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Comparison of Lipid Peroxidation and Several Antioxidants in
Blood of Normally Calved and Dystocia Affected Cows and
Their Newborn Calves
Kandemir, F.M.,1* Erisir, M.2 and Yuksel, M.3
Ataturk University, Faculty of Veterinary Medicine, Department of Biochemistry, Erzurum, Turkey.
Firat University, Faculty of Veterinary Medicine, Department of Biochemistry, Elazıg, Turkey.
3
Cumhuriyet University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Sivas, Turkey.
1
2
* Correspondence: Assoc. Prof. Fatih Mehmet Kandemir, Department of Biochemistry, Faculty of Veterinary Medicine, University of Atatürk, 25240,
Erzurum, Turkey. Tel:+90 442 2367086, Fax: +90 442 2360881. Email: fmehmet.kandemir@atauni.edu.tr
AB S T RAC T
The changes of malondialdehyde (MDA) concentrations in plasma and of enzymatic antioxidants like
glutathione peroxidase (GSHPx), catalase (CAT) activities, non-enzymatic antioxidants like glutathione
(GSH) concentrations in blood of normally calved and dystocia affected cows and their newborn calves
were investigated. The study included 8 normally calved cows, 8 dystocic cows and their neonates. Calves of
dystocic cows were delivered by traction from the birth canal. The MDA, GSH concentrations (P<0.001) and
CAT activity (P<0.01) were significantly higher in dystocia affected cows compared to the normally calved
cows. MDA concentrations were significantly higher in the calves of dystocia affected cows compared to the
calves of normally calved cows (P<0.001), but CAT activity and GSH concentration were significantly lower
(P<0.01). There were no significant differences in the GSHPx activity between the groups. In both dystocia
affected cows’ and normally calved cows’ calves, the plasma MDA concentrations were higher than in their
mothers. Moreover the CAT and GSHPx activities and GSH concentration in the normally calved cows’
calves were higher than in their mothers’. Our results suggest that lipid peroxidation and antioxidant status
change during dystocia, and these changes may affect the fetus by creating oxidative stress.
Keywords: Dystocia; Cows; Calves; Lipid peroxidation; Antioxidants.
INTRODUCTION
Free radicals or reactive oxygen metabolites (ROM), formed
during physiological and pathological conditions in the body
are extremely reactive and unstable reacting with proteins,
lipids, carbohydrates and nucleic acids (1, 2). The imbalance
between the production of ROM and their disposal leads to
free radical accumulation, deregulation of metabolic pathways and cellular damage through oxidative chain reactions
and lipid peroxidation, resulting in oxidative stress (3, 4).
Under normal conditions, free radicals are neutralized by
efficient antioxidant systems (3). Enzymatic antioxidants
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JUNE 2016.indb 19
like glutathione peroxidase (GSHPx), superoxide dismutase
(SOD), catalase (CAT), and non-enzymatic antioxidants
like vitamins A, E, and ß-carotene and glutathione (GSH)
protect living organisms against ROM (3).
Pregnancy is a state when oxidative stress can be expected
due to a high energy demand and increased oxygen requirements (5). Birth due to oxidative stresses causes changes in
free radical formation and in the antioxidant system of the
blood and other organs (6). Difficult calving, termed as dystocia, occurs in 3% to 25% of cattle pregnancies (7). Dystocia
has been a long-standing problem in both the beef and dairy
industry (7). It is one of the most serious complications of
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pregnancy in cattle (7). The process of parturition, though
physiological, is a stressful event and abnormal parturition
(dystocia) further adds to the normal stress of calving (8).
Information about the oxidant and antioxidant status
in dystocia affected cows is limited in the literature (9, 10),
and furthermore, there are no reports about the oxidant and
antioxidant status in neonates born by dystocia-affected
cows. Therefore, our aim was to investigate the changes of
MDA concentrations in plasma and of antioxidant enzyme
(GSHPx, CAT) activities, GSH concentrations in blood of
normally calved and dystocia affected cows and their newborn calves, particularly the oxidant and antioxidant status
in neonates born by dystocia-affected cows.
