Saliva is an outstanding fluid, especially in terms of research and diagnostic possibilities. Its composition – namely electrolytes, hormones and especially its proteome – contains valuable information about feeding status, nutritional requirements as well as adaptation to diet and environment. The biggest advantage of saliva as a ’research tool’, however, is the possibility to collect it on a non-invasive basis; and there is almost no need for special training. Therefore, the results of our analysis of salivary proteomes from five different herbivorous species (camel, cattle, gelada baboon, goat, hamadryas baboon) already ignited major interest in salivary research, with the future goal to maintain and improve livestock productivity on one hand and certainly zoo animal welfare on the other. Moreover, the comprehensive analysis and identification of salivary proteins is a necessary pre-requisite to better understand animal physiology and diet adaptation.

Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants (phytoliths) and environmental silicates (grit), which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly bound by bovine odorant-binding protein, bovine salivary protein 30×103 and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin β and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.

Dental microwear and 3D surface texture analyses are useful in reconstructing herbivore diets, with scratches usually interpreted as indicators of grass dominated diets and pits as indicators of browse. We conducted feeding experiments with four groups of rabbits (Oryctolagus cuniculus) each fed a different uniform, pelleted diet (lucerne, lucerne & oats, grass & oats, grass). The lowest silica content was measured in the lucerne and the highest in the grass diet. After 25 weeks of exposure to the diets, dental castings were made of the rabbit's lower molars. Occlusal surfaces were then investigated using dental microwear and 3D areal surface texture analysis. In terms of traditional microwear, we found our hypothesis supported, as the grass group showed a high proportion of (long) "scratches'' and the lucerne group a high proportion of "pits''. Regardless of the uniform diets, variability of microwear and surface textures was higher when silica content was low. A high variability in microwear and texture analysis thus need not represent dietary diversity, but can also be related to a uniform, low-abrasion diet. The uniformity or variability of microwear/texture analysis results thus might represent varying degrees of abrasion and attrition rather than a variety of diet items per se.

Mostly fed with grass in fresh or conserved form, cattle and other grazing ruminants such as goat, sheep and camels have to cope with two main disadvantages of their food. On one side, silicate defence bodies from plant cells (phytoliths), and environmental grit are supposed to abrade tooth enamel (Baker et al., 1959). Furthermore, it seems likely that these silicates could bind different important salivary proteins during mastication with yet indefinite effects on pH regulation, olfaction or anti-bacterial defence (Mau et al., 2006). However, the interactions of salivary proteins with inorganic food particles in free-ranging and livestock animals are still unknown.

Saliva is an extraordinary fluid in terms of research and diagnostic possibilities. Its composition in electrolytes, hormones and especially its proteome contains information about feeding status, nutritional requirements and adaptations to diet and environment, and also about health status of animals. It is easy to collect on a non-invasive and routine basis without any need for special training. Therefore, the analysis of salivary proteomes is going to emerge into a field of high interest with the future goal to maintain and improve livestock productivity and welfare. Moreover, the comprehensive analysis and identification of salivary proteins and peptides in whole and glandular saliva is a necessary pre-requisite to identify animal disease biomarkers and a powerful tool to better understand animal physiology.

Between 2008 and 2010 the Zoological gardens in Rheine, Stuttgart, Karlsruhe, Cologne and Zurich took part in a research project initiated by scientists of the Institute of Animal Science, University of Bonn. Saliva was collected from various zoo animals to determine certain dietary adaptations on enzymatic and physiological levels that might explain or change our current understanding of animal nutrition and the feeding in zoo environments. The interest was primarily focused on species with specialized nutritional needs or fermentation strategies such as geladas, chimpanzees, camels and zebras. The work produced highly valuable data especially on geladas, which, despite from their adaptation to grass diet, revealed to have the enzymatic precondition (salivary amylase) to use starch-rich food items very effectively. Therefore, to prevent obesity in captive geladas, the reduction of starchy food in their daily rations is mandatory. Finally, saliva sampling is an easy, cost-effective and non-invasive procedure, the use of which is both practical and attractive in a zoo environment. In the future, the world-wide emerging field of salivary research might give birth to new applications of saliva as a tool to detect biomarkers of physiology and diseases in zoo animals.

