https://doi.org/10.52973/rcfcv-e34451
Received: 14/05/2024 Accepted: 12/07/2024 Published: 13/10/2024
1 of 15
Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
ABSTRACT
Pregnancy toxemia (PT) is a metabolic disease of small ruminant that
develops during the last period of pregnancy and can cause death.
Considering the high mortality rate, early diagnosis of the disease
is important to minimize economic loss. Thus, this study aimed to
investigate the concentrations of some adipokines (Leptin, Apelin,
Resistin) and some other biochemical parameters for their role in the
diagnosis and prognosis of PT. Fifty Kangal ewes that were between
the ages of 2–4 year–old, were used in the study. The animals included
in the study were subjected to estrus synchronization. Rams were
introduced to all ewes subjected to synchronization. Pregnancies
were diagnosed with ultrasonography on the 25
th
, 60
th
, and 110
th
days
(d) following matings. Sixteen ewes bearing twin fetuses, whose
fetal viability continued, were included in the study after general
examinations. The ewes were maintained under grazing conditions
th
d, 16 twin–bearing
pregnant ewes were randomly divided into two groups (Control and
PT). Ewes in the control group were fed to meet the nutritional
requirements. the experimental PT group ewes were fed with
equivalent to 50% of the daily needs for 20 d (120–140) and then fasted
for 72 hours (141–143). During study blood samples were taken via jugular
vein every 5 d from the 120
th
d to the 140
th
d. BHBA, glucose, Leptin,
Resistin, Insulin and Apelin were measured in the blood samples.
Liver biopsy samples were collected twice from all ewes on the 120
th
and 143
rd
d. As a result, in group PT, BHBA and leptin concentrations
reduced. Resistin, Insulin and Apelin concentration were similar in
both groups. In conclusion, it was concluded that monitoring BHBA,
glucose and leptin in PT may be useful in diagnosis and prognosis.
Key words: Pregnancy toxemia; apelin; leptin; insulin; adipokines;
Kangal ewes
RESUMEN
La toxemia de la gestación (PT) es una enfermedad metabólica de
los pequeños rumiantes que se desarrolla durante el último período
de la gestación y puede provocar la muerte. Teniendo en cuenta la
alta tasa de mortalidad, el diagnóstico precoz de la enfermedad es
importante para minimizar las pérdidas económicas. Por lo tanto,
este estudio tuvo como objetivo investigar las concentraciones de
algunas adipocinas (Leptina, Apelina, Resistina) y algunos otros
parámetros bioquímicos por su papel en el diagnóstico y pronóstico
del PT. En el estudio se utilizaron 50 ovejas de la raza Kangal que
tenían entre 2 y 4 años. Los animales incluidos en el estudio fueron
sometidos a sincronización de estro. Se introdujeron carneros a todas
las ovejas sometidas a sincronización. Las hembras gestantes fueron
diagnosticadas mediante ecografía los días 25, 60 y 110 después
del apareamiento. Se incluyeron en el estudio 16 ovejas con fetos
gemelos, cuya viabilidad fetal continuaba, después de exámenes
generales. Durante los primeros 110 días de gestación, las ovejas
16 ovejas preñadas con gemelos se dividieron aleatoriamente en
dos grupos (Control y PT). Las ovejas del grupo de control fueron
alimentadas para satisfacer los requerimientos nutricionales.
Las ovejas del grupo PT experimental fueron alimentadas con el
equivalente al 50% de las necesidades diarias durante 20 días y
luego ayunaron durante 72 horas (141–143). Durante el estudio, se
tomaron muestras de sangre a través de la vena yugular cada 5 días
desde el día 120 hasta el día 140. En las muestras se midieron BHBA,
glucosa, leptina, resistina, insulina y apelina. Se recogieron muestras
de biopsia hepática dos veces de todas las ovejas los días 120 y 143.
Como resultado, en el grupo PT, las concentraciones de BHBA y leptina
Insulina y Apelina fueron similares en ambos grupos. En conclusión,
la monitorización de BHBA, glucosa y leptina en PT puede ser útil.
Palabras clave: Toxemia de la gestación; apelina; leptina; insulina;
adipocinas; Kangal ovejas
Adipokine concentrations in sheep with experimental pregnancy toxemia.
A randomized, controlled clinical trial
Concentraciones de adipocinas en ovejas con toxemia de gestación
experimental. Un ensayo clínico controlado y aleatorizado
Sefer Türk
1
* , İhsan Keleş
2
1
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Internal Medicine. Sivas, Türkiye.
2
Erciyes University, Faculty of Veterinary Medicine, Department of Internal Medicine. Kayseri, Türkiye.
*Corresponding Author: seferturk@cumhuriyet.edu.tr
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
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INTRODUCTION
Pregnancy toxemia (PT) is a metabolic disease that develops due
to negative energy balance (NEB) in small ruminants during the last
period of pregnancy and causes hyperketonemia and hypoglycemia.
The frequency of occurrence in herds can reach up to 20%. Although
result in death if not diagnosed and treated early [1]. Sheep (Ovis aries)
and lamb deaths in sheep farming cause great economic losses [2, 3].
The mortality rate in affected animals can reach 80%. Furthermore,
electrolyte, glucose, and propylene glycol), and even if they recover,
premature birth, miscarriage, and stillbirth may occur [1, 4].
Pregnancy is an important period during which sheep must
metabolism and also ensure fetal growth [4
in the diet or reduced rumen capacity due to fetal growth are two
important risk factors for PT. As a compensation mechanism, the
mobilization of fats is aimed at meeting the energy needs necessary
to maintain fetal development and meet the needs of the sheep [5].