MATERIAL AND METHODS
Animals and Samples
The study was performed on 16 multiparous cows, 3-8 years
old, brought for parturition around full term to the Clinic
of Obstetrics and Gynecology of Firat University. The study
was composed of 8 normally calved cows (3 Simmental,
4 Montafon, 1 Holstein), 8 dystocia affected cows (3
Simmental, 4 Montafon, 1 Holstein) and their neonates.
Normal calving was defined as a spontaneous calving
of normal duration. Calves of dystocia-affected cows were
delivered by traction from the birth canal after correction of
presentation, position, and posture of the fetus. The blood of
normally calved and dystocia affected cows and their newborn calves were taken from the jugular vein within the first
half hour after birth. All blood samples were collected into
sterile blood collecting heparinized tubes from all cows and
newborn calves. Whole blood was separated for GSHPx and
GSH assays. The remaining blood was immediately centrifuged at 1500 g for 5 min. The erythrocytes and plasma were
collected separately for CAT and MDA assays, respectively.
All samples were kept at -25°C pending analysis.
Biochemical assays
Lipid peroxidation in plasma was measured by the thiobarbituric acid reacting substance (TBARS) method (11) and was
expressed in terms of the MDA content, which served as the
standard of 1,1,3,3-tetraethoxypropane. The formed MDA
created a pink complex with thiobarbituric acid (TBA) and
the absorbance was read at 532 nm. Values were expressed
as MDA equivalents in ηmol/ml plasma.
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Kandemir, F.M.
JUNE 2016.indb 20
Whole blood GSHPx activity was assayed by the method
of Matkovics et al. (12) and expressed as unit per g of Hb
(U/g Hb). GSHPx activity was determined by using cumene
hydroperoxide and reduced glutathione (GSH) as co-substrates and the loss of GSH following enzymic reaction at
37oC was measured spectrophotometrically with Ellman’s
reagent at 412 nm (12).
Erythrocyte CAT activity were determined according
to the method of Aebi (13) and expressed as kat/g Hb. The
decomposition of H2O2 was directly followed by the decrease
of absorbance at 240 nm. The difference in absorbance at
240 nm per time unit allowed determining the CAT activity.
The concentration of reduced glutathione was assayed by
the method of Beutler et al. (14) and expressed as µmol/g Hb.
The method was based on the capacity of sulfhydryl groups
present in whole blood to react with 5, 5’-dithiobis-(2nitrobenzoic acid) (Ellman’s reagent) and form a yellow dye
with maximum absorbance at 412 nm.
Haemoglobin (Hb) concentration was determined according to cyanmethaemoglobin method (15) and expressed
as g/ml for GSHPx and CAT activities, as g/dl for GSH
concentration. An aliquot of whole blood was mixed with a
solution of potassium cyanide and potassium ferricyanide.
All forms of haemoglobin except sulphaemoglobin were converted to cyanmethemoglobin. The absorbance was measured
in a colorimeter at a wavelength of 540 nm.
Statistical analysis
All data were and analyzed by means of the SPSS 15.0
software (SPSS 15.0, SPSS, Inc., Chicago, IL, USA).
Results were expressed as mean ± SEM (Standard Error of
the Mean). The independent t-test was used to determine
statistically significant variations between groups and differences which were considered as significant for P values
less than 0.05.
RESULTS
The MDA, GSH concentrations (P<0.001) and CAT activity (P<0.01) were significantly higher in dystocia affected
cows compared to the cows undergoing normal parturition
(Table 1). MDA concentrations were significantly higher in
the calves of dystocia affected cows compared to the calves of
normally calved cows (P<0.001), but CAT activity and GSH
concentration were significantly lower (P<0.01) (Table 2).