Grasses, leaves and wood contain large amounts of structural carbohydrates such as cellulose, which in animals are digested with the help of symbiotic, cellulolytic microorganisms living in fermentative chambers in the stomach or gut. Although we find the identical relationship in grass eaters (ruminants), in leave-eaters (Colobus monkeys) and in wood-eaters (termites), still little is known about the evolutionary pre-requisites that enabled the development of this very complex symbiosis. Two carbonic anhydrases (CA; CA-VI and the recently described CA-II) are prominently expressed in the saliva of ruminating animals, suggesting that the enzymatic production of bicarbonate is basic for the regulation of oral pH and for maintaining a consistent milieu in the alimentary tract allowing microbial digestion of cellulose. Interestingly, the fermentative chambers of termites (Arthropoda), Colobus monkeys and ruminants show a similar slightly acidic to basic pH. Therefore, the expression of high amounts of CA-II in saliva or mucus of ruminants, camelids, Colobus monkeys and termites is considered to be an essential basis for the development of cellulose fermentation with the help of symbiotic bacteria.

Hamadryas baboons possess salivary proline-rich proteins (PRP), as indicated by the presence of pink-staining protein bands using 1D SDS gel electrophoresis and Coomassie R250 staining. The ability of these protein bands to interact with tannic acid was further examined. In a tannin-binding assay using 5 µg tannic acid mixed with hamadryas whole saliva, we recently found four distinct protein bands of apparently 72, 55, 20, and 15 kDa that were precipitated during the experiments. In this work, we were able to identify these protein bands in a follow-up analysis using MS/MS mass spectrometry after excising such bands out of air-dried gels. Albumin and α-amylase were present in the tannic acid-protein complexes, with albumin already known to nonspecifically interact with a great diversity of chemical compounds. More interesting, we also identified a basic PRP and a cystatin precursor protein. This was the first successful attempt to identify a PRP from precipitated tannin-protein complexes in hamadryas baboons using MS/MS mass spectrometry. On the other hand, the role of cystatins in tannin binding is not yet well understood. However, there are recent reports on cystatin expression in saliva of rats responding to astringent dietary compounds. In conclusion, the follow-up data on tannin-binding proteins present in salivary secretions from hamadryas baboons adds important knowledge to primate physiology and feeding ecology, in order to shed light on the establishment and development of food adaptations in primates. It also demonstrates that tannin binding is characteristic for PRP, but might not be restricted to this particular group of proteins in primate species.

As feeding strategies are typical traits reflecting the adaptation of species, studies on ingestive physiology of different trophic groups could increase knowledge on complex dynamic processes of grazing ecosystems. This article points out the potential of salivary protein profiles for non-invasively and dynamically accessing mammal feeding behavior.The oral cavity is the part of an animal's internal medium that first comes into contact with food. Numerous chemical and mechanical mouth receptors respond to chemical and physical properties of food and monitor its changes during processing. This leads to the central perception of taste and texture of food, which together with odor are important determinants in the decision for ingestion. Saliva plays an important role in the perception of taste and texture sensations: its composition can modulate food perception and can be simultaneously modulated by the type of diet.Differences in food acceptance are common among species of different trophic groups. They are thought to be related to the levels of potentially harmful compounds found in regular diets and to the behavioral and physiological adaptations of animals to these compounds. Concerning plant secondary metabolites (PSMs), such as tannins, salivary proteins are considered one of the animal's defense mechanisms. Among tannin-binding salivary proteins, proline-rich proteins are the best studied. Their presence in mammalian saliva varies according to species and dietary habits. However, various other salivary proteins may also be involved in diet selection.An extensive and quantitative comprehension of salivary protein composition is only available for humans. In the present article we present the state of the art of animal salivary protein research categorized in trophic groups, which are important to define the ecological role of mammals. For example, sheep and goats are two ruminant species, which differ in PSMs intake and salivary proteomes. In accordance to that, mice, having a different digestive morphophysiology, show significant differences in salivary proteomes both in the normal profile and after tannin consumption. Although adaptation to tannins is species-specific a common feature was found: the increase in protein expression levels usually associated with stress situations. The pro and contra of using these data in ingestive behavior research is also critically assessed

