Prevalence, risk factors for and impact of subclinical endometritis in repeat breeder dairy cows

The aim of this study was to determine the prevalence and risk factors for subclinical endometritis (SE) and its effects on fertility in repeat breeder dairy cows. Dairy cows of parity 1 to 5 that were artificially inseminated (AI) 3 or more times (selected cows were artificially inseminated an average of 3.9 times) were examined at 190 ± 40 days in milk, and clinically normal cows (n = 77) were selected based on the absence of abnormal discharges on external inspection and the absence of abnormal findings on transrectal palpation and ultrasonographic examination. Endometrial samples were collected from the uterus by using the lavage technique in the luteal phase of the estrus cycle. Collected samples were centrifuged and a drop of sediment was streaked on to a clean microscopic slide and stained with Giemsa. The percentage of polymorphonuclear cells (neutrophils) was counted for each specimen. The analysed data showed that the average amount of neutrophils was 3.1% (0-9) in the selected cows. Abnormal calving (dystocia, twin births, and abortion), retained placenta, and postpartum uterine infections were associated with an increase in prevalence of SE. Subsequently, SE was significantly associated with a decrease in conception rate in the next AI. Conception rate in the next AI was 5% for cows (n = 38) with SE (≥ 3% neutrophil), and 47% for cows (n = 34) without SE (< 3% neutrophil) (P = 0.001). The prevalence of cytologically diagnosed SE (≥ 3% neutrophil) was 52.7% (n = 38). In conclusion, abnormal calving, retained placenta, and postpartum uterine infections may be associated with an increase in prevalence of SE and subsequently, SE may decrease reproductive performance and increase the incidence of repeat breeder syndrome.     

Potential relationship between normalization of endometrial epidermal growth factor profile and restoration of fertility in repeat breeder cows

The present study characterized alterations of the endometrial epidermal growth factor (EGF) and examined potential relationships between normalization of the EGF and restoration of fertility in repeat breeder cows. In Experiment 1, endometrial tissues were obtained by biopsy on Days 3, 7 and 14 of the estrous cycle from 99 fertile dairy cows and normal ranges of uterine tissue EGF concentrations were determined. Then, eight fertile cows were examined for endometrial EGF concentrations on Days 3, 7 and 14 during the three consecutive estrous cycles. All eight cows had a normal EGF profile (EGF concentrations were within the normal ranges of all 3 days) in all three estrous cycles and endometrial EGF concentrations were similar among the three estrous cycles. In Experiment 2, 61 repeat breeder cows were examined EGF profile and 43 (70.5%) cows had altered EGF profiles compared with control profiles. A typical alteration was characterized by decreased concentrations on Days 3 and 14 and accounted for 86.0% of all alterations. When repeat breeder cows with altered EGF profiles (n=17) were left untreated, abnormality persisted in 14 (82.4%) cows at the second examination and only 3 animals became pregnant within the next two estrous cycles. Among 15 cows showing a typical alteration at the first examination, 11 (73.3%) cows had the same alteration until the third examination performed after two infertile inseminations following the second examination. When repeat breeder cows with altered EGF profiles (n=26) were given one of four therapeutic treatments, cows in which the EGF profile normalized after treatment had a greater pregnancy rate than those with altered profiles (11/14 animals versus 3/12 animals pregnant; P<0.05). In conclusion, suppressed endometrial EGF concentrations on Days 3 and 14 may be common alterations and persist between estrous cycles in repeat breeder cows. Potential relationship between normalization of the endometrial EGF profile and restoration of fertility was observed. Additional study to confirm results of the treatment is, however, necessary because the present study used a limited number of animals. Nevertheless, current data suggest that normalization of the EGF profile is one of the prerequisite factors to restore fertility.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis

The objective of this study was to compare the concentrations of inflammatory cytokines in uterine flush and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus) and subclinical endometritis was diagnosed by evaluation of uterine cytology (neutrophils >18%) at 4 weeks postpartum. Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at 1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations. Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum, concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum.     

Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows

The objectives of the study were to validate the use of endometrial cytology (EC) and ultrasonography (US) to diagnose subclinical endometritis in clinically normal postpartum dairy cows, and to measure the impact of subclinical endometritis on reproductive performance. Holstein cows from two dairy farms were examined at Visit 1 (V1) at 20-33 days in milk (DIM), and clinically normal cows (n = 228), based on the absence of abnormal discharge on external inspection and vaginoscopy, were selected. The reproductive tract of selected cows was evaluated by transrectal palpation, US and EC. All cows in the study were re-examined at Visit 2 (V2) at 34-47 DIM (2 weeks after V1) and were subsequently followed for a minimum of 8 months (until pregnant or culled). Survival analysis was used to derive a case definition of subclinical endometritis, based on factors associated with decreased relative pregnancy rate. Positive EC at V1 (>18% polymorphonuclear leukocytes; PMN) or fluid in uterus at V1 (FIU1) were associated with a significant reduction in the relative pregnancy rate and identified cows with subclinical endometritis. Similarly, a positive EC (>10% PMN) at V2 or fluid in the uterus at V2 (FIU2), identified cows with subclinical endometritis. Cows with subclinical endometritis at V1 and at V2 had a relative pregnancy rate of 41 and 51% (hazard ratio for pregnancy of 0.59 and 0.49), respectively, compared to cows without subclinical endometritis. Given EC or US findings, no diagnostic criteria based on transrectal palpation of the uterus had predictive value for risk of pregnancy. In conclusion, subclinical endometritis, diagnosed by EC or US, was associated with reduced relative pregnancy rate.     

Secretion of prostaglandins and leukotrienes by endometrial cells in cows with subclinical and clinical endometritis

The aims of this study were (1) to measure the secretion of prostaglandin F_2α (PGF_2α), prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), and leukotriene C₄ (LTC₄) by endometrial cells collected by a cytobrush from healthy cows and cows with subclinical and clinical endometritis in the fourth week postpartum, and (2) to evaluate the relationship between the mediators' levels of secretion and the number of polymorphonuclear neutrophils (PMNs) in the uterine smears of cows with subclinical endometritis. The study included cows without any signs of clinical endometritis (n = 63) and cows with clinical endometritis as a positive control (ENDOM, n = 12). Two different threshold ratios (>5% and >18% of PMNs) were used to categorize the cows without clinical signs as with or without cytologic endometritis (CE). Considering the first or second threshold, the animals with CE were included in group CE POS I or CE POS II, whereas the healthy cows were assigned to group CE NEG I or CE NEG II, respectively.     

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1α (IL-1α), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n = 9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n = 8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1α and IL-1-RN mRNA were expressed significantly higher (P < 0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P < 0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P < 0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.     

The effect of a single administration of cephapirin or cloprostenol on the reproductive performance of dairy cows with subclinical endometritis

This study examined the effect of a single administration of cephapirin iu or cloprostenol im on the reproductive performance of dairy cows with subclinical endometritis. Cows (n = 228) at 20-33 days in milk (DIM) from two commercial dairy farms, determined to be normal for clinical endometritis (based on absence of abnormal uterine discharge on vaginoscopic examination) were enrolled. At enrollment, a thorough reproductive examination was performed, including rectal palpation, ultrasonography (US) and endometrial cytology (EC). The case definition for subclinical endometritis was the presence of >18% neutrophils on EC examination or fluid in uterus (FIU) on US examination. All cows were randomly assigned to receive one of three treatments: 500 mg benzathine cephapirin iu, 500 microg cloprostenol im, or control (no treatment). Reproductive performance was monitored for a minimum of 8 months after treatment. Cows with subclinical endometritis treated with cephapirin or cloprostenol had a significantly increased relative pregnancy rate compared to control [hazard ratios 1.89 (P = 0.01) and 1.70 (P = 0.05), respectively]. In conclusion, a single treatment with cephapirin or cloprostenol at 20-33 DIM significantly improved the reproductive performance of cows with subclinical endometritis.     

Effect of source of supplemental selenium on uterine health and embryo quality in high-producing dairy cows

