Hematopoietic stem cells (HSCs) are the self‐renewing, multipotent progenitors that give rise to all types of mature blood cells. The hallmark properties of HSCs are the ability to balance self‐renewal versus differentiation cell fate decisions to provide sufficient primitive cells to sustain haematopoiesis, while generating more mature cells with specialized capacities. In the present experiment, we optimized the techniques for isolation and identification of hematopoietic stem cells from cow peripheral blood. The objective of this study was to optimize the more accurate methodology for separation of mononuclear cells (MNCs) from peripheral blood and identification of HSCs by using a specific cell surface marker i.e. CD34. A total 10 peripheral blood samples were collected from Holstein dairy cows from jugular vein. We used Ficoll 400 in different concentrations from 1 to 12% and Ficollpaque Plus (1.077 g/ml) at different centrifugation speed and time. After Giemsa staining, we found more than 98% recovery of monocytes with Ficollpaque Plus (1.077 g/ml). It was demonstrated that Ficollpaque Plus (1.077 g/ml) and centrifugation at 400xg for 30 min is the best method for separation of MNCs from bovine peripheral blood. Separated MNCs were immediately subjected for magnetic activated cell sorting (MACS) by using CD34 microbead kit. HSCs (CD34+ cells) recovery was 0.307% of peripheral blood. Peripheral blood MNCs and CD34+ cells were morphologically characterized by Giemsa staining. CD34+ cells were also confirmed by immunochemistry using FITC conjugated CD34 antibodies. HSCs were also confirmed by progenitor assay including burst forming unit‐erythroid (BFU‐E), colony forming cells‐ granulocyte (CFC‐G), colony forming cells‐ macrophage (CFC‐M), colony forming cells‐ granulocyte macrophage (CFU‐GM) and colony forming cells‐ granulocyte erythroid macrophage monocyte (CFCGEMM) on Methocult 4435.

The isolation of preantral follicles from the ovaries of bovine was performed under mechanical and enzymatic methods. A significant increase in the total number of follicles retrieved was detected when tissue chopper was used. Micro-dissection could supply good quality, larger sized follicles (400 to ) but with the lowest yield (). The isolated preantral and early antral follicles were cultured for 14 days. Follicles isolated by the mechanical method had a greater growth during a culture period than follicles collected enzymatically. Morphologically normal bovine oocytes from early antral follicles after 14 days culture were 59.6% after culture and after 24 h of maturation culture, 12.9% of in vitro-grown oocytes reached the second metaphase. In conclusion, this study showed that mechanical method can be used effectively to isolate intact preantral follicles from bovine ovaries.

Bovine mammalian gland has biopotential in therapeutic protein production and could be used as a model in further lactation researches. In this study, we have isolated bovine mammary gland-derived epithelial cells (BMECs) from Korean Holstein dairy cattle and themselves show differential dynamic ability in in-vitro culture. BMECs enables to form lobulo-alveolar structure, express milk production related gene. Functional studies indicated that BMECs secret exogeneous antibacterial fragment-Bovine Lactoferricin B (bLfcin-B), which is inserted in PiggyBac system, and this bioactive fragment inhibits the growths of Escherichia coli and Staphylococcus aureus. These data demonstrated that BMECs open new scope in either bioactive fragment, heading to prevent the spread of mastitis or post-mastitis damage in dairy graze and could be an ideal bioreactor for antibacterial proteins.