ABSTRACT
The urea nitrogen salvaging process (UNS) supports the symbiotic
relationship between ruminants and their gastrointestinal microbiome by
both supplying nitrogen and buffering bacterially-derived short chain
fatty acids (SCFAs). Studying such physiological processes via
investigation of rumen papillae morphology and functioning, as well as
western blotting to detect rumen urea transporters, allows researchers
to test hypotheses linking physiology and ecology. The goal of this
innovative approach is to indicate the way forward for future research.
Our previous studies have shown the importance of the UT-B2 urea
transporters in the rumen of wild fallow deer living in Phoenix Park,
Dublin. In this current pilot study, we investigated the effects on
these transporters of seasonal changes – linked to changes in feeding
intake during and after the mating season - and acceptance of artificial
food from park visitors in adult male bucks. Investigation of the rumen
papillae revealed that animals culled in January had significantly
longer papillae than those culled just after the rutting season in
November, when bucks interrupted feeding from a few days to weeks. In
contrast, western blotting analysis showed that there was no significant
difference in the abundance of UT-B2 transporters between these two
groups. Adult males that had displayed consistent begging behaviour to
obtain food from human visitors to the park had a higher papillae
density. Furthermore, these animals had a significantly higher abundance
of UT-B2 transporters, which was shown by immunolocalization studies to
be predominantly in the stratum basale layer of the rumen papillae. Our
research suggests that human-wildlife feeding interactions can have
subtle effects on the physiology of individual animals involved. The
findings of this novel study therefore improve our understanding of
basic rumen physiological processes, but also add insight into the
unseen effects that humans feeding wildlife may have.
Keywords: deer; rumen transporters; seasonal; artificial
feeding; human-wildlife interactions
INTRODUCTION
The process of urea nitrogen salvaging (UNS) is crucial to the
gastrointestinal functioning of ruminant species, such as deer (Zhong et
al., 2022a). The passage of urea from the bloodstream into the rumen,
where it is broken down by bacterial urease into ammonia and carbon
dioxide, was initially viewed as simply providing a valuable nitrogen
source to facilitate microbial population growth (Fuller & Reeds, 1998;
Stewart & Smith, 2005). However, more recent studies have highlighted
the importance of a second key function, namely the buffering of the
excess H+ ions formed by the bacterial-derived
production of short-chain fatty acids (SCFA) (Lu et al., 2015). Previous
studies have highlighted the key role within the UNS process played by
the UT-B2 urea transporters present in the rumen papillae epithelial
layers - recently reviewed (Zhong et al., 2022a). For example, it has
been shown that UT-B2 proteins, rather than an alternative route via
AQP3 transporters, appear to be critical to urea transport in the
developing bovine rumen (Zhong et al., 2022b).
Our recent investigations of the epithelial transporters with the rumen
of a wild population of fallow deer, living in Phoenix Park in Dublin,
have added new insight into the processes regulating these ruminal UT-B2
transporters. We have found that levels of the 50 kDa UT-B2 protein were
significantly greater in the rumen of female adults compared to male
adults that had recently been through the rutting season (Zhong et al.,
2022c). Since these males had minimal food intake during that time
(Apollonio & Di Vittorio 2004), these findings were interpreted as
further evidence that UT-B2 abundance was highly regulated by its’ role
in SCFA buffering rather than just nitrogen requirements per se – as
had been already reported in cows (Simmons et al., 2009) and goats (Lu
et al., 2015). However, since these deer are wild and their diet was not
under strict control, there may have been alternative explanations for
these differences. Fortunately, this deer population has also been
extensively studied as part of animal behaviour investigations, and
hence the general feeding patterns of each individual animal are known.
For example, the extent to which an individual animal will approach
human visitors to Phoenix Park and accept food has been recorded over
multiple years (Griffin et al., 2022a; Griffin et al., 2022b). These
kind of independent feeding interactions (i.e. where self-motivated
people feed wildlife in an unmonitored and unregulated environment) are
becoming much more common, spurring the need to unravel what the effects
on the wildlife involved are (Akinyemi, 2014; Lee & Davey, 2015;
McCance et al., 2015). By combining these studies showing that a small
proportion of the deer population (~20%) regularly
accept food from visitors with data from physical measurements of rumen
tissues obtained during the annual culling process, we have recently
reported that deer in this population that consistently accepted food
from humans have a significantly higher density of rumen papillae
(McClaughlin et al., 2022).
The aim of this pilot study was therefore to further investigate the
effect of feeding behaviour on the abundance of ruminal UT-B2 urea
transporters – at both tissue and cellular levels. Since our previous
research showed sex-related differences (Zhong et al., 2022c), this
study was restricted to focusing on the potential effects of dietary
regulation in male adult deer only. In order to achieve this, two key
comparisons were made: (i) between male adult rumen taken from animals
culled less than one month after the rutting season (i.e. November) and
three months after (i.e. January) - so to better disentangle the effect
of male hypophagia during the rut on ruminal urea transporters, and (ii)
between male adults who consistently begged food from humans (i.e.
“consistent ” beggars) and those that almost never did (i.e.
“rare ” beggars). These comparisons provide novel evidence for
dietary regulation of UT-B2 urea transporters in the rumen of adult male
fallow deer and give insights on the unseen effects that human feeding
may have on wildlife.