Lactating Holstein cows (n = 135) were randomly assigned to one of the two sources of supplemental selenium (Se), sodium selenite (SS) or Se yeast (SY), fed at 0.3 mg/kg diet dry matter from 25 d before calving to 70 d in milk (DIM), in diets not suboptimal in basal Se concentrations. Cows were evaluated for health daily in the first 10 DIM, and uterine cytology of the previously gravid uterine horn was assessed at 30 DIM. The Ovsynch protocol was initiated at 42 DIM; ovarian responses to hormonal treatments were evaluated by ultrasonography. The uteri of cows were flushed 6 d after timed AI for collection of embryos and oocytes. Plasma concentrations of Se and progesterone were measured throughout the postpartum period and during the reproductive protocol, respectively, and plasma glutathione peroxidase activity was determined 6 d after AI. Concentrations of Se in pre- and postpartum diets ranged from 0.43 to 0.56 mg/kg of dry matter. Incidence of retained placenta, fever, ketosis, mastitis, acute puerperal metritis, clinical endometritis, and subclinical endometritis were not significantly different between treatments. There were no differences between groups in concentrations of Se and progesterone or glutathione peroxidase activity in plasma. Treatment did not influence ovarian responses to the synchronization protocol, fertilization rate, number of blastomeres and live blastomeres, or proportions of grades 1 and 2, degenerated, and degenerated-unfertilized embryos/oocytes. Odds of subclinical endometritis on Day 30 postpartum more than doubled in cows with fever of unknown origin or acute puerperal metritis in the first 10 DIM. Fertilization rate tended to be reduced in cows with subclinical endometritis. In summary, replacing SS with an organic source of Se in diets not suboptimal in basal Se concentrations did not improve Se status, uterine health, fertilization, or embryo quality in early lactation dairy cows.     

A comparison of diagnostic techniques for postpartum endometritis in dairy cattle

Holstein cows (n=221) from eight commercial dairy herds were examined for endometritis between 28 and 41 days postpartum using 5 diagnostic techniques: (1) vaginoscopy; (2) ultrasonographic assessment of uterine fluid volume; (3) ultrasonographic assessment of endometrial thickness; (4) endometrial cytology collected by cytobrush; and (5) endometrial cytology collected by uterine lavage. Concordance correlation was used to evaluate the reliability of cytobrush and lavage cytology. Cytobrush cytology was found to have the greatest intraobserver repeatability (cytobrush, rho(c)=0.85 versus lavage, rho(c)=0.76) and was chosen as the reference diagnostic test. Pregnancy data at 150 days postpartum was available for 189 cows. Survival analysis was used to determine the lowest percentage of polymorphonuclear cells associated with time to pregnancy. The sensitivity and specificity of the diagnostic techniques was determined using pregnancy status at 150 days and cytobrush cytology as the diagnostic standards. The risk of non-pregnancy at 150 days was 1.9 times higher in cows with more than 8% PMNs identified using cytobrush cytology than in cows with less than 8% PMNs (P=0.04). Twenty-one cows of 189 cows (11.1%) had >8% PMNs and were considered to be positive for endometritis. Cows with endometritis had a 17.9% lower first service conception rate (P=0.03) and a 24-day increase in median days open (P=0.04). The sensitivities of all five diagnostic tests relative to 150-day pregnancy status ranged from 7.1 to 14.3% and the specificities from 84.0 to 93.3%. Relative to cytobrush cytology, the respective sensitivity and specificity values are as follows: vaginoscopy (53.9%, 95.4%); lavage cytology (92.3%, 93.9%); ultrasonographic assessment of uterine fluid (30.8%, 92.8%); and ultrasonographic assessment of endometrial thickness (3.9%, 89.2%). Endometritis impaired reproductive performance. Cytobrush cytology was the most reliable method of diagnosing endometritis in cattle.     

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum

Background  

Inflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1β (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period.     

Evaluation of cytokine expression by blood monocytes of lactating Holstein cows with or without postpartum uterine disease

Whereas neutrophils are the main phagocytic leukocytes, monocytes and macrophages are actively involved in immunomodulation after infection. Recent studies have demonstrated that neutrophil function is impaired by the state of negative energy balance around parturition, and that cows that develop uterine disease have a greater degree of negative energy balance than healthy cows. The objectives of this study were to compare monocyte gene expression and protein secretion of selected cytokines from calving to 42 d after calving in Holstein cows that did or did not develop uterine disease. Real time quantitative RT-PCR (Tumor necrosis factor-α (TNFα), Interleukin (IL)-1β, IL-6, IL-8 and IL-10) and ELISA (TNFα, IL-1β and IL-8) were used to evaluate cytokine response following in vitro stimulation of blood-derived monocytes with irradiated E. coli. Relative to unstimulated cells, E. coli-stimulated monocytes from cows with metritis had lower gene expression of key pro-inflammatory cytokines than healthy cows from calving to 14 d after calving (TNFα at 0, 7, and 14 d after calving, IL-1β and IL-6 at 7 and 14 d after calving; P < 0.05). There were no significant differences between groups for expression of IL-8 or the anti-inflammatory cytokine IL-10. This was due, in part, to higher gene expression in unstimulated monocytes (TNFα, IL-1β, IL-6 and IL-10) in early lactation from cows with metritis. Expression of mRNA in stimulated cells (relative to housekeeping genes) was lower for TNFα (7 and 14 d postpartum) and for IL-10 (7 and 14 d postpartum) in cows with metritis. Concentration of TNFα was lower in the culture medium of E. coli-stimulated monocytes from cows with metritis than healthy cows at calving and 7 and 21 d after calving (P < 0.05). Circulating cytokine concentrations were not different between groups for IL-8 and were below the limits of detection for TNFα and IL-1β. Cytokine gene expression and production were similar between healthy cows and cows that developed endometritis, diagnosed cytologically at 42 d after calving. We concluded that altered levels of expression and production of pro-inflammatory cytokines postpartum could contribute to impaired inflammatory response and predispose cows to development of metritis.     

