Heritability of subclinical endometritis in Norwegian Red cows.

Subclinical endometritis (SCE) is highly prevalent in dairy cows, causing negative effects on reproductive outcomes and the producer economy. Genetic selection for animals with better resilience against uterine disease should be prioritized due to both sustainability and animal welfare. Therefore, the aim of the present study was to estimate the heritability of SCE in the Norwegian Red (NR) population. Moreover, future perspectives of the condition as a fertility phenotype for breeding are discussed. A total of 1,642 NR cows were sampled for SCE at the time of artificial insemination, using cytotape. The percentage of polymorphonuclear cells (PMN) in each sample was established by cytology, through the counting of 300 PMN and epithelial cells. The mean percentage of PMN was 5%. Different trait definitions were examined, and SCE was defined as binary traits, based on the following cut-off levels of PMN: Cyto0 = PMN  >0, Cyto3 = PMN  >3%, Cyto5 = PMN  >5%, Cyto10 = PMN  >10%, and Cyto20 = PMN  >20%.  The mean ranged from 0.07 (Cyto20) to 0.59 (Cyto0). We also analyzed PMN as a continuous variable using percent PMN. Information on the animals and herds was obtained from the Norwegian Dairy Herd Recording System. The pedigree of cows with data included a total of 24,066 animals. A linear animal model was used to estimate the heritability. The only trait definition that had an estimated genetic variance larger than the standard error was Cyto5, with an estimated heritability of 0.04. For all other definitions, the genetic variance was not significantly different from zero. A cut-off level of 5% PMN has been established as a general threshold for the definition of SCE in earlier literature. The standard errors of the estimated variance components were relatively large, and results should be interpreted with caution. However, the current study indicates that SCE is heritable at a similar level to that of clinical endometritis and metritis, and has potential as a future fertility phenotype to be used for breeding purposes. A more feasible method to diagnose SCE is needed to establish larger data sets.

Prevalence, risk factors, and effects on fertility of cytological endometritis at the time of insemination in Norwegian Red cows.

The present study aimed to assess the occurrence of cytological endometritis (CYTO), a nonsymptomatic inflammation of the endometrium, at first artificial insemination (AI) postpartum in Norwegian Red cows. Further, risk factors for CYTO manifestation and its effect on reproductive success and late embryo loss were evaluated. In total 1,648 cows located in 116 herds were included in the study. On mainly spontaneous estrus, endometrial cytology samples were collected using a cytotape technique, and a total of 300 representative epithelial cells and polymorphonuclear neutrophils (PMN) were counted at 400× magnification. Vaginal mucus obtained by Metricheck (Simcro) and body condition score were recorded. Milk samples for progesterone analysis were collected at AI and 21 d later. Pregnancy was diagnosed by rectal palpation or analysis of pregnancy-associated glycoproteins. Based on the constructions of a receiver operator characteristics curve, the cut-off level for PMN defined as CYTO was set to 3.0%, representing the level at which the PMN occurrence affected pregnancy outcome, with the highest summation of sensitivity (32.4%) and specificity (74.9%). Three logistic models with herd included as random factor were constructed. The outcome for the first model was the likelihood for CYTO based on the endometrial samples, in the second model pregnancy to first AI, and in the third model embryo loss. The proportion of CYTO was 28.0% (461/1,648). The average interval in days to first AI was 71.7 d (standard error ± 0.7) and the overall pregnancy incidence to first AI was 59.8% (866/1,449). The likelihood for CYTO at first AI was associated with AI personnel, calving to first AI interval, vaginal mucus characteristics, amount of red blood cells in sample, season, and barn type. Pregnancy to first AI was lower in CYTO-positive cows (odds ratio = 1.51, confidence interval = 1.17-1.94). Other factors affecting pregnancy to first AI were AI personnel, test day milk yield, barn type, and obstetrical conditions or fertility treatments before first AI. The proportion of late embryo loss and abortion was 8.6% (82/948) and 2.8% (24/866), respectively. Late embryo loss was associated with treatment against fertility disorders before first AI, but not associated with CYTO. Overall, our results suggest that even if Norwegian Red cows show a fairly high prevalence of CYTO in the endometrium at first AI, it does not seem to have a major effect on the reproductive performance. The Norwegian Red breeding program has emphasized fertility and health for decades, and a genetically advantageous uterine immunology might be one of the preserved mechanisms.

