Subclinical Endometritis Cows Research Articles

Profile of uterine flush lipid mediators in cows with subclinical endometritis: pilot study.

Subclinical endometritis affects reproductive outcomes and causes economic losses in dairy cows, thus, it is important to understand disease progression mechanisms and develop diagnostic procedures for better disease management. We measured the levels of 146 lipid mediators in uterine flush samples using lipid chromatography-mass spectrometry. We detected 25 lipid mediators in the uterine flush of both the control and subclinical endometritis cows; 15 of the 25 lipid mediators were AA-derived metabolites. Among the AA-derived metabolites, cyclooxygenase (COX)-generated mediators were the most abundant. Specifically, levels of 11β-13,14-dihydro-15-keto prostaglandin (PG) F2α, PGE2, PGA2, 13-hydroxyoctadecadienoic acid, and PGD1 were elevated in all the cows with subclinical endometritis. This study may provide new insights for the management of subclinical bovine endometritis.

Progesterone-independent endometrial mRNA expression in dairy cows with clinical or subclinical endometritis

Up to 50 % of dairy cows fail to resolve uterine involution and develop chronic clinical (CE) or subclinical endometritis (SE) 21 days after calving. Clinical endometritis is associated with purulent discharge, while SE is not associated with overt clinical signs. Along with numerous knowledge gaps related to its pathogenesis, SE does not allow for a straightforward and effective therapy. Therefore, it is crucial to unravel differences in the expression of genes among healthy, CE, and SE cows. This might contribute to the discovery of new drug candidates and, in consequence, a potentially effective treatment. In the present study, cows between 21 and 28 days postpartum (PP) were examined using vaginoscopy for the presence of vaginal discharge and endometrial cytology for the determination of the endometrial polymorphonuclear cell (PMN) percentage. Next, an endometrial biopsy sample was taken to investigate the expression of 13 selected candidate genes by qPCR. Uterine health status was assigned to healthy (absence of abnormal vaginal discharge and ≤5 % PMN, n = 13), SE (absence of abnormal vaginal discharge and >5 % PMN, n = 30), and CE (mucopurulent or purulent vaginal discharge and >5 % PMN, n = 9). At the same time, a blood sample was collected to assess serum progesterone concentration and to categorize cows as low (≤1 ng/mL) or high (>1 ng/mL) in progesterone. High expression of IL1B, IL6, IL17A, CXCL8, PTGES, PTGS1, PTGS2, and INHBA genes and low expression of FST was noted in the endometrium of CE compared to healthy cows. Increased endometrial INHBA expression was observed in both SE and CE compared to healthy cows. Interestingly, greater expression of PTGES and PRXL2B genes and lower expression of PTGS2 were characteristic of SE versus CE or healthy. Among cows with no overt clinical symptoms of uterine disease (healthy and SE), the endometrial expression of IL1 B, CXCL8, and PTGES was greater in cows with high versus low serum progesterone. Several genes were differentially expressed among healthy, SE, and CE cows indicating different pathways for the development of different uterine diseases. In conclusion, we found progesterone-independent SE markers, which suggests that low endometrial PTGS2 expression may be indicative of an inadequate immune response and thus contribute to the pathogenesis of SE.

Characterization of circulating microRNA profiles of postpartum dairy cows with persistent subclinical endometritis

