KHON KAEN UNIVERSITY
Subacute Ruminal Acidosis
In vitro efficacy of absorbents to trap intrinsically generated
lipopolysaccharides in rumen fluid of dairy cows
Drs. M.G.G. Mullaert
7/1/2010
Subacute Ruminal Acidosis 2010
Drs. M.G.G. Mullaert
Studentnr. 0461636
Juli-oktober 2010
Supervisors:
Dr. J.T. (Thomas) Schonewille
Department of Farm Animal Health
Faculty of Veterinary Medicine, Utrecht University
Dr. Anantachai Chaiyotwittayakun
Department of Veterinary Medicine
Faculty of Veterinary Medicine, Khon Kaen University
Drs. Rittichai Pilachai
Department of Veterinary Medicine
Faculty of Veterinary Medicine, Khon Kaen University
Acknowlegdement
We gratefully thank Drs. D de Haan from Trouw International for his kind donation of the clay
minerals NovasilTM Plus and TOX-OTM .
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Subacute Ruminal Acidosis 2010
Abstract
Laminitis is a common health problem in dairy cows in Thailand nowadays. In order to maintain high
production, dairy cows need to be fed with a diet high in grain and low in forage. Consequently, the
diet contains a high amount of rapid fermentable carbohydrates and insufficient fiber. This results in
the production of a large quantity of volatile fatty acids, and causes a depression on the rumen pH
and associated with rumen acidosis. (Kleen et al, 2009). Subacute ruminal acidosis (SARA), the
condition were the rumen pH<5.6 for at least 180 min/d (Gozho et al., 2005), is suggested to play an
important role in the cascade.
It has been suggested that the pathogenic mechanism of laminitis may involve the activation of
systemic inflammatory responses due to the lysis of gram-negative bacteria. Rumen pH depression
during SARA increases free LPS concentration of the rumen (Emmanuel et al., 2008; Gozho et al.,
2007) and may result in the transfer of LPS from the gut into blood circulation, and activation of an
inflammatory response (Khafipour et al., 2009). Thus, materials that can bind LPS in the rumen and
can prevent the transfer of endotoxin (LPS) to the blood are potentially of interest to the prevention
of laminitis.
It is know that montmorillonite have binding affinity for LPS (Pilachai et al., 2010, unpublished data).
This trial was conducted to evaluate the efficacy of three adsorbents; montmorillonite, hydrate
sodium calcium aluminosilicate (HSCAS) and modified HSCAS, to trap intrinsically generate
lipopolysaccharides (LPS) in rumen fluid collected from SARA induced cows. In vitro, we added the
three absorbents at three levels (0, 0.5 and 1% (w/v) to the rumen fluid from SARA cows and
measured the LPS concentration.
It appear that the sequestering agents tested here had no significant absorbing effect on the LPS in
the rumen fluid of the SARA induced cows.
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Subacute Ruminal Acidosis 2010
Contents
Abstract ................................................................................................................................................... 3
Contents .................................................................................................................................................. 4
Introduction............................................................................................................................................. 5
Materials and Methods ......................................................................................................................... 12
Results ................................................................................................................................................... 14
Discussion .............................................................................................................................................. 16
Conclusion ............................................................................................................................................. 16
Acknowledgement................................................................................................................................. 17
References ............................................................................................................................................. 18
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Subacute Ruminal Acidosis 2010
Introduction
Laminitis (pododermatitis aseptic diffusa) is a common problem in dairy cattle in Thailand. Laminitis
is defined as the inflammation of the dermal layers inside the foot (Nocek, 1997). The cause of
laminitis should be considered as a combination of predisposing factors leading to an altered vascular
reactivity and microcirculation and disruption of normal cell differentiation and horn formation in the
clay epidermis (Mulling, 2002).
There are multiple causes of lameness such as infectious diseases of the interdigital space for
example foot rot or digital dermatitis. Traumatic damage to the sole can also occur when the cattle is
housed in stables with hard or rough flours or when they have to walk long distances over rough
roadways. Embedded stones for example can cause white line abcesses to form.
Laminitis is a condition where the bloodflow to the extremities is restricted which causes damage to
the claws. Laminitis can be divided in three forms. Acute, subacute and subclinical (Greenough 2007).
