Slinger, Kimberley R.
(2020)
Optimisation and application of a method designed to assess host DNA in pig and poultry faeces as a potential marker of gut cell loss.
PhD thesis, University of Nottingham.
Abstract
Maintaining and replenishing the intestinal barrier is an energy demanding process. In farmed species the excessive energy utilised by this process could be better utilised in the production of proteins for lean muscle deposition, milk synthesis or egg production. Altering the diets of farmed animals can improve how efficiently feed energy is converted into useful product. Therefore, it would be useful to examine how any nutritional changes impact upon gut function, particularly gut cell turnover which utilises such a significant proportion of dietary energy. Currently all methods to assess gut turnover in farmed species are invasive to some extent and typically very expensive, therefore, there is scope to develop a novel non-invasive method that could potentially be used routinely in a farm setting. Sloughed epithelial cells are broken down in the lumen of the intestine by endonucleases, presumably leading to fragmented host DNA being present in the faeces. This thesis developed methodologies for analysing faecal host DNA from pig and chicken faeces and the developed protocols were then applied to feeding studies where differences in gut cell sloughing were expected.
Different faecal DNA extraction protocols were developed for the pig and chicken faeces. Pig faecal DNA was extracted using a modified phenol-chloroform based method. Chicken faecal DNA was extracted using a protocol that employed bead-beating with MagNA Lyser Green Beads (Roche) prior to using the QIAamp® DNA Stool Mini Kit (Qiagen). However, the same species-specific gene target was successfully used to amplify host DNA from the extracted faeces, which was cytochrome b (CYTB) from the mitochondrial genome. For each species, the appropriate developed methodology was applied to 2 feeding studies where faecal samples had been collected.
The first pig feeding study compared a control diet with a xylanase inclusion diet and found that the level of pig CYTB DNA present in the faeces of xylanase supplemented pigs was significantly (P=0.039) lower than the levels from control fed pigs. In the second pig feeding study, pigs were either given a control diet or a treatment diet which was supplemented with yeast-enriched protein concentrate (YPC) at 1 of 3 possible inclusion levels (2.5%, 5.0% or 7.5%). Pigs fed YPC had significantly (P=0.008) reduced pig CYTB DNA in their faeces, as well as significantly improved feed conversion ratio, indicating that the YPC may have reduced gut cell losses which impacts upon pig performance. Equally in the YPC feeding study, faecal calprotectin, a marker of intestinal inflammation, was measured and this was found to only be affected by the age of the animal rather than the diet it received. Importantly, faecal pig CYTB DNA content positively correlated with feed conversion ratio, while faecal calprotectin did not. These results further illustrated that measuring host DNA content in faeces is measuring a completely different process to inflammation and may therefore be a measure of gut cell losses as anticipated.
In the first chicken feeding study, faeces were collected from control and treated birds from a phytase supplementation study. Birds were given a diet supplemented with 2500FTU of phytase, which was expected to decrease gut cell sloughing. However, there was no effect (P=0.493) of diet on the levels of chicken CYTB DNA present in the faeces. In a second feeding study, chickens were given either a control diet or a 20% wheat bran diet, which was expected to increase gut cell sloughing. Like the phytase study, there was no effect of a 20% wheat bran inclusion diet on chicken CYTB DNA levels in the faeces. Digesta from the duodenum, jejunum, ileum and colon were also collected on day of cull from birds receiving each diet. The levels of chicken CYTB DNA were highest at the duodenum and significantly (P<0.001) decreased down the gut, indicating that DNA is degraded down the gastrointestinal tract, or being diluted with other DNA sources, such as microbial DNA, as expected. In addition, the jejunum appeared to be the region where a high fibre diet had the greatest effect on gut cell loss as indicated by higher levels of chicken CYTB DNA from digesta of the wheat bran treated birds. This was in accordance with a study by Jin et al. (1994), who observed greater levels of DNA synthesis in the jejunum of pigs fed 10% wheat straw.
Taken together the results from this thesis indicate that measuring host DNA content in faeces may be a marker of gut cell loss, but further studies are required to validate this.
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