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.