Conclusions This paper explains the basis of the beneficial effect on meat and milk fatty acid composition of adding oils to the ruminant BTK signaling inhibitor diet. Ruminal biohydrogenation

is modified via differential toxicity to ruminal bacteria of different PUFA, including the fish oil fatty acids, EPA and DHA. If we can understand how selective fatty acid toxicity, or indeed other factors, affects the physiology of biohydrogenating bacteria in the rumen, we may be able to suggest new, rational dietary modifications that will eventually lead to ruminant products that are healthier for human consumption. Methods Bacteria and growth conditions Butyrivibrio fibrisolvens JW11 was originally isolated from sheep as a proteolytic species [21],

and is held in the culture collection maintained at the Rowett Institute. All transfers and incubations were carried out under O2-free CO2 and at 39°C in Hungate-type tubes [43]. Inoculum volumes were 5% (v/v) of a fresh culture. The media used in these experiments were the liquid form of M2 selleck screening library medium [44]. Fatty acids were prepared as a separate solution, sonicated for 4 min in water and added to the medium before autoclaving. Growth of bacteria was measured SB202190 from the increase in optical density (OD) at 650 nm of the control tubes, in triplicate, using a Novaspec II spectrophotometer (Amersham Biosciences, UK). The influence of fatty acids and their methyl esters was determined in two kinds of experiment. In experiments where fatty acid concentrations were measured at the end-point of the growth curve, usually in stationary phase, the tubes were freeze-dried in order to enable fatty acid extraction from the whole culture. The experiment was conducted by inoculating multiple 10-ml tubes. At each sampling time, three tubes were

removed, the turbidity was determined, and the tubes were placed in a heating block at 100°C for 5 min, left to cool and frozen. One ml was taken for protein analysis and for fatty acid extraction and derivatization. Fatty acid extraction and analysis Extraction, derivatization of fatty acids and L-gulonolactone oxidase GC analysis of methyl esters were carried out using procedures described by Wąsowska et al. [11]. The products from incubations with LNA were identified by comparing elution profiles and mass spectra with those identified previously from analysis of methyl and 4,4-dimethyloxazoline (DMOX) esters [11]. Measurement of cell integrity using propidium iodide One ml of overnight culture was inoculated into 10 ml of M2 medium and incubated at 39°C until it reached mid-exponential phase (OD650 = 0.4, approx. 4 h). The bacterial cultures were centrifuged (3000 g, 10 min, 4°C) and the pellet was washed twice with anaerobic potassium phosphate buffer (100 mM; pH 7.0) containing 1 mM dithiothreitol (DTT).