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.