Accepted Manuscript Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis Ill-Hwa Kim, Hyun-Gu Kang, Jae-Kwan Jeong, Tai-Young Hur, Young-Hun Jung PII:
S0093-691X(14)00202-7
DOI:
10.1016/j.theriogenology.2014.04.022
Reference:
THE 12787
To appear in:
Theriogenology
Received Date: 16 January 2014 Revised Date:
28 April 2014
Accepted Date: 28 April 2014
Please cite this article as: Kim I-H, Kang H-G, Jeong J-K, Hur T-Y, Jung Y-H, Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.04.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
revised
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Inflammatory cytokine concentrations in uterine flush and serum
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samples from dairy cows with clinical or subclinical endometritis
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Ill-Hwa Kima,*, Hyun-Gu Kanga, Jae-Kwan Jeonga, Tai-Young Hurb, Young-
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Hun Jungb
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College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Korea b
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National Livestock Research Institute, RDA, Cheonan, Chungnam, 330-800 Korea
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Corresponding author. Tel.: +82 43 2612571; Fax: +82 43 2673150
E-mail address: [email protected] (I.H. Kim).
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Abstract This objective of this study was to compare concentrations of inflammatory cytokines in uterine flush
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and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis.
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Clinical endometritis was diagnosed by observation of vaginal discharges (> 50% pus) and subclinical
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endometritis was diagnosed by evaluation of uterine cytology (neutrophils > 18%) at 4 weeks postpartum.
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Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β,
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IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at
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1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations.
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Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than
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in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows
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with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-
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10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical
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endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with
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subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum,
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concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant
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effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows
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with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows,
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but no differences were observed in serum.
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Key words: Endometritis; Cytokines; Uterine flush; Serum; Dairy cows
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1. Introduction
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Most cows are exposed to bacterial infection after calving [1,2]. Greater than 70% clear the uterine
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bacteria via innate immune responses; however, 17 to 37% of cows develop clinical endometritis,
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whereas14 to 53% develop subclinical endometritis, which results in reduced fertility [3–9]. Proper
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regulation of immune responses during the weeks after calving is important for subsequent uterine health
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[10–13]. The development of bovine endometritis is associated with very complex signaling processes
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involving the detection of bacterial components by innate immune cells via toll-like receptors, the
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production of tumor necrosis factor-α (TNF-α) and other pro-inflammatory cytokines (e.g., interleukins;
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IL), and the mobilization of neutrophils followed by phagocytosis of invading pathogens within the
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uterine lumen [14–21]. Pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and chemokines (e.g.,
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IL-8) stimulate neutrophil and monocyte diapedesis and chemoattraction, and promote increased
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phagocytosis [22].
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Previous studies have used uterine biopsy [23] and the cytobrush technique [24] to examine the
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expression of inflammatory cytokine mRNA in uterine tissue collected from cows with endometritis.
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Others used endometrial tissue scraping from buffalos with endometritis [25]. The results revealed that
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the expression of TNF-α, IL-1β, and IL-6 (all pro-inflammatory cytokines), as well as IL-8 (the primary
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chemokine that regulates neutrophil activity) and IL-10 (an anti-inflammatory cytokine), was related to
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the development of bovine clinical or subclinical endometritis [23,24,26]. Moreover, a recent study
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suggests that the cytokine profile in uterine tissue is associated with the severity and persistency of
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uterine inflammation [25]. In addition, other studies found that serum obtained from cows with
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endometritis and from healthy cows during the peripartum period contained different levels of
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inflammatory cytokines [26,27]. Likewise, higher expression of inflammatory cytokine genes in uterine
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tissue and/or an increase in the levels of those cytokines in the serum are thought to predict the
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development of bovine endometritis [23,24,27]. However, no study has measured the levels of
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inflammatory cytokines in uterine flush from cows with endometritis. Measuring inflammatory cytokine
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levels in uterine flush and serum from dairy cows with clinical or subclinical endometritis during the
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voluntary waiting period may provide valuable information that can serve as a diagnostic tool for the
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uterine inflammation. The objective of the present study was to compare uterine flush and serum concentrations of
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inflammatory cytokines in healthy postpartum dairy cows with cows that develop clinical or subclinical
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endometritis.
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2. Materials and methods
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2.1. Animals
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All experiments were performed with the approval of the Institutional Animal Care and Use
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Committee of Chungbuk National University, Korea. This experiment was contacted on two dairy farms
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located in Chungcheong Province, Korea, during the period from March 2012 to September 2013. Forty-
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eight and 34 Holstein cows, with 2.3 ± 1.4 lactations (mean ± standard deviation; range: 1–7 lactations),
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were used to obtain uterine flush and serum samples, respectively. The cows were maintained in a loose-
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housing system, fed a total mixed ration, and milked twice daily.