Subclinical endometritis (SCE) is an unresolved inflammation of the endometrium of postpartum dairy cows, seriously affecting fertility. Current diagnosis, which relies on uterine cytology or even more invasive biopsy sampling, would benefit from the identification of blood-based diagnostic biomarkers. Due to the known role of microRNAs (miRNAs) in other diseases, this case-control study evaluated the cell-free circulating miRNA profiles of SCE cows, and the network of transcripts predicted to interact with those miRNAs, previously identified as differentially expressed genes (DEG) in the endometrium of the same cows. Healthy (H, n = 6) and persistent SCE (n = 11) cows characterized by endometrial cytology and biopsy were blood sampled at 21 and 44 d postpartum (DPP). Following extraction of cell-free plasma miRNAs and RNA-seq analysis, differential abundance analysis of miRNAs was performed with the DESeq2 R package (adjusted p-value of 0.05), and in silico prediction of miRNA-interacting genes on a sequence complementary basis was conducted using the miRWalk database. The principal component analysis showed a clear clustering between groups of uterine health phenotypes (H vs. SCE), although the clustering between groups was less pronounced at 44 DPP than at 21 DPP. No effect of the stage (21 vs. 44 DPP) was observed. A total of 799 known circulating miRNAs were identified, from which 34 demonstrated differential abundance between H and SCE cows (12 less abundant and 22 more abundant in SCE than in H cows). These 34 miRNAs are predicted to interact with 10,104 transcripts, among which 43, 81, and 147 were previously identified as differentially expressed in, respectively, endometrial luminal epithelial, glandular epithelial, and stromal cells of the same cows. This accounts for approximately half of the DEG identified between those H and SCE cows, including genes involved in endometrial cell proliferation, angiogenesis and immune response, whose dysregulation in SCE cows may impair pregnancy establishment. From 219 miRNAs with mean normalized read counts above 100, the presence and abundance of miR-425-3p and miR-2285z had the highest discriminatory level to differentiate SCE from H cows. In conclusion, despite apparent confinement to the endometrium, SCE is associated with a distinct circulating miRNA profile, which may represent a link between the systemic changes associated with disease and the endometrial immune response. The validation of a miRNA panel consisting of circulating cell-free miR-425-3p and miR-2285z may prove a relevant advancement for the noninvasive diagnosis of persistent SCE.

Expression study of pro-inflammatory cytokines and acute phase proteins in post-partum crossbred dairy cows with subclinical endometritis

Subclinical endometritis (SCE) is one of the causes of poor reproductive performance in dairy cows. The objective of the study was to evaluate the expression of pro-inflammatory cytokines [PICs; Interleukin -1 beta (IL-1β), Interleukin -6 (IL-6), and Tumor necrosis factor- alpha (TNF-α)] and Acute phase proteins [APPs; Serum Amyloid- A (SAA) and Haptoglobin (HP)] in blood and uterine flushings of postpartum dairy cows with subclinical endometritis (SCE). Animals (n=49) were screened for SCE at 60 days postpartum (dpp) using the endometrial cytobrush cytology, and those with PMN cell percentage ≥ 18% were identified as SCE cows. The blood samples and uterine flushings from SCE (n=15) and the control group (n=15) were assessed for mRNA expressions of PICs and APPs using the Real-Time PCR method (RT-PCR). The PICs and APPs except TNF-α were non-significant (p>0.05) in blood samples whereas a significant difference (p<0.05) was found in uterine flushings. The PICs and APPs were significantly up-regulated in the uterine endometrium indicating that collection of uterine flushings at 60 dpp could be used as a diagnostic method for evaluating gene expressions of inflammatory mediators in dairy cows with SCE.

Evaluation of clinical and subclinical endometritis impacts on the reproductive performance and milk production of dairy cows in Brazilian herds.

Clinical endometritis (CE) and subclinical endometritis (SE) are two of the main uterine diseases in dairy cows during the puerperium period that can directly affect milk production and fertility in dairy herds. The first objective of this study was to investigate the effects of the CE and SE on the subsequent reproductive performance and milk production of dairy cows in Brazilian herds, and the second objective was to evaluate the reproductive performance and milk production of dairy cows according to the positive or negative isolation of bacteria in the uterine environment. A total of 279 lactating Holstein dairy cows (28-33 days in milk) from six commercial farms were studied. These were classified into three groups: healthy cows (without CE and SE, n=161), cows with CE (vaginal discharge score=3 and ≥18% PMNL, n=83) and cows with SE (absence of signs of CE and >18% PMNL, n=35). According to the presence of bacteria isolated from the uterine environment, the animals were classified as healthy cows (n=161), CE cows with positive (n=68) and negative (n=15) bacterial isolation; and SE cows with positive (n=21) and negative (n=14) bacterial isolation. Cows with CE (20.5%) and SE (31.4%) had a lower (p < .05) conception rate at first AI than healthy cows (47.8%). Furthermore, both cows with CE (3.1) and SE (2.8) needed more service to become pregnant than healthy cows (1.7) and more days to be pregnant (50.3 days in CE and 43.1 days in SE than healthy group; p < .001). Cows with CE (23.8kg) and SE (23.7kg) produced less milk than healthy cows on the day of sampling (28.2kg); however, milk production during 305 DIM of cows with CE and SE was not impaired when compared to healthy cows. The logistic regression showed that parity, body condition score, 305 days milk production, last calving (eutocia or dystocia), twin birth and stillbirth were not associated with the incidence of CE and SE. In addition, we highlight that cows with CE and SE, but with negative bacteria isolation, did not have reproductive performance and milk production impaired during the postpartum period. These results support that the use of antibiotics would not be beneficial in these cows, contributing to the strategic and rational use of antibiotic therapy on dairy farms.