Acute laminitis is a consequence of a grain overload where the cow has eaten a large amount of grain
and the pH of the rumen drops rapidly below 5.0. Affected animals show signs rapidly after
overloading and an increase in heart rate, respiratory rate and liquid, light colored feces can be
observed. Cows can become recumbent or crawl on their knees.
Subacute laminitis is a more subtle disorder were the only signs are that of a mild discomfort with
cows shifting weight from leg to leg and they place their feet on the ground very carefully. Subacute
laminitis is also associated with nutritional changes which can cause vasoactive substances to be
released into the bloodstream that alter the bloodflow in the claw causing damage and discomfort.
Subclinical laminitis or pododermatitis aseptica diffuse is a disorder without clinical signs at the
moment that structures in the claw are damaged but creates specific lesions that will show after a
period of time. These lesions are caused by two different pathological processes. 1) The claw horn is
weakened structurally and functionally and no longer able to endure loading and more vulnarabule
to environmental agents. 2) the support system of the pedal bone and the integrity of the suspensory
apparatus of the digit are weakened (Greenough 2007). Systemic vasoactive substances change the
bloodpressure in the extremities and arteriovenous shunts start to form. This increases the
bloodpressure in the claws and the vessels start to leak and are damaged and edema and
hemorraghing occurs. The opening of the shunts also causes ischemia to occur in the dermis (Nocek
et al 1997, Greenough 2007). As a result of the increased pressure and ischemia in the dermis the
degeneration of the corium and the formation of horn is impaired. This can be detected in the sole 4
to 8 weeks after the initial damage is done as sole hemorraghes, white line defects, dorsal ridging of
the wall or double soles (Nocek et al 1997). If the damage to the epidermis is severe the laminar layer
can separate completely which will cause the pedal bone to shift. This creates even more damage to
the soft tissue and necrosis can occur. The claw is tilted slightly which can be observed.
It is very difficult to distinguish between sole hemorraghes caused by subclinical laminitis or caused
by traumatic damage to the sole. Housing should always be considered when sole bruising is found
as a herd problem.
Laminitis has been associated with a reduced pH of rumen fluid: i.e. rumen acidosis. This condition
occurs both in dairy cattle as well as in beef cattle. The drop of the pH in the rumen, the formation of
endotoxins (lipopolysaccharides) and damage to the rumen wall can have a systemic effect that
could possibly have an effect on the bloodflow in the extremitities and play a role in the occurence of
laminitis. See figure 1.
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Subacute Ruminal Acidosis 2010
Figure 1. Progression
of physiological events
that link acidosis
with laminitis. CHO =
Carbohydrate.
(Modified from Nocek,
1997)
Ruminal acidosis is a bovine disease that affects feedlot as well as dairy cattle. It’s caused by the
ingestion of greater than normal quantities of ruminally fermentable carbohydrates. This results in a
decreased ruminal pH. The cascade effects of this acidosis depend upon the intensity and duration of
the insult. Most critical is the pH threshold, which not only relates to microbial growth rates and
shifts in ruminal populations but also significantly influences the systemic metabolic state (Nocek,
1997). Rumen acidosis can be divided into an acute and a subacute form. The subacute form is also
known as SARA: Subacute Rumen Acidosis. Acute acidosis results in very sick cows and is
characterized by a ruminal pH < 5.0. (Nocek, 1997), its generally linked to lactic acid production
(figure 1).
There are many definitions of when to speak of SARA based on method and location of taking rumen
fluid samples. Samples taken by stomach tube, rumenocentesis or through a fistula from the ventral
sac of the rumen differ in pH value. Samples taken by stomach tube can be diluted by saliva and are
on average 0,3 point higher than samples taken by rumenocentesis (Plaizier et al 1998). The pH of
the rumen fluctuates during the day and the most accurate way to measure the pH is by using an
indwelling pH probe in the ventral sac of the rumen. SARA is characterized by intermittent periods of
depressed rumen pH each day, e.g. <5.6 for at least 180 min/day (Kleen et al., 2003; Stone, 2004;
Gozho et al., 2005). The onset of SARA is marked by the intake of a diet low in structure and high in
energy, while the ruminal environment is not yet adapted to ferment and later absorb the arising
shortchain fatty acids (SCFA) adequately enough to keep the ruminal pH within physiological borders.
