COMPARATIVE ANALYSIS OF THE FUNCTIONAL STATE OF

LIVER AND PARAMETERS OF THE LIPID STATUS IN THE

DOMESTIC MOUNTAIN PONY DEPENDING ON THE NUTRITION
STATE

INTRODUCTION:
Due to its strategic anatomical position in the organism, liver is
permanently exposed to numerous noxious agents, which can
lead to more or less severe liver impairments that again often
lead to severe hepatic insufficiency with serious consequences
on the homeostatic stability. Various infective and toxic agents,
metabolic and nutritive disorders, immunological factors,
neoplasms participate in the development of liver impairment.
(Divers, 2005; Divers, 2008; Moore, 2008; Bandarra and ass.,
2010) Because of the relatively large functional hepatic reserves
and its pronounced capability to regenerate, at the beginning of
the process there are no clinically manifested symptoms, but in
long term when two thirds of the parenchyma get out of
function, clear signs of the insufficiency emerge, which are
known as “characteristic symptoms of the liver impairment”.
(Smith, 2002; Robinson, 2003)
Early detection of the liver impairment is based upon blood
laboratory analysis that mislim da treba which nekako mi tako
zvuci a nisam sig kad koje ide, a which moze uvijek tako da to
koristi include serum biochemistry profile which measures
serum activity of the liver enzymes like: gamma glutamyl
transferase (GGT), lactate dehydrogenase (LDH), alkaline
phosphatase (ALP), aspartate transaminase (AST), succinate
dehydrogenase (SDH), glutamate dehydrogenase (GLDH) and
total protein, albumin, glucose, bilirubin, urea and creatinine
concentrations. (Katiuska and ass, 2013)
Enzymes are normally found in the cytoplasm (AST, LDH),
mitochondria (GLDH, AST), nucleus or somatic cell membranes
(ALP, GGT) where they catalyse chemical reactions.
Cytoplasmic enzymes are released in early stages of the cell
damage, whilst mitochondrial enzymes are released in
advanced stages of hepatocytes destruction. (Hoffman and ass,
2008) Significantly higher serum activity concentrations of AST,
SDH and GLDH are primarily indicators of hepatocytes’ necrosis,
which is usually seen in toxic liver impairments or possibly in
acute serum hepatitis (Trailovic, 2016), compared to ALP and
GGT which are indicators of cholestasis. In the case of
cholestasis, higher levels of bilirubin are also noted. Cholestasis
is primarily seen in hepatitis e.g. infectious hepatitis. In chronic
liver disease, regardless the cause, lowering of albumin to
mislim da ne treba to izmedju, provjeri globulin ratio due to
hypoalbuminemia and hyperproteinaemia is often seen
(Trailovic, 2016).
Extremely rarely, low urea concentration can be a sign of
severe liver impairment due to low urea synthesis in
hepatocytes. Higher concentrations of urea and creatinine are
therefore more common and indicative of primary renal failure
that ovdje ide which sigurno (sad sam se sjetila that ne moze ici
ispred glagola) is in most cases linked to the liver damage.

Infectious equine hepatitis is not so common, at least not in our

surroundings. Acute serum hepatitis (TADV) is not confirmed,
whilst bacterial hepatitis caused by bacterial penetration from
the digestive tract into the bile duct (cholangiohepatitis) or by
hematogenous/lymphatic dissemination from the other foci in
the organism, is sporadic (Trailovic, 2016).
Toxic liver impairments are probably more common. Besides
iatrogenic hepatopathies induced by the uncontrollable
application of hepatotoxic drugs and mycotoxicosis (aflatoxin),
importance of pyrrolizidine alkaloids' poisoning due to
exposure to plants Jacobea vulgaris sin. Senecio jacobea is
often highlighted (Curran and ass, 1996).
Over the years, liver impairments were often linked to obesity
and metabolic syndrome which are definitely a big problem in
modern horsemanship due to nutrients given in excess of needs
kontam sta hoces da kazes al mi nekako cudno zvuci, al
jerovatno nisam nikad cula tu konstrukciju pa zato and
insufficient exercise. On the Old Mountain’s pastures a large
number of subclinical liver impairments cases were registered
and were all linked to pyrrolizidine poisoning (Trailovic, 2013).
On the other hand, on the same pastures metabolic syndrome
was confirmed in a few (ovdje provjeri kad se koristi samo few,
a kad A few, jer ima drugacije znacenje al ne mogu se sjetiti sta
je) Balkan donkeys (Trailovic, 2013).
In order to investigate the significance of these two factors, we
decided to make a comparative analysis of the functional state
of liver and parameters of the lipid status in two groups of
domesticated mountain ponies kept on Old Mountain’s
pastures: well-nourished and relatively flashy ponies on the
ovdje ne treba the sigurno one side and malnourished ponies
on the other side.

