- Email: [email protected]
Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis
March 2015
ScienceDirect
Vol. 22 No. 1 35-39
Journal of Northeast Agricultural University (English Edition)
Available online at www.sciencedirect.com
Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis Song Xue, Li De-jun, Feng Guo-feng, Li Bei, and Liu Yun-feng* College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
Abstract: Subclinical endometritis is a physiological inflammation that serves to clear persistent contaminants from the uterus. To investigate the alteration of antioxidant, such as vitamin E (VE) and vitamin C (VC), total oxidant capacity (TOC) and nitric oxide (NO) in cows with normal and subclinical endometritis (SCE), we examined the concentrations of NO, VC and VE, TOC and polymorphonuclear neutrophils (PMN) percentage in uterine secretion. The cows were divided into two groups, normal (n=20) and subclinical endometritis (SCE, n=60), based on endometrial cytology (presence of PMN≥5%). Uterine secretion and blood were collected as described previously. Griess reaction was used to determine the concentration of NO. The concentrations of TOC, VC and VE were detected by a commercially available assay kit. The results showed that the concentrations of NO, TOC and PMN percentage were significantly higher (P<0.01, P<0.05, and P<0.01, respectively) in uterine secretion with SCE compared to those from normal; however, the levels of VC and VE were significantly lesser (P<0.01). In conclusion, the concentrations of NO, TOC, VC, VE and PMN percentage differed between normal and SCE cows. Meanwhile, the relationship between the concentration of NO and PMN percentage from uterine secretion in cows with subclinical endometritis were positively correlated. Consequently, these alterations in NO, TOC, VC, VE levels and PMN percentage contributed to as a diagnostic index of the uterine inflammation, with the aim to increase the reproduction of the cows and the decrease economic losses. Key words: cow, subclinical endometritis, nitric oxide, antioxidant, total oxidant capacity CLC number: S823
Document code: A
Article ID: 1006-8104(2015)-01-0035-05
Ribeiro et al., 2013; Mohammad et al., 2014).
Introduction
Polymorphonuclear neutrophils (PMN) in uterine
Subclinical endometritis (SCE), as one of the most
state (Kasimanickam, 2004). Excessive amounts of
important disorders in dairy cows during the post-
neutrophilic granulocytes, macrophages, lymphocytes,
partum period, impacts reproduction (Sheldon et al.,
eosinophilic granulocytes and various epithelial cells
2006; Dubuc et al., 2010) and causes economic losses
of the uterine tissue in uterine fluid are considered as a
(Azawi, 2008). Earlier diagnose and treatment of
response to prevent exogenous pathogenic bacterium
SCE play an important role in controlling clinical
in the part of inflammation of the uterus (Singh, 2008).
endometritis, yet the index of the early diagnosis are
In addition, the level of pro-inflammatory cytokines,
still not clear (Ahmadi et al., 2005; Gilbert, 2005;
such as tumor necrosis factor α (TNF-α), interleukin
secretion is an indicator reflecting uterine health
Received 20 October 2014 Supported by Funding (RCB22) from the Doctoral Research Foundation of Northeast Agricultural University (2012); the Postdoctoral Fund of Heilongjiang Province (LBH-Z11239) Song Xue (1989-), female, Master, engaged in the research of nutrition regulation and metabolism disease. E-mail: songxue521521@163. com * Corresponding author. Liu Yun-feng, associate professor, engaged in the research of nutrition regulation and metabolism disease. E-mail: liuyunfeng@ neau.edu.cn http: //publish.neau.edu.cn
·36·
Journal of Northeast Agricultural University (English Edition)
Vol. 22 No. 1 2015
(IL) 1β, IL-6, IL-8 and some other molecules such
NO, VC and VE, total oxidant capacity (TOC) and
as nitric oxide (NO) are increased during the period
PMN percentage from uterine secretion in cows were
of infections (Sheldon et al., 2001; Li et al., 2010;
examined.
Loyi et al., 2013). As an inflammatory mediator, NO which synthesized by macrophages causes smooth muscle relaxation and mediates cytoimmunity and
Materials and Methods
inflammation toxicity. Excess NO is produced during
Experimental animals
inflammation as a primer defense system (Subandrio
Totally 80 Holstein cows from Dairy Tarm were enroll-
et al., 2000). Study showed that NO content is in-
ed in this study (Sarkar et al., 2006; Kasimanickam
creased during inflammation diseases (Rawlingson,
et al., 2004). All the Holstein cows were routinely
2003; Abdorrahman et al., 2005). In addition, sub-
examined once between 28 and 33 days after calv-
stances derived from oxidation of NO, such as pero-
ing, included inspection of the vulva, tail, and
xynitrite, changed antioxidant balance of the bacteria
perineum, vaginoscopy, and transrectal palpation
(Onur et al., 2010).