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2.2. Diagnosis of clinical and subclinical endometritis
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All of the cows were evaluated for clinical endometritis at Week 4 postpartum by examining vaginal
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discharges removed using the Metricheck tool [28]. Briefly, after cleaning the vulva with the disinfectant
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(chlorhexidine gluconate), the Metricheck device was inserted until it reached the vaginal fornix and then
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retracted for evaluation of the vaginal mucus contained in the cup. Cows with a mucopurulent uterine
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discharge (> 50% pus) were diagnosed with clinical endometritis [1,3]. At the same time, cows were
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evaluated for subclinical endometritis by uterine cytology [29]. Briefly, after cleaning the vulva, a
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cytobrush and stainless steel rod (which was guarded by a stainless steel sheath and covered with a
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protective plastic sheath) were introduced into the vagina. At the external end of the cervix, the plastic
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sheath was pulled back, and the stainless steel sheath and stainless steel rod and cytobrush were passed
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into the body of the uterus. The stainless steel sheath was then retracted to expose the cytobrush. The
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cytobrush was rotated clockwise to obtain cellular material from the endometrium. After removal from
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the vagina, the brush was rolled onto a glass slide that was allowed to air-dry. All slides were stained
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using Diff-Quick stain (Sysmex Inc., Kobe, Japan) according to the manufacturer’s guidelines. Each slide
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was examined under a microscope (× 200 magnification) by the same examiner. The numbers of epithelial
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endometrial cells and neutrophils were counted (up to 200 cells per slide) and the percentage of
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neutrophils was calculated. Subclinical endometritis was defined as a neutrophil proportion > 18% in the
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absence of clinical endometritis [1,30]. Cows not diagnosed with clinical or subclinical endometritis were
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classified as healthy controls.
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2.3 Sampling of uterine flush and blood
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Uterine flush samples were collected at Weeks 4, 6, and 8 postpartum. Briefly, after thoroughly
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cleaning the vulva with disinfectant, a two-way Foley catheter (20 French, 30 mL) was placed into the
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previously pregnant uterine horn (determined as the horn with the greater diameter and length) and
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inserted approximately 5 cm past the point of bifurcation of the uterus. The cuff of the catheter was
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inflated with 8 to 10 mL of air (depending on the diameter of the uterine horn), and 20 mL of isotonic
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saline solution was infused into the uterine horn and recovered using a 60 mL sterile syringe. Aspirated
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fluid was transferred to a sterile 50 mL sterile disposable centrifuge tube and immediately placed in an ice
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bath. The samples were then centrifuged at 2000 × g for 10 min at 4°C and the supernatant transferred to
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2 mL microcentrifuge tubes and stored frozen at -80°C until analysis.
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Blood samples were collected from the tail vein just after calving (1.0 ± 0.1 h; range: 30 min to 3 h)
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and then again at Weeks 1, 2, 4, 6, and 8 postpartum. Ten milliliters of blood were placed into a plastic
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centrifuge tube without additives and immediately placed in an ice bath. The samples were then
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centrifuged at 2000 × g for 10 min at 4°C, and the serum was harvested and frozen at -80°C until required.
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2.4. Measurement of cytokine concentrations in uterine flush and serum samples
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The concentrations of TNF-α, IL-1β, IL-6, IL-8, and IL-10 in uterine flush and TNF-α, IL-1β, and IL-6
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in serum samples were determined using commercially available kits [a bovine TNF-α ELISA kit (R&D
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Systems, Minneapolis, MN, USA), bovine IL-1β and IL-6 ELISA kits and a human IL-10 ELISA kit
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(Thermo Fisher Scientific, Rockford, IL, USA), and a bovine IL-8 ELISA kit (USCN Life Science, Hubei,
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China)]. All procedures were performed according to the guidelines provided by the manufacturers.
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Briefly, for TNF-α, 100 µL of uterine flush, serum, or standards diluted in Reagent Diluent were added to
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96-well microplates and incubated for 2 h at room temperature. After aspiration and washing, 100 µL of
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the Detection Antibody (diluted in Regent Diluent) was added to each well and incubated for 2 h. The
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plates were washed and then incubated with 100 µL of the working Streptavidin-HRP solution for 20 min,
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washed again, and then incubated with 100 µL of Substrate Solution for 20 min. Finally, 50 µL of Stop
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Solution was added. For IL-8, 100 µL of uterine flush sample or standard was added to a 96-well
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microplate, and incubated for 2 h at 37°C. After aspiration, 100 µL of Detection Reagent A (diluted in
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Regent Diluent) was added and the plates were incubated for 1 h at 37°C. The plates were washed and
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100 µL of the Detection Reagent B (diluted in Regent Diluent) was added to each well. The plates were
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then incubated for 30 min at 37°C. Substrate Solution was added (90 µL) and then the plate was incubated
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for 20 min at 37°C before the addition of 50 µL Stop Solution. For IL-1β, IL-6, and IL-10, 50 µL of
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uterine flush, serum, or standard was added to each well followed by 50 µL of Biotinylated Antibody
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Reagent. The plates were then incubated for 2 h at room temperature. After washing, 100 µL of
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Streptavidin-HRP Solution was added to each well and the plates were incubated for 30 min at room
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temperature. Finally, 100 µL of TMB Substrate Solution was added to each well for 30 min after which
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the reaction was terminated by adding 100 µL of Stop Solution. The optical density of each well was then
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measured at 450 nm in a Microplate Spectrophotometer (Emax, Molecular Devices, Sunnyvale, CA,
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USA).