MATERIALS AND METHODS

2.1. Animals
The study was carried out on 13 domestic mountain pony
mares, selected from the Old Mountain’s pastures. The animals
were aged between 5 and 22 years (ova citava recenica nisam
sigurna, al mozda se tako kaze za zivotinje) . They were reared
in a herd of approximately 50 horses kept on a 20-hectare
pasture, which was surrounded by an electrical fence. Their
nutrition was based entirely on green grass, with free access to
water.
2.2. Body Condition Score (BCS)
Body condition of the chosen mares was evaluated during the
collection of the blood, using Henneke body condition scoring
system from 1 to 9 (Henneke and ass, 1983). Based upon the
BCS, mares were divided into two groups: malnourished and
well-nourished ones, whereby mares with BCS of 2,5 or below
were characterised as malnourished (group A – n=7), whilst
mares with BCS of 3 and above where characterised as well-
nourished (group B – n=6).
2.3. Collection of the blood
Blood collection was performed on the pasture in the middle of
August. Samples of the blood were obtained by puncture of the
jugular vein in aseptic environment and kontam da ovdje fali
put ili tako nesto into serum tubes without anticoagulant. Two
hours from mislim da treba after the time of collection, samples
were centrifugated and separated serums were stored on -
20°C.
2.4. Serum biochemistry profile
Concentrations of total protein and albumin, total bilirubin,
triglycerides and cholesterol, also serum activities of AST, GGT,
ALP and LDH were analysed in the serums. Mentioned
biochemical parameters were measured by semi-automatic
biochemistry analyser Mindray, using commercial kits for
spectrophotometric analysis by the same manufacturer.
2.5. Statistical data analysis
Mean and standard deviation were calculated for each
parameter, also an analysis of variance was carried out to
examine the differences between groups using Student’s “t”
test. Complete statistical data analysis was done in the
“Microsoft office Excel 2007”.

RESULTS
The results obtained from the analysed parameters of the
functional state of liver and lipid status determine the
differences between two groups of mares: malnourished and
well-nourished, which can be seen in Table 1.
Significant deviations between the analysed parameters of the
functional state of liver and reference values were noted in
malnourished mares. Hence, serum activities of ALP, LDH and
GGT were statistically significantly higher in malnourished
mares compared to well-nourished (p=0,018, p=0,008,
p=0,017). What was interesting was that the values of enzymes
ALP and LDH were higher than the reference values for horses
in both groups of mares (Trailovic, 2016).
AST activity was mildly elevated in malnourished and on the
upper reference limit in well-nourished mares, whilst GGT
activity was higher in malnourished and normal in the well-
nourished mares. Concentration of albumin was lower than the
lower reference limit in malnourished mares compared to
normal level in well-nourished mares. This difference was
statistically significant (p=0,002).
Values of the remaining parameters were in the reference
range despite the differences between groups. Statistically
significant difference stavi was for example noted jer mi ovo
e.g. Ide vise kad se nabrajae.g. was noted while comparing
concentrations of the triglycerides between the malnourished
and well-nourished mares (p=0,001), however, obtained values
stayed in the reference range for this parameter. Differences in
the values of the remaining parameters were not statistically
significant, partially due to individual variations, primarily in the
group of the malnourished mares.