Adipose tissue is considered an important part of the body because
it works as an endocrine organ in addition to its contributions to
energy metabolism. The adipokines it secretes play a role in many
physiological processes of the body, such as nutrition, appetite,
energy balance, insulin and glucose metabolism, lipid metabolism,
6, 7]. The process,
with the discovery of adiponectin, resistin, visfatin, apelin, omentin,
chemin, nesfatin and a number of other adipokines [8]. The Apelin
]. It has been found that
AP is expressed at high levels especially in the hypothalamus. The
hypothalamus is an important part of the brain that can secrete
various appetite–regulating factors. This suggests that it may play an
important role in regulating nutrition. Various studies conducted in
mammals have also shown that AP plays an active role in food intake
[10
hormone in 2001 [11]. Studies conducted on animals; It has revealed
the relationship of RETN with obesity, metabolic syndrome and Type
2 diabetes [12]. Hyperglycemia and hyperinsulinemia stimulate RETN
secretion. In studies conducted on humans; A strong relationship
has been observed between RETN and insulin resistance, high blood
sugar and increased blood insulin levels [13]. Leptin (LP), synthesized
from cells in white adipose tissue, regulates food intake, body
mass, reproductive function, and plays a role in fetal growth, pro–
ysis [14].
Excessive mobilization of fats to meet energy needs contributes to
the formation and exacerbation of PT. Considering the high mortality
rate, early diagnosis of the disease is important to minimize economic
that animals affected by PT show in the early stages [15, 16]. Routine
measurement of biochemical parameters and their simultaneous
evaluation with clinical examination is an important tool in the
diagnosis of diseases [17]. Therefore, biomarkers that will enable
rapid decision–making are needed to enable treatment of the disease.
Thus, this study aimed to investigate the concentrations of LP, AP,
RETN and some other biochemical parameters for their role in the
diagnosis and prognosis of PT.
MATERIALS AND METHODS
The study was conducted in a commercial sheep farm located
was conducted between March and August of 2023, when sheep are
in the natural anoestrus period in the northern hemisphere.
Animals
In this study, 50 female Kangal sheep breeds that were between
the ages of 2–4–year–old, had given birth at least once, had not had
any disease in the last 2 months, and had been vaccinated within
the scope of preventive medicine activities were used. The sheep
did not receive any medication for the treatment of any disease,
information. The animals included in the study were weighed (Pinar,
determined as described by Ferguson et al. [18].
The animals included in the study were subjected to estrus
synchronization induction during the anoestrous period, as described by
Kivrak etal. []. In summary, intravaginal progesterone–impregnated
sponge (a white, 40 × 30 mm cylindrical polyurethane sponge
containing 20 mg Chronolone flugestone acetate; Chronogest®
synchronization purposes. On the 13
th
d after sponge application,
a single dose of PGF2α analogue (263 μg Cloprostenol sodium
equivalent to 250 μ
administered intramuscularly (IM) to all animals. One day after
prostaglandin application, the intravaginal sponge was removed
and 480 IU eCG (each mL of solution for injection contains 240 IU of
equine chorionic gonadotropin hormone; Chronogest/PMSG®, MSD,
removed, all animals were treated with a ram for 1 hour (h), morning
and evening, and the mated animals were taken into a separate
compartment and away from the rams. At 25
th
(±2) d after the mating
of the ram, a pregnancy examination was performed using transrectal
ultrasonography (5 MHz linear probe, E.I Medical Ibex Lite, USA) and
pregnancies, the pregnancy examination was repeated on 60
th
with transabdominal ultrasonography to determine fetal viability.
Finally, on d 110 (±2), all animals were examined transabdominally for
pregnancy, and 16 animals with twin offspring, whose fetal viability
continued, were included in the study after general examinations.
Before the start of restricted feeding, the right ribs were partially
shaved to allow biopsy.
Experimental Design
grazing conditions between 08:00 and 17:00 on medium–quality
pasture (average 1500 kg DM·year
-1
·ha
-1
). They had ad libitum access
to water and mineral salt and were housed in a barn for the rest of the
day. At the end of the 110
th
d, the ewes were examined by ultrasound
for pregnancy, and 16 twin–bearing pregnant ewes were selected and
by draw lot randomly divided into two groups (n=8). Ewes in the control
group were fed to meet the nutritional requirements of twin–bearing
pregnant ewes in late pregnancy (10.5% CP and 2.35 Mcal·kg
-1
of ME
on a dry matter basis) until their parturition [20]. To adapt ewes in
the control group to the control diet, a 10–d step–up protocol was
implemented, gradually increasing the concentrate levels from 0 to
46% between d 110 and 120, respectively (TABLE I). Following grazing,
TABLE I
Content and chemical composition of the ration given to the ewes in
the pregnancy toxemia (PT) group (n=6), which started with restricted
feeding on the 120
th
day of pregnancy and starved between the
140
th
and 143
rd
days, and the ewes in the control group (n=7), which
were adequately fed throughout pregnancy, during the study
Groups
Items Control Experimental
Ingredients, DM %
Wheat straw 7.4 20
Meadow hay 38.5 79
Barley 15.3 –
Corn 22.6 –
Wheat bran 4 –
Sunower meal, 36 % 7.2 –
Molasses 4 –
Salt 0.25 0.25
CaCO
3
0.45 0.45
Mineral–vitamin premix
1
0.30 0.30
Chemical composition, DM %
Dry matter
89.6 92.5
Crude Ash
8.7 9.6
Crude Protein
10.5 5.3
Ether extract
2.4 1.3
Neutral detergent ber (NDF)
44.6 68.7
Acid detergent ber (ADF)
31.8 44.0
Metabolisable energy
2
(Mcal·kg
-1
DM)
2.36 1.62
1
:Each kg contained 50.000 mg Fe, 50.000 mg Mn, 50.000 mg Zn, 10.000 mg Cu, 800
mg I, 150 mg Co, 150 mg Se, 8.000.000 IU vitamin A, 2.000.000 IU vitamin D3, 20.000
mg vitamin E.