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2.5. Experimental design and statistical analyses
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Uterine flush samples were obtained at 4, 6, and 8 weeks postpartum from cows with clinical
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endometritis (n = 20), subclinical endometritis (n = 14), and healthy controls (n = 14). Serum samples
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were obtained just after calving and at 1, 2, 4, 6, and 8 postpartum from cows with clinical endometritis (n
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= 15), subclinical endometritis (n = 10), and healthy controls (n = 9). Sample size calculations were done
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a priori. A minimum of seven cows per group were needed to detect statistical significance in TNF-α
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concentrations when the standard deviation was 50 pg/mL and the difference among treatment groups
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was 200 pg/mL in the repeated measures model (α = 0.01; β = 0.20).
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Statistical analyses were performed using the SAS program (version 9.2; SAS Inst., Cary, NC, USA).
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Cytokine concentrations were not normally distributed; therefore, values were transformed to their natural
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logarithms for data analysis, although non-transformed data expressed as means and SEM are presented
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herein. The effects of group (clinical endometritis, subclinical endometritis, or healthy controls), parity
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(primiparous or multiparous), sampling time (week postpartum), and two-way interactions between group,
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parity, and sampling time on inflammatory cytokines concentrations were determined using the mixed
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model. Cows were included in the model as a random effect. Duncan’s multiple range tests were used to
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locate significant main effects. A P-value ≤ 0.05 was considered significant and 0.05 < P < 0.1 was
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considered to indicate a tendency toward a significant difference.
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3. Results
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When uterine flushes were evaluated, there were group (P < 0.05) effects on TNF-α and IL-8, and
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group (P < 0.0001) and sampling time (P < 0.005) effects on IL-6 and IL-1β concentrations. There were
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tendencies (P < 0.1) for group and sampling time effects on IL-10 and group-by-sampling time interaction
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effect on IL-1β (Fig. 1). There were no effects of parity on concentrations of any cytokine. Concentrations
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of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with
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subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical
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and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10
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concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical
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endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with
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subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum,
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concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant
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main effects on serum TNF-α, IL-1β, and IL-6 concentrations (153.6 ± 18.7, 10.7 ± 0.8, and 772.8 ± 74.3
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pg/mL, respectively).
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4. Discussion
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The postpartum immune response, which is associated with the release of the several inflammatory
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cytokines followed by the mobilization of neutrophils, is important for the subsequent uterine health in
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dairy cows [16,18,22]. The present study demonstrated that higher concentrations of the inflammatory
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cytokines are present in the uterine flush, but not in the serum, of cows with clinical and subclinical
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endometritis when compared to healthy controls.
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Several recently published studies show that increased expression of pro-inflammatory cytokines in
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bovine uterine tissue is related to the development of clinical and/or subclinical endometritis [23,31]. To
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the best of our knowledge, however, the present study is the first to measure the levels of inflammatory
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cytokines in the uterine flush from dairy cows with endometritis. In the present study, we found
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differences in the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, anti-inflammatory
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cytokines IL-10, and the primary chemokine that regulates neutrophil activity, IL-8, in the uterine flush of
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cows with clinical or subclinical endometritis. Moreover, the data showed that changes in the levels of the
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five inflammatory cytokines were associated with the development of uterine inflammation and that the
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levels varied depending on the clinical or subclinical endometritis and the sampling time. Differences in
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IL-1β and IL-6 levels in the uterine flush were evident over the entire sampling period, indicating that
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these cytokines might be strongly related to the development of uterine inflammation in postpartum dairy
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cows. In addition, the remarkably higher levels of these two cytokines in the clinical endometritis group
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might enable these cows to be differentiated from cows with subclinical endometritis or healthy cows.
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Furthermore, the sharp linear decrease in the levels of IL-1β and IL-6 observed in the clinical
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endometritis group from 4 to 8 weeks postpartum, which was not observed in cows with subclinical
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endometritis or the healthy controls, may indicate progression toward the resolution of uterine
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inflammation.
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A previous study used real-time reverse transcription-polymerase chain reaction experiments to show
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that the expression of IL-1β, IL-8, and TNF-α mRNA was significantly higher in cows with subclinical or
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clinical endometritis than in healthy cows between 21 and 27 day postpartum [31]. In contrast IL-6
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mRNA expression was not influenced by uterine inflammation [31], which, in general, is in agreement
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with the results presented herein. Another study examined uterine cytology and found that dairy cows
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with endometritis, diagnosed between 5 and 7 weeks postpartum, expressed higher levels of inflammatory
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cytokine genes IL-1β, IL-6, and IL-8 than cows without endometritis [23]. This is also consistent with the
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finding of the present study. Loyi et al. [25] found that the expression of pro-inflammatory cytokine
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transcripts (e.g., TNF-α, IL-1β, and IL-6 mRNAs) increased several-fold in the uterine tissue of buffalos
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with clinical endometritis. They also detected significant up-regulation of TNF-α and IL-6 mRNA in the
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uterine tissue of buffalos with subclinical endometritis compared with normal buffalos. Taken together,
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these results indicate that greater levels of inflammatory cytokines in the uterine flush as well as higher
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gene expression of inflammatory cytokines in the uterine tissue during the postpartum period might be
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indicative of the development of clinical or subclinical endometritis. However, threshold levels for the
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diagnosis of clinical or subclinical endometritis still need to be determined. In addition, another study demonstrated that dairy cows with subclinical endometritis expressed
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higher levels of TNF-α, IL-6, and IL-8 mRNA than normal cows [24]; however, there were no differences
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in the levels of TNF-α and IL-6 in the uterine flush between the subclinical endometritis and healthy
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control groups in the present study. This discrepancy may be due to the differences between the
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measurement of pro-inflammatory cytokine mRNA in uterine tissue and the detection of the cytokines
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(protein) in the uterine flush. Another reason might be that, although synthesis of the primary RNA
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transcript (mRNA) is the first step in protein production and secretion, several factors such as
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posttranscriptional modification of mRNA, mRNA degradation, posttranscriptional modification of
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proteins, protein targeting and transport, and protein degradation can affect the amount of protein that is
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ultimately released into the circulation [32].
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The IL-10 concentration in the uterine flush from the clinical endometritis group was higher than that
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in the healthy control and subclinical endometritis groups in the present study. During acute inflammation,
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IL-10 acts as a potent anti-inflammatory cytokine that functions to dampen the inflammatory response to
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pathogen by limiting the expression of pro-inflammatory cytokines and chemokines. It thus protects the
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host from excessive inflammation [33–35], which might explain why the concentration of the IL-10
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increased during the early postpartum period in the clinical endometritis group. A previous study reported
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that bovine uterine disease was not associated with IL-10 gene expression in uterine tissue [23]; however,
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another showed that the IL-10 concentrations in serum from cows with clinical endometritis was higher
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than that in the serum of normal cows, with a peak level recorded at 30 days postpartum in cows with
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clinical endometritis [26].
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The observation in this study that the levels of TNF-α, IL-1β, and IL-6 in the serum did not differ
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between the groups is in agreement with a previous study showing no differences in IL-6 levels between
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cows with clinical endometritis and healthy cows [36]. Previous studies also reported that although IL-8
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expression in uterine tissue from cows with endometritis was higher than that in healthy controls, IL-8
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levels of in the serum were similar [23,37], which agrees with the results of the present study. However,
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another study showed that cows with clinical or subclinical endometritis had higher serum concentrations
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of TNF-α, IL-1β, and IL-6 than normal cows [27], which conflicts with the results of the present study.
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Besides, a recent study revealed that blood leukocytes from cows with subclinical endometritis expressed
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a higher level of TNF-α mRNA, but not IL-1β or IL-10, compared with those without the uterine disease
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[38]. Although significant increases in the levels of TNF-α, IL-1β, and IL-6 were observed in the uterine
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flush of cows with endometritis, there were no significant differences in the levels of these cytokines in
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serum samples from cows with endometritis (clinical or subclinical) or healthy cows in this study. This
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was unexpected. The reasons for the discrepancy between the cytokine levels in the uterine flush and
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serum samples were not clarified in the present study. However, it is assumed that the serum levels of pro-
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inflammatory cytokines were not influenced due to the local, but not systemic, actions of these cytokines
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in the uterine tissue [23]. Alternatively, it might be due to the fact that samples of the uterine flush and
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serum were not the same cows, or other confounding factors, such as inclusion of cows with retained
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placenta or septicemic metritis in the present study.
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In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum.
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Acknowledgements
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This work was carried out with the support of the “Cooperative Research Program for Agriculture
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Science & Technology Development (Project No. PJ008464)” Rural Development Administration,
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Republic of Korea.
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[37] Galvão KN, Felippe MJB, Brittin SB, Sper R, Fraga M, Galvão JS, et al. Evaluation of cytokine
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[38] Düvel A, Maaß J, Heppelmann M, Hussen J, Koy M, Piechotta M, et al. Peripheral blood leukocytes
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Figure legends
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Figure 1. Mean (± SEM) TNF-α, IL-1β, IL-6, IL-8, and IL-10 concentrations in the uterine flush from
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healthy control cows (n = 14) and cows with clinical (n = 20) and subclinical endometritis (n = 14) at 4, 6,
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and 8 weeks postpartum. G: group effect, WK: sampling period effect, G*WK: group-by-sampling period
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effect. a,bmeans with different superscripts differ (P < 0.05) among groups.