There were no significant differences in the GSHPx activity
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Table 1: GSHPx, CAT activities and MDA, GSH levels in normally calved cows (n=8) and dystocia affected cows (n=8)
Normal calved cows
Dystocia affected cows
P
MDA
(ηmol/ml)
7.38 ± 0.37
10.25 ± 0.76
***
CAT
(kat/g Hb)
21.95 ± 2.20
34.93 ± 2.77
**
GSHPx
(U/g Hb)
13.49 ± 0.43
13.48 ± 0.47
NS
GSH
(μmol/g Hb)
1.98 ± 0.31
3.60 ± 0.21
***
Table 2: GSHPx, CAT activities and MDA, GSH levels in normally calved cows’ newborn calves (n=8)
and dystocia affected cows’ newborn calves (n=8)
Normal calved cows’
newborn calves
Dystocia affected cows’
newborn calves
P
MDA
(ηmol/ml)
CAT
(kat/g Hb)
GSHPx
(U/g Hb)
GSH
(μmol/g Hb)
8.88 ± 0.32
51.79 ± 5.85
14.18 ± 0.84
4.52 ± 0.79
11.07 ± 0.23
32.07 ± 3.43
12.79 ± 0.28
3.59 ± 0.55
***
**
NS
**
** P<0.01, *** P<0.001, NS: No significant difference
between the groups (Table 1, 2). In both dystocia affected
cows’ and normally calved cows’ calves, the plasma MDA
concentrations were higher than in their mothers. Moreover
the CAT and GSHPx activities and GSH concentration in
the normally calved cows’ calves were higher compared to
their mothers’ values (Table 1, 2).
DISCUSSION
The process of parturition, though physiological, is a stressful
event and abnormal parturition (dystocia) further adds to
the normal stress of calving (8). Controversial information
has been available on the oxidative status in dystocia (9,10).
While some studies have reported an increase in the erythrocytic MDA concentrations of the dystocia-affected cows (9),
others have reported no significant changes in erythrocytic
(10) and plasma (9) MDA concentrations. However in the
present study, a significant increase in the plasma MDA
levels was observed in cows with dystocia. Higher levels of
MDA in cows with dystocia may be explained by higher
levels of glucocorticoids, eicosanoids, and adrenaline-induced
pathways of aerobic energy production associated with parturition, which generate reactive oxygen metabolites and lipid
peroxidation (9, 10).
The process of parturition potentially can induce oxidative
stress to the newborn (16). The transition from fetal to neonatal environment, exposes the newborn to a more oxidative
environment (16). Arguelles et al. (17) found higher oxidative
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stress of the newborn based on measurements in umbilical
cord blood compared to mother’s blood at the moment of
the birth. Gaal et al. (6) reported that in newborn calves, the
concentration of free radicals in blood was 30% higher than
their mother’s samples at calving. We also found that in both
dystocia affected cows and normal parturition cows’ calves,
the plasma MDA concentrations were higher than in their
mothers’ samples indicating that neonates in both normal
birth and dystocia are under an increased oxidative stress
when compared to their mothers.
In the present study it was found that MDA concentrations were significantly higher in the calves of dystocic cows
compared to the calves of normally calved cows. Hracsko et
al. (18) reported that in humans, lipid peroxidation in the
neonatal cord blood was significantly higher in caesarean
section as compared to vaginal delivery. This is probably an
indication of higher fetal oxidative stress in dystocia. The
greater the MDA elevation in neonates due to dystocia may
be the cause of some neonatal diseases. Free radicals were
reported to play an important role in the pathogenesis of
several pathological conditions such as hemolytic disease of
the newborn, broncho-pulmonary dysplasia, and retinopathy
of prematurity. Indeed, neonates born by caesarean section
have an increased incidence of these conditions (19).
Glutathione and glutathione-related enzymes, are one of
the major detoxification and free-radical scavenging systems
may play a role in controlling diseases (20). In our study,
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no significant differences were found in GSHPx activity in
dystocic cows as compared to cows calving normally, but
significantly higher CAT activity and GSH levels in cows suffering from dystocia were detected. Similarly, the enzymatic
and non-enzymatic antioxidant levels in the reports were
variable (9, 10, 21). We suggest that an increase in the GSH
concentration and CAT activity in the dystocia-affected
cows may be due to high concentrations of lipid peroxidation. Hermes-Lima et al. (22) proposed that the activation
of antioxidant defenses, in which the actual production of
oxyradicals should decrease, is a preparative mechanism
against oxidative stress caused by stress situations.
In various studies fetal oxidative stress in cord blood of
fetuses born in elective cesarean delivery (CD) was compared
to fetuses born in spontaneous vaginal delivery (VD) in humans, by measuring umbilical cord venous blood antioxidants
(16,18,23-25). Differences between elective CD and VD
groups were non-significant for SOD, CAT, GSHPx enzyme
activities, except for the GSH concentrations (18). Some
studies have reported an increase of SOD and CAT activities
(23) in the elective CD compared to the VD, yet others have
indicated a reduction of CAT and GSHPx activities (24).
GSH concentrations were significantly lower in the elective
CD than in VD (16, 18, 25).
An important point in the present study is the CAT
activity and GSH concentrations in the calves of dystocia
affected cows compared to the calves of normally calved cows
which were lower. These results indicate that the antioxidant
system may be impaired in the calves of dystocia affected
cows. Paamoni-Keren et al. (16) suggested that neonates who
were delivered by CD have a lower GSH level than delivered
by VD in human. Therefore, they appeared to be exposed to
the higher oxidative stress when compared to neonates who
were delivered by VD.
In present study it was found that the CAT, GSHPx
activities and GSH concentration in the normally delivered
cows’ calves were higher than in their mothers’ samples. Gaal
et al. (6) have suggested that both healthy cows and their
newborn calves are well prepared for the event of birth, and
that calves are able to counter effectively the oxidative stress
inevitably present in neonates after the first inhalation of
atmospheric oxygen.
In conclusion, the obtained results suggest that in their
newborn calves and their mothers in conditions of dystocia
appear to be associated with the occurrence of a systemic
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JUNE 2016.indb 22
oxidative stress evidenced by the changes of the MDA
concentration, CAT activity and GSH level. Moreover the
results of this study indicate that dystocia exposes the fetus
to a higher oxidative stress than uncomplicated parturition as
measured by MDA, GSH concentrations and CAT activities.
Based on our measurements we conclude that the neonates born under dystocic conditions might be predisposed
to pathological conditions in which the reactive oxygen
species may play a pathogenic role, due to deficient antioxidant defenses. Thus, we suggest that providing antioxidant
supplementation to newborn calves and cows following
dystocia may be helpful in reducing the oxidative damage
and improving post-parturtion convalescence.
REFERENCES
1. Nockels, C.F.: Antioxidants improve cattle immunity following
stress. Anim. Feed. Sci. Tech, 62:59–68, 1996.
2. Solomons, T.W.G. and Fryhle, C.B.: Organic chemistry. Wiley,
New York, pp. 459-501, 2011.
3. Miller, J.K., Brezezinska-Slebodzinska, E. and Madsen, F.C.:
Oxidative stress, antioxidants and animal function. J. Dairy. Sci.
76:2812–2823, 1993.
4. Berry, E.M. and Kohen, R.: Is the biological antioxidant system
integrated and regulated? Med. Hypotheses. 53:397–401, 1999.
5. Gitto, E., Reiter, R.J., Karbownik, M., Tan, D.X., Gitto, P., Barberi, S. and Barberi, I.: Causes of oxidative stress in the pre- and
perinatal period. Biol. Neonate. 81:146-157, 2002.
6. Gaál, T., Ribiczeyné-Szabó, P., Stadler, K., Jakus, J., Reiczigel, J.,
Pál, K., Mézes, M. and Sümeghy, L.: Free radicals, lipid peroxidation and the antioxidant system in the blood of cows and newborn
calves around calving. Comp. Biochem. Phys. B. 143:391–396,
2006.
7. Oakes, D.E., Parkinson, T.J. and England, G.C.W.: Dystocia
and other disorders associated with parturition. In: Oakes, D,E.
and Saunders, W.B. (Ed.): Arthur’s Veterinary Reproduction and
Obstetrics. London, pp. 205- 333, 2001.
8. Nakao, J. and Grunet, E.: Effects of dystocia on postpartum adrenocortical function in dairy cows. J. Dairy. Sci. 73: 2801–2806, 1990.
9. Yıldız, H., Şimşek, H., Saat, N. and Yüksel, M.: Effects of dystocia
on lipid peroxidation and enzymatic and non-enzymatic antioxidants in crossbred dairy cows. Bull. Vet. Inst. Pulawy. 55:135-139,
2011.
10. Sathya, A., Prabhakar, S., Sangha, S.P.S. and Ghuman, S.P.S.: Vitamin E and Selenium supplementation reduces plasma cortisol and
oxidative stress in dystocia-affected buffaloes. Vet. Res. Commun.
