158 Umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) used for the prevention of metritis in cattle

Metritis is a uterine inflammation that affects the endometrial and muscular layers of the uterus caused by bacteria that drastically reduce milk production, fertility, and costly treatment for dairy farmers. Antibiotics are used for treating metritis in cattle, but they do not regenerate the endometrial tissues, increasing antimicrobial resistance, and antibiotic residues in milk that may cause human health hazards. Mesenchymal stem cells (MSCs) as regenerative medicine and its extracellular vesicle (EV) may be an alternative treatment of metritis in cows. In the present study, we isolated, cultured, and characterized MSCs in vitro and evaluated the efficacy of MSCs for the prevention of metritis in cows. The MSCs were also differentiated into adipocytes, chondrocytes, and osteocytes. The immunological reaction safety evaluation of MSCs was carried out using fresh and cryopreserved MSCs, and EV in 2 mL of Dulbecco’s PBS was injected on Days 0 and 7 locally (as much as possible inside the vaginal region) and intravenously (IV) in each group of four healthy cows. Blood was drawn from these cows on Days 0, 1, 7, and 15 for haemogram determination, and serum was isolated from blood for immunoglobulin level determination. No significant (P > 0.05) changes were found in haematological parameters or IgA, IgG, and IgM levels in the safety trial. In the efficacy trial, a total of 40 metritic cows were selected on the basis of turbid mucopurulent uterine discharge and polymorphonuclear (PMN) cell count around 21 days after postpartum and divided in four groups, each group comprising 10 cows: antibiotic-treated (control), fresh MSC, cryopreserved MSC, and EV injected both locally and IV on Day 0 and Day 7. Blood samples were taken from each cow and pooled group-wise on Days 0, 1, 7, and 15 and total RNA was isolated for gene expression analysis. PMN cells were stained and counted from cervical vaginal fluid (CVF) of cows with metritis. The PMN cell count was reduced significantly after treatment (P < 0.05) from 13.40 ± 0.75 to 5.5 ± 0.5 in fresh-MSC, 14.10 ± 0.88 to 6.6 ± 0.58 in cryo-MSC, and 14.0 ± 0.99 to 5.2 ± 0.68 in EV-treated cows compared with the antibiotic-treated control group (16.9 ± 1.15 to 11.2 ± 1.12). All cows with metritis were observed to have clear/transparent mucus uterine discharge after the MSCs treatment. Gene expression analysis showed that there was a significant (P < 0.05) increase in anti-inflammatory cytokine IL-10, cathelicidin, cystatin, lipocalin 2, angiopoietin and decreased proinflammatory cytokine IL-6 in MSCs and EV treatment groups compared with the control group. All cows with metritis were cured within 30 days after treatment with MSCs and EV. A total of 19 cured cows were inseminated after MSC treatment and presently seven cows are diagnosed pregnant in the fresh, cryo-MSC, and EV treated groups. However, at present, none of the antibiotic-treated control group cows are pregnant. In conclusion, the present study optimised the isolation and characterisation of UCB-MSC and confirmed the safety and efficacy of UCB-MSCs for the treatment of metritis in dairy cattle; this finding may help enhance the income of the dairy farmer.