This diet will result in reduced amount of time spent chewing and thus less saliva production. Saliva
contains inorganic buffers that contribute to the neutralization of the organic acids produced during
fermentation in the rumen (Church, 1988).
The ruminal pappilae are of crucial importance in the absorption of SCFA; the proliferation of the
papillae is promoted by SCFA arising from the fermentation. If the ruminal mucosa is not adapted,
which especially is the case at the shift from a dry-period to an early lactation diet, the papillae are
too short and the resorbing surface too small to deal with the sudden increase of SCFA-levels
(Nordlund et al.,1995).
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Subacute Ruminal Acidosis 2010
Also the microbial population, which has to metabolize the lactic acid arising from the fermentation
of abundant carbohydrates of Streptococcus bovis or Dasytricha spp., is insufficiently developed
(Slyter, 1976; Dawson and Allison, 1988; Nordlund et al., 1995).
These factors together cause a transient fall of ruminal pH. The balance between production and
absorption of fermentation end products is disturbed (Garrett, 1996).
In conclusion, rumen acidosis is linked to lactic acid production and in the case of SARA, VFA
production plays an important role. That’s the difference between these two.
See figure 2.
Figure 2. Step-by-step
microbial mechanisms
of occurence of
acidosis in the rumen.
(Modified from rumenhealth.com)
Thus, subacute ruminal acidosis is a common disease in high yielding dairy cows that receive highly
digestible diets, and has a high economic impact (Plaizier et al., 2009). Especially because its most of
the time subclinical. Oetzel (2004) found that 20% of cows from commercial dairy herds sampled by
rumenocentesis during clinical herd investigations had ruminal pH of <5.5. Also Garrett et al. 1997
concluded that SARA is present in a large number of dairy herds. Costs of SARA resulting from lost
production alone were estimated to be 1.12/d per cow in a herd diagnosed with SARA (Stone, 1999).
Subacute ruminal acidosis (SARA), is often difficult to detect. The clinical signs are variable and not
specific to SARA. The related clinical signs in dairy cows can be diarrhea, rumen mucosal damage,
laminitis, inflammation throughout the body, and liver abscesses (Nocek, 1997; Kleen et al., 2003;
Stone, 2004; Alzahal et al., 2007). Herds with a high prevalence of SARA may have a high culling rate,
increased death loss, and decreased milk production (Nocek, 1997, Stone, 1999).We can speak of
two major critical situations were SARA can develop, in other words, where the non-adaptated
rumen is likely to meet a concentrate-based diet.
The most important one is the early post-partum period: a rumen non-adapted to concentrates and a
possibly high concentrate-uptake around parturition, during general low-feed intake in combination
with a considerable impact of stress and a more or less sever negative energy balance. And the midlactation period: mostly linked to managerial factors, like feeding frequency, processing of feed and
housing (Kleen, 2003).
There is substantial evidence that grain-based SARA challenge increases the content of free LPS in
the rumen due to the increase in lysis of gram-negative bacteria (Gozho et al., 2007; Nagaraja and
Lechtenberg, 2007; Plaizier et al., 2008). This increase in luminal LPS could increase permeability of
the gut for LPS (Chin et al., 2006). Also, the barrier function of rumen epithelium may be
compromised by the parakeratosis, rumenitis, and abscesses of the rumen wall that result from high
rumen acidity (Kleen et al., 2003). See figure 3.
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Subacute Ruminal Acidosis 2010
Figure 3. Proposed
relationships between
route of endotoxin
exposure, hepatic
function, biological
reponse and clinical
response in bovine
endotoxicossis.
(Modified from Nocek,
1997)
Additionally, the high
rumen osmolality (by
increase
of
VFA
(Bennink et al., 1978))
that is seen during SARA
can cause swelling and
rupture of ruminal
papillae, which will also reduce the barrier function of the rumen (Plaizier et al., 2009). This results in
translocation of immunogenic compounds into circulation, such as free LPS. Endotoxin, also known as
LPS, is a cellular component of gram-negative bacteria and is an extremely potent toxin (Andersen et
al., 2000). See figure 4. LPS interacts with a specific acute phase protein, LPS binding protein (LBP), an
increase in LBP in peripheral circulation facilitate binding of LPS to cell membrane receptors, which
then triggers a cascade of events toward a local or systemic inflammatory response and release of
other acute phase proteins (APP) into the blood (Khafipour 2009). The main stimulators of APP
production are the inflammation-associated cytokines IL-1, IL-6, tumor necrosis factor (TNF)-α, and
IFN-γ which are released during inflammatory processes (Gabay and Kushner, 1999). Two APP, serum
amyloid A (SAA) and LPS-binding protein (LBP), directly participate in the detoxification and removal
of endotoxin during an acute phase response. (Emmanuel et. al., 2008)
Figure 4. Endotoxins
(lipopolysaccharides)
are structural parts of
the bacterial cell wall
and in general
comprise 3 major
regions: the side chain,
the core
polysaccharides and
lipid A (Modified from
Rietschel 1996).
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Subacute Ruminal Acidosis 2010
The link between acidosis and laminitis appears to be associated with altered hemodynamics of the
peripheral microvasculature. The primary target of endotoxins appears to be vascular arterial tissues,
which affects the release of vasoactive mediators, thus inducing an acute inflammation of the dermis
and associated vascular responses. Histamine causes impedance of blood flow and pressure building
within the capillaries forces fluid through the vessels into the interstitial tissue spaces setting up
ischemia. When the microvasculature of the corium is affected by vasoactive substances, vascular
destruction is inevitable. When blood is not returned to circulation by the musculature of the
vascular system, seepage and hemorrhage can result. The anatomical features of the corium vascular
network play a very important role in the development of various stages of laminitis. (Nocek., 1997).
See also figure 6.
To prevent problems around laminitis, caused by SARA in dairy herds, it’s important to concentrate
on two points: prevention of a fermentation disorder or regulation of the fermentation process by
direct intervention. Alternatively, there are other options regarding the tecnonology of feedstuffs
and certain substances or organisms added. Materials that can bind LPS in the rumen and can
prevent the transfer of endotoxin to the blood. Those materials, like microbials, buffers or lactate are
potentially of interest to the prevention of SARA and thus laminitis.
It is well know that absorbent materials such as clay minerals have a high absorbing capacity for
some toxins, the layered structure can load inorganic antibacterial cations (Ag, Cu, Zn etc.) within the
interlayer space by an ion exchange reaction (Dakovic, 2008). Indeed, in vitro studies have shown
that clay minerals have the ability to bind aflatoxin B1 under different pH conditions (Diaz et al, 2003;
Spotti, 2005; Moschini, 2008). In addition, clay minerals also have a high capacity to bind mycotoxin
in rumen fluid (Diaz et al, 2003; Spotti et al, 2005). Clay minerals can bind mycotoxins, despite this,
there is no evidence that they can also bind endotoxins produced during SARA-conditions. Recently,
Pilachai et al. (2010) (unpublished data) demonstrated that both activated charcoal and
montmorrilonite are capable of absorbing LPS in a buffer as well in rumen fluid.
Furthermore, it appeared that pH had no effect on the efficacy of the clay minerals to trap LPS. In the
study of Pilachai et al. (2010) (unpublished data) montmorillonite (MONT) was the most promising
clay mineral because it showed the highest sequestering efficacy in rumen fluid. Therefore,
montmorillonite was also used in the current experiment. Montmorillonite in the pure form isn’t yet
used in animals, so we also want to evaluate hydrate sodium calcium aluminosilicate (HSCAS) and
modified HSCAS, because they are already used in animals. Montmorillonite, a smectite clay, has a
2:1 layered structure with the inner layer composed of an octahedral sheet that shares oxygen atoms
between two tetrahedral sheets. In smectites, the substitution of Al3+ for Si4+ in tetrahedral sheets
and substitution of divalent cations such as Mg2+ and Fe2+, for trivalent cations such as Al3+ and
Fe3+ in octahedral sheets give rise to a deficiency of the positive charge in the framework. This
charge is balanced by monovalent and divalent cations (usually Na+, K+, Ca2+ and Mg2+), and is
expressed as the cation exchange capacity (CEC). These cations positioned mainly in the interlamellar
space, can easily be exchanged with other inorganic and/or organic cations under aqueous
conditions (Dakovic., 2008).
