acid in maturation of oocytes and subsequent blastocyst production is largely ambiguous in different mammalian species. The aim of this study was to investigate the effect of folic acid supplementation on expression of oocyte maturation markers and folate cycle enzymes in cumulus cells in addition to blastocyst development rate in goat. Immature oocytes were isolated from ovaries, and 1600 oocytes were matured in maturation medium (medium-199) supplemented with 0 (control), 50, 100, or 150 µM folic acid (Sigma Aldrich, India), comprising total 400 oocytes in each group in 5 replicates. The oocytes were in vitro fertilized and embryos were cultured for 7 days. Blastocyst rate was calculated in all 4 groups. Cumulus cells from matured oocytes from each group were analysed for the differential expression of oocyte maturation markers: PTX3, PTGS2, CTSB, and CTSS, and folate cycle enzyme transcripts MTR, MAT2A, ACHY, DHFR, and SLC19A1, by qRT-PCR. For statistical analysis, one-way ANOVA was used. The results of present study show that blastocyst rate was significantly higher (P ≤ 0.05) at 50 µM folic acid (21.4 ± 1.2%) compared with control (13.6 ± 0.4%), 100 µM (10.4 ± 0.34%), and 150 µM (7.4 ± 0.54%). However, there was no effect on the number of cleaved embryos among control (72 ± 0.21%), 50 µM (70.6 ± 0.58%), 100 µM (73.75 ± 0.31%), and 150 µM (73.02 ± 0.04%). The expression of the oocyte maturation markers PTX3 and PTGS2 was up-regulated, whereas that of CTSS and CTSSB was down-regulated (P ≤ 0.05), in the 50 µM group, but did not change (P ≤ 0.05) among the other groups. The expression of these genes did not change significantly among the other groups. The expression of genes of the folate cycle, MTR, MAT2A, ACHY, and DHFR, increased (P ≤ 0.05) by 5.7-, 3.6-, 2.2-, and 1.7-fold, respectively, at 50 µM, but did not change (P ≤ 0.05) among the other groups. The expression of SLC19A1 did not change significantly among all the groups. The results of present study show that the supplementation of 50 µM folic acid to the maturation medium results in increased maturation of oocytes, making them more competent for the blastocyst development. Up-regulation of transcripts of folate cycle enzymes at 50 µM might explain the increase in the blastocyst production rate, which further needs to be validated.