Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares

Low-volume uterine flush (n=401) was performed in 308 infertile mares to diagnose endometritis. Mares evaluated were either barren after three or more breedings or had two or more unsuccessful embryo recovery attempts during consecutive cycles. Culture results were compared with cytological and histological findings, efflux clarity and pH to substantiate that the micro-organisms recovered were truly pathogens. Cytological specimens were evaluated for presence of epithelial and inflammatory cells, bacteria, yeast and debris. Endometrial biopsies (n=110) were examined for the presence of neutrophils in the stratum compactum. Micro-organisms were recovered in 282/401 (70%) of low-volume flushes; E. coli was most frequently isolated (42.2%), followed by beta hemolytic Streptococcus (37.6%). Efflux clarity of 318 flushes was clear (n=109), cloudy (n=149), or mucoid (n=60). Isolation of micro-organisms was highly associated with cloudy and mucoid effluxes (P<0.001), debris on cytological specimens (P<0.001), increased efflux pH (P<0.003), and neutrophils on endometrial biopsy (P<0.01). E. coli was associated with debris on cytological smear (P<0.002), whereas beta hemolytic Streptococcus was associated with increased efflux pH (P<0.002). Using the presence of neutrophils in a tissue specimen as the "best standard" for diagnosing endometritis, the sensitivity of flush culture was 0.71 and for flush cytology was 0.8, whereas the specificity was 0.86 and 0.67, respectively. Neutrophils in uterine flushes under-reported inflammation; only 86/282 positive cultures were positive on cytology. The clinical estimate of a contaminated (false positive) flush culture was 11%, if a false positive was defined as positive culture with clear efflux and no debris or neutrophils on cytology (26/228). In conclusion, a low-volume uterine flush was a rapid, accurate method for identifying mares with chronic endometritis. When micro-organisms were recovered, endometritis was confirmed by efflux clarity, pH and cytological findings of debris, bacteria, or neutrophils. E. coli was most commonly isolated and it appeared to differ in pathogenicity from beta hemolytic Streptococcus.     

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P < 0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P < 0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P < 0.0001). The DGS produced significantly more (P = 0.003) intact cells than CB and LVF. Distorted cells were significantly (P = 0.001) more frequent in smears by LVF. The CB harvested significantly (P = 0.009) more fragmented cells. CB and LVF produced significantly (P < 0.0001; P = 0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P = 0.0062; P = 0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P = 0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k ≤ 0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.     

Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy

The objective of this study was to compare results from endometrial culture swabs with results from culturing of endometrial biopsies. The culture results were related to cytological findings (polymorphonuclear; PMN-cells) and histological observations (PMN-cells). Biopsy and swab samples were smeared on the surface of a blood agar petri dish, and examined for growth of bacteria. Cytology samples were obtained from endometrial biopsies, stained and examined under microscopy for the presence of PMN-cells. Endometrial biopsies were examined for the presence of PMN-infiltration of the endometrial luminal epithelium and the stratum compactum. Using the presence of PMNs in a tissue specimen as the "best standard" for diagnosing endometritis, the sensitivity of bacterial growth from an endometrial biopsy was 0.82. The sensitivity for cytology was 0.77, and the sensitivity of bacterial growth from an endometrial surface swab was 0.34. The specificity for biopsy cultures, swab cultures, and cytology to diagnose endometritis were 0.92, 1.0, and 1.0 respectively. The positive predictive value for biopsy cultures, swab cultures, and cytology were 0.97, 1.0, and 1.0 respectively. The negative predictive value for biopsy cultures, swab cultures, and cytology were 0.67, 0.44, and 0.62 respectively. In conclusion, bacteriological culture and cytology from an endometrial biopsy provide the practitioner with the most accurate results regarding both sensitivity and positive predictive value.     

Susceptibility to persistent breeding-induced endometritis in the mare: relationship to endometrial biopsy score and age, and variations between seasons

The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10⁹ freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention and biopsy score or age. In Experiment 2, a subset of 14 mares was classified for susceptibility to persistent breeding-induced endometritis in two subsequent breeding seasons. Biopsy score and age were associated with fluid retention (P < 0.001). In addition, age was related to biopsy score (P < 0.001). Of the mares examined for susceptibility, 36% (5 of 14) changed status during subsequent seasons. Three mares changed to a more severe classification (intermediate to susceptible, or resistant to intermediate), whereas two mares changed to a less severe classification (susceptible to intermediate).     