Prevalence of cytological endometritis and effect on pregnancy outcomes at the time of insemination in nulliparous dairy heifers.

The objectives of the present study were to assess the prevalence of cytological endometritis (CYTO) at the time of artificial insemination (AI) and its effect on pregnancy outcomes in nulliparous dairy heifers. In total, 512 endometrial cytology samples were taken during AI from 351 nulliparous Holstein-Friesian heifers using cytotape (a 1.5-cm piece of paper tape rolled on the top of an AI catheter covered with a double guard sheet). After sampling, the top of the AI catheter was gently rolled onto a glass slide, air-dried, and stained using Diff-Quick (Fisher Diagnostics, Newark, DE). For each slide, 300 nucleated cells were counted, and the polymorphonuclear cell ratio (% PMN) was assessed at 400× magnification. We constructed a receiver operating characteristic curve to find the cutoff point at which sensitivity and specificity (% PMN) affected pregnancy outcomes. The receiver operating characteristic curve revealed that the threshold level for diagnosing CYTO in nulliparous dairy heifers was 1% PMN. An insemination was considered successful when pregnancy was confirmed by rectal palpation at least 45d post-AI. Heifers were considered not pregnant when they received a subsequent insemination or were diagnosed empty by rectal palpation. We built multilevel generalized mixed-effect models to test factors affecting pregnancy outcomes and the occurrence of CYTO at AI. We excluded 16 samples harvested from 12 heifers due to poor sample quality or unavailability of reproductive data. Of the 496 AI samples, the prevalence of CYTO at AI was 7.86% (n=39). The conception rate was 62.8% (n=287) in CYTO-negative samples (n=457) and 38.46% (n=15) in CYTO-positive samples. Risk factors for non-pregnancy were a previous AI (odds ratio 2.96; 95% confidence interval: 1.21-7.26) and the interaction between CYTO and previous AI. The only risk factor identified as being associated with the occurrence of CYTO was a previous AI (odds ratio 4.7; 95% confidence interval: 2.15-10.34). The performance of unsuccessful inseminations significantly affects reproductive outcomes in subsequent AI and may lead to CYTO in nulliparous dairy heifers.

Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows.

Multiparous Holstein cows (n=61) were used to determine the effects of chromium propionate (Cr-Pro) supplementation during the periparturient period and early lactation on metabolism, performance, and the incidence of cytological endometritis (CE). After a 1-wk preliminary period, cows were assigned randomly to 1 of 2 treatments from 21 d before expected calving through 63 d postpartum: (1) control (n=31) and (2) Cr-Pro (n=30) administered by daily topdress at a rate of 8 mg/d of Cr. A tendency was detected for increased dry matter intake (DMI) during the prepartum period for cows fed Cr-Pro. Moreover, cows fed Cr-Pro tended to have lower plasma concentrations of nonesterified fatty acids during the prepartum period. However, effects of Cr-Pro supplementation on postpartum DMI and milk yield were not significant. Cows fed Cr-Pro tended to have higher urea N concentrations in milk. An interaction of treatment and day existed during the postpartum period, such that cows fed Cr-Pro had lower plasma glucose concentrations within the first day postpartum compared with controls. Plasma haptoglobin concentration was not affected by treatment during the postpartum period. Blood neutrophil glycogen concentrations were not affected by treatment when sampled at either 7 d postpartum or on one day between 40 and 60 d (48 d ± 0.44 standard error) postpartum. Evaluation of endometrial cytology by low volume lavage at 7 d postpartum (first lavage) and on one day between 40 and 60 d (second lavage) postpartum revealed that cows fed Cr-Pro tended to have a higher percentage of neutrophils at first lavage and decreased incidence of CE as assessed at second lavage. In conclusion, supplementation with Cr-Pro resulted in trends for increased DMI and lower plasma nonesterified fatty acids prepartum. Postpartum production and energy metabolism were not affected by treatment; however, Cr-Pro supplementation tended to affect the postpartum influx of neutrophils into the uterus and decreased the incidence of CE, suggesting positive effects of Cr-Pro supplementation on uterine health.

Effects of hydroxy trace minerals on oxidative metabolism, cytological endometritis, and performance of transition dairy cows.