of the cervix, uterus, and ovaries. Uterine discharge
In the process of inflammation, pro-inflammatory
was classified as clear mucus, mucus with flecks of
cytokines and cytotoxic radicals which are released
pus, mucopurulentand orpurulent according to the
from macrophages and granulocytes inhibit cellular
criteria described previously (Sarkar et al., 2006;
metabolic pathways and lipid peroxidation (Ewa et al.,
Kasimanickam et al., 2004). Briefly, normal cows
2012). Several studies showed that increase in lipid
with no abnormal uterine discharge were selected
peroxidation during endometritis decreases the levels
according external inspection and vaginoscopy.
of some antioxidant molecules, such as vitamin E (VE)
SCE was diagnosed by endometrial cytology and
and vitamin C (VC), which lead to an increase in
histopathology. The cows were divided into two
oxidative stress (Kankofer et al., 2005; Lorraine and
groups, which included 20 normal cows and 60 cows
Stacey, 2009). Oxidative stress is a result of unbalance
with SCE. Blood and uterine secretion were collected
between oxidant and antioxidant levels (Lykkesfeldt
at the same time of inspection. None of cows had other
and Svendsen, 2007; Onur et al., 2010), occurs
diseases requiring systemic treatment.
different pathological events, such as mastitis, metritis, and retained fetal membranes during the periparturient
Sample collection
period in cows (Lykkesfeldt and Svendsen, 2007;
Uterine secretion was collected as described previously
Lorraine and Stacey, 2009). Due to membrane lipid
(Sioutas et al., 2008). In brief, uterine washings
peroxidation and oxidative stress, mammalian tissues
were collected aseptically using two ways 18 gauges.
such as cellular can be damaged by the accumulation
Sterilized stainless steel catheters were fixed in the
of reactive oxygen species (Lorraine and Stacey,
uterine horn with 10-15 mL air, then withdrew intro-
2009). Therefore, earlier diagnosis and treatment of
ducer, 40-50 mL of washing fluid (sterile PBS pH
the endometritis, especially SCE, are important to
7.0) was infused into the uterine horn and mixed with
minimize economic losses. Antioxidant is used to
the intrauterine contents by massaging the uterus per
treat mastitis in goats, bovines and mares, during the
rectum. 40 mL uterine washings were centrifuged at
reproductive state (Abdorrahman et al., 2005; Eyassu
1 000 g for 10 min, and the supernatant was frozen at
et al., 2007; Lorraine and Stacey, 2009); however, few
–80℃ until all the samples were collected.
reports show the change of the antioxidant in SCE.
The percentage of PMN was counted with a mini-
Thus, with the aim to reveal the early diagnosis
mum of 100 cells at 400 magnification.
index of the subclinical endometritis, the levels of
Blood sample from vena cervicalis was collected
E-mail: [email protected]
Song Xue et al. Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis
·37·
in EDTA-vacutainer tubes, and centrifuged at 4℃ for
significant difference. All the data were presented as
15 min at 3 000 g. Plasma was removed into Eppendorf
the mean±SD.
tubes by pipette and stored at –20℃ until usage.
Results
Biochemical analysis of sample NO concentration in uterine secretion was measured
Concentrations of NO (P<0.05) and TOC were signi-
by the Griess reaction according to Miranda et al (2001)
fication greater (P<0.01) for cows with SCE compared
(Nanjing Jiancheng Biology Engineering Research
with normal cows. In contrast, the levels of VC and VE
Institute, China). In order to measure TOC, VC and
were significantly lesser (P<0.01) in uterine secretion
VE accurately in uterine secretion, uterine secretion
with SCE compared to those from normal cows. The
samples were diluted 10 times with physiologic saline
percentage of PMN were significantly greater (P<0.01)
and filtered to obtain transparent secretion samples,
in uterine secretion with SCE compared to those
which was slightly different from the method of Erel
from normal cows (Table 1). Statistically significant
(2005). Then, secretion samples were centrifuged at
positive correlation (r=0.235, P<0.01) was detected
1 000 g for 10 min, and the supernatant was frozen
between PMN and NO concentrations in uterine
until all the samples were collected. The concentrations
secretion (Fig. 1).
of TOC, VC and VE were detected by a commercially available assay kit (Nanjing Jiancheng Biology
Table 1 Levels of nitric oxide, total oxidant capacity, VC, VE and percentage of PMN in uterine secretion
Engineering Research Institute, China), following manufacturer recommendations (Li et al., 2010).
Group
Parameter
Statistical analysis The data were analyzed by a one-way analysis of variance procedure followed by a Student's t-test (SPSS
Normal (n=20)
SCE (n=60)
Nitric oxide (μmol • L-1)
32.75±2.35
37.77±2.20*
Total oxidant capacity (U • mL-1)
7.894±0.396
9.037±0.478**
17.0 software; SPSS Inc., Chicago, IL, USA). Normal distribution of the data was analyzed by AndersonDarling Normality test. Statistical significance was
PMN(%)
10
0
1
same row.
