3.2. Animal Feeding Behaviour
For a second set of experiments, rumen tissue samples were compared for adult male deer with differences in the extent of their food begging behaviour (i.e. likelihood of begging food from human visitors in Phoenix Park). Animals were separated into two groups consisting of either consistent beggars (N = 4) or rare beggars (N = 7) – with respective feeding scores of +1.64 ± 0.23 and -1.88 ± 0.30 respectively (p < 0.001, N=4-7, Unpaired T-Test). Initial comparisons were made of the papillae physical measurements of tissues from both groups -see Table 2 for details. On this occasion, these data showed that there were no significant differences in either papillae length or papillae width. In contrast, the expected significant increase in papillae density in the consistent beggars was observed.
Following on from this, western blotting experiments were performed using 3 male adults from both groups. In addition, samples from two other consistent acceptor animals, one a juvenile male and one a female adult, were included in the experiments as controls – see Figure 2 . Since there was some small differences in the amounts of protein obtained for each sample, western blot signals for UT-B2 and MCT1 were evaluated as a ratio to the amount of protein. In this case, the 50 kDa UT-B2/protein signal ratio was significantly higher in consistent beggars (0.56 ± 0.10, N = 3) than it was in the rare beggars (0.13 ± 0.04, N = 3) (p = 0.0183, Unpaired T-test). Interestingly, the increased level of UT-B2 abundance in the male adult consistent beggars was similar to that observed in the other two beggars - one male juvenile and one adult female. In contrast, there was no significant difference in the 43 kDa MCT1/protein signal, which was 0.70 ± 0.11 to 0.46 ± 0.07 respectively (p = 0.1364, N=3, Unpaired T-test). Additionally, although variable, there was also no significant difference in the ages of the groups (p = 0.1380, Unpaired T-test), with consistent beggars being 8.3 ± 1.9 years (N = 4) and rare beggars being 5.7 ± 0.6 years (N = 7).
In order to identify where these transporter proteins were located and confirm their abundance, a series of immunolocalization experiments were performed. Initial studies showed that both MCT1 and UT-B2 proteins were predominantly located in the stratum basale layer of consistent acceptor male adult rumen – see Figure 3 . Further experiments were then performed with tissue sections taken from all 8 animals that had been used in the western blotting analysis. For MCT1, moderate to strong staining was observed in rare beggar adult males (M1-M3), whilst strong to very strong staining was observed in the consistent beggar adult males (M4-M6) – see Figure 4 . There was also strong staining observed in the juvenile male (J1) and adult female (F1). In all cases, the MCT1 staining was mainly found in the stratum basale layer. Finally, for UT-B2 there was very little staining in rare beggar adult males (M1-M3), in contrast to the weak-to-moderate staining in consistent beggar adult males (M4-M6) – see Figure 5 . In particular, strong staining was observed in the M6 sections. Similarly, stronger UT-B2 staining was observed in the juvenile male (J1) and adult female (F1) sections. In each case, the UT-B2 staining was located predominantly in the stratum basale layer.