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From the Department for Farm Animals and Veterinary Public Health

at the University of Veterinary Medicine, Vienna

(Department Speaker: Univ.-Prof. Dr.med.vet. Michael Hess)

Institute of Animal Nutrition and Functional Plant Compounds

(Head: Univ.-Prof. Dr.sc.agr. Qendrim Zebeli)

Effects of grain-induced subacute ruminal acidosis on rumen digesta particle distribution in dairy cattle

Diploma thesis for obtaining the dignity of

Magistra Medicinae Veterinariae

at the University of Veterinary Medicine, Vienna

submitted by

Carolin Anna Imbery

Vienna, in September 2016

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Supervisor:

Dr.Sc. Ratchaneewan Khiaosa-ard

Institute for Animal Nutrition and Functional Plant Compounds

Department for Farm Animals and Veterinary Public Health

1st Assessor:

Univ.-Prof. Dr.med.vet. Thomas Wittek, Diplomate ECBHM

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meiner Familie

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Table of contents

1. Introduction and study hypothesis ...................................................................................... 1

2. Literature ........................................................................................................................... 3

2.1. Subacute ruminal acidosis ........................................................................................... 3

2.1.1. Definition of SARA ................................................................................................ 3

2.1.2. Causes of SARA ................................................................................................... 3

2.1.3. Occurrence and importance of SARA ................................................................... 4

2.1.4. Health consequences of SARA ............................................................................. 5

2.1.5. Prevention of SARA .............................................................................................. 6

2.2. Structural carbohydrates: characterisation, measurements, ruminal digestion and the

importance for rumen health ............................................................................................... 8

2.2.1. Characterisation of carbohydrates in ruminant nutrition ......................................... 8

2.2.1.1. Definition ........................................................................................................ 8

2.2.1.2. Characterisation of plant cell walls .................................................................. 9

2.2.1.3. Chemical and physical measurements of fibre and measurement of physical

parameters of the digestive content ........................................................................... 12

2.2.2. Physiology of ruminal digestion ........................................................................... 14

2.2.3. Importance of ruminal microorganisms for fibre digestion ................................... 15

2.2.4. Regulation of ruminal pH ..................................................................................... 18

2.2.5. Effects of pH decline on ruminal fibre digestion ................................................... 19

2.2.6. Particle size, effectiveness of fibre and the importance for rumen health ............ 21

3. Materials and methods ..................................................................................................... 25

3.1. Cows, feeding and SARA challenge .......................................................................... 25

3.2. Measurement of ruminal pH ...................................................................................... 27

3.3. Measurements of dry matter intake and water intake ................................................ 27

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3.4. Rumen sampling and wet-sieving method ................................................................. 27

3.5. Calculations and statistics ......................................................................................... 30

4. Results ............................................................................................................................. 31

4.1. Intake of dry matter, concentrate and water ............................................................... 31

4.2. Ruminal pH ............................................................................................................... 31

4.3. Rumen digesta particle distribution ............................................................................ 32

4.3.1. Mean particle length ............................................................................................ 32

4.3.2. The large particle fraction (> 2 mm) .................................................................... 33

4.3.3. The medium particle fraction (1.18—2.00 mm) ................................................... 35

4.3.4. The small particle fraction (< 1.18 mm) ............................................................... 36

4.3.5. The soluble fraction (< 0.063 mm) ....................................................................... 37

5. Discussion ....................................................................................................................... 38

5.1. Dry matter, concentrate and water intake ............................................................... 38

5.2. Ruminal pH ............................................................................................................ 39

5.3. Rumen particle distribution ..................................................................................... 40

6. Conclusion ....................................................................................................................... 44

7. Zusammenfassung .......................................................................................................... 45

8. List of abbreviations ......................................................................................................... 47

9. References ...................................................................................................................... 48

10. List of tables ................................................................................................................... 56

11. List of figures ................................................................................................................. 57

1

1. Introduction and study hypothesis Subacute ruminal acidosis (SARA) is an important metabolic dysfunction in ruminants

(Enemark 2008; Kleen et al. 2003; Nocek 1997). In recent times SARA is increasingly

challenging high-yielding dairy herds (Enemark 2008) because various consequences arise

from SARA, including changes in milk production (Kleen et al. 2003; Nocek 1997; Plaizier et

al. 2008), higher culling rates and high economical impacts (Enemark 2008; Kleen et al.

2003; Nocek 1997). The threshold of the pH profile and the time of pH depression at which

SARA is defined depends on the authors. Nevertheless, pH values of 5.5 to 5.9 are often

used to define SARA (Plaizier et al. 2008) and when the pH depression (pH < 5.6) lasts for

more than 3 h/day (Gozho et al. 2005) or (pH < 5.8) for more than 5—6 h/day (Zebeli et al.

2012).

SARA arises when diets that are high in energy and low in structure are fed to ruminants

whose ruminal ecosystem has not yet been adapted (Kleen et al. 2003). However, such diets

are required due to a requested gain in energy to meet the daily requirement of high

producing dairy cows (Aschenbach et al. 2011; Kleen et al. 2003; Plaizier et al. 2008). The

rapidly fermentable carbohydrates contained in these diets are fermented to large amounts

of short chain fatty acids (SCFA) in the rumen (Dijkstra et al. 2012; Kleen et al. 2003). These

increasing amounts of SCFA cannot be absorbed appropriately (Kleen et al. 2003), which,

combined with insufficient rumen buffering, can lead to low pH values resulting in SARA

(Dijkstra et al. 2012). Therefore, the prevention of SARA involves feeding sufficient amounts

of fibre, especially physically effective fibre, since it plays an important role in feeding

management of cows to maintain a healthy rumen environment (Zebeli et al. 2006). It is well

known that physically effective fibre stimulates chewing and rumination, leading to an

increasing saliva production and therefore an increasing rumen buffering (Mertens 1997;

Yang and Beauchemin 2007a; Zebeli et al. 2006). Furthermore, longer fibre particles are

needed in the ruminal mat to stimulate rumen contractions which are important for a proper

passage and absorption of nutrients (Yang and Beauchemin 2007a).

Ruminal pH has a profound effect on the ruminal microorganisms. As a result of decreasing

pH, ruminal fibre digestibility is generally decreased during SARA (Calsamiglia et al. 2008;

Dijkstra et al. 2012). This is because ruminal bacteria, that are responsible for fibre

degradation, are sensitive to pH values lower than 6.0 (Sung et al. 2006), especially when

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the low pH is extended, since fibrolytic bacteria can survive temporary low pH values in the

rumen (Calsamiglia et al. 2002).

These implications lead to the hypothesis that as a result of SARA the decreased fibre

digestion leads to a greater retention of large particles in the rumen and changes particle

distribution of rumen digesta. This alteration is of importance because it can affect the

characteristic of the ruminal mat, ruminal passage rate, rumen fill, dietary intake and,

therefore, animal production. Currently, there is not enough data, regarding rumen particle

distribution in relation to SARA, especially regarding long-term SARA challenge. This thesis

focuses on short-term and long-term effects of diet-induced SARA on rumen particle

distribution. Results regarding ruminal pH profile and dietary intake are also included in this

thesis.

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2. Literature

2.1. Subacute ruminal acidosis

2.1.1. Definition of SARA

Throughout the literature several definitions of SARA exist. Initially, SARA has been

characterised as a periodical reduction of ruminal pH to pH levels, which are below the

physiological threshold (Kleen et al. 2003). This can be caused by the uptake of diets, based

on certain concentrates, while the ruminal microflora and ruminal mucosa are not yet

adapted (Kleen et al. 2003). More recently, certain ruminal pH thresholds have been

proposed, however, at different pH values, depending on the authors (Kleen et al. 2003;

Krause and Oetzel 2006), but often pH values of 5.5 to 5.9 are used to define SARA (Plaizier

et al. 2008). Some authors claim that if ruminal pH drops below 5.5, cows develop SARA

(Kleen and Cannizzo 2012; Krause and Oetzel 2006), while other authors describe SARA at

a threshold pH value of 5.8 (Yang and Beauchemin 2007a). These drops of ruminal pH occur

several times per day (Kleen and Cannizzo 2012). In addition to the pH threshold, the

magnitude and the duration of the pH depression are also critical for the diagnosis of SARA.

According to Zebeli et al. (2008) for SARA prevention daily mean ruminal pH should not drop

below values of 6.16 and the ruminal pH should not be lower than 5.8 for a duration of 5.24

h/day, while Gozho et al. (2005) defines SARA at a ruminal pH below 5.6 for a duration

longer than 3 h/day. However, the dimensions of SARA depend on factors like management

and feeding (Kleen and Cannizzo 2012).

2.1.2. Causes of SARA

As already mentioned above, SARA begins to arise with the intake of diets that are low in

fibrous structure and high in energy (e.g., starch) (Allen 1997; Kleen et al. 2003; Kleen and

Cannizzo 2012; Krause and Oetzel 2006). Rations that are low in fibre but rich in starch are

often fed to high producing dairy cows since these animals have a high demand in energy to

support their production (Aschenbach et al. 2011; Kleen et al. 2003; Nocek 1997; Plaizier et

al. 2008). However, not only the content of forage and concentrate have to be considered in

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diet formulation, also method of grain processing, source of concentrate and type and quality

of the forage have to be regarded because these factors can also influence ruminal pH and

SARA (Nocek 1997; Zebeli et al. 2010).

On the one hand, diets that consist of rapidly fermentable carbohydrates, like starch and

sugars, lead to an increasing production of SCFA in the rumen, which further leads to a

decline of ruminal pH (Dijkstra et al. 2012). On the other hand, diets which are high in starch

that lack structural characteristics do not adequately promote chewing activity and hence

less salivary buffers are provided for the rumen, which puts more strain on the rumen

environment, risking acidotic insults (Allen 1997).

When the ruminal pH drops below a value of 5.5 during acidotic periods, lactate production

starts which can have even a more severe impact on ruminal pH (Krause and Oetzel 2006).

However, different from acute acidosis, during SARA lactate is only present at a low

concentration in the rumen (Krause and Oetzel 2005).

2.1.3. Occurrence and importance of SARA

Generally, the occurrence of ruminal acidosis is higher in regions where grain is less

expensive and/or regions which do not have a regulated market for milk production (Krause

and Oetzel 2006). This can result in overfeeding of grains which in turn causes SARA

(Krause and Oetzel 2006). This is also relevant for German dairy herds, which are affected

by SARA in many cases (Kleen et al. 2013). Especially larger farms are at a higher risk for

SARA, as their animals show lower pH values (Kleen et al. 2013).

SARA has high economic impacts (Enemark 2008; Nocek 1997; Plaizier et al. 2008)

because it is associated with decreased milk production and higher culling rates (Enemark

2008; Kleen et al. 2003; Nocek 1997). Enemark (2008) claims that SARA generates high

losses of approximately US $ 500 million to US $ 1 billion per year and of US $ 1.12 for

individual cows per day. The diagnosis of SARA in cows and in herds is regarded as

important because the insults of SARA will get more intense, if cows have suffered from

SARA before (Dohme et al. 2008).