The functional competence of uterine-derived polymorphonuclear neutrophils (PMN) from mares resistant and susceptible to chronic uterine infection: a sequential migration analysis

The functional competence of uterine-derived polymorphonuclear neutrophils (PMNs) from 28 mares was measured for migration responsiveness by use of a chamber (filter) assay. Uterine infection was induced with Streptococcus zooepidemicus in mares considered resistant to chronic uterine infection (Grade I). In sequential analysis of uterine flushings obtained from these mares 5, 12, 15, 20, and 25 h after infection was induced, PMNs showed an initial rise at 12 h (from 5), then a general decline in migration response and in concentration of cells per ml from 12 through 25 h post-inoculation. In contrast, PMNs obtained from the uterine flushings from mares considered susceptible to chronic uterine infection (Grade III) demonstrated premature migration dysfunction 12 h after infection. Subsequent increases in functional competence of the PMNs were demonstrated at 15 and again at 25 h after induced infection. The concentration of uterine PMNs per ml from mares considered susceptible to chronic endometritis remained elevated from 12 through 25 h after inoculation, which suggests a possible continued recruitment of new PMNs from the peripheral circulation. The results of this study suggest that uterine-derived PMNs obtained from mares susceptible to chronic uterine infection have a compromised ability to migrate. This dysfunction may play an important role in rendering the endometrium (uterus) susceptible to chronic endometritis.     

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.     

Interaction between equine semen and the endometrium: the inflammatory response to semen.

Insemination of mares with bacteria-free equine spermatozoa results in an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen. In vitro studies have demonstrated that equine spermatozoa activate complement, resulting in cleavage of factors C5a and C3b. Since uterine secretion is rich in complement, it is likely that an interaction between spermatozoa and uterine secretion results in C5a-mediated chemotaxis and migration of PMNs into the uterine lumen. Once in the uterine lumen, the PMNs phagocytize bacteria and spermatozoa, which is an important part of sperm elimination from the reproductive tract. It is not clear how the spermatozoa are opsonized, or if phagocytosis of equine spermatozoa is a selective or non-selective process. Breeding-induced endometritis appears to be both up and down regulated by seminal components. A modulatory role on the inflammation has been suggested for equine seminal plasma. Seminal plasma suppressed complement activation, PMN-chemotaxis and phagocytosis in vitro. Preliminary in vivo experiments also support a suppressive role of seminal plasma in breeding-induced endometritis. The duration but not the magnitude of the PMN-influx into the uterine lumen was shortened when seminal plasma was included in an insemination dose. The presence of PMNs in the uterus affects the motion characteristics of spermatozoa in vitro. Both progressive motility and mean path velocity were impaired when spermatozoa were incubated in uterine secretion from mares with ongoing breeding-induced endometritis. The binding of spermatozoa to PMNs was prominent in all samples collected from mares with an ongoing endometritis. The motility remained impaired, but the binding of the spermatozoa to PMNs was reduced when the spermatozoa were incubated in uterine secretion in the presence of seminal plasma. Preliminary characterization of the immune-suppressive component in seminal plasma suggests that it is one or more molecule(s) with a molecular weight between 50 and 100 kDa, partially inactivated by charcoal stripping and partially heat-inactivated at 95 degrees C for 45 min.     

Use of leukocytes as treatment for endometritis in mares experimentally infected with Streptococcus equi subsp. zooepidemicus

This study compared four treatments for bacterial endometritis in mares experimentally infected with Streptococcus zooepidemicus. Twenty-five mares were used, 20 resistant and five susceptible to endometritis. Mares would be in estrus when infected. Twenty-four hours after inoculation, clinical, bacteriological and cytological examinations were performed and repeated until the first occurrence: negative cytology and no Streptococcus growth or the seventh day post-infection. All mares showed clinical signs of endometritis and were assigned to one of the following treatments: (1) intrauterine infusion of fresh leukocytes; (2) intrauterine infusion of frozen-thawed leukocytes; (3) intrauterine infusion of lysed leukocytes; (4) intrauterine infusion of recombinant human interleukin-8 (rhIL-8); (5) control. Mares were submitted to all treatments, with at least a 14-day interval between treatments in a Latin square design. Treatment did not affect (P=0.121) time needed for resistant mares to eliminate bacteria. Time needed for elimination of bacteria was similar in susceptible mares treated with fresh and frozen leukocytes (P=0.333). Susceptible mares treated with frozen leukocytes also did not differ from those treated with lysed leukocytes (P=0.227) for time to eliminate bacteria, but were significantly different (P>0.02) from those treated with rhIL-8 and control. In resistant mares, physical clearance ability was probably the responsible for bacterial elimination. Intrauterine infusions in susceptible mares with viable or lysed leukocytes associated or not to opsonizing factors, reduced the time to elimination of bacteria. Infusions with bactericidal effect (functional neutrophils and granules) was likely effective and responsible for the more rapid elimination of bacteria in susceptible mares.     