2
:Calculated according to (NRC, 2007)
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
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the PT group did not receive concentrate feed (10.5% CP and 2.35
Mcal·kg
-1
of ME on a dry matter basis); consequently, no adaptation
protocol was implemented. On the other hand, to produce pregnancy
toxemia–inducing environment, the experimental PT group ewes were
fed with a mixture of meadow hay and wheat straw diet (equivalent to
50% of the daily needs, 5.3% CP 1.62 Mcal·kg
-1
of ME on a dry matter
basis; TABLE I) for 20 d (120–140) and then fasted for 72 h (141–143).
After 72 h of fasting, the study was terminated. TABLE I presents
the contents and chemical compositions of the diets provided to
the ewes during the 110 to d 143.
Blood Sampling and Analysis
For biochemical analyses, blood samples were taken from the Vena
jugularis with 18–gauge sterile syringe every 5 d from the 120
th
d to
the 140
th
d. Blood samples were always taken in the morning before
th
d,
blood was taken every 24 h. For serum biochemical analysis, blood
were removed by centrifugation for 10 min, distributed into labeled
the day of measurements. β–Hydroxy Butyric Acid (BHBA) levels
were measured immediately from the samples taken. BHBA was
detected and recorded with an electronic ketone meter (Precision
23]. Mindray BS–200
(Shenzhen Mindray Animal Medical Technology, China) biochemistry
analyzer was used for glucose, alkaline phosphatase (ALP), total
bilirubin, direct bilirubin, indirect bilirubin, magnesium, phosphorus,
total protein, creatinine, and aspartate transaminase (AST) analyses.
Hormone analysis
The serum samples obtained and stored in labeled microtubes at
thawed, and then vortexed (Hettich Zentrifugen Tuttlingen, D–78532
Eba 20, Germany). Analyzes of LP, RETN, Insulin and AP from serum
samples are as follows; Sheep (LP) Elisa Kit (Catalog no: 201–07–006),
Sheep (RETN) Elisa Kit (Catalog no: 201–07–0053), Sheep (Insulin)
Elisa Kit (Catalog no: 201–07–002) and Sheep (AP) Elisa Kit (Catalog
no: 201–07–368) using commercial test kits through sandwich ELISA
ELISA device. The standard curve range and sensitivity of the tests
were 0.1–30 mU·L
-1
and 0.0866 mU·L
-1
for Insulin; 2–600 ng·mL
-1
and
1.857 ng·mL
-1
for AP; 0.25–70.0 ng·mL
-1
and 0.244 ng·mL
-1
for RETN,
and 0.05–10 ng·mL
-1
and 0.05 ng·mL
-1
for LP, respectively.
Biopsy acquisition and analysis
Liver biopsy samples were taken twice from the animals in the
control and PT groups, on the 120
th
and 143
rd
after the animals were restrained, liver biopsies were performed
with an 18 G (1.2 mm), disposable soft tissue biopsy needle inserted
space on the right side of the sheep, as described in Ferreira et al.
[24]. (Medax, Italy) accompanied by ultrasound (2 Mhz Convex Probe,
Mindray DC–N3 Vet, China).
in 10% neutral formalin solution for 24–48 h. Then, it was washed
under running water for 8 h to remove the formalin present in the
tissue. Tissues were subjected to routine alcohol–xylene follow–up
stained with hematoxylin–eosin. The histopathological changes seen
in the tissues were evaluated under a light microscope (Zeiss Primo
Star with an integrated Carl Zeiss Axiocam ERc 5s; Carl Zeiss AG,
no changes (0), mild (1), moderate (2) and severe (3).
Chemical analysis
The dry matter, crude ash, ether extract, and crude protein were
(AOAC) procedure [21
Mertens (2002) [22
the AOAC procedure [21]. The analysis of both NDF and ADF were
Corp., USA) and expressed exclusive of residual ash.
FIGURE 1. A – Control group, 120
th
day of pregnancy, normal histological appearance. B – Control group, 143
rd
day of pregnancy, normal histological appearance.
C – Pregnancy toxemia (PT) group, 120
th
day of pregnancy, normal histological appearance. D – PT group, 143
rd
day of pregnancy, fatty vacuoles in the cytoplasm of
hepatocytes and nuclei pushed towards the cell wall (Arrows). Liver – H&E. 40×
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
4 of 15
Statistical analysis
The number of animals used in the study was determined using
G*Power 3.1 software, with an effect size of 0.3, α<0.05 and 1 – β
and 8 repeated measurements in 2 groups. The data obtained in
the study were used in SPSS 25.0. version (IBM, Armonk, NY, USA)
(Graphpad Software, Inc., CA, USA) []. For comparisons of means,
repeated analysis of variance with one of the factors in factorial
order was used. The suitability of the data for repeated measures
If it did not meet the prerequisites of parametric tests, the degrees
of freedom were corrected, and Greenhouse and Geisser tests were
applied. Bonferroni correction was made for multiple comparisons.
normality and homogeneity using the Shapiro wilk and Levene tests.
live weight data. In histopathological measurements, the difference
U test. Pearson correlation analysis was performed for parametric
data to determine the relationship between the parameters. The
cut off scoreswere evaluated by receiver operating characteristic
values were calculated. Cutting points were evaluated according to
the Youden Index. Values of P<0.05 and P<0.01 were accepted for the
the tests.