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S0093-691X(14)00202-7
DOI:
10.1016/j.theriogenology.2014.04.022
Reference:
THE 12787
To appear in:
Theriogenology
Received Date: 16 January 2014 Revised Date:
28 April 2014
Accepted Date: 28 April 2014
Please cite this article as: Kim I-H, Kang H-G, Jeong J-K, Hur T-Y, Jung Y-H, Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.04.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Inflammatory cytokine concentrations in uterine flush and serum
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samples from dairy cows with clinical or subclinical endometritis
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Ill-Hwa Kima,*, Hyun-Gu Kanga, Jae-Kwan Jeonga, Tai-Young Hurb, Young-
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Hun Jungb
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College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Korea b
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National Livestock Research Institute, RDA, Cheonan, Chungnam, 330-800 Korea
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Corresponding author. Tel.: +82 43 2612571; Fax: +82 43 2673150
E-mail address: [email protected] (I.H. Kim).
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Abstract This objective of this study was to compare concentrations of inflammatory cytokines in uterine flush
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and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis.
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Clinical endometritis was diagnosed by observation of vaginal discharges (> 50% pus) and subclinical
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endometritis was diagnosed by evaluation of uterine cytology (neutrophils > 18%) at 4 weeks postpartum.
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Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β,
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IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at
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1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations.
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Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than
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in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows
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with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-
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10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical
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endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with
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subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum,
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concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant
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effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows
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with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows,
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but no differences were observed in serum.
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Key words: Endometritis; Cytokines; Uterine flush; Serum; Dairy cows
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1. Introduction
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Most cows are exposed to bacterial infection after calving [1,2]. Greater than 70% clear the uterine
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bacteria via innate immune responses; however, 17 to 37% of cows develop clinical endometritis,
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whereas14 to 53% develop subclinical endometritis, which results in reduced fertility [3–9]. Proper
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regulation of immune responses during the weeks after calving is important for subsequent uterine health
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[10–13]. The development of bovine endometritis is associated with very complex signaling processes
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involving the detection of bacterial components by innate immune cells via toll-like receptors, the
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production of tumor necrosis factor-α (TNF-α) and other pro-inflammatory cytokines (e.g., interleukins;
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IL), and the mobilization of neutrophils followed by phagocytosis of invading pathogens within the
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uterine lumen [14–21]. Pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and chemokines (e.g.,
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IL-8) stimulate neutrophil and monocyte diapedesis and chemoattraction, and promote increased
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phagocytosis [22].
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Previous studies have used uterine biopsy [23] and the cytobrush technique [24] to examine the
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expression of inflammatory cytokine mRNA in uterine tissue collected from cows with endometritis.
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Others used endometrial tissue scraping from buffalos with endometritis [25]. The results revealed that
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the expression of TNF-α, IL-1β, and IL-6 (all pro-inflammatory cytokines), as well as IL-8 (the primary
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chemokine that regulates neutrophil activity) and IL-10 (an anti-inflammatory cytokine), was related to
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the development of bovine clinical or subclinical endometritis [23,24,26]. Moreover, a recent study
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suggests that the cytokine profile in uterine tissue is associated with the severity and persistency of
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uterine inflammation [25]. In addition, other studies found that serum obtained from cows with
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endometritis and from healthy cows during the peripartum period contained different levels of
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inflammatory cytokines [26,27]. Likewise, higher expression of inflammatory cytokine genes in uterine
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tissue and/or an increase in the levels of those cytokines in the serum are thought to predict the
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development of bovine endometritis [23,24,27]. However, no study has measured the levels of
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inflammatory cytokines in uterine flush from cows with endometritis. Measuring inflammatory cytokine
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levels in uterine flush and serum from dairy cows with clinical or subclinical endometritis during the
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voluntary waiting period may provide valuable information that can serve as a diagnostic tool for the
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uterine inflammation. The objective of the present study was to compare uterine flush and serum concentrations of
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inflammatory cytokines in healthy postpartum dairy cows with cows that develop clinical or subclinical
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endometritis.
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2. Materials and methods
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2.1. Animals
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All experiments were performed with the approval of the Institutional Animal Care and Use
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Committee of Chungbuk National University, Korea. This experiment was contacted on two dairy farms
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located in Chungcheong Province, Korea, during the period from March 2012 to September 2013. Forty-
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eight and 34 Holstein cows, with 2.3 ± 1.4 lactations (mean ± standard deviation; range: 1–7 lactations),
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were used to obtain uterine flush and serum samples, respectively. The cows were maintained in a loose-
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housing system, fed a total mixed ration, and milked twice daily.
