Factors affecting the production and composition of saliva in

ruminants

By: ali kahyani

Function of saliva

Lubrication and taste- facilitates mastication and swallowing of the bolus

Enzymatic activity- contains lipases or pregastric esterases that preferentially hydrolyze shortchain fatty acids from triglycerides- functional only in suckling calves

Source of nutrients for ruminal microorganisms- mucoprotein and urea serve as N source- electrolytes, particularly sodium are growth factors for ruminal bacteria Influence nutrient removal rate from the rumen- important contributor to the fluidity of ruminal contents

Buffering capacity- major function of saliva- biocarbonate and phosphate buffers

Antifoaming agent- mucin is an effective antifoaming agent- role in bloat

Source of antibodies- conveys antibodies to the rumen- significance is not known

Classes of salivary glandsSerous glandsInclude:

Parotid glandsInferior molar glands

PropertiesSaliva is quite fluid

Parotid glands secrete ½ of all salivaSaliva is isotonic with plasma

Saves osmotic workSaliva is strongly buffered with HCO3- and HPO4-2Secrete continuously, but increased with eating and ruminating

Mucus glandsInclude:

Palatine glandsBuccal glandsPharyngeal glands

PropertiesVary mucus salivaIsotonic with plasmaSaliva is strongly buffered with HCO3- and HPO4-2Low flow when not stimulated

Mixed glandsInclude

SubmaxillarySublingualLabial

PropertiesVery mucus salivaHypotonic to plasmaPoorly bufferedVariable flow

Chemical composition of saliva:

Saliva’s role in recycling N and minerals

NitrogenIn a 24 hour period, a 700 kg cow receiving a mixed hay:grain diet with

secrete:190 l saliva30 to 80 gm total N50-130 gm urea

N recycling

Will be important on low protein dietsAn important consideration in minimizing N excretion

Protein

Microbialprotein

Metabolizableprotein

Dietary protein

NPN

NH3

Urea

Amounts recycledo General estimates

% dietary N recycled = 15-20% (Approximately ½ as urea)o CNCPS program

% N recycled = (121.7 – 12.02 x %CP + .3235 x %CP2)/100 % CP in diet % N recycled 6 61 8 46 10 34 12 24 14 17 16 12 18 10o Marini et al. (2003)

Holstein heifers fed a corn meal-molasses- citrus pulp diet fed at 1.8 x maintenance

% CP in diet % N recycled 9.1 30 11.8 37 15.7 25 18.6 22

Intake control (?)

Saliva infused into the abomasum increased reticular contractions and DM intake in sheep

Infused into the abomasum, ml/hr Saliva:McDougall’s solution 0:1000 250:750 500:500 0:1000DMI, % BW 1.23 3.5 5.1 1.23Reticular contractions, 1.4 5.7% increase over no infusion

Activity of animalActivity % of saliva flowResting 36Eating 27Ruminating 37

Factors affecting saliva flow

Feed consumption(DMI)Increased DM intake increases saliva flow

stage of lactation

Feeding Management

Multiparous cows (MP) primiparous (PP)

INTAKE, diet and Feeeding patternON Salivary production in Cattel

SALIVA PRODUCTION AND RATE OF DIETARY INTAKE

SALIVA PRODUCTION IN CATTLE AS INFLUENCED BY TYPE OF FEED AND PHYSICAL FORM

FEED INTAKE AND SALIVARY PRODUCTION IN STEERS

Intake of physically effective fiber stimulates chewing activity (Mertns, 1997)

often assumed that time spent chewing is a good indication of rumen health because chewing stimulates saliva secretion

Saliva contributes approximately half the bicarbonate entering the which helps buffer the acids produced during fermentation rumen(Owens et al., 1998)

need to understand the relationships between chewing and salivary secretion for dairy cows because

the incidence of subclinical ruminal acidosis is high when diets contain high proportions of concentrates,particularly when concentrates are allocated separately from forage (Østergaard and Gro¨hn, 2000)

The objectives of this experiment were to determine the effects of the proportion of forage in the TMR on chewing activity, saliva production, and ruminal pH of lactating dairy cows, and to evaluate the effects of feeding a TMR compared with separate ingredients (SI) on these same variables