RESULTS AND DISCUSSION
The mean body weight was 58 ± 4,5 kg (BCS –body condition score–)
group, at the beginning of the study. The mean age of the animals in
the PT and control groups was 3 ± 1 old year. No statistical difference
was observed in body weight, BCS, or age between the two groups
at the beginning of the study (P<0.05).
On the 130
th
d of the study, one animal in the control group was
removed from the control group due to widespread infection
symptoms such as high fever, leukocytosis, and cessation of eating
and drinking water. Two animals in the study group were excluded
from the study because they would not be suitable for repeated
measurement analysis due to abortion on the 136
th
and 138
th
d,
respectively. In the study group, on the second day of fasting (d 142)
in six animals, standing up with help, licking the wall, leaning their
as clinical symptoms. On the third day of starvation (d 143), 4 animals
aborted, the study was terminated, and the animals were treated.
During the study, no clinical signs of PT were observed in the animals
in the control group.
In the histopathological examination of liver tissues, the liver had a
normal histological appearance on the 120
th
and 143
rd
d of the control
group and the 120
th
d of the PT group, while the nucleus was seen to be
pushed towards the cell wall due to severe lipidosis in the hepatocytes
on the 143
rd
d of the PT group (FIG.1, TABLE II).
A B
C D
TABLE II
Results of liver histopathological examination of ewes in
the pregnancy toxemia (PT) group (n=6), which started with
restricted feeding on the 120
th
day of pregnancy and starved
between days 140 and 143, and ewes in the control group
(n=7), which were adequately fed throughout pregnancy
Days Fatty Liver Degree
Control group 120
th
day 0.16 ± 0.40
a
Control group 143
rd
day 0.25 ± 0.46
a
PT group 120
th
day 0.33 ± 0.51
a
PT group 143
rd
day 2.83 ± 0.40
b
a,b
: Indicates the dierence between times (P<0.05)
FIGURE 2. Correlations of the data obtained from the ewes in the pregnancy toxemia (PT) group (n=6), which started with restricted feeding on the 120
th
day of
pregnancy and starved between the 140
th
and 143
rd
day, and the ewes in the control group (n=7), which were adequately fed throughout pregnancy, and the heat
map representation of r values. BHBA: beta hydroxy butyric acid, Indir. Bil.: indirect bilirubin, AST: aspartate transaminase, ALP: alkaline phosphatase, Total Bil.: total
bilirubin, Direct Bil.: direct bilirubin and Total Prot.: total protein
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
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Since PT is a metabolic disease that occurs as a result of disorders
in carbohydrate and fat metabolism due to negative energy balance
and progresses with hypoglycemia and hyperketonemia, liver biopsy
is considered the gold standard in detecting fatty liver [28]. Previous
studies have reported that starvation or restricted feeding causes
the cytoplasm of hepatocytes to fill with fat and their nuclei to
be pushed towards the cell wall [28, , 30]. In this study, similar
control group led to the conclusion that this condition was associated
with the progress of pregnancy.
Correlation analysis was performed to determine the relationship
between the parameters in the data set. According to this analysis, a
between BHBA with AP and RETN (r = 0.25 and r = 0.16), respectively.
A moderate correlation between BHBA and LP with rate of r = 0.61
were determined. A weak correlation was observed between glucose
and AP with a rate of r = -0.13, a moderate correlation with leptin with
correlations obtained in the study are shown in Fig. 2 and in TABLE III.
Pregnancy toxemia is known as a serious metabolic condition that
of the liver and bile, mineral metabolism, glucose homeostasis, and
renal function, as well as in the connections between these biochemical
indicators [1, 6, 7
the theory that pregnancy toxemia is a complex metabolic illness.
Serum BHBA, AP, LP, insulin, RETN, glucose, ALP, total bilirubin,
magnesium, direct bilirubin, indirect bilirubin, creatinine, phosphorus,
AST, and total protein concentration and statical analysis results are
given in FIGS. 3, 4, 5, 6.