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2.2. Diagnosis of clinical and subclinical endometritis
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All of the cows were evaluated for clinical endometritis at Week 4 postpartum by examining vaginal
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discharges removed using the Metricheck tool [28]. Briefly, after cleaning the vulva with the disinfectant
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(chlorhexidine gluconate), the Metricheck device was inserted until it reached the vaginal fornix and then
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retracted for evaluation of the vaginal mucus contained in the cup. Cows with a mucopurulent uterine
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discharge (> 50% pus) were diagnosed with clinical endometritis [1,3]. At the same time, cows were
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evaluated for subclinical endometritis by uterine cytology [29]. Briefly, after cleaning the vulva, a
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cytobrush and stainless steel rod (which was guarded by a stainless steel sheath and covered with a
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protective plastic sheath) were introduced into the vagina. At the external end of the cervix, the plastic
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sheath was pulled back, and the stainless steel sheath and stainless steel rod and cytobrush were passed
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into the body of the uterus. The stainless steel sheath was then retracted to expose the cytobrush. The
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cytobrush was rotated clockwise to obtain cellular material from the endometrium. After removal from
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the vagina, the brush was rolled onto a glass slide that was allowed to air-dry. All slides were stained
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using Diff-Quick stain (Sysmex Inc., Kobe, Japan) according to the manufacturer’s guidelines. Each slide
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was examined under a microscope (× 200 magnification) by the same examiner. The numbers of epithelial
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endometrial cells and neutrophils were counted (up to 200 cells per slide) and the percentage of
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neutrophils was calculated. Subclinical endometritis was defined as a neutrophil proportion > 18% in the
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absence of clinical endometritis [1,30]. Cows not diagnosed with clinical or subclinical endometritis were
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classified as healthy controls.
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2.3 Sampling of uterine flush and blood
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Uterine flush samples were collected at Weeks 4, 6, and 8 postpartum. Briefly, after thoroughly
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cleaning the vulva with disinfectant, a two-way Foley catheter (20 French, 30 mL) was placed into the
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previously pregnant uterine horn (determined as the horn with the greater diameter and length) and
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inserted approximately 5 cm past the point of bifurcation of the uterus. The cuff of the catheter was
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inflated with 8 to 10 mL of air (depending on the diameter of the uterine horn), and 20 mL of isotonic
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saline solution was infused into the uterine horn and recovered using a 60 mL sterile syringe. Aspirated
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fluid was transferred to a sterile 50 mL sterile disposable centrifuge tube and immediately placed in an ice
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bath. The samples were then centrifuged at 2000 × g for 10 min at 4°C and the supernatant transferred to
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2 mL microcentrifuge tubes and stored frozen at -80°C until analysis.
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Blood samples were collected from the tail vein just after calving (1.0 ± 0.1 h; range: 30 min to 3 h)
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and then again at Weeks 1, 2, 4, 6, and 8 postpartum. Ten milliliters of blood were placed into a plastic
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centrifuge tube without additives and immediately placed in an ice bath. The samples were then
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centrifuged at 2000 × g for 10 min at 4°C, and the serum was harvested and frozen at -80°C until required.
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2.4. Measurement of cytokine concentrations in uterine flush and serum samples
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The concentrations of TNF-α, IL-1β, IL-6, IL-8, and IL-10 in uterine flush and TNF-α, IL-1β, and IL-6
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in serum samples were determined using commercially available kits [a bovine TNF-α ELISA kit (R&D
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Systems, Minneapolis, MN, USA), bovine IL-1β and IL-6 ELISA kits and a human IL-10 ELISA kit
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(Thermo Fisher Scientific, Rockford, IL, USA), and a bovine IL-8 ELISA kit (USCN Life Science, Hubei,
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China)]. All procedures were performed according to the guidelines provided by the manufacturers.
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Briefly, for TNF-α, 100 µL of uterine flush, serum, or standards diluted in Reagent Diluent were added to
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96-well microplates and incubated for 2 h at room temperature. After aspiration and washing, 100 µL of
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the Detection Antibody (diluted in Regent Diluent) was added to each well and incubated for 2 h. The
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plates were washed and then incubated with 100 µL of the working Streptavidin-HRP solution for 20 min,
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washed again, and then incubated with 100 µL of Substrate Solution for 20 min. Finally, 50 µL of Stop
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Solution was added. For IL-8, 100 µL of uterine flush sample or standard was added to a 96-well
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microplate, and incubated for 2 h at 37°C. After aspiration, 100 µL of Detection Reagent A (diluted in
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Regent Diluent) was added and the plates were incubated for 1 h at 37°C. The plates were washed and
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100 µL of the Detection Reagent B (diluted in Regent Diluent) was added to each well. The plates were
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then incubated for 30 min at 37°C. Substrate Solution was added (90 µL) and then the plate was incubated
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for 20 min at 37°C before the addition of 50 µL Stop Solution. For IL-1β, IL-6, and IL-10, 50 µL of
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uterine flush, serum, or standard was added to each well followed by 50 µL of Biotinylated Antibody
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Reagent. The plates were then incubated for 2 h at room temperature. After washing, 100 µL of
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Streptavidin-HRP Solution was added to each well and the plates were incubated for 30 min at room
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temperature. Finally, 100 µL of TMB Substrate Solution was added to each well for 30 min after which
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the reaction was terminated by adding 100 µL of Stop Solution. The optical density of each well was then
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measured at 450 nm in a Microplate Spectrophotometer (Emax, Molecular Devices, Sunnyvale, CA,
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USA).