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Subacute Ruminal Acidosis 2010
Figure
5.
Zn-MONT
structure. (
Modified
from Taylor
et al., 2000)
Because it was demonstrated that clay mineral could trap added LPS under in vitro conditions, it was
hypothesized that clay mineral could also trap intrinsically generated LPS in rumen fluid of cows
suffering from rumen acidosis.
The objective in this in vitro study is to evaluate the efficacy of three adsorbents; montmorillonite,
hydrate sodium calcium aluminosilicate (HSCAS) and modified HSCAS, to trap intrinsically generate
lipopolysaccharides (LPS) in rumen fluid collected from SARA induced cows.
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Subacute Ruminal Acidosis 2010
Figure 6. The phasic
progression of laminitis
development. Phase 1,
initial activation; phase
2, local mechanical
damage; phase 3, local
metabolic insult; and
phase 4, progressive
local damage of the
bone structure. AV =
Arteriovenous.
(Modified from Nocek,
1997)
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Subacute Ruminal Acidosis 2010
Materials and Methods
Animals, experimental treatment and feeding
Cows used in this study were housed in individual 3 × 3 m2 pens with concrete floors and natural
ventilation in the Ruminant Research Unit at Udon Thani Rajabhat University, Thailand, in accordance
with the guidelines of the Animal Ethics Committee of Khon Kaen University, based on the Ethics of
Animal Experimentation of National Research Council of Thailand. Four ruminally fistulated, adult,
non-pregnant and non-lactating cows were used in this study. The cows were used during two
experimental periods. These consisted of a 21 day run-in period, followed by a 7 day rumen acidosis
induction according to the method of Pilachai et al. (2010) (unpublished). Briefly, cows were fed 6 kg
of concentrate and rice straw ad libitum during the run-in period of 21 days (no SARA). Then, SARA
was induced with increasing the amount of concentrate to 10.5 kg DM/cow/day in combination with
a restriction of the amount of rice straw to 1.5 kg DM/cow/day. The ration was offered daily in two
equal portion at 08:00 and 17:00 h and the animals had unlimited access to water throughout the
experiment.
Table 1 Ingredient composition of experimental rations
Ingredients, % of DM
Experimental ration
Rice straw
12.5
Rice bran
15.0
Soybean meal
30.5
Ground cassava chips
40.5
a
Dairy premix minerals
1.5
a
Dairy premix minerals consisted per kg DM; 6 g Zn, 8 g Mn, 10 g Fe, 1.6 g Cu, 0.10 g I, 0.02 g Co,
0.06 g Se, 10 g Mg, 2,000,000 IU vitamin A, 400,000 IU vitamin D and 3,000 IU vitamin E (TS Dairy
mix®, Thailand)
Absorbents
Three mineral clays (product 1, 2 and 3), were used in this study. Product 1: montmorillonite was
purchased from Sigma-aldrich inc.. Product 2: Hydrated Sodium Calcium Aluminosilicate (NovasilTM
Plus) was donated by Trouw Nutrition inc., and product 3: a modified Hydrated Sodium Calcium
Aluminosilicate (TOX-OTM) was also donated by Trouw Nutrition inc. Product 3 consisted of a
combination of montmorillonite and Yeast Cell Walls (raw material). The three absorbents were
sterilized by using UV light for 10 min.
In vitro experimental design
Three absorbents (product 1, 2 and 3) at three levels (0, 0.5 and 1% (w/v)) were conducted in
triplicate to evaluate the capacity of them to bind LPS in rumen fluid during acidosis challenge.
In vitro trial
Rumen fluid was collected from 4 different cows 7 days after the induction of SARA. Rumen fluid
samples were collected from the ventral sac of the rumen at 4 h after the morning feeding, using the
cannula tube fitted with a strainer and a 20-ml syringe. Immediately after collection, pH of ruminal
fluid was recorded (Mettler-Toledo GmBh 8603, Schwerzenbach, Switzerland) whereafter the rumen
content was filtrated through four layers of cheesecloth and transferred into 1.8-ml pyrogen free
tubes. The samples were kept on ice until transported to the laboratory for the initial processing.