TABLE III
P-values of the data obtained from the ewes in the pregnancy toxemia (PT) group (n=6), which started with restricted feeding on the 120
th
day of
pregnancy and starved between the 140
th
and 143
rd
days, and the ewes in the control group (n=7), which were adequately fed throughout pregnancy
BHBA Apelin Leptin Insulin Resistin CRE IND.BIL. Mg AST ALP TOT.BIL DIR.BIL Phos Glu TP
BHBA mmol·L
-1
Apelin (ng·L
-1
) 0.552
Leptin (ng·mL
-1
) 0.110 0.818
Insulin (U·L
-1
) 0.036 0.390 0.274
Resistin (ng·mL
-1
) 0.698 0.908 0.481 0.993
CRE (mg·dL
-1
) 0.000 0.678 0.233 0.040 0.595
IND.BIL. (mg·dL
-1
) 0.000 0.472 0.035 0.100 0.860 0.007
Mg (mg·dL
-1
) 0.003 0.612 0.037 0.138 0.915 0.015 0.000
AST (U·L
-1
) 0.027 0.565 0.048 0.167 0.298 0.068 0.012 0.009
ALP (U·L
-1
) 0.002 0.888 0.052 0.065 0.536 0.009 0.001 0.000 0.055
TOT.BIL (mg·dL
-1
) 0.000 0.733 0.029 0.044 0.874 0.010 0.000 0.000 0.016 0.000
DIR.BIL (mg·dL
-1
) 0.044 0.475 0.117 0.005 0.926 0.046 0.093 0.169 0.310 0.076 0.052
Phos (mg·dL
-1
) 0.017 0.996 0.039 0.098 0.706 0.024 0.012 0.006 0.002 0.017 0.012 0.212
Glu (mg·dL
-1
) 0.001 0.763 0.172 0.040 0.296 0.004 0.004 0.023 0.194 0.003 0.003 0.022 0.099
TP (g·dL
-1
) 0.168 0.451 0.572 0.243 0.238 0.137 0.281 0.440 0.069 0.636 0.357 0.340 0.176 0.460
Creatinine (CRE), indirect bilirubin (IND.BIL.), magnesium (Mg), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TOT.BIL), direct bilirubin (DIR.BIL),
phosphorus (Phos), glucose (Glu) and total protein (TP)
120
125
130
135
140
F1
F2
F3
6.5
7.0
7.5
8.0
8.5
Time (days of pregnancy and fasting)
Total protein (g·dL
-1
)
Control
Pregnancy toxemia
(time P=0.14 | group P=0.01 | time × group P=0.01)
*
*
*
*
*
*
120
125
130
135
140
F1
F2 F3
0
20
40
60
80
Time (days of pregnancy and fasting)
Glucose mg·dL
-1
Control
Pregnancy toxemia
(time P=0.01 | group P=0.01 | time × group P=0.01)
*
*
*
*
*
*
*
a
b
b
c
bc
bc
bc
abc
FIGURE 3. Changes in total protein and glucose concentrations in the study group twin pregnant ewes, which were subjected to restricted feeding from 120
th
.days of
pregnancy to 140
th
days and starved after 140
th
days of pregnancy, and in the control group twin pregnant ewes, which were fed with healthy food during the same
period. Error bars show SEM, PT: Pregnancy toxemia F: Fasting, *: P<0.05, abc: Varied characters under the same line are statically dierent. (Time = Eect of time
independent of groups, Group = Eect of group independent of time, Time × Group = Interaction between groups over time)
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
6 of 15
P<0.05)
between them and the control group. The BHBA concentration of the
(P>0.05).
The BHBA concentration started to increase after 5 d of restricted
feeding and soon exceeded 1 mmol·L
-1
in PT group. It reached the
critical concentration of 1.6 mmol·L
-1
at the end of 10 d and rose above
3.2 mmol·L
-1
on the 143
rd
d when the study was terminated. (FIG. 4).
Based on the 0.8 mmol·L
-1
BHBA threshold reported in the literature
Duehlmeier et al. [25], ROC analyzes of the variables in the data set
ratio and negative likelihood ratio (+, – ) were calculated. ROC analysis
results presented in TABLE IV. and FIGS. 7, 8.
120 125 130 135 140 F1 F2 F3
1.0
1.5
2.0
2.5
3.0
Time (days of pregnancy and fasting)
Mg mg·dL
-1
Control
Pregnancy toxemia
(time P=0.01 | group P=0.02 | time × group P=0.01)
120
125
130 135 140 F1 F2 F3
0
1
2
3
4
BHBA mmol·L
-1
Control
Pregnancy toxemia
Time (days of pregnancy and fasting)
(time P=0.01 | group P=0.01 | time × group P=0.01)
120 125 130 135 140 F1 F2 F3
25
50
75
100
125
150
Apelin ng·L
-1
ControlPregnancy toxemia
(time P=0.84 | group P=0.60 | time × group P=0.84)
Time (days of pregnancy and fasting)
120 125 130 135 140 F1 F2 F3
0
1
2
3
4
5
Leptin ng·mL
-1
Control
Pregnancy toxemia
(time P=0.54 | group P=0.27 | time × group P=0.008)
Time (days of pregnancy and fasting)
120 125 130 135 140 F1 F2 F3
0
5
10
15
20
25
30
35
Insulin IU·L
-1
Control
Pregnancy toxemia
Time (days of pregnancy and fasting)
(time P=0.50 | group P=0.27 | time × group P=0.16)
120
125
130
135
140
F1
F2
F3
0
5
10
15
20
25
30
35
Resistin ng·mL
-1
Control
Pregnancy toxemia
Time (days of pregnancy and fasting)
(time
P
=0.50 | group
P
=0.27 | time × group
P
=0.16)
FIGURE 4. Changes in magnesium (Mg), beta hydroxy butyric acid (BHBA), apelin, leptin, resistin, insulin concentrations in the study group twin pregnant ewes, which
were subjected to restricted feeding from 120
th
.days of pregnancy to 140
th
days and starved after 140
th
days of pregnancy, and in the control group twin pregnant ewes,
which were fed with healthy food during the same period. Error bars show SEM, PT: Pregnancy toxemia F: Fasting, *: P<0.05, abc: Varied characters under the same line
are statically dierent. (Time = Eect of time independent of groups, Group = Eect of group independent of time, Time × Group = Interaction between groups over time)
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
7 of 15
120
125
130
135
140
F1
F2
F3
0.5
0.6
0.7
0.8