The diagnosis and prevalence of subclinical endometritis in cows evaluated by different cytologic thresholds

The aims of our study were to determine (1) how the prevalence of cytologically determined subclinical endometritis varies when using three different cytological threshold ratios to categorize cows as either with or without endometritis, (2) how the number of animals categorized as having endometritis changes from the fourth to the sixth wk postpartum when using each threshold, (3) how subclinical endometritis influences the number of days open, and (4) how the results of cytological and bacterial examinations correlate. To answer these questions, 222 clinically healthy cows in two herds were examined in the fourth (Exam 1) and the sixth wk (Exam 2) postpartum, when endometrial surface scrapings for bacteriologic and cytologic examination were collected by cytobrush from their uterine horns. After each examination, all cows were categorized using three different thresholds: (1) > 18% polymorphonuclear leucocytes in Exam 1 and > 10% in Exam 2, (2) > 8% in both exams, and (3) > 5% in both exams. It was found that: (1) The number of cows categorized as having endometritis increased as the threshold was lowered, and ranged from 18.9% to 75.4% according to herd, time of examination, and the threshold used; (2) with all three thresholds and in both herds, the number of cows categorized as having endometritis in Exam 1 was approximately double that in Exam 2; whereas depending on the herd and the threshold used, 6.1% to 17.0% of the cows that were negative in the first exam were positive in the second, and 7.4% to 33.3% were positive in both exams; (3) cows were open for a significantly greater number of days if categorized as having endometritis with the first threshold in Exam 1 (mean ± SEM 151.5 ± 9.5 vs. 115.9 ± 7.8; P < 0.01), or with either the first or the second threshold in Exam 2 (mean ± SEM 155.0 ± 15.0 vs. 125.1 ± 6.6; P < 0.05); and (4) the most common bacteria were Streptococcus acidominimus and Escherichia coli, and the correlation between cytologic and bacteriologic findings was low (Φ = 0.08 to 0.17 for different tested thresholds). Subclinical endometritis seems to be associated more with the postpartum recovery of the endometrium than with bacterial infection.     

Comparison of ceftiofur hydrochloride and estradiol cypionate for metritis prevention and reproductive performance in dairy cows affected with retained fetal membranes

The objective of this study was to compare the effect of ceftiofur hydrochloride and estradiol cypionate (ECP) administration for metritis prevention and reproductive performance in dairy cows affected with retained fetal membranes (RFMs). After parturition, 97 dairy cows affected with RFM from a single dairy herd were randomly allocated to 1 of 3 treatment groups. Cows in-group 1 (n=31) were treated daily for 5 days with ceftiofur hydrochloride (2.2mg/kg, i.m.); cows in group 2 (n=33) were treated once with ECP (4 mg, i.m.); and cows in group 3 (n=33) were not treated. The proportion of cows with metritis, uterine involution patterns and the calving-to-conception interval were compared between groups. The proportion of cows that developed metritis was significantly different (P<0.05) in cows treated with ceftiofur hydrochloride (13%), compared with cows treated with ECP (42%) or cows that received no treatment (42%). Uterine involution patterns (i.e. median time to complete retraction of the uterus and mean diameter measure of cervix and uterine horns) were not significantly different between groups. Cows treated with ECP were 0.40 times as likely to conceive as control cows (P=0.05); median time to conception in cows treated with ECP (192 days) was longer, compared to control cows (124 days). We conclude that systemic administration of ceftioufur hydrochloride is beneficial for prevention of metritis, but its effect on reproductive performance was not significantly different to that of ECP or no treatment. In addition, administration of ECP did not have beneficial effects on metritis prevention and reproductive performance.     

