4. DISCUSSION
The initial part of this pilot study investigated whether the month of the cull affected the male deer rumen samples. We showed that adult males culled in January had a greater papillae length than those culled in November, although papillae width and density were unaffected. Since January was about 3 months after the rutting season, the simplest explanation for these findings is that as the males return to feeding more normally (in agreement with Apollonio & Di Vittorio 2004), the necessity grows for larger papillae to reabsorb products of digestion, such as bacterially derived SCFAs. In contrast, we found no significant changes in the protein abundance of either MCT1 or UT-B2 transporters, strongly suggesting that regulatory changes were occurring at the tissue rather than the cellular level. Regulation in papillae size and density are characteristic of winter-acclimatized wild ruminants (Arnold, 2020), so these data are as expected. Wild ruminants face more pressure on their dietary intake during the winter season than domestic animals (Kay et al., 1980). This is particularly true of male adult deer, who severely reduce their dietary intake while defending females during the October-early November rutting season (Yoccoz et al., 2002). Overall, these findings highlight the dangers in focusing solely on cellular changes when undertaking rumen physiology research.
The second, more substantial part of this study was the investigation comparing adult male deer who were either rare beggars or consistent beggars of food from humans (McLaughlin et al., 2022). Whilst there were no differences in papillae length or width, significantly higher papillae density was observed in consistent beggars. This is in complete agreement with our previous overall findings for the Phoenix Park fallow deer population (McLaughlin et al 2022), so was an expected result. It is known that deer that consistently accept food from humans have a highly variable, acidogenic diet (Bevans et al., 2005). As such, there will be an increased need for UT-B2-mediated urea transport to assist in the SCFA buffering process (Lu et al., 2019). We indeed found increased 50 kDa UT-B2 abundance in consistent beggars, but not for MCT1. These findings appear to agree with the idea that UT-B2 is predominantly regulated by SCFA concentration – as shown in previous studies in a variety of ruminant species (Simmons et al., 2009; Lu et al 2015; Zhong et al., 2022a).
Next, immunolocalization studies confirmed that both MCT1 and UT-B2 transporter proteins were predominantly located in stratum basale, in agreement with our previous studies (Zhong et al., 2022c). For MCT1, our staining confirmed MCT1 protein was found in the stratum basale of all the male adult deer rumen, except M3. In agreement with the western blotting data, no major difference between rare and consistent beggar animals was observed. Interestingly, MCT1 levels in male adults were similar to those observed in the juvenile male and the adult female. In comparison, we firstly showed that UTBc19 appeared not to work as well on immunolocalization slides (c.f. MCT1) despite the high 1:250 concentration that was used. In other words, it did not detect UT-B2 native protein as effectively as when detecting UT-B2 via the western blot technique. However, clearly the same pattern of UT-B2 abundance being higher in consistent beggars than in rare beggars was observed. Indeed, in the latter case, virtually no staining could be detected.
At this point, it is worthwhile noting that two other interesting observations can be made. Firstly, the strength of staining in consistent beggar animals matched that observed in western blotting (i.e. strongest signal in M6 animal). Secondly, UT-B2 levels in male adult consistent beggars are similar to those in the juvenile male and adult female. In other words, when there is sufficient dietary intake in adult males, the cellular transport levels match those of the rest of the population. We therefore interpret this as strong evidence that UT-B2 levels in adult male deer are indeed highly regulated by dietary intake.
Future studies into the Phoenix Park fallow deer population, and, more in general, ungulate populations living within human-dominated landscapes or engaging in interactions with humans should investigate whether similar differences are observed in rare versus consistent beggars in the female adult population. Furthermore, since MCT1 transporter abundance was relatively unaffected in these current studies, additional investigation of alternative SCFA transporter mechanisms is required, such as DRA and MCT4 (Stumpff, 2018). For example, other recent studies in goat rumen have shown that female SCFA transporter expression is greater than male SCFA transporter expression at the RNA level (Guo et al., 2022). Intriguingly, older studies in reindeer rumen have also reported MCT1 and MCT4, but not MCT2 (Koho et al., 2005). It has also been shown that MCT1 was more abundant in free-ranging animals than captive ones (Koho et al., 2005), which would be an interesting area of further investigation.
In conclusion, this pilot study has shown that recovery of male adult deer rumen after the rutting season appears to predominantly occur at the tissue level. In contrast, begging food from humans appears to alter rumen functioning at the cellular level (e.g. UT-B2 urea transporter abundance). These novel findings therefore illustrate that (i) rumen physiology studies should always investigate dietary effects at both the cellular and tissue level, (ii) that the wild fallow deer population of Phoenix Park can provide unique insights that may help us better understand ruminant physiology, and (iii) human-wildlife-interactions may have as yet previously unreported detrimental impacts on rumen tissue.