0.9
1.0
Time (days of pregnancy and fasting)
Creatinine mg·dL
-1
Control
Pregnancy toxemia
*
*
*
*
*
*
(time
P
=0.54 | group
P
=0.01 | time × group
P
=0.10)
120 125 130 135 140 F1 F2 F3
60
70
80
90
100
110
120
130
140
150
Time (days of pregnancy and fasting)
AST U·L
-1
Control
Pregnancy toxemia
(time P=0.01 | group P=0.36 | time × group P=0.01)
120
125
130
135
140
F1
F2
F3
0
25
50
75
100
125
150
Time (days of pregnancy and fasting)
ALP U·L
-1
Control
Pregnancy toxemia
*
*
(time
P
=0.02 | group
P
=0.36 | time × group
P
=0.01)
120 125 130 135 140 F1 F2 F3
0.0
0.5
1.0
1.5
Time (days of pregnancy and fasting)
Tot
al
Bili
rub
in
mg·
dL
-1
Control
Pregnancy toxemia
(time P=0.01 | group P=0.01 | time × group P=0.01)
*
*
*
*
*
*
*
120 125 130 135 140 F1 F2 F3
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Time (days of pregnancy and fasting
Direct Bilirubin mg·dL
-1
ControlPregnancy toxemia
*
*
*
*
(time P=0.10 | group P=0.01 | time × group P=0.07)
120 125 130 135 140 F1 F2 F3
0.0
0.2
0.4
0.6
0.8
1.0
Time (days of pregnancy and fasting
Indirect Bilirubin mg·dL
-1
Control
Pregnancy toxemia
(time P=0.01 | group P=0.01 | time × group P=0.01)
*
*
*
*
*
*
FIGURE 5. Changes in creatinine, aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin, direct bilirubin and indirect bilirubin concentrations in the
study group twin pregnant ewes, which were subjected to restricted feeding from 120 d of pregnancy to 140 d and starved after 140 d of pregnancy, and in the control
group twin pregnant ewes, which were fed with healthy food during the same period. Error bars show SEM. PT: Pregnancy toxemia F: Fasting *: P<0.05(Time = Eect
of time independent of groups, Group = Eect of group independent of time, Time x Group = Interaction between groups over time)
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
8 of 15
120 125 130 135 140 F1 F2 F3
0
2
4
6
8
10
12
Time (days of pregnancy and fasting
Phosphorus mg·dL
-1
Control
Pregnancy toxemia
*
*
FIGURE 6. Changes in phosphorus concentrations in the study group twin pregnant
ewes, which were subjected to restricted feeding from 120 d of pregnancy to 140 d
and starved after 140 d of pregnancy, and in the control group twin pregnant ewes,
which were fed with healthy food during the same period. Error bars show SEM.
PT: Pregnancy toxemia F: Fasting *: P<0.05(Time = Eect of time independent of
groups, Group = Eect of group independent of time, Time × Group = Interaction
between groups over time)
TABLE IV
Optimum cut o values and respective sensitivity, specicity, area under the curve, SE, positive likelihood
ratio and negative likelihood ratio of the Pregnancy toxemia (PT) (beta hyrdoxy butyric acid (BHBA)
threshold is 0.8 mmol/l) prediction in last trimester pregnancy in Akkaraman–Kangal ewes
Parameters Cuto AUC SE %95 CI for AUC P
Sensitivity%
(%95 CI)
Spesicity%
(%95 CI)
LR+ LR–
Resistin (ng·mL
-1
) 8.740 0.555 0.0672 0.423 – 0.686 =0.386 55.56 60.00 1.38 0.74
Insulin (U·L
-1
) 5.971 0.589 0.0657 0.460 – 0.718 =0,158 52.78 60.00 1.31 0.78
Apelin (ng·L
-1
) 87.60 0.529 0.0657 0.400 – 0.658 =0.642 52.78 62.00 1.38 0.76
Leptin (ng·mL
-1
) 1.386 0.644 0.0607 0.525 – 0.763 =0.023* 69.44 56.00 1.57 0.63
IND.BIL. (mg·dL
-1
) 0.465 0.929 0.0322 0.866 – 0.992 <0.0001** 81.58 97.56 33.45 0.19
CRE (mg·dL
-1
) 0.735 0.915 0.0306 0.855 – 0.975 <0.0001** 81,58 87.23 6.39 0.21
TOT.BIL (mg·dL
-1
) 0.420 0.975 0.0130 0.950 – 1.000 <0.0001** 97.37 82.98 5.72 0.03
DIR.BIL (mg·dL
-1
) 0.165 0.957 0.0199 0.918 – 0.996 <0.0001** 89.19 89.13 8.20 0.12
TP (g·dL
-1
) 7.435 0.874 0.0380 0.799 – 0.948 =0.0001** 84.21 82.98 4.94 0.19
Glu (mg·dL
-1
) 48.50 0.988 0.0087 0.971 – 1.000 <0.0001** 94.74 95.74 22.26 0.05
Mg (mg·dL
-1
) 2.020 0.762 0.0523 0.659 – 0.864 <0.0001* 71.05 63.83 1.96 0.45
Phos (mg·dL
-1
) 6.495 0.577 0.0639 0.452 – 0.703 =0.219 52.63 53.19 1.12 0.89
ALP (U·L
-1
) 56.75 0.815 0.0482 0.721 – 0.910 <0.0001** 78.95 74.47 3.09 0.28
AST (U·L
-1
) 83.00 0.636 0.0626 0.513 – 0.759 =0.031* 57.89 57.45 1.36 0.73
AUC: area under the curve, LR+: positive likelihood ratio, LR-: negative likelihood ratio, SE: standard error, CI: condence interval,
P: signicance,
indirect bilirubin (IND.BIL.), Creatinine (CRE), magnesium (Mg), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TOT.BIL),
direct bilirubin (DIR.BIL), phosphorus (Phos), glucose (Glu) and total protein (TP)
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
Although creatinine levels increased over time, no difference was
detected between groups. There was no difference in AP, RETN and
Insulin levels between the groups in the measurements. At the LP
groups, especially after the fasting application.
The frequently preferred method to establish experimental PT is to
fast for 72 h on the 140
th
d of pregnancy [26, 27]. In previous study with
study team Kivrak et al. [
of fasting were applied to singleton pregnant sheep to create PT. This
sheep, the fasting period was planned as 72 h, unlike previous study,
anticipating that the symptoms would occur more quickly.
