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Introduction: The period around calving forms the most vulnerable time in the life of a dairy cow, characterized by an increased prevalence of metabolic and infectious diseases. Most of these disorders originate from defective liver function due to disturbances of its glucose, fat and protein metabolism or an insufficient generation of an inflammatory response. However, the mechanisms underlying the defective liver function in dairy cows are not completely understood yet, mainly due to the inaccessibility of the bovine liver in vivo.
Methods: A part of the liver from a freshly slaughtered cow is used in a perfusion system to isolate and culture primary bovine hepatocytes for in vitro studies on the effect of systemic stimuli or drugs which specifically strain or alleviate the cellular stress response of liver cells. Before and after stimulation samples of the cells were taken, from which total RNA is isolated (RNeasy kit, QIAGEN) and transcribed into cDNA (StrataScript First-Strand Synthesis System, STRATAGENE). Different expression of Hsp70 and IL-1β, IL-6, IL-10, INF-γ and TNF-α is calculated against house-keeping genes using Real Time-PCR (Brilliant SYBR Green QPCR Master Mix, STRATAGENE).
Results: The new established liver perfusion technique delivered viable hepatocytes which survived in culture for two weeks. After being exposed to different stimuli (heat, NEFA’s, keton bodies) the hepatocyte RNA was isolated for further analysis.
Conclusion: By using the in vitro--experiments on bovine hepatocytes we expect to gain insights into the genetic and molecular basis of the cellular stress response in the bovine liver and to contribute to the reduction of animal experiments.