Cytological diagnosis of endometritis in the mare: investigations of sampling techniques and relation to bacteriological results

Aim of this study was to compare uterine smears made using the Knudsen catheter, the cytology brush and a uterine culture swab with regard to diagnostic usefulness and the occurrence of neutrophils. Additionally correlation between culture results and the occurrence of neutrophils in uterine smears was investigated. Samples were collected from 340 mares, 81.5% of which were in estrus. Smears made using the cytology brush yielded more endometrial cells per high-power field than those made using the other two instruments (p<0.0001), and a larger proportion had PMNs compared with smears made using the uterine swab (p<0.0001). For smears made with the cytology brush, cultures of β-hemolytic streptococci were more often (p=0.002) accompanied by PMNs than cultures of bacteria other than β-hemolytic streptococci, and there was a positive correlation (r(s)=0.2 p=0.01) between the number of PMNs in smears and the number of colonies of β-hemolytic streptococci. The cytology brush was superior to the other methods because it generated a larger proportion of diagnostic useful smears and the occurrence of PMNs in smears was significantly correlated with the occurrence of cultures of β-hemolytic streptococci.     

The effects of EDTA-Tris infusion on the equine endometrium

Four groups of five pony mares each were used to determine if the intrauterine infusion of EDTA-Tris solution caused adverse effects on the endometrium. The uteri of mares were infused with either saline or EDTA-Tris solution or biopsied or sham-biopsied without infusion. Acute endometritis developed in one (20%) to three (60%) mares in each group during the seven days following treatment, but there were no differences (P > 0.05) in the incidence of endometritis among the groups. Endometrial fibrosis was not evident in biopsies taken on days 14, 30 and 60 following infusion of saline or EDTA-Tris. It was concluded that the endometrial response to saline and EDTA-Tris was not different and that EDTA-Tris may be a useful adjunct to treatment of uterine infections in the mare.     

Effect of oxytocin and flunixin meglumine on uterine response to insemination in mares

The most probable reason for persistent postbreeding endometritis in mares is weak myometrial contractility. The influence of oxytocin (OT; an ecbolic agent) and flunixin meglumine (FLU; a prostaglandin inhibitor serving as a model for mares with decreased uterine contractility) on uterine response to artificial insemination (AI) was studied in mares with no history of reproductive failure. The mares were treated intravenously with 10 mL saline (Group C, n = 10) or 0.01 IU/kg OT (Group OT, n = 10) 2, 4, 8, and 25 h after AI. Group FLU (n = 11) was treated with 1.1 mg/kg FLU 2 h after AI and with saline thereafter. The mares received the same treatments in the first and third cycles but were sampled either at 8 or 25 h. The amount of intrauterine fluid (IUF) and edema and the number of uterine contractions were recorded before AI and 10 min after the treatments using transrectal ultrasonography. At 8 h after AI, the mares were treated with human chorionic gonadotropin, and, after 8-h or 25-h scans, a 500-mL uterine lavage and a biopsy were performed. Ovulation was confirmed at 48 h and pregnancy 14 to 17 d after AI. No manipulations were done during the second estrus. At 8 h after AI, Group FLU had more polymorphonuclear leukocytes (PMNs) in the uterine lavage fluid than did Group OT (P < 0.05), but uterine contractions did not differ significantly. At 25 h, the PMN concentrations were low in all groups. Group OT rarely showed IUF. The uterine biopsy specimens of Group FLU showed less inflammation of the stroma but more PMNs in the uterine lumen 8 h after AI than that of the control group (P < 0.05). The pregnancy rates did not differ between the groups (63% C, 53% OT, and 50% FLU). Oxytocin rapidly and effectively removed IUF and PMNs after AI and thereby shortened the duration of postbreeding inflammation.     