Food components and salivary hormones modulate the function of various tissues in the oral cavity. However, the mechanisms underlying such interactions are poorly understood. This study aimed at the detection of GPR30 and GPR43 in oral epithelia. Although unknown yet, the expression of these receptors is hypothesized to be fundamental for the actions of salivary oestrogens, dietary isoflavones and short chain fatty acids (SCFA) in the oral environment. Either immunoblotting or RT-PCR techniques were used for receptor detection in bovine and primate oral tissues. Here we show for the first time that mRNA of the G-protein-coupled oestrogen receptor, GPR30, and the short chain fatty acid receptor, GPR43, are expressed in bovine parotid glands. Furthermore, GPR30 protein is expressed in bovine parotid gland and the tongue of the primate Theropithecus gelada. With GPR30 being a target for dietary isoflavones and GPR43 being a suggested target for short chain fatty acids, we propose new hypotheses concerning the receptors' roles in salivary gland physiology and pathology. Our findings may trigger more detailed studies on GPRs to unravel their role in regulatory mechanisms in the oral cavity as well as in cancer development in relation to diets or biologically active compounds like soy isoflavones.

Hindgut fermentation has been suggested to contribute significantly to the digestive process in the gelada (Theropithecus gelada). We therefore hypothesized that in an in vitro fermentation test (Hohenheim gas test, using gas production as measure of microbial digestion) inoculum based on fresh gelada feces would degrade grass to a similar degree as zebra (Equus burchelli chapmani) feces and to a higher degree than that of hamadryas baboons (Papio hamadryas). Additionally, morphology of gelada tongue, salivary glands, stomach, and intestine were examined in this study. Gas production was measured between 4 and 96 hr using animal feces incubated with 200 mg of air-dry hay or mixed concentrate sample. For grass hay, 12-hr gas production was as follows: T. gelada (19.9 ml)>Papio (18.4 ml)>Equus (15.7 ml). After 24 hr, gas production changed: Papio (35.1 ml)>T. gelada (31.9 ml)>Equus (27.9 ml). At 96 hr, Papio was unexpectedly the most effective species with the highest gas production (53.1 ml)>zebra (51.2 ml)>gelada (49.4 ml). With a concentrate standard, 12-hr gas production was as follows: T. gelada (38.5 ml)>Equus (36.8 ml) = Papio (36.4 ml). At 24 hr, gas production differed: Papio (51.7 ml)>Equus (47.0 ml) = T. gelada (46.8 ml). At 96 hr, zebra was the most effective species with the highest gas production (63.9 ml)>Papio (60 ml) = T. gelada (59.9 ml). In conclusion, the results show that the microbial population present in gelada feces is able to ferment forage and concentrate substrates in vitro, although this fermentation did not occur with the expected effectiveness. Future studies should therefore focus also on the bacteria species involved.

Besides carbonic anhydrase VI (CA-VI), CA-II is suggested to be a second secreted isoenzyme in ruminant saliva. Therefore, the aim of the present study was to investigate the expression of salivary CA-II in bovine parotid glands at the protein level. Moreover, we intended to identify the cells which secrete the enzyme into the saliva. Two commercially available CA-II specific antibodies were tested for use in immunohistochemistry on frozen sections of bovine parotid tissue. Intense positive staining for CA-II was found in luminal duct cells and for the first time also inside the duct lumen, clearly demonstrating the expression and secretion of salivary CA-II in bovine parotid glands. The presence of CA-II protein was verified for parotid tissue and whole saliva using immunoblot analysis. Both salivary CA-II and CA-VI are highly active in supplying the alimentary tract with bicarbonate. It is suggested that a decrease in the expression of either one of these enzymes might severely disturb digestion and/or increase susceptibility to acidosis in ruminants.

BackgroundLittle is known about salivary alpha-amylase expression in primates.MethodsWe compared saliva of gelada and hamadryas baboons, chimpanzees and humans using SDS-PAGE and immunoblotting.Results and conclusionsAmylase expression was increased in hamadryas baboons (P = 0.0376) compared to humans and might indicate dietary starch use in Cercopithecines.

The “expensive tissue hypothesis” states that large brains are active at high metabolic rates, which have to be financed by a significant trade-off with other organs such as the alimentary tract. Recent morphological findings on primate brains and guts support this idea also considering the importance of high-energy diets as a possible driving power of this process. However, the trade-off correlation between brain and alimentary tract, the essence of the “expensive tissue hypothesis”, has not yet been tested using molecular data to complement morpho-functional findings. We therefore hypothesize that the activity of marker proteins expressed both in brain and alimentary tract should parallel functional morphology in organs at the molecular level. Thus, in animals feeding on hard to digest diet, we would expect a high concentration per unit mass of that marker protein in the digestive tract and reversely a lower concentration in the brain. In contrast, in animals feeding on easily-digested, high-energy food we would expect the reverse pattern. Recent preliminary studies suggest that carbonic anhydrase II (CA-II) could act as a marker. The enzyme concentration was found to increase in the brain with higher cerebral activity from cattle to humans and to reversely decrease in salivary secretions. The reverse concentration of CA-II in saliva and brains of cattle and primates might be the first molecular evidence of the validity of the “expensive tissue hypothesis”.

