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Background for the ph.d.-project

The handling of pigs prior to slaughter is critical for obtaining good pork quality. It is recognized that stress exposure during this process is a key factor causing huge variations in meat quality, which cause economic loss for the meat industry. An understanding of how the muscle cells respond to stress exposure before slaughter as well as the post mortem conditions would be extremely helpful for the overall understanding of how these conditions affect the meat quality.

Using primary porcine muscle cell cultures, the present study aims at investigating the cellular responses to stress exposure. In this model system it is possible to manipulate the involved factors such as temperature, hormones, and oxygen and energy supply under strictly controlled conditions. Thus, it is possible to study these mechanisms comprehensively.

Hypothesis

The stressors in connection with slaughter are hypothesised to trigger:

1. Cellular acidification.

2. Perturbations in intra-and extra-cellular ion localisation (Ca2+, Na+).

3. Changes in mitochondrial activity.

4. Expression of specific heat shock proteins.

5. Influence cellular energy metabolism.

Two techniques will primarily be used to investigate the cellular processes; confocal microscopy and nuclear magnetic resonance (NMR)-based metabolic profiling. CLSM provides information on the presence and localisation of specific components in the cell and the biophysical microenvironment, and NMR spectroscopy provides information on the concentration of significant metabolites in the cell.

Combining the two techniques gives a unique opportunity in the direction of “whole systems biology”.

Materials and methods

In this ph.d.-project confocal microscopy and NMR-based metabolic profiling will be used to investigate the influence of stress on primary muscle cell cultures isolated from pigs.

The following stress factors will be investigated:

- H2O2 stress

- Hormonal stress (adrenaline, cortisol)

- Temperature stress (heat, cold)

- Oxygen deprivation

- Glucose deprivation

By use of confocal microscopy the cellular response to these stressors will be analysed:

- Cellular acidification as a course of stress can be quantitatively measured by use of dyes that are pH-sensitive within the physiological ranges.

- Perturbations in intra-and extra-cellular ion localisation (Ca2+, Na+) will be determined using confocal microscopy together with two calcium-sensitive dyes.

- Changes in mitochondrial activity can be measured by use of dyes that selectively label mitochondria and dyes that measure mitochondrial transmembrane potential.

- Expression of specific heat shock proteins such as HSP70, HSP27, and HO1 will be investigated by use of specific antibodies.

Confocal microscopy is combined with NMR-based metabolic profiling to investigate the effect of the different stressors on the cellular energy metabolism.