Multiparous Holstein cows (n=60) were used to determine effects of supplementing hydroxy forms of Zn, Cu, and Mn compared with 2 other common supplementation strategies on oxidative metabolism, cytological endometritis, and performance of transition cows. After a 1-wk pretreatment period, cows were assigned randomly to 1 of 3 dietary treatments from 21 d before expected calving through 84 d postcalving. Dietary treatments administered by daily top-dressing included (1) inorganic sulfate forms of Zn, Cu, and Mn (ITM); (2) a blend (75:25) of sulfates and organic complexes of Zn, Cu, and Mn (ITM/OTM); and (3) hydroxy trace minerals (HTM) of Zn, Cu, and Mn. The resulting dietary concentrations of supplemental Zn, Cu, and Mn were similar among treatments and averaged 40, 10, and 27 mg/kg, respectively, before calving and 59, 15, and 40 mg/kg, respectively, after calving. Total concentrations of Zn, Cu, and Mn averaged 80, 16, and 62 mg/kg during the prepartum period and 102, 23, and 75 mg/kg, respectively, during the postpartum period. Overall, effects of treatment on milk yield and milk composition were not significant. Cows fed HTM during the prepartum period had higher body weight (BW) than those fed ITM during the prepartum period and had higher BW during the postpartum period than those fed the other treatments; however, BW change, body condition score, and body condition score change were not affected by treatment. Plasma total antioxidant capacity was lower in cows fed HTM than ITM but was not different from cows fed ITM/OTM. Cows fed HTM tended to have lower concentrations of plasma thiobarbituric acid reactive substances than those fed ITM during the whole study period, but plasma thiobarbituric acid reactive substances were not different between HTM and ITM/OTM. Plasma haptoglobin was lower in cows fed HTM than ITM/OTM at 1 wk postpartum. Endometrial cytology 7d postcalving and cytological endometritis as assessed on 1d between 40 and 60 d postcalving was not affected by treatment. In conclusion, supplementation with HTM sources of Zn, Cu, and Mn modulated plasma variables related to oxidative metabolism compared with supplementation with ITM; however, HTM and ITM/OTM resulted in similar responses. Furthermore, the source of trace minerals did not affect performance or uterine health in this experiment.

Individual and combined effects of anovulation and cytological endometritis on the reproductive performance of dairy cows.

The objective was to evaluate the individual and combined effect of anovulation and cytological endometritis (CTE) on the reproductive performance of dairy cows. A total of 1,569 cows from 3 data sets were used. In data set 1, 403 Holstein cows from 5 dairies in New York were used. In data set 2, 750 Holstein cows from 2 dairies, one in Florida and one in California were used. In data set 3, 416 dairy cows, 165 Holsteins, 36 Jerseys, and 215 Holstein-Jersey crossbreeds from a grazing dairy in Florida were used. Cyclicity and CTE was determined at 35±3 (data set 2) or 49±3 d in milk (data sets 1 and 3). A variable (VarCycCTE) containing all 4 possible permutations between cyclicity (cyclic = Cyc; anovular = Anov) and CTE (present = CTE; absent = Healthy) was created. In the combined data set (sets 1, 2, and 3), pregnancy per artificial insemination (P/AI) diagnosed at 30 to 38 d after first AI was affected by VarCycCTE, with AnovCTE cows having decreased P/AI compared with CycHealthy cows (21.3 vs. 46.7%), whereas AnovHealthy (37.9%) and CycCTE cows (36.0%) had intermediate P/AI. Pregnancy per artificial insemination for the individual data sets and for pregnancy diagnosed at 63 to 74 d after artificial insemination followed a similar pattern. Pregnancy loss was not affected by VarCycCTE. Hazard of pregnancy up to 300 d in milk was affected by VarCycCTE in the combined data sets 1 and 2, with AnovCTE [hazard ratio (HR)=0.55], AnovHealthy cows (HR=0.71), and CycCTE (HR=0.8) having decreased hazard of pregnancy compared with CycHealthy cows. Median days open were 200, 159, 145, and 121 for AnovCTE, AnovHealthy, CycCTE, and CycHealthy, respectively. Hazard of pregnancy for the individual data sets followed a similar pattern. In summary, both anovulation and CTE were negatively associated with reproductive performance and, when combined, they had an additive negative effect.

Proteomic analysis of uterine lavage fluid of dairy cows at different time after delivery by mass spectrometry.

Endometritis is a common disease in the reproductive system, which is the infection and inflammation of the endometrium. In severe cases, it can affect the myometrium and adversely affect the subsequent fertility of dairy cows. We used a mass spectrometry-based technique to compare proteomics of uterine lavage fluid between healthy cows and cows with cytological endometritis classified according to 100-day postpartum pregnancy results and diagnosis result. The uterine lavage fluid of dairy cows collected at 15 and 30 days after delivery was analyzed. 15 days postpartum, we identified a total of 1129 proteins in the control and cytological endometritis (CEM) groups. Among them, 160 proteins were accurately screened out. 30 days postpartum, we identified a total of 846 proteins in the control and cytological endometritis (CEM) groups. Among them, 186 proteins were accurately cytological endometritis (CEM). Endometritis is a costly reproductive disease in lactating cows, which needs to be diagnosed in time. Using proteomics method based on gel mass spectrometry, we compared the proteome of uterine lavage fluid of dairy cows with and without cytological endometritis to characterize the changes of proteomic characteristics associated with postpartum uterine disease. To provide reference for clinical application and basic research.

Local and systemic inflammatory response to the intrauterine infusion of enzymes during estrus in water buffaloes with subclinical endometritis.

Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1ß and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.

Individual and Combined Effects of Diseases and Cytological Endometritis on Reproductive Performance and Culling of Dairy Cows: Preliminary Results.

The objective of this study was to evaluate the effect of calving-related disorders/clinical diseases (DIS) and cytological endometritis (CYTO) on the reproductive performance and culling of dairy cows. In a total of 119 multiparous Holstein cows, DIS were recorded and CYTO was diagnosed at 40 ± 2 d in milk. Onset of luteal activity was defined as the first postpartum measurement of milk progesterone >5 ng/mL. A dummy variable containing all four possible permutations between DIS and CYTO statuses was created. The pregnancy rates after first artificial insemination were 57.7, 42.9, 23.0 and 15.8% for the DIS−/CYTO−, DIS+/CYTO−, DIS−/CYTO+ and DIS+/CYTO+ groups, respectively. The hazard of pregnancy was affected by DIS−/CYTO+ and DIS+/CYTO+ health statuses (hazard ratio (HR) = 0.43 and 0.29, respectively), whereas DIS+/CYTO− and DIS−/CYTO− cows had a similar hazard to pregnancy. The hazard of onset of luteal activity was affected by DIS+/CYTO+ health status (HR = 0.45), but not by DIS+/CYTO− and DIS−/CYTO+ health statuses. Compared with DIS−/CYTO− cows, DIS−/CYTO+ and DIS+/CYTO+ cows had 4.24 and 5.57 times the odds of being culled, respectively. Culling was not affected by DIS+/CYTO− health status. In conclusion, CYTO+ status, irrespective of DIS status, decreased the pregnancy rate. When DIS+ and CYTO+ were combined, they had an additive negative effect. Our preliminary findings suggest that the far-reaching consequences of clinical diseases on fertility and culling can be mediated through the development of CYTO. Further investigations including a larger number of cows are required to confirm these results.

Effects of intra-uterine infusion of proteolytic enzymes on selected cytokine concentrations, uterine inflammation, and fertility in postpartum water buffalo cows with subclinical endometritis.

The study was conducted to evaluate effects of intrauterine administration of proteolytic enzymes on endometrial inflammation and reproductive performance in postpartum water buffalo cows with subclinical endometritis (SCE). Cows (n = 38) with SCE (≥ 18 % PMN i.e.; polymorphonuclear cells) on day 21 postpartum (21 dpp), were allocated into treatment (TR; n = 19; intrauterine infusion of trypsin, chymotrypsin and papain in 20 ml normal saline on 21 dpp) and control (PC; n = 19; intrauterine administration 20 ml saline) groups. Cows without SCE (< 18 % PMN) were not treated and served as the negative control (NC; n = 30). Ultrasonography and sampling (endometrial cytology, uterine flushing, blood) were conducted on day 21 (before treatment) and 28 postpartum (28 dpp). The PMN % and uterine horn diameter were less on 28 dpp (compared with 21 dpp) in NC and TR group only. Cows with SCE had greater uterine concentrations of interleukin (IL)-1ß, IL-8 and tumor necrosis factor (TNF)-α; but lesser IL-10 than NC group on 21 and 28 dpp. There were greater serum IL-1ß and TNF-α concentrations on 28 dpp in cows with SCE than NC group. Uterine concentrations of IL-1ß were less, whereas IL-6 was greater following enzymatic treatment. Proteolytic enzyme treatment did not result in improvement in pregnancy rate compared with the PC group; however, days to conception were less in TR compared with the other two groups. In conclusion, results indicated a reduction in endometrial inflammation and days nonpregnant after proteolytic enzyme treatment in buffalo cows with SCE.