Immediately after arriving at the lab, the samples were centrifuged at 10,000×g at 4 ºC for 15 min
and autoclaved at 121 ºC for 20 minutes.
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Subacute Ruminal Acidosis 2010
Ten-ml of the sterile supernatant, was placed into 15-ml pyrogen-free tubes with 0, 50 and 100 mg to
reach final concentrations 0, 0.5 and 1 % (w/v) of each adsorbent and then thoroughly mixed by
using a vortex mixer. The solution was incubated at 39 ºC for 1 h. Each 15 min the tubes were gently
turned over to prevent too much deposit and so achieve a totally mixed solution. After incubation
1.8-ml of the solution was transferred into 1.8-ml pyrogen-free tube and centrifuged for 15 min at
10,000×g at 4 ºC to deposit any sequestering absorbent-endotoxin complex as a pellet. The
supernatant was aspirated gently to prevent its mixing with the pellet and passed through a
disposable 0.22 µm sterile filter (Sartorius Stedim Biotech Minisart syringe filter) and stored in 1.8 ml
pyrogen-free tube at -20 ºC for subsequent LPS analysis.
Throughout the experiment all cows were clinically examined daily for any signs related to sub-acute
laminitis. They were observed for hearth rate, respiration rate, rectal temperature, rumen
contractions, fecal score and claw inflammation and foot pain according to the definition of subacute laminitis described by Greenough et al. (2007). Weight shifting was defined as a shifting of
weight laterally from one leg to another in a monotonous manner as 0 = no reaction, and 1 = marked
reaction.
LPS analysis
The concentration of free LPS in the rumen fluid was determined by a chromogenic kinetic Limulus
amebocyte lyase (LAL) according to Ghozo et al. (2005).
Table 2 Experimental design
Treatment
Positive control; sample at day 7, SARA
Product 1
Product 2
Product 3
0
3
0
0
0
Absorbent concentration, %
0.50
0
3
3
3
1
0
3
3
3
Statistic analysis and calculations
LPS concentrations were logarithmically transformed before statistical analysis. The data were
subjected to one-way ANOVA with cow as factor (SPSS® , version 16). Fishers least-significantdifference-test was used to identify treatment with different effects. Throughout, significance was
declared at P<0.05.
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Results
In vivo
After a 21-day run-in period in which the cows were fed 6 kg of concentrates, concentrate supply was
abruptly increased to 12 kg / cow /day (equal to 10.5 kg DM). At the same time the supply of rice
straw was changed from ad. Lib. to a restricted amount of 2 kg/cow/day. The first days a sharp
decline in especially the concentrate intake . After four days the feed intake increases again, but the
level of feed intake doesn’t reach the same amount as before the induction period.
Figure 7. Mean feed intake during the last 7 days of the experimental period. (DMI= dry matter
intake)
The pH of the rumen fluid collected on day 7 of each cow is shown in Table 3 and it is clear that the
cows suffered from rumen acidoses.
Table 3 Clinical examination, d7
Heart
Respiration
Rumen
Rec.Temp,
rate,
rate,
contraction,
Cow ID
ºC
beats/min breaths/min
/5 min
1
3
5
6
38.8
38.6
38.6
39.7
92
76
75
96
44
43
37
79
9
6
8
6
Faeces
score
4
3
2
3
Weight pH
shifting rumenfluid
0
2
0
2
4.84
4.75
5.33
4.56
During the trial, the cows were also clinically evaluated on day 7, the sampling day (Table 3). Cow 6
suffered from a light fever, an increased heart rate and respiration rate and shows some signs of
weight shifting. Thus, this cow showed clinical signs of laminitis.
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In vitro
The outcome of the in-vitro experiment is shown in figure 10. Although there are some suggestive
trends, the data clearly show that the LPS concentrations in rumen fluid were not significantly
affected by the various treatments. It is clear that the analysis of LPS in rumen fluid is subject to high
variation.