The contribution of complement to opsonic activity in the uterine secretions of mares free of endometritis

The aim of the present study was to investigate if complement contributes to opsonic activity in the uterine secretions of mares with normal reproductive functions. Five mares with a mean age of 9 years were used in the study. The mares were considered to be free of endometritis based upon clinical history, palpation per rectum and ultrasonogaraphy of the genital tract, videoendoscopic inspection of the uterus, electronmicroscopy of endometrial biopsies, and bacteriological and cytological examination of swabs from the endometrium. The hormonal status of the mares was also determined. Uterine secretions were collected by a tampon inserted into the uterus during estrus. Secretion and serum samples were divided in half, one half of which was subjected to heat treatment (56 degrees C in 30 minutes) in order to inactivate the complement. Opsonic activity in the samples was then determined in a chemiluminescence assay using zymosan as the target particle. Opsonic activity was expressed as peak chemiluminescence and the time to peak chemiluminescence. Heat treatment of the secretions reduced peak luminescence significantly (P<0.01), and it prolonged the time to peak luminescence significantly (P<0.05). These data show that complement contributes a considerably to opsonic activity in uterine secretions prior to an inflammatory response in the equine uterus.     

Effect on fertility of human chorionic gonadotrophin and uterine lavage with oxytocin performed after mating in Arabian barren mares

The objective of the present study was to evaluate the beneficial effect of hCG injected immediately after mating in Arabian barren mares treated with uterine lavage and oxytocin. Arabian barren mares (n = 36) with PMIE were subjected to detailed clinical examinations including palpation per rectum, vaginoscopy, and cytological examination. After mating the 36 mares were randomly divided into four groups. The mares in group 1 (n = 10) were immediately after breeding injected with hCG 3000 IU IM. Uterine lavage with 1 L of N-saline containing 4 million IU of crystalline penicillin and 4 g of streptomycin sulphate was performed 4 h after breeding. Then mares received two injections of oxytocin 40 IU IM 2 h apart after 6 h of mating. Mares in group 2 (n = 10) treated with uterine lavage and oxytocin as group 1. While mares in group 3 (n = 10) received uterine lavage only. A control group (n = 6) as group 4 did not received any treatment. The results of clinical examination indicated that 69.4% of PMIE mares were harboring severe endometritis and 30.6% with a moderate form of endometritis. Significant (P < 0.01) increase in lymphocytes were founded in barren mares included in this study. Higher pregnancy rate (P < 0.01) was founded in Arabian barren mares 80% injected with hCG immediately after breeding and uterine lavage and oxytocin. No significant difference was found in mares received uterine lavage and oxytocin and uterine lavage only. In a conclusion, administration of hCG immediately after mating and intrauterine lavage containing antibiotics performed 4 h and two injections of oxytocin 40 IU IM 2 h apart after 6 h of mating had improved fertility of Arabian barren mares.     

Biodegradable estradiol microspheres do not affect uterine involution or characteristics of postpartum estrus in mares

Quarterhorse mares were used to investigate effects of estradiol-17beta on uterine involution, duration of estrus, interval to ovulation, and fertility achieved by breeding on the first postpartum estrus. On the day of foaling, mares were injected with biodegradable poly (DL-lactide) microspheres containing either 100 mg estradiol-17beta (25 mares) or no drug (27 mares). The treatment period was considered to last for 12 to 15 d. Estrus was determined by teasing mares (n=16) with a stallion. Ovulation was detected by transrectal ultrasonographic examination of ovaries (n=48). On Days 6, 11 and 16 post partum, transrectal ultrasonography was used to measure cross-sectional diameters of the uterine body, uterine horns, and fluid within the uterine lumen (n=28). Uteri were swabbed for bacteriologic culture, and uterine biopsies were obtained from the previously gravid uterine horn on Days 11 and 16 post partum, for assessment of endometritis and morphometric analysis of endometrial histioarchitecture (n=19). Twenty-two mares were bred on foal-heat, and pregnancy was determined by transrectal ultrasonography on 14 to 16 and 30 to 35 d after breeding. With only one exception (diameter of previously gravid uterine horn on Day 11), mean values for all measures of uterine involution did not differ between treatment groups (P > 0.05). No differences were detected between treatment group means for length of estrus or interval to ovulation (P > 0.05). No differences were detected between treatment group liklihoods for recovery of potential bacterial pathogens, presence of endometritis, or presence of intrauterine fluid at 11 or 16 d post partum (P > 0.05). Pregnancy rate of mares treated with estradiol (5 11 ; 45%) was not different from that of control mares (9 11 ; 82%; P > 0.05). Estradiol treatment did not hasten uterine involution, increase duration of estrus, delay ovulation, or increase fertility in these postpartum mares.     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

Does artificial insemination with chilled, extended semen reduce the antigenic challenge to the mare's uterus compared with natural service?