Purpose:

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Eating and Resting Salivation in Early Lactation Dairy CowsK. A. CASSIDA 2 and M. R. STOKESDepartment of Animal and Veterinary SciencesUniversity of MaineOrono 04469

Forage type an important factor affecting salivation rate

stage of lactation is an important factor affecting salivation rate

Multiparous cows (MP) consume more feed per day than do primiparous (PP) cows, and this is usually reflected in higher milk production (Dado and Allen, 1994)

Chewing activity is usually a good indication of rumen health because chewing stimulates saliva secretion. Saliva has been estimated to supply about 70 to 90% of the fluid and buffering capacity entering the rumen (Mertens 1997)

Silanikove and Tadmor (1989) reported a linear relationship between feed intake and salivary secretionrate.

Dry matter intake was higher for MP cows than for PP cows, butMPcows were heavier than PP cows

Multiparous cows produced 4.3 kg/d (17%) more milk than PP cows

Multiparous cows spent 47 min/d longer eating than PP cows Multiparous cows also tended (P =0.07) to spend 1.4 min more eating per kilogram of DM than PP cows

As a result of longer eating time and longer rumination time, MP cows spent more than 1.5 h longer each day chewing:Consequently, MP cows spent less time resting compared with PP cows

salivation rate during resting tended (P = 0.06) to be higher for MP cows than for PP cows

Daily total secretions of saliva during resting and rumination were similar for both groups

MP cows secreted more saliva per day during eating

MP cows had only numerically higher total saliva secretion per day than PP cows (P = 0.16).

22%

Beauchemin (1991) reported that the amount of saliva secreted daily was proportional to feed intake

relationship between DMI and the total amount of saliva produced during eating (r = 0.58, P = < 0.01)

The higher salivation rate during resting for MP cows may be an involuntary response to reduced time spent resting and the necessity to buffer the high level of fermentation acids

There is increasing evidence that exogenous fibrolytic enzymes improve fiber digestion within the rumen, thereby increasing feed utilization of ruminants (Lewis,1999; Rode, 1999).

applying fibrolytic enzymes to feed before feeding may decrease both chewing time and saliva output and increase the risk of acidosis

There was a numerical increase in eating salivation rate (P = 0.07) with enzyme supplementation compared with the CTRL

Salivation rates (ml/min) while eating (P = 0.15) and resting (P = 0.10) tended to be higher for multiparous cows than for primiparous cows

Total daily saliva production was 5.7 to 16.5% higher for cows receiving enzyme treatments compared with CTRL (P = 0.03; total daily saliva output was higher (P = 0.05) for multiparous cows compared with primiparous cows

Much of the increase in total saliva production for multiparous cows was due to higher (P = 0.09) saliva production during ruminating

physically effective fiber to account for the effects of particle size and the intrinsic properties of fiber on chewing (Mertens, 1997).

forages vary in physically effective fiber content and the extent to which they promote chewing , and chewing promotes salivation

The objectives of this study were to determine whether rate of salivation during eating differs for different feeds.

Salivation rate (213 g/min) during forage meals was not affected by source of forage

about 80% more saliva was added to a gram of straw compared with the other forages

In fact, ensalivation of NDF was greater for alfalfa hay than for the other forages. The higher ensalivation of alfalfa NDF may have been related to its longer particle length, even though the barley straw and silages were chopped coarsely

On a fresh basis, silages were consumed 6 to 7 times faster than hay and straw

dry basis,silages were consumed about 2.5 times faster than hay and straw The ability of cattle to eat silage NDF quicker than hay or straw NDF was likely due to the shorter particle size of the silages, although fragility of the fiber may also have been a factor (Iwaasa et al., 1996)

The numerically faster eating rate of hay vs. straw was attributed to the higher fiber content of the straw because eating rates were similar for these feeds when expressed on the basis of NDF

In conclusion, cows consumed concentrate about 3 to 12 times faster than the various forages offered

Ensalivation of concentrate was much lower (1.12 g of saliva/ g of DM) than for forages (3.40 to 7.23 g of saliva/g of DM)

characteristics such as particle size, DM, and NDF content affect salivary output during eating, mainly by Increasing the daily time spent eating either through increased meal frequency or by longer meals (by reducing the rate of intake during meals)