20
23.4±1.87**
60
30
10.9±1.15
15 10
5
4
7
10
20 PMN (%)
NO (μmol • L-1)
NO
PMN (%)
25
70
40
5.698±1.25**
** refers to statistical significance between the groups (P<0.01) in the
and P-values of <0.01 were considered markedly
50
1.017±0.093**
8.837±1.16
statistical significance between the groups (P<0.05) in the same row and
of <0.05 were considered to be statistically significant
80
1.516±0.081
VE (μg • mL-1)
Mean ± SD is shown to indicate each group variation. * refers to
indicated in two levels: P<0.05 and P<0.01. P-values
90
VC (μg • mL ) -1
13 16
19
22
25
28 31 34
37
40
43 46 49
52
55
58
0
Distribution of uterine secretion
Fig. 1 Correlation between PMN and NO concentrations from uterine secretion in cows with SCE http: //publish.neau.edu.cn
·38·
Journal of Northeast Agricultural University (English Edition)
Vol. 22 No. 1 2015
from cows with SCE indicated that SCE induced the
Discussion
imbalance of oxidant/antioxidant and the decrease
SCE is caused by the calving environment, diet,
to increased oxidant levels, oxidative stress changed
inadequate hygiene, stress, improper breeding and
metabolism and physiological functions (Zhong and
management conditions (Azawi, 2008). NO is an
Zhou, 2013). Early studies suggested that dairy cows
important product during the developmental process
in oxidative stress, supplement certain antioxidant
of inflammation (Riku and Tuula, 2001; Mukherjee,
could ameliorate metabolism and guard against
2008). Study showed that NO is an inflammatory
infectious diseases (Lorraine and Stacey, 2009).
mediator that causes inflammation toxicity and
Therefore, we deduced that SCE induced oxidative
mediates cytoimmunity and smooth muscle relaxation
stress, which damaged the physiological function of
(Li et al., 2010; Kamran et al., 2014). SCE leads to
the uterine.
increase the level of NO in uterine sections from cows,
In conclusion, SCE increased NO concentrations,
therefore, the increased NO concentration in uterine
leading to oxidative stress and oxidative damage.
sections and serum is speculated as an inflammatory
In addition, the positive correlation between PMN
response of uterine. In this regard, alteration in uterine
percentage and NO concentration indicated that the
section NO levels could be used as a diagnostic index
alteration of NO concentration in uterine sections
to detect inflammation during SCE. The percentage
could be used as an early diagnosis index of SCE.
of the antioxidant property. Studies showed that due
of PMN indicated the level of inflammation with SCE (Anna et al., 2014). In the present study, there was a
References
statistically significant positive correlation (P<0.05)
Abdorrahman S A, Douglas N F, Cathy S C, et al. 2005. Nitric oxide
between PMN percentage and NO concentration,
levels and nitric oxide synthase expression in uterine samples from
which indicated the positive relationship between
mares susceptible and resistant to persistent breeding-induced
them.
endometritis. American Journal of Reproductive Immunulogy, 53:
During inflammation, increased NO reacted with
230-237.
superoxide anions leading to formation of peroxide
Ahmadi M R, Khodakaram T A, Nazifi S, et al. 2005. The comparative
nitrite radical (Savenkova et al., 2002; Cross and
evaluation of uterine and cervical mucosa cytology with endometrial
Wilson, 2003), which led to the increase in lipid
histopathology in cows. Comp Clin Path, 14: 90-94.
peroxidation and formation of the free radicals (Kumar
Anna D, Janine M, Maike H, et al. 2014. Peripheral blood leukocytes
et al., 2014). Studies showed that TOC was important
of cows with subclinical endometritis show an altered cellular
components of the antioxidant defense system that
composition and gene expression. Theriogenology, 81: 906-917.
could efficiently protect against oxidantive damage (Vessey, 1993; Steenvoorden and Beijersbergen, 1997). Therefore, in this study, increased TOC in uterine secretion from cows with SCE indicated that SCE induced oxidative damage by increasing NO
Azawi O I. 2008. Postpartum uterine infection in cattle. Animal Reproduction Science, 105(3/4): 187-208. Cross R K, Wilson K T. 2003. Nitric oxide in inflammatory bowel disease. Inflamm Bowel Dis, 9: 179-189. Dubuc J, Duffield T F, Leslie K E, et al. 2010. Randomized clinical
concentration.
trial of antibiotic and prostaglandin treatments for uterine health and
VC is the most effective antioxidant in plasma. VE,
reproductive performance in dairy cows. J Dairy Sci, 94: 1325-1338
as a mundificant, cleans single oxygen and superoxide
Erel O. 2005. A new automated colorimetric method for measuring total
anion radical, more importantly, as a blocking agent,
oxidant status. Clinical Biochemistry, 38: 1103-1111.
intercepts lipid peroxidation. Therefore, in this study,
Ewa G C, Katarzyna H, Sigurd L. 2012. Is there a role for neuronal
the decreased VC and VE levels in uterine secretion
nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta
E-mail: [email protected]
Song Xue et al. Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis cells? Nitric Oxide, 27(4): 235-241. Eyassu S, Donkin E F, Buys E M. 2007. Potential of lactoperoxidase to diagnose subclinical mastitis in goats. Small Ruminant Research, 69: 154-158. Gilbert R O, Shin S T, Guard C L, et al. 2005. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology, 64: 1879-1888. Kankofer M, Lipko J, Zdunczyk S. 2005. Total antioxidant capacity of bovine spontaneously released and retained placenta. Pathophysiology, 11(4): 215-219. Kasimanickam R, Duffield T F, Foster R A, et al. 2004. Endometrial cytology and ultrasonography for the detection of subclinical
·39·
Onur A, Hasan O, Emine A, et al. 2010. Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk. Research in Veterinary Science, 89(1): 10-13. Rawlingson A. 2003. Nitric oxide, inflammation and acute burn injury. Burns, Nov 29: 631-40. Ribeiro E S, Lima F S, Greco L F, et al. 2013. Prevalence of periparturient disease and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates. Journal of Dairy Science, 193(1): 5682-5697. Riku A, Tuula M. 2001. Nitrous oxide use in paediatric surgery. Best Practice & Research Clinical Anaesthesiology, 15(3): 467-475. Sarkar P, Kumar H, Rawat M. 2006. Effect of administration of garlic
endometritis in postpartum dairy cows. Theriogenology, 62: 9-23.
extract and PGF2α on hormonal changes and recovery in endometritis
Kumar A, Chen S H, Kadiiska M B, et al. 2014. Inducible nitric oxide
cows. Asian-Australasian Journal of Animal Science, 19(7): 964-969.
synthase is key to peroxynitrite-mediated, LPS-induced protein
Savenkova L, Gercberga Z, Muter O, et al. 2002. PHB-based films as
radical formation in murine microglial BV2 cells. Free Radical Biologyand Medicine, 73: 51-59. Li D J, Liu Y F, Liu Y, et al. 2010. Significance of nitric oxide concentration in plasma and uterine secretes with puerperal endometritis in dairy cows. Vet Res Commun, 34: 315-321. Lorraine M S, Stacey L A. 2009. Immpact of oxidatice stress on the health and immune function of dairy cattle. Veterinary Immunology and Immunopathology, 128(1/2/3): 104-109. Loyi T, Kumar H, Nandi S, et al. 2013. Differential expression of proinflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis. Research in Veterinary Science, 4(94): 336-340 Lykkesfeldt J, Svendsen O. 2007. Oxidants and antioxidants in disease: oxidative stress in farm animals. Veterinary Journal, 173: 502-511.
matrices for pesticides. Process Biochemistry, 37(7): 109-114. Singh J, Murray R D, Mshelia G, et al. 2008. The immune status of the bovine uterus during the peripartum period. The Veterinary Journal, 175: 301-309. Sioutas A, Ehren I, Lundberg J O, et al. 2008. Intrauterine nitric oxide in pelvic inflammatory disease. Fertility and Sterility, 89: 948-952. Sheldon I M, Noakes D E, Rycroft A, et al. 2001. Acute phase protein response to postpartum uterine bacterial contamination in cattle. Vet Rec, 148: 172-175. Sheldon I M, Lewis G S, LeBlanc S, et al. 2006. Defining postpartum uterine disease in cattle. Theriogenology, 65: 1516-1530 Steenvoorden D P T, Beijersbergen van Henegouwen G M J. 1997, The use of endogenous antioxidants to improve photoprotection. Journal of Photochemistry and Photobiology Part B: Biology, 41: 1-10.
Miranda K M, Espey M G, Wink D A. 2001. A rapid, simple
Subandrio A L, Sheldon I M, Noakes D E. 2000. Peripheral and
spectrophotometric method for simultaneous detection of nitrate and
intrauterine neutrophil function in the cow: the influence of
nitrite. Nitric Oxide: Biology and Chemistry, 5: 62-71.
endogenous and exogenous sex steroid hormones. Theriogenology,
Mohammad R A, Arsalan H, Hamid R G, et al. 2014. Preliminary trial in treatment of postpartum endometritis with intrauterine application of hyperimmune serum in dairy cows. Asian Pacific Journal of Tropical Disease, 4(Supplement 1): S360-S365. Mukherjee R. 2008. Selenium and vitamin E increases polymor-
53(8): 1591-1608. Kamran A, Suna D M, Lain C. 2014. Uterine physiology. Anaesthesia & Intensive Care Medicine, 15(3): 133-135. Vessey D A. 1993. The cutaneous antioxidant system. Clinical Dermatology, 8: 81-103.
phonuclear cell phagocytosis and antioxidant levels during acute
Zhong R Z, Zhou D W. 2013. Oxidative stress and role of natural plant
mastitis in riverine buffaloes. Veterinary Research Communications,
derived antioxidants in animal reproduction. Journal of Integrative
32: 305-313.
Agriculture, 12(10): 1826-1838.
http: //publish.neau.edu.cn
ScienceDirect
Vol. 22 No. 1 35-39
Journal of Northeast Agricultural University (English Edition)
Available online at www.sciencedirect.com
Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis Song Xue, Li De-jun, Feng Guo-feng, Li Bei, and Liu Yun-feng* College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
Abstract: Subclinical endometritis is a physiological inflammation that serves to clear persistent contaminants from the uterus. To investigate the alteration of antioxidant, such as vitamin E (VE) and vitamin C (VC), total oxidant capacity (TOC) and nitric oxide (NO) in cows with normal and subclinical endometritis (SCE), we examined the concentrations of NO, VC and VE, TOC and polymorphonuclear neutrophils (PMN) percentage in uterine secretion. The cows were divided into two groups, normal (n=20) and subclinical endometritis (SCE, n=60), based on endometrial cytology (presence of PMN≥5%). Uterine secretion and blood were collected as described previously. Griess reaction was used to determine the concentration of NO. The concentrations of TOC, VC and VE were detected by a commercially available assay kit. The results showed that the concentrations of NO, TOC and PMN percentage were significantly higher (P<0.01, P<0.05, and P<0.01, respectively) in uterine secretion with SCE compared to those from normal; however, the levels of VC and VE were significantly lesser (P<0.01). In conclusion, the concentrations of NO, TOC, VC, VE and PMN percentage differed between normal and SCE cows. Meanwhile, the relationship between the concentration of NO and PMN percentage from uterine secretion in cows with subclinical endometritis were positively correlated. Consequently, these alterations in NO, TOC, VC, VE levels and PMN percentage contributed to as a diagnostic index of the uterine inflammation, with the aim to increase the reproduction of the cows and the decrease economic losses. Key words: cow, subclinical endometritis, nitric oxide, antioxidant, total oxidant capacity CLC number: S823
Document code: A
Article ID: 1006-8104(2015)-01-0035-05
Ribeiro et al., 2013; Mohammad et al., 2014).
Introduction
Polymorphonuclear neutrophils (PMN) in uterine
Subclinical endometritis (SCE), as one of the most
state (Kasimanickam, 2004). Excessive amounts of
important disorders in dairy cows during the post-
neutrophilic granulocytes, macrophages, lymphocytes,
partum period, impacts reproduction (Sheldon et al.,
eosinophilic granulocytes and various epithelial cells
2006; Dubuc et al., 2010) and causes economic losses
of the uterine tissue in uterine fluid are considered as a
(Azawi, 2008). Earlier diagnose and treatment of
response to prevent exogenous pathogenic bacterium
SCE play an important role in controlling clinical
in the part of inflammation of the uterus (Singh, 2008).
endometritis, yet the index of the early diagnosis are
In addition, the level of pro-inflammatory cytokines,
still not clear (Ahmadi et al., 2005; Gilbert, 2005;
such as tumor necrosis factor α (TNF-α), interleukin
secretion is an indicator reflecting uterine health
Received 20 October 2014 Supported by Funding (RCB22) from the Doctoral Research Foundation of Northeast Agricultural University (2012); the Postdoctoral Fund of Heilongjiang Province (LBH-Z11239) Song Xue (1989-), female, Master, engaged in the research of nutrition regulation and metabolism disease. E-mail: songxue521521@163. com * Corresponding author. Liu Yun-feng, associate professor, engaged in the research of nutrition regulation and metabolism disease. E-mail: liuyunfeng@ neau.edu.cn http: //publish.neau.edu.cn
·36·
Journal of Northeast Agricultural University (English Edition)
Vol. 22 No. 1 2015
(IL) 1β, IL-6, IL-8 and some other molecules such
NO, VC and VE, total oxidant capacity (TOC) and
as nitric oxide (NO) are increased during the period
PMN percentage from uterine secretion in cows were
of infections (Sheldon et al., 2001; Li et al., 2010;
examined.
Loyi et al., 2013). As an inflammatory mediator, NO which synthesized by macrophages causes smooth muscle relaxation and mediates cytoimmunity and
Materials and Methods
inflammation toxicity. Excess NO is produced during
Experimental animals
inflammation as a primer defense system (Subandrio
Totally 80 Holstein cows from Dairy Tarm were enroll-
et al., 2000). Study showed that NO content is in-
ed in this study (Sarkar et al., 2006; Kasimanickam
creased during inflammation diseases (Rawlingson,
et al., 2004). All the Holstein cows were routinely
2003; Abdorrahman et al., 2005). In addition, sub-
examined once between 28 and 33 days after calv-
stances derived from oxidation of NO, such as pero-
ing, included inspection of the vulva, tail, and
xynitrite, changed antioxidant balance of the bacteria
perineum, vaginoscopy, and transrectal palpation
(Onur et al., 2010).
of the cervix, uterus, and ovaries. Uterine discharge
In the process of inflammation, pro-inflammatory
was classified as clear mucus, mucus with flecks of
cytokines and cytotoxic radicals which are released
pus, mucopurulentand orpurulent according to the
from macrophages and granulocytes inhibit cellular
criteria described previously (Sarkar et al., 2006;
metabolic pathways and lipid peroxidation (Ewa et al.,
Kasimanickam et al., 2004). Briefly, normal cows
2012). Several studies showed that increase in lipid
with no abnormal uterine discharge were selected
peroxidation during endometritis decreases the levels
according external inspection and vaginoscopy.
of some antioxidant molecules, such as vitamin E (VE)
SCE was diagnosed by endometrial cytology and
and vitamin C (VC), which lead to an increase in
histopathology. The cows were divided into two
oxidative stress (Kankofer et al., 2005; Lorraine and
groups, which included 20 normal cows and 60 cows
Stacey, 2009). Oxidative stress is a result of unbalance
with SCE. Blood and uterine secretion were collected
between oxidant and antioxidant levels (Lykkesfeldt
at the same time of inspection. None of cows had other
and Svendsen, 2007; Onur et al., 2010), occurs
diseases requiring systemic treatment.
different pathological events, such as mastitis, metritis, and retained fetal membranes during the periparturient
Sample collection
period in cows (Lykkesfeldt and Svendsen, 2007;
Uterine secretion was collected as described previously
Lorraine and Stacey, 2009). Due to membrane lipid
(Sioutas et al., 2008). In brief, uterine washings
peroxidation and oxidative stress, mammalian tissues
were collected aseptically using two ways 18 gauges.
such as cellular can be damaged by the accumulation
Sterilized stainless steel catheters were fixed in the
of reactive oxygen species (Lorraine and Stacey,
uterine horn with 10-15 mL air, then withdrew intro-
2009). Therefore, earlier diagnosis and treatment of
ducer, 40-50 mL of washing fluid (sterile PBS pH
the endometritis, especially SCE, are important to
7.0) was infused into the uterine horn and mixed with
minimize economic losses. Antioxidant is used to
the intrauterine contents by massaging the uterus per
treat mastitis in goats, bovines and mares, during the
rectum. 40 mL uterine washings were centrifuged at
reproductive state (Abdorrahman et al., 2005; Eyassu
1 000 g for 10 min, and the supernatant was frozen at
et al., 2007; Lorraine and Stacey, 2009); however, few
–80℃ until all the samples were collected.
reports show the change of the antioxidant in SCE.
The percentage of PMN was counted with a mini-
Thus, with the aim to reveal the early diagnosis
mum of 100 cells at 400 magnification.
index of the subclinical endometritis, the levels of
Blood sample from vena cervicalis was collected
E-mail: [email protected]
Song Xue et al. Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis
·37·
in EDTA-vacutainer tubes, and centrifuged at 4℃ for
significant difference. All the data were presented as
15 min at 3 000 g. Plasma was removed into Eppendorf
the mean±SD.
tubes by pipette and stored at –20℃ until usage.
Results
Biochemical analysis of sample NO concentration in uterine secretion was measured
Concentrations of NO (P<0.05) and TOC were signi-
by the Griess reaction according to Miranda et al (2001)
fication greater (P<0.01) for cows with SCE compared
(Nanjing Jiancheng Biology Engineering Research
with normal cows. In contrast, the levels of VC and VE
Institute, China). In order to measure TOC, VC and
were significantly lesser (P<0.01) in uterine secretion
VE accurately in uterine secretion, uterine secretion
with SCE compared to those from normal cows. The
samples were diluted 10 times with physiologic saline
percentage of PMN were significantly greater (P<0.01)
and filtered to obtain transparent secretion samples,
in uterine secretion with SCE compared to those
which was slightly different from the method of Erel
from normal cows (Table 1). Statistically significant
(2005). Then, secretion samples were centrifuged at
positive correlation (r=0.235, P<0.01) was detected
1 000 g for 10 min, and the supernatant was frozen
between PMN and NO concentrations in uterine
until all the samples were collected. The concentrations
secretion (Fig. 1).
of TOC, VC and VE were detected by a commercially available assay kit (Nanjing Jiancheng Biology
Table 1 Levels of nitric oxide, total oxidant capacity, VC, VE and percentage of PMN in uterine secretion
Engineering Research Institute, China), following manufacturer recommendations (Li et al., 2010).
Group
Parameter
Statistical analysis The data were analyzed by a one-way analysis of variance procedure followed by a Student's t-test (SPSS
Normal (n=20)
SCE (n=60)
Nitric oxide (μmol • L-1)
32.75±2.35
37.77±2.20*
Total oxidant capacity (U • mL-1)
7.894±0.396
9.037±0.478**
17.0 software; SPSS Inc., Chicago, IL, USA). Normal distribution of the data was analyzed by AndersonDarling Normality test. Statistical significance was
PMN(%)
10
0
1
same row.