Assessment of the main pathogens associated with clinical and subclinical endometritis in cows by culture and MALDI-TOF mass spectrometry identification

Clinical endometritis (CE) and subclinical endometritis (SCE) are diseases that affect dairy cows during the puerperium, causing negative effects on the animals' milk production and fertility. The objective of this study was to assess the main bacteria related to cases of CE and SCE from uterine samples of dairy cows in Brazilian herds. Selective and differential media were used for isolation of aerobic and anaerobic bacteria and further MALDI-TOF mass spectrometry (MS) identification. A total of 279 lactating dairy cows with 28 to 33 d in milk from 6 commercial farms were evaluated. Initially, cows were classified in 3 groups: cytologic healthy cows (n = 161), cows with CE (n = 83), and cows with SCE (n = 35). Healthy animals presented 97 species, followed by the CE group with 53 identified species, and SCE cows presented only 21 bacterial species. We found a significantly higher isolation rate of Trueperella pyogenes in CE (26.5%) cows compared with healthy and SCE cows. Some anaerobic species were exclusively isolated from the CE group, even though they presented lower frequency. Interestingly, 18.1% of samples from CE cows and 40% of SCE cows were negative to bacterial isolation. Despite the use of culture-dependent methods instead of molecular methods, the present study enabled the identification of a complex community of 127 different species from 48 genera, composed of aerobic and anaerobic bacterial species among the 3 different animal groups. The method of sample collection, culture, and identification by MALDI-TOF MS were essential for the success of the analyses.

The effect of subclinical endometritis on reproductive performance in postpartum Bos indicus multiparous beef cows

The objective was to investigate the prevalence of subclinical endometritis (SE) in postpartum Bos indicus multiparous beef cows using different polymorphonuclear cells (PMN) threshold ratios, and to evaluate the impacts of SE on their reproductive performance. A total of 689 postpartum Nellore cows (45.2 ± 7.8 days postpartum) were submitted to an estrus synchronization protocol + timed-artificial insemination (TAI). Endometrial cytology samples were collected by cytobrush before the beginning of the protocol. Cows were considered positive for SE if: (PMN) ≥ 3% (PMN3), PMN ≥ 5% (PMN5) and PMN ≥ 7% (PMN7). Pregnancy diagnosis was performed at 30 and 100 d after TAI. The prevalence of cows categorized as positive for SE increased (P < 0.05) as the threshold was lowered (PMN7 = 3.9%; PMN5 = 5.5%; PMN3 = 9.1%). Positive SE cows had similar (P > 0.41) BCS, days postpartum, and expression of estrus at TAI compared with negative SE cows. Positive SE cows had decreased (P = 0.04) pregnancy rates compared to negative SE cows in the PMN5 threshold (26.3 ± 8.7% vs 44.5 ± 4.1%); however, no difference (P > 0.45) was observed between positive and negative SE cows in the PMN3 and PMN7 thresholds. Embryonic mortality between days 30 and 100 was not affected by SE (P > 0.16). In conclusion, the prevalence of SE varies based on the PMN threshold used, whereas SE at the beginning of the TAI protocol decreased the pregnancy rates in postpartum Bos indicus beef cows when the PMN5 was used.

MRNA Expressions of Candidate Genes in Gestational Day 16 Conceptus and Corresponding Endometrium in Repeat Breeder Dairy Cows with Suboptimal Uterine Environment Following Transfer of Different Quality Day 7 Embryos