Herds confronted with SARA show different signs hinting at ruminal acidosis. For instance, a

depression of dry matter intake (DMI), an increase of diarrhoea, laminitis, higher culling

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rates, poor performance or poor body condition may occur (Kleen et al. 2003; Nocek 1997;

Plaizier et al. 2008). To diagnose SARA monitoring and recording of health consequences,

paraclinical signs and clinical signs are required (Enemark 2008). Attention should be paid to

diseases such as rumenitis, rumen tympani, abomasal displacement, abomasal ulcers,

laminitis, metabolic acidosis and also to changes in feed intake, feeding patterns, faecal

characteristics, fertility and calf health (Enemark 2008). Moreover, parameters of ruminal

fluid, especially ruminal pH, but also milk, blood and urine need to be monitored, as well as

feeding patterns, faecal particle size, epistaxis and culling rate (Enemark 2008). However,

the diagnosis of SARA is challenging because the clinical symptoms are delayed in time and

difficult to detect (Enemark 2008) and because the threshold of ruminal pH for SARA and

method of collecting ruminal fluid are not yet standardised (Plaizier et al. 2008).

2.1.4. Health consequences of SARA

Recently SARA is claimed to be an increasing problem in high-yielding dairy herds (Enemark

2008). It leads to several symptoms, diseases and far-reaching consequences affecting

cow’s health and productivity (Enemark 2008; Kleen et al. 2003; Nocek 1997; Plaizier et al.

2008). However, the effects of SARA on individual cows can vary (Kleen and Cannizzo 2012)

because they depend on the duration and the magnitude of ruminal pH depression (Nocek

1997).

As already mentioned above, SARA is associated with symptoms and diseases such as

rumenitis, diarrhoea, laminitis and liver abscess (Kleen et al. 2003; Nocek 1997; Plaizier et

al. 2008). Additionally, Kleen et al. (2003) mentions the clinical picture of SARA is formed by

a complex of parakeratosis, liver abscess and rumenitis. Initially, inflammation of the ruminal

epithelium can lead to further health upsets because ruminal cells are not covered by mucus

and are in danger of being damaged when SCFA accumulate in the rumen (Krause and

Oetzel 2006). As a result of SARA potentially pathogenic bacteria can prevail and release

immunogens, such as microbial endotoxin, into the rumen (Zebeli and Metzler-Zebeli 2012).

Ruminal epithelial cells begin to differentiate and proliferate at a greater rate during SARA

(Penner et al. 2011; Zebeli and Metzler-Zebeli 2012) and ruminal cellular junctions can be

damaged (Plaizier et al. 2008), which further leads to a decrease of cellular adhesion (Zebeli

and Metzler-Zebeli 2012). Therefore, it is possible for toxins (Zebeli and Metzler-Zebeli 2012)

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and for bacteria to filtrate from the rumen into the bloodstream (Krause and Oetzel 2006).

Bacteria and toxins increasingly leaking into prehepatic circulation might be the triggers for

systemic inflammation (Gozho et al. 2005), inducing inflammatory responses in multiple

organs causing peritonitis, liver abscess and other inflammations (Krause and Oetzel 2006).

Even acute phase responses can arise during SARA, particularly when specific feed

materials, like grains, are fed (Gozho et al. 2005; Khafipour et al. 2009a; Plaizier et al. 2012).

A decrease in feed intake and feed conversion efficiency might also be related to SARA and,

somehow, might even be directly related to rumenitis (Kleen and Cannizzo 2012; Krause and

Oetzel 2006). Consequently, cows suffering from SARA often experience a loss of body

condition (Kleen et al. 2003; Nocek 1997) and SARA-affected herds have a higher annual

turnover and higher culling rates within the herd (Kleen et al. 2003; Nocek 1997; Plaizier et

al. 2008).

In terms of production, research shows that SARA has a negative impact on milk production

(Kleen et al. 2003; Nocek 1997; Plaizier et al. 2008) and can even result in a depression of

milk fat (Allen 1997; Kleen et al. 2003). Other studies, on the other hand, show that SARA

does not have an effect on milk fat (Kleen et al. 2013; Krajcarski-Hunt et al. 2002). We can

expect that the effects of SARA on milk production and composition depend on feeding

conditions and the severity of SARA.

2.1.5. Prevention of SARA

It can be generalised that SARA is mainly diet-dependent. Cows are put at a higher risk for

developing ruminal acidosis when their diets contain high starch contents and are low in fibre

(Allen 1997; Kleen et al. 2003; Kleen and Cannizzo 2012; Krause and Oetzel 2006). To

prevent ruminal acidosis a suitable diet formulation and feeding management, monitoring of

ruminal pH and fermentation processes are important (Calsamiglia et al. 2008). Diets should

be formulated in a way that allows microbial flora a proper growth and production, which

should prevent excessive production of SCFA (Allen 1997), and in a way to stimulate the

ruminal epithelium to absorb and to metabolise the arising fermentation products (Zebeli et

al. 2012). Besides the diet formulation, feeding management and cows’ steady feed intakes

have to be taken care of because cycles of feed deprivation and the subsequent overeating

are contributing to drops of ruminal pH and thus to SARA (Krause and Oetzel 2006). Also, a

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reduced feed intake depresses energy metabolism of cows and their ruminal

microorganisms, which in turn promotes SARA (Nocek 1997).

Furthermore, SARA seems to be a herd problem, especially as a consequence of incorrect

herd management (e.g., animal observation, stress and varieties in feeding management),

with larger herds being more prone to SARA (Kleen et al. 2013). To lower the risk of SARA,

herds and even individual cows which have a higher DMI should receive diets with lower

contents of non-fibre carbohydrates (NFC) (Krause and Oetzel 2006). Diets of lactating dairy

cows should not contain more than 30–40 % starch and 35–40 % non-structural

carbohydrates (NSC) (Nocek 1997). The National Research Council (NRC) (2001)

recommends a NFC content in the ration being 2–3 % higher than the NSC content

recommended by Nocek (1997). The content of neutral detergent fibre (NDF) is

recommended to be at 25–30 % (Nocek 1997; NRC 2001). In addition to the content of

dietary fibre, the effectiveness of the fibre is equally important for prevention of SARA

because of its influence on ruminal physiology (see 2.2.5.). To minimise the risk of SARA

while not impairing the production, the physically effective fibre content in total mixed rations

(TMR) should be about 30–33 % to maintain ruminal pH in the physiological range (Zebeli et

al. 2008; Zebeli et al. 2010). The effects rapidly fermentable carbohydrates have on cow

health underline the crucial role of dietary fibre on rumen health and on the general health of

the animal.

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2.2. Structural carbohydrates: characterisation, measurements, ruminal digestion and the importance for rumen health

2.2.1. Characterisation of carbohydrates in ruminant nutrition

2.2.1.1. Definition

Carbohydrates in ruminant nutrition can be classified in different ways depending on their

occurrence, their chemical composition, their chemical and physical characteristics or their

function (Hall 2003; Kamphues et al. 2014; Kirchgeßner et al. 2014). Plant carbohydrates

can be differentiated according to their location, whether they occur in the cell wall or inside

the cell (Fig. 1) (Hall 2003), and according to their chemical composition into

monosaccharides, disaccharides, oligosaccharides and polysaccharides (Kamphues et al.

2014; Kirchgeßner et al. 2014). Disaccharides (e.g., sucrose, maltose) are composed of two

sugar monomers, like glucose or fructose, whereas oligosaccharides (e.g., raffinose) consist

of three to ten sugar monomers (Kirchgeßner et al. 2014). Polysaccharides are characterised

through a composition of several sugar monomers (Kirchgeßner et al. 2014). Regarding their

function, dietary polysaccharides can be divided into the fractions of structure

polysaccharides (e.g., cellulose), water regulating polysaccharides (e.g., pectin) and

polysaccharides used for the plant’s energy metabolism (e.g., starch) (Kirchgeßner et al.

2014). Another classification of plant carbohydrates mentions the group of non-starch-

polysaccharides (NSP) (Fig. 1) (Kamphues et al. 2014; Kirchgeßner et al. 2014), which

includes detergent fibre (e.g., pectins and other polysaccharides) and non-detergent fibre

(e.g., cellulose, hemicelluloses and β-glucans) (Kamphues et al. 2014). Non-fibre

carbohydrates (NFC) (Fig. 1) can be estimated by the subtraction of DM and the percentages

of crude protein, crude fat, ash and NDF (Hall 2003; NRC 2001). Sources of NFC are starch,

sugars, organic acids and substances addressed as neutral detergent soluble fibre like

fructans, pectic substances, galactans, β-glucans (Hall 2003). Different from NFC, NSP is the

fibrous fraction, which can be grouped into NDF (i.e., cellulose, hemicellulose), acid

detergent fibre (ADF) (i.e., cellulose and lignin) and acid detergent lignin (ADL) (Kamphues

et al. 2014; Kirchgeßner et al. 2014). The ADL represents approximately the lignin content

(Kamphues et al. 2014; Kirchgeßner et al. 2014). The fraction of NDF, furthermore, can be

classified depending on its degree of digestibility, into digestible NDF and indigestible NDF

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(Lund et al. 2007). However, it has to be mentioned that lignin is not a polysaccharide, but

rather a hydrocarbon polymer (Heldt and Heldt 2003; Kirchgeßner et al. 2014; Orpin 1984).

Fig. 1: Plant carbohydrate fractions (Adapted after Hall (2003))

2.2.1.2. Characterisation of plant cell walls

The cell wall provides the plant cell protection against being physically damaged or damaged

by infections (Kindl 1994) and helps to maintain the cell’s water balance (i.e., cell volume)

(Heldt and Heldt 2003; Kindl 1994). Cell wall composition and structure are different for

various plants, depending on their species and their age, and even different types of plant

cells show variations within their cell walls (Orpin 1984).

Beginning to describe the plant cell wall from the centre outwards, the innermost layer is the

secondary cell wall, followed by the primary cell wall and the middle lamella, which lays

between neighbouring plant cells (Orpin 1984). The surface of some plant cell walls is

covered with a hydrophobic layer of wax and cutin, which is called the cuticle (Heldt and

Heldt 2003). Plant cell walls are mainly composed of carbohydrates (such as cellulose,

hemicelluloses and pectin) and to a lesser extent of glycoproteins (Heldt and Heldt 2003;

Kindl 1994; Orpin 1984). Plant cell walls consist out of 40–50 % cellulose, but some plants

even have a higher content of cellulose in their cell walls (Kirchgeßner et al. 2014).

Plant carbohydrates

Cell contents

Organic acids

Sugars Starches Fructans

Cell wall

β-glucans, Pectic

substances, Galactans

Hemicellulose Cellulose

NFC

Neutral detergent soluble fibre

ADF

NDF

NSP

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During the plant cell’s growth, the primary cell wall is formed into a matrix by hemicellulose,

pectin and glycoproteins in which microfibrils of cellulose are embedded (Heldt and Heldt

2003; Kindl 1994; Orpin 1984). After cell growth is complete, the secondary cell wall,

primarily made up of cellulose, is formed and is later lignified (Heldt and Heldt 2003; Orpin

1984). The middle lamella consists of pectin and lignin (Orpin 1984).

The polysaccharides of plant cell walls are formed by various sugars such as glucose,

galactose, mannose, xylose, arabinose, fucose, rhamnose, galacturonate and glucoronate

(Kindl 1994; Kirchgeßner et al. 2014; Wang and McAllister 2002). These are synthesised

within the plant cells (Kindl 1994). Digestibilities of plant cell carbohydrates vary from rapidly

fermentable to hardly or even non digestible (Kamphues et al. 2014). Because ruminants

lack of enzymes to digest plant cell wall carbohydrates (Russell and Rychlik 2001), fructans,

pectin, hemicelluloses and cellulose are fermented by microbial enzymes in the rumen

(Kamphues et al. 2014).

Cellulose is a long water-insoluble polysaccharide consisting of linear chains of thousands of

glucose molecules, linked together by 1,4-β-glycosidic bonds (Fig. 2) (Heldt and Heldt 2003;

Kindl 1994; Kirchgeßner et al. 2014; Moreira et al. 2013). The chains of cellulose are bound

together and form microfibrils (i.e., crystalline cellulose) (Heldt and Heldt 2003; Kindl 1994),

which come together to form fibres and then the fibres bind together into groups (Orpin

1984).

Fig. 2: Molecule of cellulose (Heldt and Heldt 2003)

Hemicellulose is not a unique polysaccharide, but rather is a group of several

polysaccharides, which are made up of glucose, mannose, galactose, fucose, xylose and

arabinose (Fig. 3) (Heldt and Heldt 2003; Kirchgeßner et al. 2014). The arrangement of these

sugars differs between plant species (Kirchgeßner et al. 2014; Orpin 1984).

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Fig. 3: Molecule of xyloglucan (hemicellulose) (Heldt and Heldt 2003)

Pectin is formed by multiple sugar acids, mostly D-galacturonate, linked by α-1,4-glycosidic

bonds (Fig. 4) (Heldt and Heldt 2003; Wang and McAllister 2002) and is an amorphous and

water regulating molecule (Heldt and Heldt 2003; Kirchgeßner et al. 2014).

Fig. 4: Molecule of D-galacturonate (Heldt and Heldt 2003)

Lignin is a polymer formed by derivates of phenyl propan and is found in the secondary cell

wall (Heldt and Heldt 2003; Orpin 1984). Usually, lignin is hardly digestible (Heldt and Heldt

2003; Kamphues et al. 2014; Kirchgeßner et al. 2014). Also, lignin limits the digestion

process in the rumen because a supramolecular net formed out of lignin and the cell wall’s

polysaccharides (Moreira et al. 2013), which prohibits the enzymes’ access to the contents in

the inner of cell (Kirchgeßner et al. 2014; Wang and McAllister 2002).

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2.2.1.3. Chemical and physical measurements of fibre and measurement of physical

parameters of the digestive content

Fibre can be measured with different methods to show its characteristics. In general, it can

be determined chemically (Kamphues et al. 2014; Kirchgeßner et al. 2014; van Soest 1963;

van Soest and Wine 1967) and physically (Mertens 1997; Yang and Beauchemin 2009;

Zebeli et al. 2006).

Measuring fibre with chemical methods, crude fibre, NDF, ADF and ADL can be determined

(Kamphues et al. 2014; Kirchgeßner et al. 2014). In terms of animal nutrition, the chemical

analysis of diets is common. Crude fibre, which is determined using Weende analysis,

includes insoluble cell wall components such as cellulose, hemicelluloses, pectin, lignin, cutin

and others (Kamphues et al. 2014). However, using Weende analysis, the soluble parts of

cellulose, hemicellulose, lignin and pectin attribute to the content of nitrogen-free extractives

(NFE), which further includes mono-, disaccharides and starch (Kamphues et al. 2014;

Kirchgeßner et al. 2014). The more precise system to determine fibre contents in terms of

ruminant nutrition is the detergent fibre system. Using this method, the fraction of NDF

contains cellulose, hemicellulose and lignin, while ADF includes only cellulose and lignin

(Kamphues et al. 2014; Kirchgeßner et al. 2014).

As mentioned before, physical parameters are also used to characterise fibre of diets.

Particle size determines the effectiveness of fibre (Mertens 1997). Particle size of diets are

widely determined by using the Pennstate Particle Separator (PSPS) (Zebeli et al. 2010).

Subsequently, the concept of physically effective fibre (termed as peNDF in research papers)

unites chemical factors (NDF concentration) and the proportion of the particles retaining at

different PSPS sieves (Mertens 1997; Yang and Beauchemin 2009; Zebeli et al. 2010).

Physical parameters of the digestive content (e.g., masticate, rumen digesta, faeces) are

evaluated using wet-sieving technique (Allen and Grant 2000; Tafaj et al. 2005; Teimouri

Yansari et al. 2007). A nested column of sieves (wire mesh) is used for a typical sieve

analysis (Fig. 5).

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Fig. 5: Wet-sieving device used in the present study (Vibratory sieve shaker AS 200 digit 2, Retsch,

Germany)

A wet-sieving analysis is carried out like a dry sieving process by using the same apparatus,

the sample being placed on the top sieve. But to support the sieving process, the wet-sieving

approach involves the vertical flow of water in addition to the sieving motion. According to

Uden and Van Soest (1982), wet-sieving is a more logical method compared to dry sieving to

study digestive movement in the gastrointestinal tract. Consistently, a wet-sieving analysis

has been used in research to quantitatively measure breakdown processes of forages in the

digestive tract of ruminants as a means to indicate quality of forages (Moseley and Jones

1984) and to study particle size distribution of the digestive content (Allen and Grant 2000;

Teimouri Yansari et al. 2007), as well as to measure critical particle size for ruminants

(Oshita et al. 2004). As mentioned in Teimouri Yansari et al. (2007), the particle size of

rumen digesta could affect ruminal passage rate, ruminal mat consistency and may also

affect the ruminal digestion at all. A decrease in mean particle length and proportion of large

particle size of rumen digesta can suggest an increased fibre digestion in the rumen (Tafaj et

al. 2005).

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2.2.2. Physiology of ruminal digestion

The rumen is the one of four stomach compartments in ruminants (Nickel et al. 2004) in

which fermentation of feed materials takes place (Allen 1997; Dijkstra et al. 1993). Feed

materials are broken down as a result of microbial fermentation, chewing and rumination

(Ishler et al. 1996). Hence, the rumen contains feed particles with different sizes and different

densities which create different layers in the rumen (Tafaj et al. 2004). Near the bottom of the

rumen small and dense feed particles can be found in the ruminal fluid, while the next layer

consists of larger and less dense particles that form the ruminal mat (Ishler et al. 1996). The

gases that derive from the fermentation process, such as carbon dioxide, methane, nitrogen,

oxygen and hydrogen, accumulate at the top of the rumen (Ishler et al. 1996). The gases that

derive during fermentation leave the rumen via expelling, this process is called eructation

(Russell and Rychlik 2001).

Zebeli et al. (2012) claims the ruminal mat is responsible for proper rumen functions, like

rumination and rumen motility, and for retaining feed particles to be digested in the rumen.

The ruminal mat stimulates contractions of the rumen (Yang and Beauchemin 2007a),

thereby mixing the ingested feedstuffs with the ruminal microorganisms and promoting

absorption of the nutrients and fermentation products (Russell and Rychlik 2001). However,

feeding ground materials inhibits a proper formation of the ruminal mat (Allen and Mertens

1988). When there are less muscle contractions of the rumen the risk of ruminal acidosis is

increased partly because the absorption of the SCFA decreases (Yang and Beauchemin

2007a). Generally, particles have to remain in the rumen until they are comminuted to a

certain size because particles can only pass out of the rumen when being smaller than 1.18

mm (Maulfair et al. 2011). Some forage types remain in the rumen longer than others, for

example corn silage remains longer in the rumen than grass silage (Krämer et al. 2013).

Concentrate usually passes the rumen faster than forage (Colucci et al. 1990). Different

parameters determine ruminal degradation and retention of feed materials including

composition of cell walls, particle size and density of the particles (Colucci et al. 1990).

Diverse populations of bacteria, protozoa and fungi (Krause et al. 2003; Russell and Rychlik

2001; Zhang et al. 2007) as well as viruses (Klieve and Bauchop 1988) are accommodated

in the rumen and normally live in symbiosis with the ruminant host that offers an appropriate

habitat for the microorganisms (Krause and Combs 2003; Russell and Rychlik 2001). A

synergistic relationship between ruminal bacteria, protozoa and fungi exists (Zhang et al.

15

2007) and normally ruminal microflora is quite stable (Russell and Rychlik 2001). The various

microorganisms hosted in the rumen secrete enzymes for the digestion of feedstuffs (Krause

et al. 2003; Russell and Rychlik 2001; Wang and McAllister 2002) such as cellulose,

hemicellulose, pectin, starch, sugars, amino acids and others (Russell and Rychlik 2001).

Depending on the substrates, a broad range of products derive from microbial fermentation

(Tab. 1), the most common fermentation products being organic acids such as acetate,

butyrate, propionate, lactate and gasses like methane, carbon dioxide and hydrogen (Russell

and Rychlik 2001).

Tab. 1: The characteristics of predominant ruminal bacteria (Russell and Rychlik 2001)

Species Ruminal niche Fermentation products Fibrobacter succinogenes CU S, F, A Ruminococcus albus CU, HC A, F, E, H2 Ruminococcus flavefaciens CU, HC S, F, A, H2 Eubacterium ruminantium HC, DX, SU A, F, B, L Ruminobacter amylophilus ST S, F, A, E Streptococcus bovis ST, SU L, A, F, E Succinomonas amylolytica ST S, A, P Prevotella ruminocola, albensis, brevis and bryantii

ST, PC, XY, SU S, A, F, P

Butyrivibrio fibrisolvens ST, CU, HC, PC, SU B, F, A, H2 Selenomonas ruminantium ST, DX, SU, L, S L, A, P, B, F, H2 Megasphaera elsdenii L, SU P, A, B, Br, H2 Lachnospira multiparus PC, SU L, A, F, H2 Succinivibrio dextrinosolvens PC, DX, SU S, A, F, L Anaerovibrio lipolytica GL, SU A, S, P Peptostreptococcus anaerobius AA Br, A Clostridium aminophilum AA A, B Clostridium sticklandii AA A, Br, B, P Wolinella succinogenes OA, H2, F S Methanobrevibacter ruminantium H2, CO2, F CH4

Abbreviations are as follows: CU, cellulose; HC, hemicellulose; DX, dextrins; SU, sugars; ST, starch;

PC, pectin; XY, xylans; L, lactate; S, succinate; GL, glycerol; AA, amino acids; OA, organic acids; H2,

hydrogen; F, formate; CO2, carbon dioxide; A, acetate; E, ethanol; B, butyrate; L, lactate; P,

propionate; Br, branched-chain volatile fatty acids; CH4, methane (Russell and Rychlik 2001)

2.2.3. Importance of ruminal microorganisms for fibre digestion

Ruminal cellulolytic microorganisms are very important in ruminal digestion, since mammals

are not able to produce enzymes to digest cellulose (Russell and Rychlik 2001; Russell and

16

Wilson 1996). Different species of bacteria, protozoa and fungi contribute to the degradation

of cell wall components (Tab. 2) (Krause et al. 2003; Wang and McAllister 2002; Zhang et al.

2007). In the rumen several enzymes such as cellulase, xylanases, β-glucanases, pectinase,

amylases, proteases, phytases and even enzymes that degrade plant toxins (Wang and

McAllister 2002) are produced by various microorganisms (Krause et al. 2003). Individual

ruminal microorganisms are able to produce several different enzymes that degrade different

plant cell wall components (Krause et al. 2003). Most of the ruminal bacterial species that

utilise polysaccharides, like starch or cellulose, can also utilise mono- and disaccharides

such as glucose, cellobiose, xylose, sucrose, fructose and others (Weimer 1996).

Microbial enzymes work synergistically in the rumen to completely hydrolyse plant cell

components. For example, for digesting cellulose, multiple enzymes such as endonucleases,

exonucleases and β-glucosidases exist in the rumen (Krause et al. 2003). For other cell wall

components, other specific enzymes are needed, for example cellobiose is hydrolysed by

β-1,4-glucosidase, while xylan is hydrolysed by endo-β-1,4-xylanase (Wang and McAllister

2002). Among fibrolytic species, Fibrobacter succinogenes, Ruminococcus albus and

Ruminococcus flavefaciens are the most important ones in the rumen (Krause et al. 2003).

They are able to degrade cellulose, hemicelluloses and pectin (Tab. 2) (Wang and McAllister

2002). Other important bacteria species are Butyrivibrio fibrisolvens and Prevotella spp. who

produce xylanolytic enzymes (Krause et al. 2003).

The ruminal protozoa also contribute to the degradation of plant cell walls, as they produce a

wide range of enzymes like cellulase, xylanase, xylodextrinase, arabinofuranosidase,

pectinesterase, polygalacturonase and endopectate lyase (Orpin 1984). Fungi, like

Neocallimastix patriciarum and Piromyces communis are also important players in ruminal

fibre digestion because fungi help to fragment the plant tissue and are able to degrade a

great variety of cell wall components, mainly cellulose, hemicellulose, pectin and lignin (Orpin

1984). However, they have a slower degradation process compared to bacteria (Krause et al.

2003).

17

Tab. 2: Identity and enzyme activities of ruminal microbes involved with degradation of plant cell walls

in the rumen (Wang and McAllister 2002)

Degradative activity Organism Cellulolytic Hemicellulolytic Pectinolytic Bacteria Fibrobacter succinogenes + + + Ruminococcus albus + + + Ruminococcus flavefaciens + + + Butyrivibrio fibrisolvens + + + Eubacterium cellulosolvens + + Clostridium longisporum + Clostridium locheadii + + Prevotella ruminantium + + Eubacterium xylanophilum + Ruminobacter amylophilus + Succinimonas amylolytica + Succinivibrio dextrinosolvens + Selenomonas ruminantium + Selenomonas lactilytica + Lachnospira multiparus + + Streptococcus bovis + + Megasphaera elsdenii +

Protozoa Eudiplodinium maggii + + + Ostracodinium dilobum + + + Epidinium caudatum + + Metadinium affine + + + Eudiplodinium bovis + + + Orphryoscolex caudatus + + + Polyplastron multivesiculatum + + + Diplodinium pentacanthum + Endoploplastron triloricatum + Orphyroscolex tricoronatus + Ostracodinium gracile + Entodinium caudatum + + Isotricha intestinalis + + + Isotricha prostoma + + +

Fungi Neocallimastix frontalis + + + Neocallimastix patriciarum + + + Neocallimastix joyonii + + Caecomyces communis + + + Piromyces communis + + + Orpinomyces bovis + + Ruminomyces elegans + +

18

Because of fibre’s physical and chemical characteristics nutrients can only be digested to

certain extents (Weimer 1996). As a result of lignification, approximately 10–40 % of

cellulose and hemicellulose are not digestible by ruminal microorganisms (Moreira et al.

2013). The number of fibre degrading microorganisms and the surface for attachment of

microorganisms and their enzymes also affect the rate of fibre digestion (Allen and Mertens

1988). Microbial attachment is crucial for fibre digestion (Allen and Mertens 1988) because it

enables microorganisms to remain longer in the rumen and facilitates the hydrolysis of cell

wall components, which requires contact between the enzyme and the feed particles (Wang

and McAllister 2002).

2.2.4. Regulation of ruminal pH

Over the course of the day ruminal pH changes within a physiological range of 5.5 to 7.0

(Krause and Oetzel 2006) in response to eating, rumination, ruminal digestion and SCFA

absorption (Dohme et al. 2008). After feeding, ingested feed materials are fermented in the

rumen which usually takes place at a pH lower than 7 (i.e., the physiological pH range)

(Aschenbach et al. 2011). Ruminal fermentation of carbohydrates and proteins produces

SCFA and branched-chain SCFA, respectively (Allen 1997; Dijkstra et al. 1993; Dijkstra

1994, 1994), and protons (Aschenbach et al. 2011) which cause ruminal pH to decline

(Dijkstra et al. 2012). However, different SCFA are derived from the fermentation process,

the major SCFA being produced in the rumen are acetate, propionate and butyrate (Dijkstra

1994). Acetate is the most abundant SCFA produced in the rumen and the rate of absorption

is different for acetate, propionate and butyrate depending on their lipophilicity (Allen 1997).

Further acids that are produced during the fermentation process are isobutyrate, valerate,

isovalerate and others (Dijkstra 1994; Zhang et al. 2007).

Ruminal pH can be kept within the physiological range by absorption, passage and

increasing neutralisation of SCFA (Allen 1997). Neutralisation can be achieved by ruminal

buffering using buffer systems like the carbonate system, the phosphate system, other

organic salts, ammonia, feed ingredients, proteins and, as mentioned before, the SCFA

themselves (Aschenbach et al. 2011). The SCFA can bind and release H+ depending on the

present ruminal pH values (Aschenbach et al. 2011). At a ruminal pH below 5.5 the SCFA

are mainly found in their protonated form, reducing H+ concentration in the rumen (Krause

19

and Oetzel 2006). The protonated SCFA (i.e., undissociated form) can be absorbed

passively by the ruminal epithelium (Aschenbach et al. 2011; Krause and Oetzel 2006), while

dissociated SCFA can be absorbed by the ruminal epithelium in an exchange with

bicarbonate (Aschenbach et al. 2011). At low pH values (pH = 4.5) the absorption rates of

the ruminal SCFA increase, particularly for butyrate and propionate (Dijkstra et al. 1993).

This does not seem to be true for lactate, which accumulates in the rumen at low pH (Krause

and Oetzel 2006). Besides absorption, SCFA can be removed by passage from the rumen to

the omasum (Allen 1997; Aschenbach et al. 2011; Dijkstra et al. 1993), even if much of the

SCFA are already absorbed in the rumen (Aschenbach et al. 2011).

Saliva plays a crucial role in ruminal buffering because with saliva bicarbonate and hydrogen

phosphate ions are secreted into the rumen (Allen 1997). Bicarbonate does not only reach

the rumen via saliva, but it is also secreted by the ruminal epithelium due to bicarbonate

dependent uptake of SCFA in the ruminal cells (Aschenbach et al. 2011; Dijkstra et al. 2012).

In addition, hydrogen ions are incorporated into water and the carbon dioxide, which is

formed by dehydration of carbonate is then expelled by eructation of the cow (Allen 1997).

Ammonia, which is produced during the digestion process of proteins, is also able to buffer

ruminal pH (Dijkstra et al. 2012). The originating ammonia can be absorbed as ammonium

through the epithelial rumen wall or be absorbed as ammonia, however, this majorly takes

place at a pH above 7 (Aschenbach et al. 2011).

2.2.5. Effects of pH decline on ruminal fibre digestion

Depression of fibre degradation is one of the major consequences with respect to pH decline

in the rumen. Low ruminal pH, especially if the pH depression lasts for a longer time, affects

the host (e.g., feed intake), the microbial metabolism (Nocek 1997), the nutrient degradation

of feeds (Calsamiglia et al. 2002; Sung et al. 2006) and the ruminal ecosystem (Zebeli and

Metzler-Zebeli 2012). The ruminal microbial ecosystem shows alterations in its composition

during SARA (Zebeli and Metzler-Zebeli 2012), however, it is affected in different ways

depending on the severity of SARA and on how SARA is induced (Khafipour et al. 2009b).

Fibre digestion is decreased at a low pH (Allen and Mertens 1988; Calsamiglia et al. 2008;

Dijkstra et al. 2012; Sung et al. 2006), especially when the low pH is maintained for a long

period of time (Calsamiglia et al. 2002). This is the outcome of a reduced number of fibre

20

digesting bacteria that are vulnerable to changes in pH, especially to reduced ruminal pH

(Sung et al. 2006). For instance, an in vitro study showed that at a pH below 6.0 hardly any

growth of the cellulolytic bacterium F. succinogenes was observed (Russell and Wilson

1996). Another in vitro study indicated that at a pH of 5.7 the growth of the important fibrolytic

bacteria species F. succinogenes, R. flavefaciens, R. albus was reduced (Fig. 6) (Sung et al.

2006). The decline in fibrolytic bacteria at low pH is caused by a reduced attachment to fibre,

by a higher energy use that is necessary for the bacteria to persists at reduced pH and by

altered microbial metabolism (Allen and Mertens 1988; Sung et al. 2006).

Fig. 6: The relationship between initial pH, DM digestibility and bacterial attachment (Sung et al. 2006)

Not only fibre digestibility decreases at a low ruminal pH, digestibility of other nutrients may

also be reduced. This was supported by the finding that DM digestibility decreased with

decreasing pH (Fig. 6) (Sung et al. 2006). Also a reduction of truly digested organic matter

has been recognised at a low pH (mean pH of 5.95) (Calsamiglia et al. 2008). The reduced

digestibility of fibre at low pH values leads to nutrients remaining in the undigested fibre

passing the gastrointestinal tract with the ruminant not being able to absorb these (Dijkstra et

al. 2012).

21

2.2.6. Particle size, effectiveness of fibre and the importance for rumen health

Not only the amount of dietary fibre but also the physical form of the fibre contained in diets

are essential to maintain proper ruminal function, animal health and the composition of milk

(Mertens 1997; Zebeli et al. 2006). Large feed particles are more physically effective

because large particles promote chewing, rumination and salivary buffer secretion (Mertens

1997) which consequently reduces the acid production in the rumen (Allen 1997; Mertens

1997). Therefore, providing an adequate content of physically effective fibre in diets is

important to reduce the incidence of SARA (Dohme et al. 2008; Plaizier et al. 2008; Yang

and Beauchemin 2009; Zebeli et al. 2010; Zebeli et al. 2012). Increased saliva secretion

while eating forages is the reaction to forage’s physical and chemical characteristics, which

lead to cows having a slower eating rate and making single meals last longer (Beauchemin

et al. 2008).

Moreover, particle length is necessary for rumen physiology because of its positive influence

on ruminal contractions and rumen motility (Yang and Beauchemin 2007a, 2009; Zebeli et al.

2006). By increasing particle size the consistence of the ruminal mat is improved (Zebeli et

al. 2007). On the contrary, fine particles having a low physically effectiveness tend to leave

the rumen earlier and rumen motility is decreased (Zebeli et al. 2012). Furthermore, when

there is too little fibre and the rumen becomes stagnant due to insufficient contractions of the

rumen, the absorption and passage of SCFA is decreased, putting cows at a higher risk for

ruminal acidosis (Yang and Beauchemin 2007a).

The content of physically effective fibre in diets can be altered by changing the particle size

of forage and changing diet’s forage to concentrate ratio. The diet’s alterations will change

particle distribution of the digestive content. Teimouri Yansari et al. (2007) showed that the

proportion of small particles in rumen digesta increased with decreasing particle size of

forage accomplished by chopping process. The authors also observed that the proportion of

large (≥ 4.00 mm) and medium (< 4.00 mm and ≥ 1.18 mm) particles decreased while the

proportion of small particles (< 1.18 mm and ≥ 0.05 mm) increased with time after feeding

which indicates a high breakdown of forage particles in the reticulorumen (Teimouri Yansari

et al. 2007). Consistently, Maulfair et al. (2011) reported an increase of coarse rumen digesta

particles (> 3.5 mm) with increasing ration particle size. Increasing concentrate amount in the

diet could negatively affect chewing activity, ruminal solid passage rate and fibre digestibility

and therefore increase mean particle length and proportion of large particle of rumen digesta

22

(Tafaj et al. 2005). Because particles above 1.18 mm cannot pass through the reticulo-

omasal orifice, it is believed to be the critical particle size for ruminal passage in cattle

(Oshita et al. 2004). A study by Maulfair et al. (2011) suggest a larger size than 1.18 mm to

be the critical particle size for modern dairy cows. Supporting this notion, the proportion of

rumen digesta particle retaining on the sieve with 1.18 mm mesh size did not well reflect a

reduction of silage particle, but the proportion of very large particles did (> 6.7 mm) (Kononoff

and Heinrichs 2003a).

Tafaj et al. (2005) demonstrate that decreasing mean particle length and of large (> 1.0 mm)

to small (< 1.0 mm) particle ratio in the digestive content can be interpreted as a result of an

increased fibre digestion in the rumen. However, studies investigating the influence of the

effectiveness of dietary fibre on digestibilities are not conclusive. For instance, some studies

show that increasing particle length of chopped forages increased the fibre digestibilities

(Tafaj et al. 2001; Yang and Beauchemin 2007b). Whereas, other workers report no effect

(Kononoff and Heinrichs 2003a; Zebeli et al. 2007) or an adverse effect of increased particle

length on nutrient digestibilities in total tract (Yang and Beauchemin 2006). Presumably, the

negative effect could be due to a decreased area which is available for microbial attachment

(Yang and Beauchemin 2006) but the authors, however, did not determine the digesta

particle lengths. Zebeli et al. (2012) states that by a slight decrease of fibre particle length in

the diet fibre digestion can be stimulated while sorting of cows against NDF in diet can be

reduced at the same time, and thus the risk for ruminal acidosis is decreased.

The impacts of an increased forage to concentrate ratio on total tract fibre digestibility are

also inconclusive, some studies reported a positive effect (Tafaj et al. 2001; Tafaj et al. 2006;

Yang and Beauchemin 2007b) or no effect (Zebeli et al. 2007). In line with that, there is a

discrepancy between studies regarding effects of concentrate levels on rumen particle size

and distribution (e.g., Tafaj et al. 2004; Tafaj et al. 2005). The diet formulation and

interactions between different nutrients make studies concerning particle distribution difficult

to compare (Maulfair et al. 2011). In addition, it must be underlined that effects of forage to

concentrate ratio on ruminal digestion and on particle distribution in the digestive content can

be associated with factors beyond the level of concentrate in the diet. For example, the effect

of concentrate level may depend also on the quality of forages fed along (Tafaj et al. 2005).

Tafaj et al. (2005) showed that feeding of low quality hay (high-fibre hay) combined with a

high concentrate diet (50 % of diet DM) decreased ruminal microbial digestion and

considerably increased accumulation of large particles in rumen digesta and faeces. The

23

same concentrate level but combined with high quality hay (low-fibre hay) partly alleviated

the negative effects, while a high quality hay and a low concentrate diet (20 % of diet DM)

provided even better ruminal conditions for fibre digestion. Furthermore, Tafaj et al. (2006)

demonstrated that the efficiency of particle communition is determined by correlating

processes as chewing, ruminal fermentation and other digestive factors like the quality of

rumen digesta stratification and ruminal passage rate, especially when a high concentrate

diet is fed to ruminants. When particle distribution in the rumen is altered, the formation of the

ruminal mat can also be changed because larger particles remaining in the rumen contribute

to an enhanced ruminal mat formation (Teimouri Yansari et al. 2004). The ruminal mat is

responsible for feed particle retention (Zebeli et al. 2012) and feed particles need to be

comminuted to a certain extent (i.e., critical size for ruminal escape) to be able to pass from

the rumen to the omasum (Maulfair et al. 2011; Oshita et al. 2004). On the one hand, large

particles stimulate ruminal mat formation, rumen contraction and motility, and chewing and

rumination of the animal. On the other hand, as a result of larger particles retaining in the

rumen, rumen fill may be prolonged, and, further, the filling effect will limit dry matter intake

(Allen 2000; Zebeli et al. 2006). Consequently, this could lead to a decreased animal

production (Allen 2000). In diet formulation the oppositional characteristics of fibre regarding

maintenance of ruminal pH and reduction of DMI have to be considered (Zebeli et al. 2012).

Summing up we can say that increasing fibre content of diets and the effectiveness of fibre

can reduce the risk for acidosis (Yang and Beauchemin 2007a; Zebeli et al. 2010).

Alterations of diet composition (i.e., forage to concentrate ratio), forage quality and forage

chop length can influence the functions of cows’ digestive tract and therefore cows’ health

and nutrient utilisation (Mertens 1997; Nocek 1997). Diets, which do not meet cows’ optimal

requirements for fibre, can lead to an altered rumen particle distribution and changes in

rumen particle size (Maulfair et al. 2011; Tafaj et al. 2005; Teimouri Yansari et al. 2007).

During SARA, being caused by such diets, fibre digestibility is usually reduced (Allen and

Mertens 1988; Calsamiglia et al. 2008) which may result in larger particles remaining a

longer time in the rumen as they would normally. SARA is often accompanied by reduced

feed intake (Kleen et al. 2003; Nocek 1997; Plaizier et al. 2008) which may be attributed by

changes in decreased ruminal digestion and passage rate and increased rumen fill as

described above. If the particle communition is reduced attention has to be paid to a reduced

utilisation of nutrients which subsequently afflicts animal health and production. Changes in

24

particle size therefore deliver information about the ruminal particle breakdown and a proper

function of the digestive tract and hints about diseases like SARA.

25

3. Materials and methods

3.1. Cows, feeding and SARA challenge The feeding and sampling experiment was conducted at the research dairy farm

“Kremesberg” of Vetmeduni Vienna, Austria. Handling and treatment procedures applied to

animals were approved by the institutional ethics committee of the Vetmeduni Vienna and

the national authority according to §26ff of the Law for Animal Experiments,

Tierversuchsgesetz 2012-TVG (GZ 68.205/0093-II/3b/2013). Eight non-lactating Holstein

cows that were ruminally cannulated (100 mm i. d.; Bar Diamond Inc., USA) with an initial

body weight of 710 ± 118 kg were kept in one group in a loose-housing stable with straw

bedding. The experiment was carried out in two consecutive periods each one lasting 34

days (six days gradual adaptation, 28 days of SARA challenge). In between the two runs a

break of eight weeks was conducted to allow cows to recover from the high-concentrate diet

they were given before. At the start of the experiment the cows were at baseline (BASE) and

were fed a forage mix for two to three weeks, followed by six days of adaption during which

the concentrate ratio in the diet was increased daily by 10 %, starting at 0 % up to 60 % (DM

basis). In each run, after the adaptation a 60 % concentrate diet was fed to cows to induce

SARA for 28 days. During the challenge period four cows were induced transiently (they

were off concentrate in the second week) while the other four cows were induced

continuously. In the next run, cows were swapped for the challenge method. For all cows and

each run rumen digesta were sampled at baseline, after seven days of the challenge

(SARA1) and on the last day of challenge (SARA2) (Fig. 7) for the analysis of particle

distribution (more details see 3.4.).

SARA challenge period

BASE

day -7 day 1 day 7 day 14 day 21 day 28 Sampling Points

SARA1 SARA2

baseline

adaption

26

Fig. 7: Timeline of SARA challenging-experiment and sampling points

The forage mix fed during no challenge periods consisted of 50.0 % grass silage and 50.0 %

second-cut meadow hay (DM basis). The SARA challenge diet was composed of 20.0 %

grass silage, 20.0 % second-cut meadow hay, 19.8 % barley grain, 18.0 % wheat, 10.2 %

rapeseed meal, 9.0 % corn, 1.9 % dried beet pulp, 0.6 % mineral-vitamin premix, 0.3 %

calcium carbonate and 0.2 % sodium chloride (DM basis). Chemical composition of the

forage mix and the SARA challenge diet is shown in Tab. 3.

Tab. 3: Chemical composition of the SARA challenge diet and the forage mix (DM basis)

Diet composition Forage mix SARA challenge diet

% of DM % of DM

DM 54.4 74.5

OM 91.6 94.1

Crude protein 12.8 15.4

NDF 51.7 31.8

ADF 36.2 19.9

Ether extract 1.50 1.71

Ash 8.36 5.86

NFC1 25.6 45.2 1 NFC = 100 – (% ash + % crude protein + % NDF + % ether extract)

During baseline and until the first four days of the adaption period, the cows were fed 1.5 %

(DM basis) of their body weight. Thereafter, the diet was offered at 2.0 % of cows’ body

weight. The forage mix and the concentrate diets were offered separately. The cows had

access to the forage mix from 08:00 a.m. on, whereas the concentrate diet was offered from

10:00 a.m. on. Fresh water was provided ad libitum. A salt licking stone was provided

throughout the experiment.

27

3.2. Measurement of ruminal pH

To measure the pH continuously, the cows were equipped with an indwelling wireless

ruminal pH sensor (smaXtec Heat, Health & Feed Management, smaXtec animal care sales

GmbH, Austria) one week before the experiment started. For each of the two experimental

periods new sensors were used because their maximal guaranteed working time was 50

days. These sensors were put into the ventral rumen via the rumen cannula. Before use,

they were calibrated as the company’s instruction protocol recommended by a calibration

buffer of pH 7.0. The data of pH which were measured every ten minutes were transmitted in

real time to a base station via three antennas installed in the barn. For each period (BASE,

SARA1 and SARA2), representative pH data of the last five days were used for statistical

analysis.

3.3. Measurements of dry matter intake and water intake

Daily data of feed and water intake were electronically measured. To control distribution and

individual feed intake of the cows the feeding troughs were equipped with electronic weighing

scales and computer-regulated access gates (RIC system, Insentec B.V., The Netherlands).

Depending on the amount of daily forage consumption, the corresponding unconsumed

concentrate, which was checked twice a day, was given through the rumen cannula, to keep

the forage and concentrate ratio of DMI constant. During the four weeks of SARA challenge,

the concentrate amount that had to be delivered via the cannula was an average of 2.48 ±

3.66 kg DM, accounting approximately for 30 % of daily total concentrate intake. Water

consumption of individual cows was measured through a computer-regulated water trough

(RIC system, Insentec B.V., The Netherlands). Fresh feed was provided throughout the day.

Like pH data, the data of the last five days of each period were statistically analysed.

3.4. Rumen sampling and wet-sieving method

28

As mentioned before, rumen samples were taken for three different periods: Baseline

(BASE), short-term SARA (SARA1) and long-term SARA (SARA2). Samples for BASE were

taken before the adaption period (day -7), samples for SARA1 were taken in the first week

after SARA induction (day 7). Samples for SARA2 were taken in the fourth week of SARA

(day 28) (Fig. 7). The rumen sampling was conducted at different hours (0 h, 4 h, 8 h) after

morning feeding. Through the rumen cannula approximately 200 g of solid digesta from the

ruminal mat were manually taken, squeezed and were immediately stored at −20 °C until

wet-sieving analysis. Prior to wet-sieving analysis, the rumen digesta samples were

defrosted. Each sample was analysed in duplicate. For the wet-sieving 40 g of each sample

were treated with 400 ml of distillated water for 30 min at room temperature. Afterwards, the

sample was processed using an analytic sieve shaker (Vibratory sieve shaker AS 200 digit 2,

Retsch Technology GmbH, Germany), which contained seven sieves with decreasing mesh

size, beginning at 5 mm followed by 4 mm, 2 mm, 1.18 mm, 0.5 mm, 0.15 mm and ending

with a mesh size of 0.063 mm. Particles escaping the sieve with a mesh size of 0.063 mm

were addressed as the soluble fraction. The analytic sieve shaker was set with a running

time of 12 min at a shaking frequency of 5 shakes/min. The water flowed through the

separator with a constant flow speed of 1.5—2.0 L/min and the particles separated via the

vertical oscillating wet-sieving technique. The particles remaining on each of the sieves were

removed and the content was then washed with water and filtered through a pre-weighed

filter paper (General use filter paper DP 400 185, Hahnemühle FineArt GmbH, Germany).

The filters containing the particles retained on the different sieves of the analytic sieve shaker

were put into an oven (Universal Wärmeschrank UFE, Memmert GmbH + Co.KG, Germany)

for 48 h at 65 °C and were weighed thereafter. Subsequently, they were put into an oven

again (Brutschrank, Memmert GmbH + Co.KG, Germany) for 24 h at 100 °C and were

weighed again. To estimate the weight of the contents the filters’ empty weight was

subtracted from the weight of the filters containing the dried rumen particles. Also the soluble

portion was calculated from the difference between the initial amount and the sum of retained

amounts on all sieves.

Simultaneously, another portion of the rumen digesta samples was used to measure the DM

content. To do so, 30 g of the defrosted sample were put into petri dishes, which were

weighed before. The petri dishes containing the samples then were put into an oven

(Universal Wärmeschrank UFE, Memmert GmbH + Co.KG, Germany) for 48 h at 65 °C. After

29

48 h the samples were weighed and put into another oven (Brutschrank, Memmert GmbH +

Co.KG, Germany) for 12 h at 100 °C. Afterwards the samples were weighed again.

30

3.5. Calculations and statistics Percentage of the particle fractions retained on each sieve was calculated based on DM

basis. Subsequently, cumulative fractions were calculated, meaning the sum of the fraction

with those of larger fractions (e.g., cumulative fraction of sieve 2 mm (%) = % sieve 2 mm +

% sieve 4 mm + % sieve 5 mm). Therefore, the soluble fraction always yielded 100 %.

Afterwards, the size of the sieve and the cumulative fractions were used to estimate mean

particle length of the rumen digesta (Fisher et al. 1988).

Pre-evaluation of the particle distribution data showed no large difference between the

challenge methods (transient versus continuous), therefore their data were pooled together

and SARA challenge (BASE, SARA1, SARA2), time relative to feeding (0 h, 4 h and 8 h) and

their interaction and experimental run were included as fixed effects in the statistical model

while cows were considered as a random effect. The data of intake and pH (also pooled

between two challenge models) were analysed considering fixed effects of SARA challenge

and experimental run, a random effect of the cow and repeated measures of measurement

days. Superscripts were given to the least square means to underline their differences. The

significant level was deemed at P < 0.05 according to the Tukey’s method.

31

4. Results

4.1. Intake of dry matter, concentrate and water

Dry matter intake (DMI), concentrate intake and water intake are shown in Tab. 4. The intake

of DM (kg/day) was significantly increased (P < 0.001) when SARA was induced using the

60 % concentrate diet. During BASE DMI was about 10.97 kg/day, which significantly

increased by 37.01 % for SARA1 (15.03 kg/day) and by 47.22 % for SARA2 (16.15 kg/day).

However, regarding DMI there was no significant difference between SARA1 and SARA2. As

expected, when SARA was induced the concentrate intake (kg/day) significantly increased

(P < 0.001). The concentrate intake showed significantly differences between SARA1 and

SARA2 (+ 1.04 kg, P < 0.05). The water intake (kg/day) was not affected and cows had on

average 48.83 kg of daily water consumption (Tab. 4).

Tab. 4: Feed and water intake as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

Item BASE SARA1 SARA2 SEM P value

Dry matter intake, kg/day 10.97b 15.03a 16.15a 0.476 < 0.001

Concentrate intake, kg DM/day 0.20c 8.91b 9.95a 0.260 < 0.001

Water intake, kg/day 47.3 48.3 50.9 3.21 0.722 Uncommon superscripts indicate significant differences among means (P < 0.05).

4.2. Ruminal pH

Results regarding ruminal pH profile in response to SARA challenge are shown in Tab. 5.

There was an effect of SARA recognised on both, mean ruminal pH (P < 0.001) and time of

pH depression (pH < 5.8) (P < 0.001). Before SARA was induced mean ruminal pH during

BASE was about 6.42, which was significantly higher than mean ruminal pH during SARA1

(P < 0.05) and also during SARA2 (P < 0.05). However, mean ruminal pH decreased slightly

from SARA1 (6.10) to SARA2 (5.97), but no significant difference between SARA1 and

SARA2 was observed.

32

In addition to the pH depression described above, the duration of pH depression (pH < 5.8)

expressed as minutes/day was also affected by SARA (P < 0.001). There was a linear

increase in the duration of the pH depression with feeding periods. Initially, during BASE the

pH depression lasted only for 0.25 min/day, while the duration increased to 236.8 min/day

during SARA1 and to 467.0 min/day during SARA2, leading to their significant differences as

indicated by different superscripts (P < 0.05).

Tab. 5: Ruminal pH changes as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

Item BASE SARA1 SARA2 SEM P value

Mean pH 6.42a 6.10b 5.97b 0.047 < 0.001

Time (pH < 5.8), min/day

0.25c 236.8b 467.0a 52.57 < 0.001 Uncommon superscripts indicate significant differences among means (P < 0.05).

4.3. Rumen digesta particle distribution

4.3.1. Mean particle length

Changes in mean particle length (mm) in response to morning feeding (i.e., time effect) and

SARA are illustrated in Fig. 8. There was no effect of SARA found (P = 0.361), but there was

a time effect (P < 0.001) and interaction between SARA and time (P = 0.007) detected.

During BASE mean particle length increased significantly from 1.36 mm before morning

feeding (0 h) and showed 2.35-fold higher values at the sampling point of 4 h and 2.44-fold

higher values at 8 h after morning feeding. The difference between 4 h and 8 h was not

significant. Overall, mean particle lengths during SARA1 and SARA2 were constant over

feeding time, ranging from 2.15–2.69 mm.

As indicated by the significant interaction, there was a clear difference in mean particle

length between BASE and both SARA periods at 0 h (i.e., before morning feeding) but not at

later hours. At 0 h relative to morning feeding the mean particle length of BASE was 1.36 mm

while mean particle length at 0 h for SARA1 and SARA2 was 58.09 % and 61.76 % larger,

respectively (P < 0.05). At the sampling point of 4 h the mean particle lengths for SARA1 and

SARA2 were 15.67 % and 27.27 %, respectively, slightly smaller than for BASE. At 8 h

33

relative to morning feeding the mean particle length decreased from 3.32 mm during BASE

by 20.48 % and by 26.81 % for SARA1 and SARA2, respectively. However, the differences

found at 4 h and 8 h relative to morning feeding did not reach statistical significance.

4.3.2. The large particle fraction (> 2 mm)

The large particle fraction, expressed as the sum of particles larger than 2 mm (% of total

particle fraction), showed a time effect (P < 0.002) and time x SARA interaction (P = 0.007)

but no SARA effect (P = 0.313) (Fig. 9). The percentage of the large particle fraction of BASE

increased significantly from 26.04 % before feeding (0 h) to 38.13 % at 4 h after feeding and

stayed constant until 8 h after feeding (39.19 %). With slight variations, the large particle

fraction during SARA1 and SARA2 did not response to feeding time. Accordingly, at 0 h

relative to morning feeding the large particle accounted for about 31.23 % and 31.24 % of

total particle fractions of SARA1 and SARA2, respectively, making SARA had 19.93 % and

1,36

2,15

2,20

3,19

2,69

2,32

3,32

2,64

2,43

00,5

11,5

22,5

33,5

4

BASE SARA1 SARA2

Mea

n pa

rtic

le le

ngth

(mm

)

Feeding period

Mean particle length

P values SARA Time Interaction

0.361 < 0.001 0.007

0 h

4 h

8 h

Fig. 8: Mean particle length (mm) as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

34

19.97 % more large particle fraction than BASE before feeding (P < 0.001). After feeding, the

large particle fractions of SARA1 and SARA2 were about 35.58 % and 32.72 %, respectively,

at 4 h after feeding, and 34.61 % and 33.06 %, respectively, at 8 h after feeding. As opposed

to before feeding, the contents of this particle fraction found with SARA1 after 4 h and 8 h

after feeding were in a similar range as BASE as the proportion of the large particle was

slightly decreased by both SARA periods.

Fig. 9: Content of large particles (> 2 mm) as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

26,0

4 31,2

3

31,2

4 38,1

3

35,5

8

32,7

2 39,1

9

34,6

1

33,0

6

0

5

10

15

20

25

30

35

40

45

BASE SARA1 SARA2

% o

f lar

ge p

artic

les

(> 2

mm

)

Feeding period

Content of large particles (> 2 mm)

P values SARA Time Interaction

0.313 < 0.002 0.007

0 h

4 h

8 h

35

4.3.3. The medium particle fraction (1.18—2.00 mm)

For the medium particle fraction of 1.18—2.00 mm, expressed in % of total particle fraction, a

significant effect of SARA was found (P < 0.001) (Fig. 10). This particle fraction showed no

significant time effect (P = 0.458) and only a tendency for interaction between SARA and

time relative to feeding (P = 0.103). The percentages of this fraction of SARA1 and SARA2

were always greater than that of BASE. Before feeding, the content of this particle fraction of

BASE was 3.04 % but SARA1 and SARA2 had 5.14 % and 5.91 %, respectively. At 4 h

relative to morning feeding, the contents of this fraction found with SARA1 and SARA2 were

1.41- and 1.27-fold of BASE, respectively. At 8 h relative to feeding SARA1 and SARA2 were

1.10- and 1.33-fold of BASE, respectively. As a result, the average content of this particle

fraction over feeding hours significantly increased from BASE (3.48 %) to SARA1 (4.82 %)

and SARA2 (5.19 %). However, the increase from SARA1 to SARA2 was not significant

(P > 0.05). A reduction of this particle fraction in response to feeding hours seemed to be

related to the SARA challenge.

Fig. 10: Content of medium particles (1.18–2.00 mm) as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

3,04

5,14

5,91

3,72

5,25

4,74

3,69

4,07

4,92

3,48

4,82

5,19

0

1

2

3

4

5

6

7

8

BASE SARA1 SARA2

% o

f med

ium

par

ticle

s (1

.18–

2.00

mm

)

Feeding period

Content of medium particles (1.18–2.00 mm)

P values SARA Time Interaction

< 0.001 0.458 0.103

0 h

4 h

8 h

Average

36

4.3.4. The small particle fraction (< 1.18 mm)

The content of the small particles (the sum of particles < 1.18 mm, soluble fraction was

excluded), expressed as % of total particle fraction, was affected by SARA (P = 0.002) and

time (P < 0.001), while there was no interaction between SARA and time (P = 0.663). On

average across feeding hours, the content of the small particle fraction was lowest in SARA2

(32.70 %) and highest for BASE (36.70 %) while SARA1 (34.25 %) was intermediate (Fig.

11). According to Turkey’s test, SARA2 showed a significant decrease compared with BASE

(P < 0.05), while SARA1 was not significant with BASE or with SARA2.

In general, there was a decrease of the content of the small particle fraction with feeding

hours. With respect to the significant time effect, on average of all periods, at 0 h relative to

feeding this small particle fraction accounted for 38.93 % of total fraction, while it significantly

decreased to 33.19 % and 31.54 % at 4 h and 8 h after feeding, respectively (P < 0.05).

Fig. 11: Content of small particles (< 1.18 mm) as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

41,3

6

37,4

9

37,9

3

35,1

7

32,5

8

31,8

1

33,5

7

[WER

T]0

28,3

6

[WER

T]0

34,2

5

[WER

T]0

0

5

10

15

20

25

30

35

40

45

BASE SARA1 SARA2

% o

f sm

all p

artic

les

(< 1

.18

mm

)

Feeding period

Content of small particles (< 1.18 mm)

P values SARA Time Interaction

0.002 < 0.001 0.663

0 h

4 h

8 h

Average

37

4.3.5. The soluble fraction (< 0.063 mm)

The soluble fraction (i.e., particles < 0.063 mm) expressed in % of total particle fraction was

significantly affected by SARA (P = 0.016, Fig. 12) and showed a significant interaction of

SARA and time (P = 0.002), while there was no time effect (P = 0.521). On average across

feeding hours, the soluble fraction significantly increased due to the effect of SARA, so that

SARA2 resulted in approximately 17.66 % higher soluble fraction compared with BASE

(25.37 %, P < 0.05), while SARA1 showed an intermediate value (27.13 %).

As indicated by the significant interaction, the changes in the soluble fraction in relation to

feeding hours depended on SARA challenge. Accordingly, BASE showed a pattern of

decrease soluble fraction with feeding hours and that the 8 h value was 79.63 % of 0 h value.

By contrast, SARA2 and, to a lesser extent, SARA1 showed a pattern of increase and that

the 8 h value accounted for 1.35-fold and 1.10-fold of the 0 h value, respectively.

Fig. 12: Soluble fraction (< 0.063 mm) as affected by SARA challenge (BASE = no SARA, SARA1 = 1-week challenge, SARA2 = 4-week challenge) and time relative to feeding

29,5

6

26,1

5

[WER

T]0

23,0

2 26,5

9 30,9

8

23,5

4 28,6

4 33,6

7

25,3

7

27,1

3

29,8

5

0

5

10

15

20

25

30

35

40

BASE SARA1 SARA2% s

olub

le fr

actio

n (<

0.0

63 m

m)

Feeding period

Soluble fraction (< 0.063 mm)

P values SARA Time Interaction

0.016 0.521 0.002

0 h

4 h

8 h

Average

38

5. Discussion

5.1. Dry matter, concentrate and water intake

In this study SARA was induced by feeding a high concentrate diet (60 % of diet DM) while

during BASE the cows received only a forage-mix. It was obvious that the intake of DM

(kg/day) was significantly increased when SARA was induced due to increased concentrate

intake at the expense of the forage intake. The lower DMI during BASE could be the result of

the forage-mix containing more forage and therefore more NDF (51.7 % of DM) compared to

the SARA challenge diet (31.8 % of DM) and of the lower feed allowance at 1.5 % of body

weight which was elevated to 2 % DM during SARA periods. Consistent with the findings of

the present study, other authors noted that feeding diets with different ratios of forage to

concentrate (35:65 versus 60:40) increased DMI as the fibre content was decreased (Yang

and Beauchemin 2007a, 2009). This is because grains are more readily digestible than

forages (NRC 2001) and, therefore, concentrate usually passes the rumen faster (Colucci et

al. 1990). Even forages with high fermentabilities are degraded more slowly than grains

(Zebeli et al. 2012). Diets that show a slower degradability in the rumen have a slower

ruminal passage rate, further leading to decreasing DMI (Tafaj et al. 2005), what may be a

reason for lower DMI observed at BASE in the present study. It is known that high NDF

contents in diets cause distension of the rumen because they have a slow ruminal passage

rate and therefore lead to reduced DMI, this effect is known as rumen filling effect (Allen

2000). Therefore, it is being discussed that at a certain extent of increasing the forage level

in the diet (> 14.9 % physically effective fibre in the diet) cows’ DMI may be reduced (Zebeli

et al. 2012). In fact, animals experiencing SARA may reduce their intake (Kleen et al. 2003;

Plaizier et al. 2008), partly because low ruminal pH can also prolong rumen filling effect

(Allen 2000; Zebeli et al. 2006) as a result of decreased fibre digestibility (Allen and Mertens

1988; Calsamiglia et al. 2008; Dijkstra et al. 2012; Sung et al. 2006) due to reduced number

of fibrolytic bacteria (Allen and Mertens 1988; Sung et al. 2006) and reduced attachment of

bacteria to their substrates at low ruminal pH (Sung et al. 2006). However, the feeding

management coupled with the intensity of SARA induced in this experiment did not lead the

cows to go off feed. Besides, Allen (2000) states that in diets with high concentrate (> 50 %

of diet DM) and low forage content DMI is less limited by rumen fill effect and so DMI

increases until being limited by energy requirement.

39

Furthermore, studies dealing with the effects of SARA on DMI may not be conclusive

because there is too much discrepancy in how SARA is induced, in the severity of SARA and

also because of variances in individual cows (Guo et al. 2013). In this study no significant

difference in DMI between short-term SARA (SARA1) and long-term (SARA2) was observed

and the DMI was similar for both SARA models (transient versus continuous, data not

shown).

However, the concentrate intake (kg/day) in the present study increased significantly

(+ 1.04 kg/day) from short-term SARA to long-term SARA. It is important to note that in order

to keep the forage and concentrate ratio of DMI constant the concentrate intake was

controlled. Relative to the daily forage ingested the proportion of unconsumed concentrate

was delivered via the rumen cannula. Actually, cows did refuse to consume as much

concentrate as they usually did with prolonged SARA challenge and that more concentrate

had to be delivered via the rumen cannula on the last few days (SARA2) of the first run. The

number of days of concentrate refusal was even higher in the second run. A study by

Hendriksen et al. (2015) showed that cows sorted against short particles when they were

affected by SARA. They suspected cows tried to consume more large particles in order to

reduce SARA-conditions.

The water intake (kg/day) was not affected by SARA in the present study. This in conformity

with a study of Cottee et al. (2004) showing that the impact of SARA on the water intake

(L/day) was not significant.

5.2. Ruminal pH

Before SARA was induced the mean ruminal pH of BASE was 6.42, which is within the

physiological ruminal pH values (Krause and Oetzel 2006), and cows spent only

0.25 min/day below pH 5.8. During SARA mean ruminal pH was significantly decreased to

about 6.0 with no different mean pH values between the short-term (SARA1, 6.10) and long-

term SARA (SARA2, 5.97). It must be noted that day-to-day variation in mean pH existed

and that there were days that the mean pH was below 5.8 (data not shown, more details see

Pourazad et al., (2016)). Nevertheless, the time of pH depression (pH < 5.8) significantly

increased from 237 min/day during the short-term SARA to 467 min/day during the long-term

40

SARA. In the present study a pH threshold of 5.8 for at least 330 min/day (Zebeli et al. 2008)

was used for diagnosing SARA, accordingly SARA was successfully induced in the present

study. The general pH depression (i.e., BASE versus SARA) was caused by increased DMI

and the alterations of the diets components with increased concentrate portion of the diet to

60 % of diet DM. As a result, during SARA periods the NDF content of the diet was

decreased while the content of NFC was increased, thereby the feed fermentability was

presumably increased in the rumen (Dijkstra et al. 2012) which was supported by the findings

that more fermentation acids were produced during SARA compared to BASE (Pourazad et

al., 2016). It is known that forages, with high physical effectiveness (i.e., high NDF content

and long particle length) are important for maintaining a physiological ruminal pH (Mertens

1997; Zebeli et al. 2010) because forage prolongs chewing time and improves ruminal

buffering (Mertens 1997).

The increase in time of pH depression from the short-term SARA to the long-term SARA was

also found significant. This was partly a result of higher concentrate intake in the long-term

SARA. In addition, the increased time of pH depression during the long-term SARA found

here was mainly an influence of the transient challenge which was not distinguished in this

thesis but described in Pourazad et al. (2016). This prolonged duration of SARA bouts could

also be seen in a study by Dohme et al. (2008) in which SARA became increasingly severe

with repeated exposure to high grain challenge.

5.3. Rumen particle distribution In this study, both long-term and short-term SARA periods exhibited similar changes in

particle distribution in the rumen. Therefore, their effects are considered together as an

overall SARA effects. The data showed that there was a noticeable increase in the content of

large particles and of the mean particle size with the time after feeding during BASE. But

such increase could not been noticed for both, short-term and long-term SARA periods. The

more obvious increase of large particles and the mean particle length after feeding during

BASE was related to cows consuming only long-chopped forages. Compared to concentrate,

forage is more slowly digestible, because structural carbohydrates as NDF, which are

contained in forages to a higher content, are less digestible than NSC (NRC 2001).

Therefore, fibre usually remains longer in the rumen than concentrate (Allen 2000) until the

feed particles are comminuted to the critical size to pass the rumen (Maulfair et al. 2011;

41

Oshita et al. 2004). For this reason, large feed particles may have accumulated over the

course of 8 h after morning feeding during BASE. It is thinkable that such accumulation of

large particles would be less evident when cows receive high amounts of concentrate with

smaller particles and which are more rapidly degradable than forage. This may explain why

the mean particle length and the large particle fraction stayed stable over time during SARA.

In addition, when considering the particle distribution before feeding, it became clear that

during BASE although a higher content of large particles accumulated in the rumen during

the day, and that the particles were intensively comminuted in the night resulting in lesser

content of large particles and smaller mean particle length at 0 h than post-feeding hours.

Whereas during the SARA periods the particle breakdown was more constantly reduced

throughout the day and night keeping the constant mean particle length of the rumen digesta

across sampling hours.

The SARA challenge in the present study increased the content of medium particles (1.18–

2 mm) and the content of soluble fraction (< 0.063 mm) but decreased the content of small

particles (< 1.18 mm).The retention of medium particles as a result of SARA may be

explained by reduced fibre digestion that may occur during pH depression (Allen and

Mertens 1988; Russell and Wilson 1996), and so more larger particles are likely to

accumulate in the rumen. It is widely accepted that fibrolytic microbes are sensitive to low pH

by inhibiting their growth (Russell and Wilson 1996; Sung et al. 2006) and lowering their

attachment to their substrates (Sung et al. 2006). Presumably, the degradation of non-fiber

carbohydrates was not suppressed, because ruminal starch digestion is not reduced at a pH

below 5.6 (Yang et al. 2002), which is lower than the ruminal pH found in the present

experiment. Similar to our results, in a study by Tafaj et al. (2006) the content of particles

larger than 1.0 mm was significantly increased with high dietary concentrate contents

(> 50 % concentrate of DM). They expected fibre digestion being negatively influenced by the

concentration of rapidly fermentable carbohydrates and therefore a reduced or delayed

particle communition (Tafaj et al. 2006).

The increase of the content of the soluble fraction during high-concentrate feeding found in

this study was inconsistent with Tafaj et al., (2005) and Tafaj et al. (2006) who found a

decreased content of soluble particles (< 0.063 mm) with increasing concentrate levels (20 %

versus 50 % concentrate of DM and > 50 % concentrate of DM, respectively). These

contradictions may be caused by differences in diet composition. It is noteworthy that

digestion process is not only determined by chemical and physical factors like ruminal pH,

42

but also by chemical composition of fibre, the activity of fibre degrading microorganisms and

the surface for microbial attachment (Allen and Mertens 1988).

Chewing is generally believed to be the major mechanism in particle communition in the

rumen (Kononoff and Heinrichs 2003b). Forages have a positive effect on the ruminal

environment by promoting chewing and rumen motility which therefore may enhance particle

breakdown (Tafaj et al. 2006; Zebeli et al. 2006). Tafaj et al. (2006) demonstrated that the

efficiency of particle communition is determined by correlating processes as chewing,

ruminal fermentation and other digestive factors like the quality of rumen digesta satisfaction

and ruminal passage rate, especially when a high concentrate diet is fed to ruminants. A

negative effect of SARA-inducing diets (i.e., high concentrate diets) on chewing activity has

been reported (Maekawa et al. 2002; Sudweeks et al. 1975). However, Suarez-Mena et al.

(2013) reported unaffected chewing activity but tendentially decreased eating time with

decreasing forage to concentrate ratio. In addition, feed fermentability and microbial

degradation capacity are being discussed to contribute greatly to the particle communition in

the rumen (Kononoff and Heinrichs 2003b; Moseley and Jones 1984; Tafaj et al. 2006).

Accordingly, in the current study the SARA-inducing diet might have decreased chewing

activity and saliva production in comparison to BASE and may have had a consequence for

the particle distribution in the rumen digesta. Nevertheless, it has also been discussed that

ruminants would try to compensate low ruminal fibre breakdown by increasing ruminating

(Tafaj et al. 2005; Zebeli et al. 2007).

Furthermore, it should be underlined that the strategy for concentrate feeding itself (e.g.,

separate feeding or mixed with forage, frequency and allowance) can play a role the

development of SARA (Maekawa et al. 2002) and perhaps in diurnal particle distribution in

the rumen. This might have contributed to a discrepancy among studies about effects of

concentrate levels on rumen particle size and distribution (e.g., this thesis, Maulfair et al.

2011; Tafaj et al. 2005; Tafaj et al. 2006). In addition, diet formulation and interactions

between different nutrients make studies concerning particle distribution difficult to compare

(Maulfair et al. 2011).

In the context of the duration of SARA challenge, it was observed that the duration of pH

depression was found to be more severe during the long-term SARA than during the short-

term SARA. Consequently, less particle breakdown in the rumen would be expected for the

43

long-term SARA period. However, this was not the case. The rumen digesta particle

distribution was similar between the two SARA periods. Therefore, it is obvious the severity

of pH depression having potential to affect ruminal degradation cannot be the only reason.

Other key explanations for the longer retention of large particle such as feeding behaviour,

ruminal passage rate and the quality of digesta stratification have to be found.

44

6. Conclusion

In high-producing dairy farms cows are frequently confronted with subacute ruminal acidosis

(SARA) and resulting health consequences triggered by heavy concentrate feeding going

hand in hand with an insufficient content of fibre (e.g., physically effective fibre) in the diet.

Because grains are rapidly fermentable, diets with high grain contents lead to increasing

concentrations of short-chain fatty acids in the rumen. This, combined with inadequate

dietary fibre contents can lead to a declining ruminal pH, since dietary fibre is crucial for

promoting buffering (i.e., chewing, rumination, saliva production) of the rumen contents.

Decreasing ruminal pH can increase the risk of subacute ruminal acidosis, which

compromises cow’s health and productivity.

The impact of high-grain feeding on ruminal pH is supported by the results of this thesis,

showing that feeding the 60 % concentrate diet led to subacute ruminal acidosis in dairy

cattle, which proceeded for the four weeks of the challenge. The results of this thesis

regarding particle distribution of rumen digesta suggest that particle breakdown in the rumen

was decreased, because the fraction of small particles (< 1.18 mm) decreased, while the

fraction of medium particles 1.18–2 mm and the soluble fraction (< 0.063 mm) increased due

to the SARA challenge. The increased content of particles 1.18–2 mm during subacute

ruminal acidosis was already noticeable before morning feeding. The intensity of pH

depression was higher during long-term subacute ruminal acidosis compared to short-term

subacute ruminal acidosis but there was no significant difference with respect to the rumen

digesta particle distribution.

Summing up, this study proves the importance of maintaining physiological ruminal pH for

ruminal digestion and shows the negative impact of subacute ruminal acidosis on ruminal

particle breakdown. It seems that not only the pH depression but multiple factors like ruminal

digestion, chewing activity, ruminal passage rate and the quality of digesta stratification have

to be considered in order to evaluate an effect of high-grain feeding on rumen digesta

particle distribution.

45

7. Zusammenfassung

In Hochleistungsmilchbetrieben sind Kühe häufig von subakuter Pansenazidose (SARA) und

daraus resultierenden Gesundheitsschäden betroffen, ausgelöst durch eine kraftfutterreiche

Fütterung, die mit einen unzureichenden Gehalt an Fasern (insbesondere physikalisch

effektive Fasern) einhergeht. Da Getreide leicht vergärbar ist, führen Futterrationen mit

hohem Getreideanteil zu steigenden Konzentrationen kurzkettiger Fettsäuren im Pansen.

Zusammen mit einer unzureichenden Faserration im Futter kann dies zu einem sinkenden

pH-Wert im Pansen führen, da Fasern ausschlaggebend für eine geförderte Pufferung (durch

Kauen, Wiederkäuen, Speichelproduktion) des Panseninhalts sind. Ein sinkender pH-Wert

im Pansen kann das Riskio von subakuter Pansenazidose erhöhen, welche die Gesundheit

und Leistung betroffener Kühen beeinträchtigt.

Die Auswirkung getreidelastiger Fütterung auf den pH-Wert des Pansens wird auch durch

die Ergebnisse dieser Diplomarbeit gestützt. Diese zeigen, dass die Fütterung einer

Futterration mit einem Gehalt von 60 % Kraftfutter zu subakuter Pansenazidose bei

Milchkühen führte, die über die gesamte Dauer des Experiments von vier Wochen anhielt.

Die Ergebnisse der vorliegenden Diplomarbeit hinsichtlich der Futterpartikelverteilung im

Pansen legen nahe, dass die Verdauung der Futterpartikel auf Grund der SARA-Challenge

vermindert war. Denn der Anteil an kleinen Partikeln (< 1,18 mm) ging zurück, während der

Anteil an Partikeln größer als 2 mm und die lösliche Phase (< 0,063 mm) anstieg. Der

erhöhte Anteil an mittleren Partikeln mit einer Größe von 1,18–2 mm war während subakuter

Pansenazidose bereits vor der morgendlichen Fütterung beachtlich. Das Ausmaß des pH

Abfalls war während Langzeit-SARA höher als der pH Abfall während Kurzzeit-SARA, jedoch

konnte kein signifikanter Unterschied im Hinblick auf die Verteilung der Futterbreipartikel im

Pansen festgestellt werden.

Damit hebt vorliegende Diplomarbeit die Wichtigkeit der Aufrechterhaltung eines

physiologischen pH-Werts im Pansen für die Verdauung im Pansen hervor und stellt die

negative Wirkung der subakuten Pansenazidose auf die Futterpartikelverdauung im Pansen

dar. Es scheint, dass nicht nur der pH-Abfall sondern mehrere Faktoren wie die Verdauung

im Pansen, die Kauaktivität, die Passagerate durch den Pansen und die Qualität der

Faserschichtung mit einbezogen werden müssen, um den Effekt von getreidelastiger

Fütterung auf die Verteilung der Futterpartikel im Pansen beurteilen zu können.

46

47

8. List of abbreviations

ADF Acid Detergent Fibre

ADL Acid Detergent Lignin

BASE Baseline

DMI Dry Matter Intake

NDF Neutral Detergent Fibre

NFC Non-Fibre Carbohydrates

NFE Nitrogen-free Extractives

NRC National Research Council

NSC Non-Structural Carbohydrates

NSP Non-Starch-Polysaccharides

peNDF Physically Effective Neutral Detergent Fibre

PSPS Pennstate Particle Separator

SARA Subacute Ruminal Acidosis

SARA1 Short-term SARA

SARA2 Long-term SARA

SCFA Short Chain Fatty Acids

TMR Total Mixed Ration

48

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55

56

10. List of tables

Tab. 1: The characteristics of predominant ruminal bacteria (Russell and Rychlik 2001) ..... 15

Tab. 2: Identity and enzyme activities of ruminal microbes involved with degradation of plant

cell walls in the rumen (Wang and McAllister 2002) ............................................................. 17

Tab. 3: Chemical composition of the SARA challenge diet and the forage mix (DM basis) .. 26

Tab. 4: Feed and water intake ............................................................................................. 31

Tab. 5: Ruminal pH changes ............................................................................................... 32

57

11. List of figures

Fig. 1: Plant carbohydrate fractions (Adapted after Hall (2003)) ............................................ 9

Fig. 2: Molecule of cellulose (Heldt and Heldt 2003) ............................................................ 10

Fig. 3: Molecule of xyloglucan (hemicellulose) (Heldt and Heldt 2003) ................................ 11

Fig. 4: Molecule of D-galacturonate (Heldt and Heldt 2003)................................................. 11

Fig. 5: Wet-sieving device used in the present study (Vibratory sieve shaker AS 200 digit 2,

Retsch, Germany) ................................................................................................................ 13

Fig. 6: The relationship between initial pH, DM digestibility and bacterial attachment (Sung et

al. 2006)............................................................................................................................... 20

Fig. 7: Timeline of SARA challenging-experiment and sampling points ............................... 26

Fig. 8: Mean particle length (mm) ........................................................................................ 33

Fig. 9: Content of large particles (> 2 mm)........................................................................... 34

Fig. 10: Content of medium particles (1.18–2.00 mm) ......................................................... 35

Fig. 11: Content of small particles (< 1.18 mm) ................................................................... 36

Fig. 12: Soluble fraction (< 0.063 mm) ................................................................................. 37