Feeding forages that are consumed more slowly may help prevent ruminal acidosis by increasing the total daily salivary secretion.

the purpose of this study was to investigate the acute and medium-term impact of tannins on salivation (parotid saliva secretion and composition and total saliva production) in sheep and goats

Tannins are a complex group of secondary metabolites, which occur in different plant species

Binding dietary tannins with salivary proteins has been suggested as a means to alleviate the adverse effects of the ingestion of condensed tannins by herbivore(Shimada, 2006; Waghorn, 2008)

Herbivores have developed different mechanisms to counteract the negative effects of tannins (Estell, 2010)

saliva has been considered a first line defense against tannins ingested although the studies are not conclusive, especially in relation with sheep and goats (Lamy et al., 2011)

browsers have a better ability to deal with tannins than grazers. Browsers have larger salivary glands than grazers, suggesting a potential to yield larger amounts of saliva However, these size differences are not always related to saliva production (Robbins et al., 1995; Hofmann et al., 2008).

sheep fed the Tannin diet produced 55, 73, and 107% of the amount of saliva recorded in sheep fed the Control diet on P1, P2, or P3, respectively. Corresponding values in goats were 88, 130, and 134% on P1, P2, or P3, respectively

Both results could indicate a better ability of the parotid gland of goats than the parotid gland of sheep in responding to the ingestion of condensed tannins(Marsh et al., 2006)

Salivary pH was decreased (P = 0.03) on P2 in comparison with P1 and P3 but was not affected (P = 0.11) by the diet

The concentration of Na was greater (P = 0.05) and the concentration of K was reduced (P = 0.04) in sheep

there was a decrease in protein content observed in P3 when compared with P1 (P = 0.02)

The pH of the saliva was decreased (P = 0.05) when goats received the Control diet and greater on P1 than on P2 (P < 0.001) and P3 (P < 0.001)

The concentration of Ca was unaffected by the diet (P = 0.82) but increased from P1 to P2 (P = 0.02)

and also from P1 to P3 (P = 0.05) without differences between P2 and P3 (P = 0.51). There was an increase in the concentration of Na in saliva from P1 to P2 (P = 0.01) and P3 (P = 0.01) without differences (P = 0.35)between diets.

Saliva can involve these information: feeding status nutritional requirements adaptations to diet and

environment health status of animals systemic biomarkers hormones(e.g. progesterone,

cortisol) Proteins(immunoglobulins)

The connection between local (salivary glands) and systemic (blood) sources makes saliva an important fluid to search for biomarkers of diseases or to study a physiological status in particular

advantage of saliva over blood is that it is easily accessible and can be obtained non-invasively and relatively stress-free

focus on three main sections: considerations about animal saliva collection, handling

and storage discuss and review the potential of salivary proteomes to

understand and to monitor ingestive behaviour and nutritional status.

Advantages and disadvantages of current proteomic platforms will be discussed and the potential of animal saliva proteomics to monitor the health status will be pointed out

considerations about animal saliva collection, handling and storage

• Salivary proteins are synthesised in the serous and mucous acinar cells of salivary glands

A recent study using porcine saliva showed that changes in protein composition occur, when saliva is stored at −20 °C. Therefore, working on ice for not more than 1 h and subsequent freezing of samples at −80 °C for long-term storage are considered the safest and most practical handling protocol to date

Another issue in regard to biomarker search is the origin of the collected secretions, i.e. whether it is glandular or whole saliva and if stimulated or unstimulated saliva is being used

For example:

parotid glands:secrete continuously during resting, eating and rumination

Submandibular glands:increase their output volume during feeding

Saliva composition is mainly under autonomic nervous control, causing rapid changes in answer to a wide range of different chemical andmechanical stimuli

The fast compositional change of saliva in response to certain stimuli makes it an important fluid in order to adapt to diets.

saliva characteristics considerably vary within and between individuals and thus might lead to important differences in food perception and consequently in food choices

Recent studies suggested that salivary protein composition is closely related to the species as well as food preferences in animals.