Simple SummaryThe mRNA expression of Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator-activated receptors (PPARA, D, and G), and Retinoid X receptors (RXRA, B, and G) genes and proteins (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) were lower and MUC1 at mRNA and protein levels, was greater in gestation day (GD) 16 embryo and corresponding endometrium of subclinical endometritis cows, and in cows following transfer of poor quality embryo (Grade 3). All genes and proteins but MUC1 expression was lower in GD16 tubular conceptus and corresponding endometrium vs. GD16 filamentous conceptus and matching endometrium in cows with SCE and in cows following the transfer of Grade 3 embryo. Disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor GD7 embryo negatively programs the conceptus development and plausibly affects the conceptus survival. Effect of the gestational day (GD) 7 embryo quality grade (QG) and subclinical endometritis (SCE) on mRNA and protein expressions of candidate genes [Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoid X receptors (RXRA, B, and G), and Mucin-1 (MUC1)] in GD16 conceptus and corresponding endometrium were evaluated. After screening of performance records (n = 2389) and selection of repeat breeders (n = 681), cows with SCE (≥6% polymorphonuclear neutrophils—PMN; n = 180) and no-SCE (<6%PMN; n = 180) received GD7 embryos of different QGs. Based on GD16 conceptus recovery, cows with SCE (n = 30) and No- SCE (n = 30) that received GD7 embryos QG1 (good, n = 20), 2 (fair, n = 20), and 3 (poor, n = 20) were included for gene analysis. mRNA and protein expressions (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) differed between SCE and embryo QG groups. All genes but MUC1 and all proteins but MUC1 expression was greater in filamentous conceptus and corresponding endometrium vs. tubular conceptus and matching endometrium in SCE and embryo QG groups. In conclusion, disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor embryo negatively programs the conceptus development and plausibly affects conceptus survival.

Day 7 embryo quality and suboptimal uterine environment influence morphometry of Day 16 conceptus in dairy cows

The objectives of this study were to investigate the effect of Day 7 embryo quality and subclinical endometritis (SCE) in repeat breeder recipient cows on morphometry of Day 16 embryo and to determine the association of %PMN, serum progesterone and Day 16 conceptus length. Holstein dairy cows that failed to conceive at least 3 times, (parity, 3 and 4; body condition score, 3 to 3.5 out of 5) with subclinical endometritis (n = 180; SCE, >6% PMN on endometrial cytology) or without subclinical endometritis (n = 180; No-SCE, ≤ 6% PMN) were selected. Cows in each group received single, frozen-thawed, quality 1 (n = 60), 2 (n = 60) and 3 (n = 60) embryos (compact morula or early blastocyst) on Day 7 post estrus in the uterine horn ipsilateral to the ovary containing a corpus luteum, using standard nonsurgical techniques. Only cows that expressed estrus (Select-Synch protocol) and with acceptable corpus luteum (≥1.5 cm in size) were included. Conceptuses were collected on Day 16 from all recipient cows by standard non-surgical uterine flushing technique, using an 18-g embryo collection catheter with Phosphate Buffered Saline (pH 7.4). Blood samples were collected on Day 16 to determine serum progesterone concentrations. After collection, conceptuses were weighed and measured, and were categorized as tubular (underdeveloped, 10–20 mm) or filamentous (normal, >25 mm). Between cows with SCE and No-SCE, mean (±SEM) width (1.68 ± 0.13 mm vs. 1.84 ± 0.16 mm), length (34.4 ± 9.6 mm vs. 55.8 ± 13.4 mm) and weight (22.3 ± 3.7 vs. 40.6 ± 6.4 mg) of Day 16 conceptuses differed (P < 0.05). The mean width (1.75 ± 0.19 mm vs. 1.81 ± 0.22 mm), length (57.7 ± 11.2 vs. 51.1 ± 13.6 mm) and weight (34.3 ± 6.4 vs. 38.5 ± 8.2 mg) of Day 16 embryo following transfer of Day 7 embryo quality grade 1 and grade 2 embryos were not different (P > 0.1), but both differed from the mean width (1.59 ± 0.11 mm), length (28.9 ± 9.7 mm) and weight (25.3 ± 4.6 mg) of Day 16 embryo from Day 7 embryo quality grade 3 (P < 0.05). Total percentage of embryos recovered differed between SCE and No-SCE groups (P < 0.05; 36.1 vs 48.9%). Total percentage of embryos recovered on Day 16 following transfer of grade 1 (53.3%) and 2 (44.2%) Day 7 embryos were greater (P < 0.05) compared with transfer of grade 3 embryos (29.2%) (P < 0.001). Total percentage of filamentous embryos recovered was lower for SCE cows compared with No-SCE cows (P < 0.01; 15.0 vs. 25.6%). Total percentage of tubular embryos recovered did not differ between SCE and No-SCE cows (P > 0.1; 21.1% vs. 22.8%). Filamentous embryo recovered for grade 3 was lower (P < 0.05) compared with grade 1 in both SCE (8.3 vs. 21.7%) and No-SCE groups (15.0 vs. 33.3%). The mean (±SEM) CL volume (cm3; 11.8 ± 0.29 vs. 15.9 ± 0.31) and progesterone concentrations (ng/mL; 5.17 ± 1.8 vs. 8.2 ± 1.2) on Day 16 differed between SCE and No-SCE groups (P < 0.05) but not among Day 7 embryo grade groups (P > 0.1). The mean (±SEM) CL volume (cm3; 15.6 ± 0.28 vs 12.1 ± 3.9) and serum progesterone concentrations (ng/mL; 8.6 ± 1.4 vs. 4.9 ± 1.9) on Day 16 differed (P < 0.05) between cows yielded filamentous and tubular embryos. When all cows were considered, multiple regression analysis showed that the %PMN (P < 0.0001), progesterone concentrations (P < 0.0001), embryo qulaity (P < 0.05) and %PMN by progesterone interactions (P < 0.0001) influenced the length of Day 16 conceptus. Among cows without subclinical endometritis, only progesterone concentrations (P < 0.0001) and among cows with subclinical endometritis, only %PMN (P < 0.04) influenced the length of Day 16 conceptus. Progesterone concentrations (P < 0.0001) influenced the length of Day 16 conceptus in cows that received embryo quality 1 and 2. Progesterone concentration by %PMN interaction (P < 0.05) also influenced the length of Day 16 conceptus in cows that received embryo quality 2. The %PMN (P = 0.05) influenced the length of Day 16 conceptus in cows that received embryo quality 3. In conclusion, poor quality Day 7 embryo and presence of SCE negatively influenced early embryo development between Days 7 and 16 of gestation probably by dysregulated embryo-maternal interactions due to lower progesterone, prompting loss of the conceptus in sub-optimal uterine environment.