β–Hydroxy Butyric Acid levels resulting from the mobilization of
fats are considered a good indicator in showing NEB (Negative energy
balance) [1
th
d of pregnancy), the
BHBA level was below 0.8 mmol·L
-1
in both groups. Although there was
the control group, the BHBA level exceeded 3 mmol·L
-1
on the 141
st
d in
the PT group. The development of clinical symptoms, the occurrence
of abortion, and the buildup of fat in the liver were assessed as proof
that the technique employed in this study was appropriate for inducing
PT in twin pregnant sheep.
are not compatible. In some studies, it has been reported that clinical
symptoms begin to appear between BHBA levels of 0.8 mmol·L
-1
and
-1
[31], and in others, they are observed only after the level
of 3 mmol·L
-1
is exceeded [32]. In the current study, abortion was
-1
sheep in the PT group with restricted feeding and starvation. In
liver biopsy analysis, steatosis was evident in sheep in the PT group.
Glucose, magnesium, total protein and ALP levels of biochemical
bilirubin, indirect bilirubin, AST and phosphorus levels in the PT group.
FIGURE 7. ROC analysis of leptin, apelin, aspartate transaminase (AST), creatinine, direct bilirubin, indirect bilirubin, total bilirubin, glucose, insulin, alkaline phosphatase
(ALP), magnesium, phosphorus in Akkaraman–Kangal breed sheep in the last trimester of pregnancy, based on the 0.8 mmol threshold
0 20 40 60 80 100
0
20
40
60
80
100
Leptin
100% - Specificity%
Sensitivity %
0 20 40 60 80 100
0
20
40
60
80
100
Creatinine
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Direct Bilirubin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Indirect Bilirubin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Total Bilirubin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Glucose
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Insulin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
ALP
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Magnesium
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Phosphorus
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Apelin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
AST
100% - Specificity%
Sensitivity%
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
10 of 15
FIGURE 8. ROC analysis of resistin and total protein in Akkaraman–Kangal breed sheep in the last trimester of
pregnancy, based on the 0.8 mmol threshold.
0 20 40 60 80 100
0
20
40
60
80
100
Resistin
100% - Specificity%
Sensitivity%
0 20 40 60 80 100
0
20
40
60
80
100
Total Protein
100% - Specificity%
Sensitivity%
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34451
11 of 15
-1
and 1.5 mmol·L
-1
). In other sheep, clinical symptoms
began to appear only when the BHBA level increased to 3 mmol·L
-1
and
31]
and Sargison [33
-1
clinical signs (such as abortion) can be observed in animals.
Glucose level is one of the frequently measured parameters in PT.
Researchers have found different results regarding glucose levels
in PT studies. Some researchers reported hypoglycemia, some
reported normoglycemia, and some reported hyperglycemia [1, 34,
35]. Additionally, it has been reported that it is not compatible with
BHBA due to the variability of glucose level [36]. In the current study,
it was observed that glucose levels gradually decreased with the onset
of restricted feeding; It was observed that when fasting started, it
started to increase, but this increase did not reach hyperglycemia
or even normoglycemia. On the other hand, this study revealed
that glucose showed a very strong negative correlation with BHBA
level. The following explanation can be used to describe the glucose
starts. Long–term lipomobilization and hypoglycemia lead to insulin
resistance. Tissues that are resistant to insulin are less sensitive to
it. Insulin causes a decrease in glucose transport to tissues and a
rise in blood glucose levels. For this reason, studies have reported
that normoglycemia or hyperglycemia may occur [1].
Adult sheep have a limited ability to circulate body magnesium
reserves. Therefore, they need to take magnesium daily to meet their
requirements [37]. Decreased magnesium concentration may cause
[38]. Additionally, hypomagnesemia impairs both insulin secretion
and the effect of insulin on peripheral tissues, thus affecting the
development of poor insulin resistance. In this study, the magnesium
level decreased gradually with 20 d of restricted feeding and became
evident with 3 d of complete starvation. Additionally, in this study, a
strong negative correlation was observed with BHBA level. On the other
hand, magnesium levels were reported as normal in studies conducted
on sheep with subclinical PT [36, ]. However, in clinical PT cases
[40], it has been reported that magnesium levels may decrease due to
decreased food intake in the last period of pregnancy [38]. Therefore,
with the current study, it was concluded that the severity of PT and
magnesium concentration were compatible.
Since the organ most affected by gestational toxemia is the liver,
tests showing the status of the liver have been shown in many studies
[28, 35, 41, 42]. In the current study, total protein, total bilirubin,
indirect bilirubin, direct bilirubin, AST and ALP levels were measured
previous studies, it was stated that total protein decreases because
the decrease in hepatic metabolism and impairment of its function
causes a decrease in protein synthesis [41, 42]. As a matter of fact,
similar results were obtained in this study. In the current study, it
was determined that total bilirubin, indirect bilirubin and direct
bilirubin levels were high. Constable et al. [43], suggested that the
amounts of fat. AST enzyme is considered the most sensitive enzyme
to histopathological changes in the liver. Studies on the subject have
shown that AST increases due to fat accumulation in hepatocytes,
accompanied by mitochondrial dysfunction and destruction of cellular
organelles [41, 44]. In this study, the data obtained regarding AST
the PT group compared to the control group. Sargison et al. [45],
stated that high ALP levels may be an indicator of poor prognosis
for the mother. There are studies also reporting that low ALP levels
indicate fetal growth retardation or a high–risk pregnancy [46, 47].