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2.5. Experimental design and statistical analyses
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Uterine flush samples were obtained at 4, 6, and 8 weeks postpartum from cows with clinical
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endometritis (n = 20), subclinical endometritis (n = 14), and healthy controls (n = 14). Serum samples
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were obtained just after calving and at 1, 2, 4, 6, and 8 postpartum from cows with clinical endometritis (n
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= 15), subclinical endometritis (n = 10), and healthy controls (n = 9). Sample size calculations were done
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a priori. A minimum of seven cows per group were needed to detect statistical significance in TNF-α
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concentrations when the standard deviation was 50 pg/mL and the difference among treatment groups
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was 200 pg/mL in the repeated measures model (α = 0.01; β = 0.20).
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Statistical analyses were performed using the SAS program (version 9.2; SAS Inst., Cary, NC, USA).
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Cytokine concentrations were not normally distributed; therefore, values were transformed to their natural
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logarithms for data analysis, although non-transformed data expressed as means and SEM are presented
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herein. The effects of group (clinical endometritis, subclinical endometritis, or healthy controls), parity
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(primiparous or multiparous), sampling time (week postpartum), and two-way interactions between group,
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parity, and sampling time on inflammatory cytokines concentrations were determined using the mixed
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model. Cows were included in the model as a random effect. Duncan’s multiple range tests were used to
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locate significant main effects. A P-value ≤ 0.05 was considered significant and 0.05 < P < 0.1 was
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considered to indicate a tendency toward a significant difference.
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3. Results
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When uterine flushes were evaluated, there were group (P < 0.05) effects on TNF-α and IL-8, and
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group (P < 0.0001) and sampling time (P < 0.005) effects on IL-6 and IL-1β concentrations. There were
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tendencies (P < 0.1) for group and sampling time effects on IL-10 and group-by-sampling time interaction
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effect on IL-1β (Fig. 1). There were no effects of parity on concentrations of any cytokine. Concentrations
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of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with
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subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical
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and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10
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concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical
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endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with
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subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum,
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concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant
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main effects on serum TNF-α, IL-1β, and IL-6 concentrations (153.6 ± 18.7, 10.7 ± 0.8, and 772.8 ± 74.3
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pg/mL, respectively).
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4. Discussion
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The postpartum immune response, which is associated with the release of the several inflammatory
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cytokines followed by the mobilization of neutrophils, is important for the subsequent uterine health in
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dairy cows [16,18,22]. The present study demonstrated that higher concentrations of the inflammatory
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cytokines are present in the uterine flush, but not in the serum, of cows with clinical and subclinical
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endometritis when compared to healthy controls.
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Several recently published studies show that increased expression of pro-inflammatory cytokines in
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bovine uterine tissue is related to the development of clinical and/or subclinical endometritis [23,31]. To
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the best of our knowledge, however, the present study is the first to measure the levels of inflammatory
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cytokines in the uterine flush from dairy cows with endometritis. In the present study, we found
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differences in the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, anti-inflammatory
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cytokines IL-10, and the primary chemokine that regulates neutrophil activity, IL-8, in the uterine flush of
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cows with clinical or subclinical endometritis. Moreover, the data showed that changes in the levels of the
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five inflammatory cytokines were associated with the development of uterine inflammation and that the
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levels varied depending on the clinical or subclinical endometritis and the sampling time. Differences in
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IL-1β and IL-6 levels in the uterine flush were evident over the entire sampling period, indicating that
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these cytokines might be strongly related to the development of uterine inflammation in postpartum dairy
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cows. In addition, the remarkably higher levels of these two cytokines in the clinical endometritis group
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might enable these cows to be differentiated from cows with subclinical endometritis or healthy cows.
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Furthermore, the sharp linear decrease in the levels of IL-1β and IL-6 observed in the clinical
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endometritis group from 4 to 8 weeks postpartum, which was not observed in cows with subclinical
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endometritis or the healthy controls, may indicate progression toward the resolution of uterine
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inflammation.