Nutritional condition in the dry period is related to the incidence of postpartum subclinical endometritis in dairy cattle.

ObjectiveEndometritis is a major disease, that causes infertility in cattle, and is usually categorized as clinical or subclinical endometritis (SCE). The nutritional condition during the dry period is important for recovery after the last stage of the lactation period, and for postpartum production and reproduction. This study aimed to clarify the relationship between nutritional and metabolic characteristics in the dry period, and the risk of postpartum SCE.MethodsMultiparous Holstein dairy cows (n = 25, raised in a tied stall) were used. Endometrial cytological analysis was performed around 30 days post-partum, with 5% to 14% polymorphonuclear (PMN) as a cut-off point to define SCE. Serum levels of glucose, non-esterified fatty acids, β-hydroxybutyric acid (BHBA), blood urea nitrogen, total cholesterol, aspartate aminotransferase, γ-glutamyl transpeptidase, calcium, phosphorus, and magnesium were measured in the cows at the dry period to evaluate energy status, protein metabolism, and mineral metabolism.ResultsThe incidence of SCE in the cows was 60.0% (n = 15/25) and the mean PMN% in postpartum cows diagnosed as SCE was 8.05%±2.6%. Overall, 17 and 8 samples were collected from the cows in the far-off and close-up periods, respectively. The serum concentration of BHBA in the far-off period and serum glucose concentration in the close-up period were correlated with postpartum PMN% (r = 0.62, p<0.01; r = −0.74, p<0.05, respectively). Serum levels of calcium and magnesium in the dry period were associated with the incidence of postpartum SCE (healthy vs SCE cows, p<0.05).ConclusionBlood levels of glucose, BHBA, calcium, and magnesium in dry periods could be useful parameters for predicting the risk of postpartum SCE. The present study also suggests that management in the close-up period is essential for promoting recovery from calving fatigue.

Subclinical endometritis in dairy cattle is associated with distinct mRNA expression patterns in blood and endometrium.

Cattle with subclinical endometritis (SCE) are sub-fertile and diagnosing subclinical uterine disease remains a challenge. The hypothesis for this study was that endometrial inflammation is reflected in mRNA expression patterns of peripheral blood leucocytes. Transcriptome profiles were evaluated in healthy cows and in cows with SCE using circulating white blood cells (WBC) and endometrial biopsy samples collected from the same animals at 45–55 days postpartum. Bioinformatic analyses of microarray-based transcriptional data identified gene profiles associated with distinct biological functions in circulating WBC and endometrium. In circulating WBC, SCE promotes a pro-inflammatory environment, whereas functions related to tissue remodeling are also affected in the endometrium. Nineteen differentially expressed genes associated with SCE were common to both circulating WBC and the endometrium. Among these genes, transcript abundance of immune factors C3, C2, LTF, PF4 and TRAPPC13 were up-regulated in SCE cows at 45–55 days postpartum. Moreover, mRNA expression of C3, CXCL8, LTF, TLR2 and TRAPPC13 was temporally regulated during the postpartum period in circulating WBC of healthy cows compared with SCE cows. This observation might indicate an advantageous modulation of the immune system in healthy animals. The transcript abundance of these genes represents a potential source of indicators for postpartum uterine health.