DISCUSSION
Results of the functional analysis of liver in horses kept in a
semi free system on the Old Mountain’s pastures, point to a
subclinical liver impairment, which partly coincides with
findings obtained a few years earlier (Trailovic, 2013).
In the latter case, summer was dry and grass scarce which
served as basis for suspicion that due to the lack of grazing
areas, horses were obliged to consume toxic plants (Jacobea
vulgaris) which were mislim da je bolje could be found umjesto
were in abundance on the pasture.
Summer of the 2018. was also dry, primarily in August when
grass was of bad quality.
That was the reason why many of the horses were in poor
condition, nearly half of the horses were malnourished,
whereby 20% of them were thin with body condition score
below 2,5. Obesity therefor, could not be linked to the liver
impairment, although the owner claimed that horses were in
better condition in June and in July.
Autochthonous pony breeds such as domestic mountain pony,
kept their seasonality in appetite and body condition.
In free pasture keeping system, these horses during the
summer months when there is an abundance of food, gain
weight and form regional adipose tissue deposits, which are
again lost during winter (Frank and ass, 2016). Loss of the body
weight in obese ponies can result in hyperlipidaemia with
increase in serum concentrations of triglycerides, unsaturated
fatty acids and cholesterol during intensive-rapid mobilisation
from adipose tissue deposits (Brinkmann and ass. 2013,
Dugdale and ass. 2010). Consequences of hyperlipidaemia
include impaired circulation and function of organs (primarily
liver and kidneys) as a result of fatty infiltration (Durham and
ass. 2010).
Liver function impairments can be detected by measuring the
serum activity of the liver enzymes, such as: gamma-glutamyl
transferase (GGT), lactate dehydrogenase (LDH), alkaline
phosphatase (ALP), aspartate transaminase (AST) and total
protein, albumin, glucose, bilirubin and creatinine
concentrations (Divers 2008, Katiuska and ass. 2013).
Based upon experimental evaluations, it was determined that
in horses, GGT primarily originated from bile duct, AST and LDH
were of hepatocellular origin, whilst ALP was both of the
hepatocellular and of bile duct origin (Durham and ass, 2003,
Durham, 2008).
In the liver, GGT is found on the canalicular surface of the
hepatocytes and in epithelial cells of the bile ductulus.
GGT location in the liver has special significance, because most
of the enzyme’s concentration in serum if not overall jesi ovdje
mislila vecina ako ne i svi emzimi, ako da onda kontam da je
bolje all of it umjesto overall, jer mislim da i overall znaci isto
kao most, originates from liver (Hoffman and ass, 2008, Lassen,
2004).
In an evaluation that included 50 horses with liver disease, only
serum activity of GGT was increased in all cases (McGorum and
ass, 1999). Increased serum activity of this enzyme is there for
therefore characteristically to subclinical liver impairments in
horses exposed to pyrrolizidine alkaloids (Curran and ass,
1996).
Over the previous years and in 2018, on the pastures where
horses were kept, were nemoj were poisonous plants that
contained stavi containig umjesto ovog da nemas previse that
pyrrolizidine and bloomed blooming from June to October i sad
ovdje dodaj could be seen, found sta god. Due to that there is a
big possibility that horses, while grazing, also consumed a fair
amount of pyrrolizidine alkaloids during this year and previous
ones and that probably in most cases there are chronic liver
damages with consequential cirrhosis.
Changes in the concentration of albumin are also in favour ako
je vezano za ovo gore dodaj of this statement. Albumins, as it is
known, are synthesised entirely in the liver, however,
hypoalbuminemia is rare in acute liver impairment due to
relatively long half-life of the albumin in the blood (McGorum
and ass, 1999, Durham and ass, 2003, Moore and ass, 2008).
This information confirms the assumption of the presence of
chronic liver impairment (cirrhosis hepatis) which is caused by
many years' exposure to pyrrolizidine.
This assumption can be confirmed by the liver biopsy and
histopathological findings of megalocytes, specific for liver
impairments due to pyrrolizidine alkaloids (Trailovic, 2016).
Low albumin levels in thin mares, mislim bez zareza surely
attribute to liver insufficiency, though hypoalbuminemia can be
a consequence of malnourishment or kidney disease. In two
thin mares e.g. increases in urea and creatinine concentrations
were found along hypoalbuminemia. Reduction in weight and
cachexia are symptoms of chronic kidney disease as well as liver
cirrhosis.
In thin mares, concentration of total bilirubin was on the upper
reference limit, whereby individual values in few horses where
slightly above the upper reference limit, which indicated the
presence of subclinical cholestasis.
Cholestasis is thereby the most common cause of increasing
serum activities of both GGT and ALP (Hoffman and ass, 2008).
Parameters of the lipid status stayed in the reference range in
both groups of mares, which excluded obesity as being stavi
one of the factors the factor responsible for the presence of
liver impairment in domestic mountain ponies on the Old
Mountain.
Some of the changes could rather be a consequence of the liver
impairment.
In the majority of cases, good nutritional state coincides with
normal values for both parameters of the functional state of
liver and parameters of the lipid status.
Weak nutritional state and reduction in weight can there for
therefore be considered as a consequence of the liver
impairment, which does not exclude other chronic diseases,
such as chronic kidney disease.

CONCLUSION
Based upon the obtained results of the functional state of liver
and lipid status in domestic mountain ponies on the Old
Mountain, it can be concluded that the majority of horses
suffer from subclinical liver impairments, most likely caused by
pyrrolizidine plants poisoning, whereby low body condition and
cachexia are consequences of the liver disease.

ACKNOWLEDGMENS...