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A previous study used real-time reverse transcription-polymerase chain reaction experiments to show
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that the expression of IL-1β, IL-8, and TNF-α mRNA was significantly higher in cows with subclinical or
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clinical endometritis than in healthy cows between 21 and 27 day postpartum [31]. In contrast IL-6
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mRNA expression was not influenced by uterine inflammation [31], which, in general, is in agreement
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with the results presented herein. Another study examined uterine cytology and found that dairy cows
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with endometritis, diagnosed between 5 and 7 weeks postpartum, expressed higher levels of inflammatory
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cytokine genes IL-1β, IL-6, and IL-8 than cows without endometritis [23]. This is also consistent with the
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finding of the present study. Loyi et al. [25] found that the expression of pro-inflammatory cytokine
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transcripts (e.g., TNF-α, IL-1β, and IL-6 mRNAs) increased several-fold in the uterine tissue of buffalos
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with clinical endometritis. They also detected significant up-regulation of TNF-α and IL-6 mRNA in the
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uterine tissue of buffalos with subclinical endometritis compared with normal buffalos. Taken together,
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these results indicate that greater levels of inflammatory cytokines in the uterine flush as well as higher
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gene expression of inflammatory cytokines in the uterine tissue during the postpartum period might be
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indicative of the development of clinical or subclinical endometritis. However, threshold levels for the
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diagnosis of clinical or subclinical endometritis still need to be determined. In addition, another study demonstrated that dairy cows with subclinical endometritis expressed
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higher levels of TNF-α, IL-6, and IL-8 mRNA than normal cows [24]; however, there were no differences
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in the levels of TNF-α and IL-6 in the uterine flush between the subclinical endometritis and healthy
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control groups in the present study. This discrepancy may be due to the differences between the
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measurement of pro-inflammatory cytokine mRNA in uterine tissue and the detection of the cytokines
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(protein) in the uterine flush. Another reason might be that, although synthesis of the primary RNA
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transcript (mRNA) is the first step in protein production and secretion, several factors such as
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posttranscriptional modification of mRNA, mRNA degradation, posttranscriptional modification of
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proteins, protein targeting and transport, and protein degradation can affect the amount of protein that is
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ultimately released into the circulation [32].
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The IL-10 concentration in the uterine flush from the clinical endometritis group was higher than that
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in the healthy control and subclinical endometritis groups in the present study. During acute inflammation,
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IL-10 acts as a potent anti-inflammatory cytokine that functions to dampen the inflammatory response to
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pathogen by limiting the expression of pro-inflammatory cytokines and chemokines. It thus protects the
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host from excessive inflammation [33–35], which might explain why the concentration of the IL-10
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increased during the early postpartum period in the clinical endometritis group. A previous study reported
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that bovine uterine disease was not associated with IL-10 gene expression in uterine tissue [23]; however,
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another showed that the IL-10 concentrations in serum from cows with clinical endometritis was higher
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than that in the serum of normal cows, with a peak level recorded at 30 days postpartum in cows with
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clinical endometritis [26].
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The observation in this study that the levels of TNF-α, IL-1β, and IL-6 in the serum did not differ
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between the groups is in agreement with a previous study showing no differences in IL-6 levels between
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cows with clinical endometritis and healthy cows [36]. Previous studies also reported that although IL-8
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expression in uterine tissue from cows with endometritis was higher than that in healthy controls, IL-8
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levels of in the serum were similar [23,37], which agrees with the results of the present study. However,
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another study showed that cows with clinical or subclinical endometritis had higher serum concentrations
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of TNF-α, IL-1β, and IL-6 than normal cows [27], which conflicts with the results of the present study.
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Besides, a recent study revealed that blood leukocytes from cows with subclinical endometritis expressed
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a higher level of TNF-α mRNA, but not IL-1β or IL-10, compared with those without the uterine disease
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[38]. Although significant increases in the levels of TNF-α, IL-1β, and IL-6 were observed in the uterine
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flush of cows with endometritis, there were no significant differences in the levels of these cytokines in
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serum samples from cows with endometritis (clinical or subclinical) or healthy cows in this study. This
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was unexpected. The reasons for the discrepancy between the cytokine levels in the uterine flush and
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serum samples were not clarified in the present study. However, it is assumed that the serum levels of pro-
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inflammatory cytokines were not influenced due to the local, but not systemic, actions of these cytokines
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in the uterine tissue [23]. Alternatively, it might be due to the fact that samples of the uterine flush and
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serum were not the same cows, or other confounding factors, such as inclusion of cows with retained
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placenta or septicemic metritis in the present study.
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In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum.
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Acknowledgements
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This work was carried out with the support of the “Cooperative Research Program for Agriculture
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Science & Technology Development (Project No. PJ008464)” Rural Development Administration,
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Republic of Korea.
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Figure 1. Mean (± SEM) TNF-α, IL-1β, IL-6, IL-8, and IL-10 concentrations in the uterine flush from
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healthy control cows (n = 14) and cows with clinical (n = 20) and subclinical endometritis (n = 14) at 4, 6,
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