Effects of follicular fluid of preovulatory follicles of repeat breeder dairy cows with subclinical endometritis on oocyte developmental competence

The aims of the present study were to determine the concentrations of lipopolysaccharide (LPS), hormonal progesterone, estradiol-17β, insulin growth factor (IGF-1) and magnesium in the serum and the preovulatory follicle follicular fluid (FF) in repeat breeder (RB) cows without (nSCE) or with subclinical endometritis (SCE), and further to examine the effects of this FF on developmental competence of cattle oocytes. In Experiment 1, 13 of 23 clinically healthy Holstein RB cows were identified (uterine PMNs) to have SCE. The cows were estrous synchronized, and 6–12 h after detection of standing estrus, FF and blood of the preovulatory follicles were collected. The mean (±SD) LPS (862.3 ± 148.1 compared with 1063.4 ± 262.8 EU/ml, P = 0.04) and estradiol-17β (188.9 ± 15.8 compared with 162.0 ± 31.5 ng/ml, P = 0.02) concentrations of FF was different between nSCE and SCE cows. In Experiment 2, FF of RB cows with relatively lesser (nSCE, n = 4) and greater (SCE, n = 4) percentages of uterine PMNs was separately added to the oocyte maturation medium for in vitro embryo production. Addition of FF from SCE cows to the oocyte maturation medium resulted in a lesser rate of development to the blastocyst stage than that of the nSCE cows (21.9 ± 1.8 compared with 27.8 ± 2.5%, P < 0.05). Results of the present study indicate greater FF LPS concentration may result in a lesser quality microenvironment milieu for the final stages of oocyte maturation in RB dairy cows with subclinical endometritis. In addition, supplementation of oocyte maturation medium with FF of preovulatory follicles from RB cows with subclinical endometritis resulted in a lesser potential of in vitro oocyte developmental competence.

Effects of prepartum oilseed supplements on subclinical endometritis, pro- and anti-inflammatory cytokine transcripts in endometrial cells and postpartum ovarian function in dairy cows.

Postpartum uterine infections affect ovarian function and delay ovulation in cattle. As dietary fats can affect immune cell function, we investigated the influence of prepartum diets on postpartum uterine inflammatory status (UIS) as assessed 25±1 days postpartum by endometrial cytology (normal: ≤8% polymorphonuclear cells (PMN) vs subclinical endometritis (SCE): >8% PMN) and associations between SCE, pro- and anti-inflammatory cytokine gene expression and ovarian function. During the last 5 weeks of gestation, dairy cows received a diet supplemented with 8% rolled sunflower (n=10) or canola seed (n=9) or no oilseed (n=9). Ovaries were scanned until 35 days postpartum. Prepartum diets did not influence SCE, but a preovulatory-size follicle developed sooner (P≤0.05), the interval to first ovulation was shorter and the proportion of cows ovulating within 35 days postpartum was greater in the sunflower seed group. Although mRNA expression of cytokines was not affected by diet, cows with SCE had higher (P≤0.05) expression of interleukin-1β (IL1B), interleukin-8 (CXCL8), IL10 and tumour necrosis factor-α (TNF) than normal cows. The interval (mean ± s.e.m.) from calving to preovulatory-size follicle was shorter (P≤0.05) in normal (13.2±0.9 days) than SCE cows (18.7±1.4 days). In summary, a prepartum diet supplemented with sunflower seed positively influenced postpartum ovarian function without affecting UIS or pro- and anti-inflammatory cytokine gene expression in endometrial cells.

Expression of Pathogen Recognition Receptors and Pro-Inflammatory Cytokine Transcripts in Clinical and Sub-Clinical Endometritis Cows

The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with SCE.

Modulation of the immune system during postpartum uterine inflammation.