Use of plasma concentrations of 13,14-dihydro,15-keto-PGF2 alpha (PGFM) in the diagnosis of sub-clinical endometritis and its relationship to fertility in the postpartum dairy cow

The objective of this study was to determine the value of using plasma concentrations of PGFM to diagnose subclinical endometritis in the dairy cow, and its relationship to subsequent fertility. A total of 274 cows between 24 to 29 d post partum was divided into 4 groups on the basis of clinical features of the uterus and ovary. Cows in Group 1 (n = 74) had a normal, involuting uterus and a CL on the ovary; cows in Group 2 (n = 51) had a normal, involuting uterus but no CL on the ovary; cows in Group 3 (n = 83) did not have a normal, involuting uterus but had a CL on the ovary; and cows in Group 4 (n = 66) did not have a normal, involuting uterus or a CL on the ovary. A blood sample was obtained from each cow on the day they were placed on the study, and plasma concentrations of PGFM and P4 were determined using RIA. Cows were artificially inseminated (AI) at the first observed estrus after Day 60 post partum, and pregnancy was determined by palpation of the uterus per rectum between 45 and 50 d postAI. Reproductive responses evaluated were conception rate to first service, days open, and percentage of cows pregnant by 90, 120, 150 and 180 d post partum. Data were analyzed using GLM procedures of SAS and a 2 x 2 factorial with contrast procedures. Polynomial regression analysis was used to determine the shape of the PGFM, P4 and fertility curves. There was no difference among mean PGFM concentrations of cows in each group. The rate of decline of plasma PGFM concentrations was lower in cows with an abnormal uterus and a CL on the ovary compared with those without a CL. A lower percentage of cows with abnormal uteri was pregnant by 90 d post partum compared with cows with normal uteri. From the results of this study, it was concluded that plasma PGFM concentrations between Days 24 to 29 post partum were not effective in identifying cows with subclinical endometritis.     

Changes in EGF concentrations during estrous cycle in bovine endometrium and their alterations in repeat breeder cows

The objective of the present study was to determine if abnormalities in the cyclic changes of endometrial EGF concentrations can be a diagnostic tool for repeat breeder cows. First, the profile of EGF concentrations during the estrous cycle was determined using endometrial tissues obtained from 31 Holstein cows after slaughter. Cyclic cows had two peaks of EGF concentrations. Then, endometrial tissues were obtained from 12 control and 20 repeat breeder cows by biopsy on Days 3, 7, and 14 of the same estrous cycle. Endometrial EGF concentrations in biopsied samples of the controls were similar to those found in slaughterhouse materials; they were high on Days 3 and 14 (9.2 and 10.4 ng/g tissue, respectively) and low on Day 7 (3.8 ng/g tissue). Concentrations of EGF in repeat breeder cows had a different profile; they were similar on Days 3, 7, and 14 (4.4, 3.4, and 4.0 ng/g tissue, respectively). In conclusion, changes in endometrial EGF concentrations were altered in repeat breeders; these alterations may be a potential diagnostic marker for repeat breeder cows.     

Effect of presence of clinical and subclinical endometritis at the initiation of Presynch-Ovsynch program on the first service pregnancy in dairy cows

