Technical note: stearidonic acid metabolism by mixed ruminal microorganisms in vitro.

Dietary supplementation of stearidonic acid (SDA; 18:4n-3) has been considered a possible strategy to increase n-3 unsaturated fatty acid content in ruminant products; however, little is known about its metabolism in the rumen. In vitro batch incubations were carried out with bovine ruminal digesta to investigate the metabolism of SDA and its biohydrogenation products. Incubation mixtures (4.5 mL) that contained 0 (control), 0.25, 0.50, 0.75, 1.00, 1.25, or 1.50 mg of SDA supplemented to 33 mg (DM basis) of commercial total mixed ration based on corn silage, for dairy cows, were incubated for 72 h at 39°C. The content of most fatty acids in whole freeze-dried cultures was affected by SDA supplementation. Branched-chain fatty acids decreased linearly (P < 0.01), and odd-chain fatty acids decreased quadratically (P < 0.01), particularly from 1.00 mg of SDA and above, whereas most C18 fatty acids increased linearly or quadratically (P ≤ 0.04). Stearidonic acid concentrations at 72 h of incubation were very small (<0.6% of total fatty acids and ≤0.9% of added SDA) in all treatments. The apparent biohydrogenation of SDA was extensive, but it was not affected by SDA concentration (P > 0.05). Biohydrogenation followed a pattern similar to that of other C18 unsaturated fatty acids up to 1.00 mg of SDA. Stearic acid (18:0) and vaccenic acid (18:1 trans-11) were the major fatty acids formed, with the latter increasing 9-fold in the 1.00 mg of SDA treatment. At greater inclusion rates, 18:0 and 18:1 trans isomers decreased (P ≤ 0.03), accompanied by increases in unidentified 18:3 and 18:4 isomers (P = 0.02), suggesting that the biohydrogenation pathway was inhibited. The present results clearly indicate that SDA was metabolized extensively, with numerous 18:4 and 18:3 products formed en route to further conversion to 18:2, 18:1 isomers, and 18:0.

Comparison of cultured and uncultured keratinocytes seeded into a collagen-GAG matrix for skin replacements.

A well-characterised collagen-glycosaminoglycan (CG) matrix functions as an extracellular matrix analogue (ECMA) of dermis on full-thickness wounds. The epidermis can be reconstituted by seeding autologous uncultured keratinocytes into the matrix prior to grafting. We hypothesised that seeding the CG matrix with keratinocytes cultured to sub-confluence may provide the ECMA with more proliferating keratinocytes than with uncultured keratinocytes. Autologous cells were isolated from split-thickness skin grafts and cultured to sub-confluence. ECMAs were seeded by centrifuging cultured (n = 8) or uncultured (n = 8) autologous keratinocytes into a CG matrix at a density of 100,000 cells/cm2, then applied onto full-thickness wounds on Yorkshire pigs. Gross and histologic observations were made up to 21 days post-grafting. At 14 days, a fully differentiated epidermis was present on all graft sites, but the epidermis of the cultured-cell-seeded matrices was thicker, 180 (19) microns, than the uncultured-cell-seeded matrices, 110 (18) microns. The epidermis of cultured-cell-seeded matrices was acanthotic, containing 14 (4) cell layers, as compared to uncultured-cell-seeded matrices, 9 (1) cell layers. The number of subepithelial keratinocyte cysts/cm cross-section present in the neodermis was also greater in cultured-, 1.35 (0.37), than in uncultured-cell-seeded matrices, 0.47 (0.35). Epidermal confluence on day 14 was 96 (3)% on cultured-cell-seeded grafts and 50 (17)% on uncultured-cell-seeded grafts. These results are consistent with the hypothesis that the process of in vitro cell cultivation increases the proportion of dividing cells in preference to differentiated cells. This technology may be useful in reconstruction of specialised bilayer tissues with minimal donor sites.