Postpartum uterine inflammation (endometritis) in the dairy cow is associated with lower fertility at both the time of infection and after the inflammation has resolved. We hypothesized that aberrant DNA methylation may be involved in the subfertility associated with uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with subclinical endometritis (SCE). Endometrial tissues were obtained at 29 days postpartum (n = 12), and microarrays were used to characterize transcription and DNA methylation. Analyses revealed 1,856 probes differentially expressed in animals with SCE (n = 6) compared with controls (CON, n = 6, P < 0.05, Storey Multiple testing correction) and 2,976 probes with significant correlation between gene expression and bacteriology score. No significant associations among DNA methylation and gene expression were detected. Analysis of transcription data using the Database for Annotation, Visualization, and Integrated Discovery and Gene Set Enrichment Analysis identified several pathways and processes enriched in SCE cows, with the majority related to the immune response. Furthermore, the top ontology terms enriched in genes that had expression data correlated to bacteriology score were: Defense response, inflammatory response, and innate immune response. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local proinflammatory environment in the uterus. If this period of inflammation is prolonged it could result in tissue damage or failure to complete involution of the uterus, which may create a suboptimal environment for future pregnancy.

Dynamic Analysis of Nitric Oxide and Total Oxidant Capacity in Cow Uterine Secretion with Subclinical Endometritis

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.

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45–55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown.

Postpartal subclinical endometritis alters transcriptome profiles in liver and adipose tissue of dairy cows.

Transcriptome alterations in liver and adipose tissue of cows with subclinical endometritis (SCE) at 29 d postpartum were evaluated. Bioinformatics analysis was performed using the Dynamic Impact Approach by means of KEGG and DAVID databases. Milk production, blood metabolites (non-esterified fatty acids, magnesium), and disease biomarkers (albumin, aspartate aminotransferase) did not differ greatly between healthy and SCE cows. In liver tissue of cows with SCE, alterations in gene expression revealed an activation of complement and coagulation cascade, steroid hormone biosynthesis, apoptosis, inflammation, oxidative stress, MAPK signaling, and the formation of fibrinogen complex. Bioinformatics analysis also revealed an inhibition of vitamin B3 and B6 metabolism with SCE. In adipose, the most activated pathways by SCE were nicotinate and nicotinamide metabolism, long-chain fatty acid transport, oxidative phosphorylation, inflammation, T cell and B cell receptor signaling, and mTOR signaling. Results indicate that SCE in dairy cattle during early lactation induces molecular alterations in liver and adipose tissue indicative of immune activation and cellular stress.

The responsiveness of subclinical endometritis to a nonsteroidal antiinflammatory drug in pasture-grazed dairy cows

The objective of this study was to determine if the inflammation associated with subclinical endometritis (SCE) is a part of the mechanism by which reproductive performance is reduced in cows with this disease. If it is, reducing inflammation associated with SCE with a nonsteroidal antiinflammatory drug (NSAID) should reduce the severity [as measured by average polymorphonuclear cell (PMN) percentage] of uterine pathology and improve reproductive performance. It was also investigated whether the NSAID treatment reduced metabolic indicators of systemic inflammation previously reported to be altered in cows with SCE. Holstein-Friesian and Friesian-Jersey cross dairy cows (n=213) were paired by calving date and d-14 uterine PMN percentage and randomly assigned to 3 injections at intervals of 3 d of an NSAID (1.4 mg of carprofen/kg; n=104) between 21 and 31 d postpartum or left as untreated controls (n=109). Cows with ≥14% PMN (upper quartile of PMN percentage) in the cytological sample collected at d 14 postpartum were defined as having SCE. The average d-14 PMN percentage was low (9.9%) and a high self-cure rate of SCE (>90%) at d 42 was observed. Treatment with an NSAID reduced plasma concentrations of aspartate aminotransferase and increased pregnancy rate in SCE cows. However, no effect of the NSAID treatment was observed on PMN percentage at d 42, postpartum anovulatory interval, or milk production. Compared with cows without SCE, cows with SCE had lower plasma albumin concentration, albumin:globulin ratio, and body condition score, but higher nonesterified fatty acids on the day of calving. These results indicate that cows with SCE are experiencing a physiological dysfunction, including lower body condition, liver dysfunction, and greater metabolic challenge during the periparturient period. Further research is required to determine the effect of NSAID on SCE and to evaluate the influence of timing of drug application on treatment effectiveness.

Acute-phase protein concentration and metabolic status affect the outcome of treatment in cows with clinical and subclinical endometritis

The aim of this study was to investigate the role of acute-phase protein concentration and metabolic status in the establishment and resistance of clinical endometritis (CE) and subclinical endometritis (SE)...