This study was conducted to investigate whether the isoflavones genistein and daidzein, which are components of soy-based diets, and the estrogen 17beta-estradiol affect differentiation and protein metabolism of porcine skeletal muscle cells in vitro. Serum-free porcine myotube cultures expressing the estrogen receptors ERalpha and ERbeta were treated with various concentrations of genistein, daidzein, or 17beta-estradiol for 26 h. The degree of differentiation by creatine phosphokinase activity was not altered by treatment. At 100 micromol/L both genistein and daidzein caused decreases in protein amount due to cell loss. In addition, 100 micromol/L genistein reduced protein synthesis rate of the surviving cells (P < 0.05) measured as [3H]-phenylalanine incorporation. Interestingly, genistein (0.1 micromol/L), daidzein (10, 100 micromol/L), and 17beta-estradiol (0.1, 1 nmol/L) slightly reduced protein degradation (P < 0.05). The results suggest that both genistein and daidzein affect protein metabolism in a dose-dependent manner and that estrogenic actions may play a role in decreasing protein degradation in porcine skeletal muscle.

Gelada baboons are the sole survivors of the genus Theropithecus and the only known graminivorous primates. They developed special adaptations to their diet such as high-crowned teeth for processing hard and abrasive feed. The fine-tuning of salivary protein composition might be another key mechanism that is used by species for adapting to the environment and competing with rivals for exploiting new ecological niches. In order to test whether gelada (graminivorous) and hamadryas baboons (omnivorous) differ in their salivary protein composition, we compared whole saliva samples of captive Theropithecus gelada and Papio hamadryas using gel electrophoresis and tannin-binding assay. We hypothesized that the amount of proline-rich salivary proteins with tannin-binding capacity is higher in baboons consuming a feed with high dicot/monocot rations. Dicots produce tannins as a chemical defense system, discouraging animals from eating them. In contrast to dicots, monocots do not synthesize tannins. The presence of tannin-binding proteins in saliva should effectively inactivate the dicot tannin-based defense mechanism and increase the dietary breadth and/or the capability to switch between monocots and dicot leaves. The lack of such tannin-binding proteins in saliva would indicate a narrow dietary spectrum more restricted to monocots. We found T. gelada to completely lack proline-rich proteins (PRPs) and tannin-binding capacity similar to a great variety of other grazing mammals. In contrast, P. hamadryas does possess PRPs with tannin-binding activity. The findings support a growing body of evidence suggesting a high-level specialization of T. gelada to grass diets. However, it remains unclear, whether loss of salivary tannin-binding capacity drove the gelada into its narrow feeding niche, or whether this loss is the result of a long process of increased specialization. Thus, from an ecological point of view, T. gelada appears to be more vulnerable to environmental changes than other baboon species owing to its narrow dietary traits.

Salivary glands are highly variable in composition of their secretions and thus could be one of the primary ways by which species adapt or react to their environments. It has been hypothesized that feeding adaptation correlates with saliva composition. Hence, animals of different families using identical feeding niches should possess similar salivary proteins. For the first time, salivary secretions of grass-eating cattle, goat, camel and gelada baboon were compared by SDS-gel electrophoresis and immunoblotting. Salivary protein patterns were similar among individuals of the same species but varied largely among species. However, all samples showed proteins of apparently 29 and 42 kDa, identified as carbonic anhydrases (CA) by immunoblotting. The CA-VI (42 kDa) was highly expressed in cattle and camel saliva, but showed lower expression in goat saliva and could not be detected in gelada baboons. The CA-II (29 kDa) was found in saliva of all species tested and was shown in ruminating animals not to originate from cellular debris of the oral mucosa or ingested food. The results demonstrate that besides CA-VI, CA-II is another CA isoform secreted especially in ruminant saliva. Furthermore, the two CA isoenzymes detected may form a complementary system, protecting mucosa from acidity and helping to maintain a constant bicarbonate concentration in the animal's mouth and digestive tract.