The present study examined the effect of presence of clinical or subclinical endometritis at the initiation of Presynch-Ovsynch estrous synchronization program on the first service pregnancy rate in dairy cows. Lactating Holstein cows (N=275) were given a thorough reproductive examination at 32-38 days in milk, 3 days prior to the scheduled start of Presynch-Ovsynch program. Based on the reproductive exam findings the cows were diagnosed and classified into three groups as clinical endometritis, subclinical endometritis and normal. All cows received two set-up injections of 25mg PGF(2alpha) (Lutalyse((R)), Pfizer Animal Health, New York, NY, USA) i.m., 14 days apart starting at 35-42 days in milk (DIM). All cows received 75microg of GnRH (Cystorelin, Merial, Iselin, NJ, USA) i.m. 14 days after the second pre-synchronization injection of PGF(2alpha), followed by a third injection of 25mg PGF(2alpha) i.m. 7 days later. Cows received a second injection of 75microg of GnRH i.m. 54h after the third PGF(2alpha), and received timed artificial insemination at the time of the second GnRH injection or 24h later. Multivariate logistic regression was used to analyze the odds of pregnancy at the first service. Variables included in the model were endometritis status (clinical endometritis, subclinical endometritis and normal), farm (two), presence of corpus luteum (CL, yes or no), timing of second GnRH in relation to AI (0 or 24h), sire fertility (bulls with greater compared with lesser estimated relative conception rates), parity (primiparous and multiparous) and their interactions. Of all variable included in the model, cows with corpus letuem (OR=1.83 versus OR=1.00; P=0.05) 3 days prior to the scheduled start of Presynch-Ovsynch program and primiparous cows (OR=1.00 versus OR=0.55; P=0.04) had increased odds of becoming pregnant at the first service. No differences were found in the odds of first service pregnancy among clinical, subclinical endometritis and normal cows (P>0.1). In summary, presence of clinical or subclinical endometritis at the initiation of Presynch-Ovsynch estrous synchronization program does not harm the first service pregnancy rate in dairy cows.     

Prevalence of endometritis and its effects on reproductive performance of dairy cows

Hostein cows (n=141) in five commercial dairy herds in central New York were examined for endometritis by examination of endometrial aspirates for presence of inflammatory cells, principally neutrophils, by endometrial cytology at 40-60 days postpartum. The prevalence of cytologically-diagnosed endometritis was 53%; within herds the prevalence varied from 37 to 74% (P=0.02). There was excellent agreement between two examiners (Kappa=0.864; P<0.0001). Parity did not influence prevalence of endometritis (P=0.53). Cytologically diagnosed endometritis was associated with profoundly impaired reproductive performance; Kaplan-Meier survival analysis revealed lower overall pregnancy rate (P<0.0001). Median days open was 206 for cows with endometritis and 118 for cows free of the condition. Overall, 76% of cows in this study became pregnant by 300 days postpartum; 63% of cows with endometritis and 89% of cows without endometritis were confirmed pregnant by 300 days postpartum (P<0.003). (For these two groups, 69, and 90% respectively, became pregnant during the duration of the study). Pregnancy to first service percentage was lower (11 versus 36%; P=0.001) for cows with than without endometritis, and these cows required more services before 50% became pregnant (3 versus 2; P=0.006). In a second study using 22 cows in a university-owned herd, the prevalence of cytological evidence of inflammation was 100% at 2 weeks postpartum, and dropped to 89, 58, and 41% at 4, 6, and 8 weeks, respectively. Endometritis diagnosed by endometrial cytology late in the voluntary waiting period was highly prevalent and exerted a profoundly detrimental effect on subsequent reproductive performance, making this condition potentially extremely costly to the North American dairy industry.     

Lipopolysaccharide (LPS) suppresses follicle development marker expression and enhances cytokine expressions,which results in fail to granulosa cell proliferation in developing follicle in cows

Postpartum endometritis is known to be associated with ovarian dysfunction in cows. Lipopolysaccharide (LPS) generated by Gram-negative bacteria is recognized by toll-like receptor 4 (TLR4), which leads to an inflammatory response by the generation of cytokines such as tumor necrosis factor-α (TNF-α) and interleukins. In this study, we investigated the effect of endometrial LPS on granulosa cell functions during early follicular development in cows. Uteri and follicles were obtained from a slaughterhouse and classified into either clinical endometritis (CE) or normal groups by vaginal mucus test. TLR4 mRNA and protein in normal cows were expressed in granulosa cells collected from follicles measuring 1–3 and 4–7 mm in a diameter, respectively. LPS content in endometrium and follicular fluid of CE cows was significantly higher than that in normal cows. Compared to normal cows, CE cows showed lower expression of follicular development markers (FSHR, CYP19A1, CCND2, and LHCGR) in granulosa cells, lower estradiol-17β concentrations in follicular fluid, and lower granulosa cell proliferation. CE contraction significantly increased cytokine expressions (TNF, IL-1A, and IL-1B) in granulosa cells and suppressed apoptosis of granulosa cells compared to normal cows. LPS significantly suppressed the expression of follicular development markers and the production of estradiol-17β in granulosa cells and reduced granulosa cells proliferation compared to cells cultured without LPS. LPS significantly increased cytokine expressions and suppressed granulosa cell apoptosis. Thus, the present results suggest that the existence of LPS in developing follicles is one of the causes of ovarian quiescence in cows.