Proteins such as amylase or cystatins are absent in ruminant parotid saliva proteomes

The lack of salivary amylase in ruminating species appears to be related to their starch-free dietary regimens, and as a matter of fact the expression level of the enzyme in saliva correlates well with the level of starch in a species' diet

Even when looking at closely related ruminant species, such as sheep and goat, differences in salivary proteomes are evident

the size of salivary glands in ruminants relates to the levels of secondary metabolites (e.g. tannins) in certain food plants

Parotid glands of browsers (which consume highly digestible diets with high amounts of tannins) were reported to be larger than those of grazers (which chose plants with high fibre content and less or no tannins

In adaptation to their main diet, browsers secrete salivary proline rich proteins, which build a first defense line against tannins

In sheep and goats changes could be observedwith several proteins showing variations in answer to tannin-rich food. For example, increases in the expression levels of cytoplasmic actin and of annexin A1 were reported

Morphological and biochemical studies indicated that protein deficiency resulted in impaired conditions for salivary protein secretion. For example, in humans (Indian children) a reduction of salivary amylase activity has been observed under protein malnutrition

vitamin D is necessary for the synthesis of salivary proteins, and a deficiency in this vitamin affects salivary secretion in rats

iron deficiency in rats lowers the salivary peroxidase activity

ascorbate-deficiency significantly reduced total salivary protein concentration and especially the expression of specific salivary proteins, such as amylase and proline-rich proteins in guinea-pigs

saliva might be a useful tool to monitor nutritional status and nutrient deficiency also in farm animals.

Saliva, proteomics and the search for animal disease biomarkers

Saliva, with its content of hundreds of proteins and peptides, hormones

electrolytes and other chemical compounds,might be a source for biomarkers that help assessing stress factors such as malnutrition as well as diseases or ongoing viral infections in farm animals

However, saliva shows a great inter- and intra-individual variability suggesting that it is difficult to obtain a normal salivary profile for a single species

Summary and future tasks:

Saliva contains thousands of proteins and peptides of glandular or blood origin and therefore is suggested to hold physiological and pathological information accessible by proteomic research. Saliva helps to study the influence of diet on taste perception, food choice or avoidance as well as to obtain the nutritional status of an animal. It is furthermore a pool of putative diagnostic information encrypted in proteins (e.g. B SP30,immunoglobulins), hormones (e.g. progesterone, cortisol) and nucleic acids (e.g. viral RNA), all possibly helping us to secure health in farmanimals.However, especially the salivary proteome is highly variable, dependent on mechanical and chemical stimulants and vulnerable to proteolysis. Therefore, today's saliva sampling and preservation techniques highly rely on freezing the samples to −80 °C for long-time storage to reduce degradation to a minimum. Nevertheless, future strategies also need to cope with saliva sampling in farm animals from tropical or desert areas, where the cooling chain is often unsustainable. Only by developing robust techniques, we will be able to meet the future challenges of the nutritional needs of a growing world population under ongoing climate change. In achieving this goal, we might be able to support health monitoring and livestock production worldwide.

Thanks for your attention

Composition of saliva Composition from different glands

HCO3- HPO4-2 Cl- Na+ K+Parotid 95 75 13 186 5 Inferior molar 134 48 10 175 9Palatine and Buccal 109 25 25 179 4

Submaxillary 6 54 6 15 26 Composition control

Adrenal cortexAldosterone

KidneyRenin

Factors affecting saliva compositionSodium deprivation

As concentration of Na decreases, the concentration of K increases to maintain concentration of total cations

Rate of saliva secretionAs rate of secretion increases

[Na+] and [HCO3-] increases [K+] and [HPO4-2] decreases

The objective of this study was to investigate the effects of enzyme supplementation on the chewing and feeding behavior, saliva secretion, and ruminal pH of lactating dairy cows. A second objective was to determine whether the response to enzyme supplementation differed for multiparous and primiparous cows

differences in saliva production between parity groups have been reported (Maekawa et al., 2002), with multiparous cows having a higher salivation rate than primiparous cows.

Alterations in mechanical processing and chemical properties of feed can significantly alter chewing behavior, and consequently saliva production (Beauchemin and Buchanan-Smith, 1989)