20
23.4±1.87**
60
30
10.9±1.15
15 10
5
4
7
10
20 PMN (%)
NO (μmol • L-1)
NO
PMN (%)
25
70
40
5.698±1.25**
** refers to statistical significance between the groups (P<0.01) in the
and P-values of <0.01 were considered markedly
50
1.017±0.093**
8.837±1.16
statistical significance between the groups (P<0.05) in the same row and
of <0.05 were considered to be statistically significant
80
1.516±0.081
VE (μg • mL-1)
Mean ± SD is shown to indicate each group variation. * refers to
indicated in two levels: P<0.05 and P<0.01. P-values
90
VC (μg • mL ) -1
13 16
19
22
25
28 31 34
37
40
43 46 49
52
55
58
0
Distribution of uterine secretion
Fig. 1 Correlation between PMN and NO concentrations from uterine secretion in cows with SCE http: //publish.neau.edu.cn
·38·
Journal of Northeast Agricultural University (English Edition)
Vol. 22 No. 1 2015
from cows with SCE indicated that SCE induced the
Discussion
imbalance of oxidant/antioxidant and the decrease
SCE is caused by the calving environment, diet,
to increased oxidant levels, oxidative stress changed
inadequate hygiene, stress, improper breeding and
metabolism and physiological functions (Zhong and
management conditions (Azawi, 2008). NO is an
Zhou, 2013). Early studies suggested that dairy cows
important product during the developmental process
in oxidative stress, supplement certain antioxidant
of inflammation (Riku and Tuula, 2001; Mukherjee,
could ameliorate metabolism and guard against
2008). Study showed that NO is an inflammatory
infectious diseases (Lorraine and Stacey, 2009).
mediator that causes inflammation toxicity and
Therefore, we deduced that SCE induced oxidative
mediates cytoimmunity and smooth muscle relaxation
stress, which damaged the physiological function of
(Li et al., 2010; Kamran et al., 2014). SCE leads to
the uterine.
increase the level of NO in uterine sections from cows,
In conclusion, SCE increased NO concentrations,
therefore, the increased NO concentration in uterine
leading to oxidative stress and oxidative damage.
sections and serum is speculated as an inflammatory
In addition, the positive correlation between PMN
response of uterine. In this regard, alteration in uterine
percentage and NO concentration indicated that the
section NO levels could be used as a diagnostic index
alteration of NO concentration in uterine sections
to detect inflammation during SCE. The percentage
could be used as an early diagnosis index of SCE.
of the antioxidant property. Studies showed that due
of PMN indicated the level of inflammation with SCE (Anna et al., 2014). In the present study, there was a
References
statistically significant positive correlation (P<0.05)
Abdorrahman S A, Douglas N F, Cathy S C, et al. 2005. Nitric oxide
between PMN percentage and NO concentration,
levels and nitric oxide synthase expression in uterine samples from
which indicated the positive relationship between
mares susceptible and resistant to persistent breeding-induced
them.
endometritis. American Journal of Reproductive Immunulogy, 53:
During inflammation, increased NO reacted with
230-237.
superoxide anions leading to formation of peroxide
Ahmadi M R, Khodakaram T A, Nazifi S, et al. 2005. The comparative
nitrite radical (Savenkova et al., 2002; Cross and
evaluation of uterine and cervical mucosa cytology with endometrial
Wilson, 2003), which led to the increase in lipid
histopathology in cows. Comp Clin Path, 14: 90-94.
peroxidation and formation of the free radicals (Kumar
Anna D, Janine M, Maike H, et al. 2014. Peripheral blood leukocytes
et al., 2014). Studies showed that TOC was important
of cows with subclinical endometritis show an altered cellular
components of the antioxidant defense system that
composition and gene expression. Theriogenology, 81: 906-917.
could efficiently protect against oxidantive damage (Vessey, 1993; Steenvoorden and Beijersbergen, 1997). Therefore, in this study, increased TOC in uterine secretion from cows with SCE indicated that SCE induced oxidative damage by increasing NO
Azawi O I. 2008. Postpartum uterine infection in cattle. Animal Reproduction Science, 105(3/4): 187-208. Cross R K, Wilson K T. 2003. Nitric oxide in inflammatory bowel disease. Inflamm Bowel Dis, 9: 179-189. Dubuc J, Duffield T F, Leslie K E, et al. 2010. Randomized clinical
concentration.
trial of antibiotic and prostaglandin treatments for uterine health and
VC is the most effective antioxidant in plasma. VE,
reproductive performance in dairy cows. J Dairy Sci, 94: 1325-1338
as a mundificant, cleans single oxygen and superoxide
Erel O. 2005. A new automated colorimetric method for measuring total
anion radical, more importantly, as a blocking agent,
oxidant status. Clinical Biochemistry, 38: 1103-1111.