Uterine response in infection-resistant mares (n = 5) at 48 h after AI was compared with that following natural service in these same mares, and after AI in infection-susceptible mares (n = 6). In the resistant mares, small amounts of uterine fluid were detected and bacteria were isolated infrequently at 48 h after breeding, but cytological examination of uterine flushes revealed that a significant degree of endometritis was present. There was no difference in the degree of inflammatory response by 48 h after AI or natural mating. In 4 of the 6 susceptible mares moderate to large accumulations of intrauterine fluid were detected at 48 h after AI, and massive uterine neutrophilia was present in all 6 mares. It was concluded that there was no evidence that using conventional AI techniques limited the inflammatory response of the mare's uterus.     

High concentrations of myeloperoxidase in the equine uterus as an indicator of endometritis

Intraluminal fluid and excessive abnormal hyperedema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears. Studies show a relation between neutrophils and myeloperoxidase (MPO), an enzyme contained in and released by neutrophils during degranulation or after cell lysis. This enzyme has been found in many fluids and tissues, and associated with different inflammatory pathologies in the horse. The aims of this study were to assess the presence and concentration of MPO in the equine uterus, and to investigate its relation with neutrophils, and other clinical signs of endometritis. Mares (n = 51) were evaluated for the presence of intraluminal fluid and excessive endometrial edema before breeding, and a small volume lavage and cytology samples were obtained. From 69 cycles, supernatant of the uterine flushes was analyzed with a specific equine MPO ELISA assay to measure MPO concentration. Cytology samples were used for the diagnosis of endometritis. Myeloperoxidase was present in the uterus of all estrus mares in highly variable concentrations. Myeloperoxidase concentrations were significantly (P < 0.05) higher in samples with positive cytologies and in the presence of intraluminal fluid. Occasionally, some samples with negative cytologies showed high MPO concentration, but the opposite was never observed. Cycles presenting hyperedema weren't associated with high concentration of MPO, intraluminal fluid, or positive cytology, making it a poor diagnostic tool of endometritis.     

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows

Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes.Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.     

Postpartum endometrial cytology in beef cows

The objectives were to characterize postpartum endometrial cytology and to determine the prevalence of subclinical endometrial inflammation and its impact on reproduction in beef cows. Samples for endometrial cytology (low-volume uterine lavage) were obtained from 135 of 137 Angus cows (2-87 d postpartum) in northern Minnesota, 26 d before breeding started. Agreement between examiners for subjective inflammation scores was very high (kappa = 0.971); the correlation between these scores and PMN counts was high (r = 0.83; P < 0.001), validating subjective categorization. The proportion of PMN and large mononuclear cells (principally macrophages) declined with postpartum interval (P < 0.001), whereas small mononuclear cells were consistently present (and not significantly affected by postpartum interval). Pregnancy rate to fixed-time AI was 29% and overall pregnancy rate was 89%. There was no association between cell type and ultimate pregnancy status or day of conception (P > 0.10). Although inflammation later in the postpartum period apparently impaired subsequent reproduction in dairy cows, in cows >50 d postpartum at sample collection in the present study, no cytological parameter significantly predicted final pregnancy status or day of conception. Previous twinning increased the risk of subclinical endometritis (P = 0.02), but not the probability of becoming pregnant (P = 0.14). In conclusion, we inferred that beef cows had the ability to clear uterine inflammation after resumption of ovarian cyclicity.     

Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice

Endometrial cytology and culture specimens (n=2123) were collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during three breeding seasons (2001-2004). Results were compared to the 28-d pregnancy rate for the cycle from which the samples were taken. Cytological smears were evaluated for inflammation at x100 and graded as: not inflammatory (0-2 neutrophils/field), moderate inflammation (2-5 neutrophils/field), severe inflammation (>5 neutrophils/field), or hypocellular (scant epithelial cells and no neutrophils). Uterine culture swabs were plated within 6h, incubated for 72 h and results determined at 24, 48, and 72 h. Approximately, 20% (n=423) cytology samples were positive for inflammation (>2 neutrophils), whereas approximately 11% (n=231) of cultures had microorganisms recovered. A majority (64%) of the positive cultures (147/231) had inflammation on cytology smears. Streptococcus equi subsp. zooepidemicus was associated with more positive cytology results than coliforms (P<0.01). Mares with positive cytology or culture had lower pregnancy rates than mares with normal findings (P<0.01). Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (21%, versus moderate inflammation 48%). Isolation of a microorganism from mares with endometrial inflammation was not associated with a further reduction in pregnancy rates. In barren, foaling and maiden mares, cytology was positive in 28, 17, and 5%, respectively, and culture was positive in 12.2, 11.1, and 3.2%. Foaling and maiden mares had higher pregnancy rates than barren mares (62, 69, and 44%, respectively, P<0.001). In conclusion, a positive cytology was twice as common as a positive culture, and isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation.