Post on 18-Dec-2021
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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
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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