31:809–818, 2007.
11. Placer, Z.A., Cushman, L.L. and Johson, B.C.: Estimation of
product of lipid peroxidation (malonyl dialdehyde) in biochemical
systems. Anal. Biochem. 16:359–364, 1966.
12. Matkovics, B., Szabo, I. and Varga, I.S.: Determination of enzyme
activities in lipid peroxidation and glutathione pathways. Laboratoriumi Diagnosztika. 15:248–249 (in Hungarian), 1988.
Israel Journal of Veterinary Medicine  Vol. 71 (2)  June 2016
31/05/2016 13:35:51
Research Articles
13. Aebi, H.: Catalase. In: Bergmeyer, H.U. (Ed.): Methods of Enzymatic Analysis. Academic press, London, pp. 671-684, 1984.
14. Beutler, E., Duron, O., Kelly, B.M.: Improved method for the
determination of blood glutathione. J. Lab. Clin. Med. 61:882888, 1963.
15. Fairbanks, V.F. and Klee, G.G.: Biochemical aspects of hematology. In: Tietz, N.W. (Ed.): Textbook of clinical chemistry. WB
Saunders Company, Philadelphia, pp. 1532-1534, 1986.
16. Paamoni-Keren, O., Silberstein, T., Burg, A., Raz, I., Mazor, M.,
Saphier, O. and Weintraub, A.Y.: Oxidative stress as determined
by glutathione (GSH) concentrations in venous cord blood in
elective cesarean delivery versus uncomplicated vaginal delivery.
Arch. Gynecol. Obstet. 276:43-46, 2007.
17. Auguelles, S., Markado, M.J., Ayala, A., Machado, A. and Hervias,
B.: Correlation between circulating biomarkers of oxidative stress
of maternal and umbilical cord blood at birth. Free. Radic. Res.
40,565–570, 2006.
18. Hracsko, Z., Safar, Z., Orvos, H., Novak, Z., Pal, A. and Varga,
I.S.: Evaluation of oxidative stress markers after vaginal delivery
or Caesarean section. In Vivo 21(4):703-706, 2007.
19. Halliwell, B. and Gutteridge, J.M.C.: Free radicals in biology and
medicine. Oxford University Press, New York, 1999.
20. Balal, M., Canacankatan, N., Paydas, S., Seyrek, N., Karayaylalı,
Israel Journal of Veterinary Medicine  Vol. 71 (2)  June 2016
JUNE 2016.indb 23
I. and Kayrın, L.: Oxidative-antioxidative system in peripartum
acute renal failure and preeclampsia-eclampsia. Ren. Fail. 24:625632, 2004.
21. Erisir, M., Akar, Y., Gurgoze, S.Y. and Yuksel, M.: Changes in
plasma malondialdehyde concentration and some erythrocyte antioxidant enzyme in cows with prolapsus uteri, caesarean section,
and retained placenta. Revue. Med. Vet. 157:80-83, 2006.
22. Hermes-Lima, M., Storey, J.M. and Storey, K.B.: Antioxidant
defenses and metabolic depression. The hypothesis of preparation
for oxidative stress in land snails. Comp. Biochem. Physiol. BBiochem. Mol. Biol. 120:437-448, 1998.
23. Inanc, F., Kilinc, M., Kiran, G., Guven, A., Kurutas, E.B.,
Cikim, I.G. and Akyol, O.: Relationship between oxidative
stress in cord blood and route of delivery. Fetal. Diagn. Ther.
20:450-3, 2005.
24. Georgeson, G.D., Szony, B.J., Streitman, K., Varga, I.S., Kovacs,
A., Kovacs, L. and Laszlo, A.: Antioxidant enzyme activities are
decreased in preterm infants and in neonates born via caesarean
section. Eur. J. Obstet. Gyn. R. B. 103:136–139, 2002.
25. Raijmakers, M.T., Roes, E.M., Steegers, E.A., Van der Wildt, B.
and Peters, W.H.: Umbilical glutathione levels are higher after
vaginal birth than after cesarean section. J. Perinat. Med. 31:520522, 2003.
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