Figure 10 Measured LPS concentrations in rumen fluid (EU/ml) after supplementation of the various
absorbents. (Bar represents mean +/- SD)
Figure 11 shows the relationship between the log-transformed LPS concentration and rumen pH (n =
25). It appears that the variation in rumen pH accounted for 60.3% of the observed variation in LOG
LPS values ( P < 0.001). Clearly, the LPS values measured at pH 5 can be considered as outlier.
Figure 11 Relation between Log LPS and pH
6,000
Log LPS
5,000
4,000
3,000
2,000
1,000
0,000
4,40
4,60
4,80
5,00
5,20
5,40
Rumen pH
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Discussion
It is clear that we had to reject our hypothesis that intrinsically generated LPS could be trapped by
clay minerals. The observed mean LPS concentrations were subject to a high variation which may be
caused by the fact that conditions were not optimal during sample collection. Indeed, in the air and
on our clothes, there is LPS. While collecting the rumen fluid we didn’t wear special clothes. After
collecting the samples we brought the samples to the office to centrifuge, again wearing the same
clothes and not in a special room. These conditions can play a role in the scattered results.
The LPS concentration in the positive control does not match the values found in the literature
(Plaizier et al 1998). These should be much higher. Clearly high LPS concentrations were not
prevented by the pH value of the rumen fluid. Cows were clearly suffering from rumen acidois.
Interstingly, the highest LPS were found in combination with the highest rumen pH values, although
the pH values was lower than 5.5. Therefore, it can be concluded that there is large between- animal
variation in rumen LPS.
Another thing that strikes, is the high LPS concentration in the sample with 1% of Montmorillonite.
That does not match our expectations. This is most likely due the fact that while mixing the clay
minerals with the rumen fluid in the laboratories, it was very difficult to dilute the 1% of Mont. It was
almost impossible to dilute all of the Mont and after a couple of minutes it precipitated again. So it’s
possible that there wasn’t enough time and absorption surface to bind the LPS.
During this trial one cow suffered from laminitis. However, it seems unlikely that the occurrence of
laminitis was related to the LPS concentrations in the rumen because LPS concentration was around
11.500 EU/ml. Thus, it could be speculated that the occurrence of laminitis is associated with other
causative agents, such as histamine (Greenough 2007).
Conclusion
In conclusion, our results indicate that the three adsorbents we tested; montmorillonite, hydrate
sodium calcium aluminosilicate (HSCAS) and modified HSCAS, do not significantly trap intrinsically
generated LPS in rumen fluid collected from SARA induced cows.
Montmorillonite in a concentration of 0.5% appears to have the most promise as it shows the highest
absorption capacity in rumen fluid.
Nevertheless, further in vitro and in vivo studies are required to test the absorption capacity from
these clay minerals.
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Acknowledgement
I am heartly thankful to dr. Rittichai Pillachai for his effort, guidance and support and the
opportunity to fulfill my research. He was a great help and I enjoyed working together with him. I
would also like to thank him for everything he arranged for me and made my stay very pleasant.
I would also like to thank dr. Anantachai Chaiyotwittayakun for the opportunity to do my research
project at the university of Khon Kaen. His effort to make this project and my stay in Khon Kaen to a
success.
I thank dr. Thomas Schonewille for his backup during my stay abroad and his advice.
Besides, I would like to thank the Asian Link Project for providing the facilities for carrying out the
work. I express my gratitude to the University of Khon Kaen for providing a place to work and sleep.
I would like to express my gratitude to the workers on the farm in Udon Thani, to help us with the
practical side of the experiment and taking care of the cows. They were great people to work with
and I enjoyed playing soccer with them a lot.
I am grateful to the students at the university of Khon Kaen and especially Oat, Joe and Souk for their
great help and pleasant times. Their hospitality was great and therefore I had a very nice time
working and living in Khon Kaen.
I would like to express my gratitude to my friends and family for contacting me in all kind of ways
while my stay abroad. It’s very pleasant to hear from the people at home.
Finally I would like to thank my parents, Hugo en Gonny Mullaert, my boyfriend, Frank van de Vijver
and my best friend, Maartje Timmers, for their encouragement and support throughout my studies,
anywise.
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