intercepts lipid peroxidation. Therefore, in this study,
Ewa G C, Katarzyna H, Sigurd L. 2012. Is there a role for neuronal
the decreased VC and VE levels in uterine secretion
nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta
E-mail: [email protected]
Song Xue et al. Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis cells? Nitric Oxide, 27(4): 235-241. Eyassu S, Donkin E F, Buys E M. 2007. Potential of lactoperoxidase to diagnose subclinical mastitis in goats. Small Ruminant Research, 69: 154-158. Gilbert R O, Shin S T, Guard C L, et al. 2005. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology, 64: 1879-1888. Kankofer M, Lipko J, Zdunczyk S. 2005. Total antioxidant capacity of bovine spontaneously released and retained placenta. Pathophysiology, 11(4): 215-219. Kasimanickam R, Duffield T F, Foster R A, et al. 2004. Endometrial cytology and ultrasonography for the detection of subclinical
·39·
Onur A, Hasan O, Emine A, et al. 2010. Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk. Research in Veterinary Science, 89(1): 10-13. Rawlingson A. 2003. Nitric oxide, inflammation and acute burn injury. Burns, Nov 29: 631-40. Ribeiro E S, Lima F S, Greco L F, et al. 2013. Prevalence of periparturient disease and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates. Journal of Dairy Science, 193(1): 5682-5697. Riku A, Tuula M. 2001. Nitrous oxide use in paediatric surgery. Best Practice & Research Clinical Anaesthesiology, 15(3): 467-475. Sarkar P, Kumar H, Rawat M. 2006. Effect of administration of garlic
endometritis in postpartum dairy cows. Theriogenology, 62: 9-23.
extract and PGF2α on hormonal changes and recovery in endometritis
Kumar A, Chen S H, Kadiiska M B, et al. 2014. Inducible nitric oxide
cows. Asian-Australasian Journal of Animal Science, 19(7): 964-969.
synthase is key to peroxynitrite-mediated, LPS-induced protein
Savenkova L, Gercberga Z, Muter O, et al. 2002. PHB-based films as
radical formation in murine microglial BV2 cells. Free Radical Biologyand Medicine, 73: 51-59. Li D J, Liu Y F, Liu Y, et al. 2010. Significance of nitric oxide concentration in plasma and uterine secretes with puerperal endometritis in dairy cows. Vet Res Commun, 34: 315-321. Lorraine M S, Stacey L A. 2009. Immpact of oxidatice stress on the health and immune function of dairy cattle. Veterinary Immunology and Immunopathology, 128(1/2/3): 104-109. Loyi T, Kumar H, Nandi S, et al. 2013. Differential expression of proinflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis. Research in Veterinary Science, 4(94): 336-340 Lykkesfeldt J, Svendsen O. 2007. Oxidants and antioxidants in disease: oxidative stress in farm animals. Veterinary Journal, 173: 502-511.
matrices for pesticides. Process Biochemistry, 37(7): 109-114. Singh J, Murray R D, Mshelia G, et al. 2008. The immune status of the bovine uterus during the peripartum period. The Veterinary Journal, 175: 301-309. Sioutas A, Ehren I, Lundberg J O, et al. 2008. Intrauterine nitric oxide in pelvic inflammatory disease. Fertility and Sterility, 89: 948-952. Sheldon I M, Noakes D E, Rycroft A, et al. 2001. Acute phase protein response to postpartum uterine bacterial contamination in cattle. Vet Rec, 148: 172-175. Sheldon I M, Lewis G S, LeBlanc S, et al. 2006. Defining postpartum uterine disease in cattle. Theriogenology, 65: 1516-1530 Steenvoorden D P T, Beijersbergen van Henegouwen G M J. 1997, The use of endogenous antioxidants to improve photoprotection. Journal of Photochemistry and Photobiology Part B: Biology, 41: 1-10.
Miranda K M, Espey M G, Wink D A. 2001. A rapid, simple
Subandrio A L, Sheldon I M, Noakes D E. 2000. Peripheral and
spectrophotometric method for simultaneous detection of nitrate and
intrauterine neutrophil function in the cow: the influence of
nitrite. Nitric Oxide: Biology and Chemistry, 5: 62-71.
endogenous and exogenous sex steroid hormones. Theriogenology,
Mohammad R A, Arsalan H, Hamid R G, et al. 2014. Preliminary trial in treatment of postpartum endometritis with intrauterine application of hyperimmune serum in dairy cows. Asian Pacific Journal of Tropical Disease, 4(Supplement 1): S360-S365. Mukherjee R. 2008. Selenium and vitamin E increases polymor-
53(8): 1591-1608. Kamran A, Suna D M, Lain C. 2014. Uterine physiology. Anaesthesia & Intensive Care Medicine, 15(3): 133-135. Vessey D A. 1993. The cutaneous antioxidant system. Clinical Dermatology, 8: 81-103.
phonuclear cell phagocytosis and antioxidant levels during acute
Zhong R Z, Zhou D W. 2013. Oxidative stress and role of natural plant
mastitis in riverine buffaloes. Veterinary Research Communications,
derived antioxidants in animal reproduction. Journal of Integrative
32: 305-313.
Agriculture, 12(10): 1826-1838.
http: //publish.neau.edu.cn