In other words, it can be said that high ALP levels are an indicator of
a bad prognosis for the mother and low ALP levels are an indicator
this study, the pattern of abortion in the PT group was found to be
compatible with the literature information in question. In addition,
in this study, it was concluded that BHBA and liver panel tests were
highly correlated and were effective auxiliary parameters in reaching
the diagnosis, in line with the literature.
phosphorus concentrations in previous studies [27, 32, , 40, 48].
In the current study, although serum creatinine levels differed on
the d when they were measured (130
th
, 135
th
, 140
th
, 141
st
and 142
nd
),
there was no statistical difference between the groups. It is reported
in the literature that the increase in creatinine level is a result of
protein catabolism and renal failure [40
75%, creatinine levels rise. In cases of renal failure with less nephron
Adipokine concentrations in sheep with experimental pregnancy toxemia / Türk and Keleş _________________________________________
12 of 15
loss, the creatinine level may be normal [48]. Contrary to studies in
which high creatinine levels were associated with death [36, 41],
creatinine levels remained normal between groups in this study.
In the current study, it was observed that the phosphorus level
that phosphorus level increases due to hyperketonemia, decreased
calcium absorption, increased parathormone activity and decreased
35, , 50]. It is known that hypocalcemia and
hyperketonemia are common in PT. In the current study, it was
concluded that the formation of hyperphosphatemia, especially
during the fasting period, is an indicator of impaired phosphorus
balance caused by hyperketonemia.
Previous studies reported that insulin levels decreased in sheep
affected by PT [35]. On the other hand, in a study conducted on pregnant
sheep where 50% of their energy requirements were met, it was reported
that the insulin level was not affected [51]. As a matter of fact, in this
study, there was no difference between groups in insulin concentration.
In the light of these studies, it has been evaluated that hypoinsulinemia
is more prominent in severe PT cases accompanied by hyperglycemia.
Hyperglycemia did not occur in animals with PT during the current study.
Therefore, it suggests that the insulin level remains at basal level.
In previous studies, there are different results regarding the RETN
level in situations of restricted feeding or starvation. In a study using
fat–tailed sheep, it was found that the RETN level increased 5.2 times
compared to the control group after 4 weeks of fasting [52]. On the
contrary, a decrease in RETN level has been reported in rats fasted
for a long time [53]. In another study, RETN levels were shown to be
stable during fasting [54]. In the current study, no difference was seen
between the groups in the RETN level. It has been reported that high
levels of insulin, the regulator of serum RETN expression, may increase
RETN concentration [55]. In this study, it was concluded that there
was no change in RETN level as there was no change in insulin level.
Apelin, secreted from adipocytes, is found in the heart, lung,
kidney, liver, adipose tissue, digestive system, brain, adrenal
glands, endothelium and plasma and plays an important role in the
physiology of the control of feeding behavior, energy expenditure
56]. In previous studies,
it was reported that high levels of insulin, which is the regulator of
serum AP expression, decreases AP concentration, while low insulin
levels increase AP levels [57]. In the current study, no change in AP
level was observed between groups. In this study, it was concluded
that there was no change in AP level between the groups in parallel
with the insulin level.
Previous studies have reported that plasma LP level decreases due
to increased energy need [58, ]. Consistent with this information,
in the current study, a gradual decrease in LP level was observed
in the control group as birth approached. In the control group, due
to the increase in energy needs, the BHBA level was measured as
0.64 mmol·L
-1
on the 120
th
d of pregnancy, while the average was
0.76 mmol·L
-1
on the 143
rd
d of pregnancy. In the PT group, although
the LP level was similar to the control group during the restricted
feeding period, it was determined that it decreased statistically
reported that the LP level decreased due to negative energy balance
[60, 61]. On the other hand, another study reported that there was
no change in the serum LP level of sheep after 5 days of fasting [62].
In a different study on LP level, the plasma LP level of sheep in the
anoestrus period and the mating season was compared and it was
reported that it was 180% higher in the anoestrus period [63]. High
LP levels were expressed as LP resistance and were reported to be
a result of seasonal adaptation, which provides sheep with more
nutrition opportunities and contributes to energy storage [64].
Prolonged hypoglycemia and correspondingly increased cortisol
levels in pregnancy toxemia are well known [65, 66]. Cortisol hormone
and leptin levels show an inverse correlation in acute and subacute
cases. However, long–term exposure to cortisol increases leptin
synthesis [67, 68]. Although the process was short in previous studies,
in the present study it was concluded that it covered a 23 d period
and was carried out during the anoestrus period, contributing to the
increase in LP level.
CONCLUSION
β–Hydroxy Butyric Acid and glucose are simple and effective
biomarkers of maternal energy deprivation. This study revealed
that while glucose levels decreased and BHBA levels increased with
restricted feeding, there was no change in AP, RETN and Insulin
concentrations. This study showed that monitoring LP, liver panel
tests, kidney function tests, and minerals in addition to BHBA and
glucose in PT may aid diagnosis. Low ALP concentration may be a sign
that the health of the fetus is in danger in sheep. More experimental
animals and more detailed studies are needed to fully understand the
role of adipokines, especially LP, in pregnancy toxaemia of sheep.
ACKNOWLEDGMENTS
and Stem Cell Research Center for assitance and contributions with
the laboratory analysis. (GENKÖK), Kayseri,
Funding
Erciyes University (Project No. TDK–2022–12347).
Conict of Interest
to the research, authorship, and/or publication of this article.
Ethics Approval
The study protocol was approved by the Animal Experiments Local
Ethics Committee of Sivas Cumhuriyet University (Approval number
656; Approval date, 17.06.2022).
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