Cd hydrolyzed

Commercially available culture media were evaluated for B. bovis in vitro growth, including VP-SFM AGT™ (VP-SFM) (GIBCO^®, Grand Island, NE, USA), CD-CHO (GIBCO^®), CD-Hydrolyzed (Sigma-Aldrich), CD-CHO (Sigma-Aldrich), Hybrigro SF Medium (SFM) (CORNING^® Inc., Kansas city, KS, USA), and Advanced-DMEM/F12 (ADMEM/F12) (GIBCO^®). Each culture medium was buffered with 25 mM 2-[(2-hydroxy-1, 1-bis(hydroxymethyl) ethyl) amino] ethane sulfonic, N-[Tris (hydroxymethyl) methyl]-2-aminoethanosulphonic (TES) (Sigma-Aldrich), and 25 mM L-glutamine (GIBCO^®) was added. A mixture of antioxidants was added to all culture media as described above and supplemented with a commercially available chemically defined lipid concentrate (CD-lipid mixture, GIBCO^®). The CD lipid mixture contained 2 μg/mL arachidonic and 10 μg/mL each of linoleic, linolenic, myristic, oleic, palmitic, and stearic acids diluted in the media at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1000. The media were adjusted to pH 6.8 and filter sterilized through a 0.22-µm pore size membrane (Millipore St. Louis, MO, USA).

Four culture media free of animal components were used in this study, including VP-SFM (Gibco^®), CD-CHO (Gibco^®), CD-Hydrolyzed (Sigma-Aldrich, St. Louis, MO, USA), and CD-CHO (Sigma-Aldrich St. Louis, MO, USA). The Advanced-DMEM/F12 (Gibco^®) that contains animal components was used as the control for B. bigemina growth (Table 1).
All culture media were buffered with 25 mM 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino] ethane sulfonic, N-[Tris(hydroxymethyl)methyl]-2-aminoethanosulphonic (TES) (Sigma-Aldrich, St. Louis, MO, USA). An antioxidant mixture was added to 2 mM of L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) (GIBCO^®). The pH was adjusted (6.8) for all culture media and sterilized by filtration with a 0.22 µm membrane (Millipore). All media were supplemented with different concentrations of CD-Lipid (CORNING^®) (Table 2).