Cytobrush cytology patterns of early postpartum dairy cows.

Endometrial cytobrush cytology has been recommended as a reliable method for determining the percentage of polymorphonuclear leukocytes (PMN%) in cattle smears to diagnose cytological endometritis (CE). In this study, the clarity of cytobrush　cytological smears and the influence of different sample evaluation methods (number　and types of cells counted) on CE diagnosis were evaluated. Samples from 28 lactating　Holstein cows were collected weekly between 3 and 7 weeks postpartum. Smear clarity,　based on cell density, quality of cell morphology, and red blood cell contamination,　was significantly poorer at 3 weeks than between 5 and 7 weeks postpartum. Five　different cell counting methods (C100, C200, C300, C400, and C500) were used,　where 100-500 nucleated cells (endometrial epithelial cells, PMN consisting of neutrophils, eosinophils and basophils, lymphocytes, and macrophages) were counted. Agreement of diagnostic results for CE between C300 and C500 and between C400 and C500 was excellent at all observation times. In calculations of the PMN% based on whether the number of lymphocytes and macrophages were or were not excluded in the denominator, exclusion of these cells in the calculations did not affect the diagnosis of CE. While reduced clarity in earlier stage samples might interfere with the accuracy of cytobrush cytology, C300 can be recommended to determine the endometrial PMN%.

Cytological endometritis in dairy cows: diagnostic threshold, risk factors, and impact on reproductive performance.

We determined the threshold proportion of polymorphonuclear leukocytes (PMNs) for a diagnosis of cytological endometritis (CEM), the risk factors for this condition, and its impact on reproductive performance in dairy cows. Uterine cytology was performed on 407 Holstein cows 4 weeks postpartum to determine the proportions of endometrial cells and PMNs. A receiver operator characteristics curve was used to determine the threshold above which the PMN proportion affected the likelihood of cows conceiving by 200 days postpartum. The optimal threshold was ≥ 14% PMN (sensitivity, 31.3%; specificity, 81.7%; p ＜ 0.05). The farm identity, retained placenta (odds ratio [OR] = 1.87), and septicemic metritis (OR = 3.07) were risk factors for CEM (p ＜ 0.05). Cows with CEM were less likely to resume cyclicity (OR = 0.58) and to conceive by 200 days postpartum (hazard ratio = 0.58). Cows with CEM tended (p ＜ 0.1) to be less likely to become pregnant after their first insemination (OR = 0.65) and to require a greater number of inseminations per conception (2.3 vs. 2.2). In conclusion, a PMN threshold of 14% defined the presence of CEM at 4 weeks postpartum. The farm, retained placenta, and septicemic metritis were risk factors for CEM, which reduces subsequent reproductive performance.

Relationships between plasma insulin-like growth factor-1 and insulin concentrations in multiparous dairy cows with cytological endometritis.

Cytological endometritis (CYTO) is a uterine inflammation characterised by the percentage of polymorphonuclear neutrophils in endometrial cytology. This observational study evaluated the association of blood plasma insulin-like growth factor-1 (IGF-1) and insulin concentrations at -2 weeks prepartum, +1 week and +3 weeks postpartum with the development of CYTO in 119 multiparous Holstein cows. Overall CYTO prevalence was 30.3 per cent. Multivariable logistic regression model revealed the odds of developing CYTO were 3.54 times (P=0.018) greater in cows with week -2 prepartum IGF-1 concentrations less than 74.6 ng/ml and 4.41 times (P=0.004) higher in cows with week +1 postpartum IGF-1 concentrations less than 13.2 ng/ml than the odds of this health outcome in cows with IGF-1 concentrations at least 74.6 and13.2 ng/ml, respectively. Additionally, cows with body condition score (BCS) up to 2.75 at week -2 prepartum and cows experiencing calving-related disorders and/or ill health had higher risk for CYTO compared with cows with BCS=3.50-3.75 (OR=6.8, P=0.049) and cows without health complications (OR=3.1, P=0.030). Insulin was not a significant predictor for CYTO in the model. Our findings provide further evidence that reduced dairy cow fertility associated with low plasma concentrations of IGF-1 is in part mediated through the inflammatory status of the uterus.

Risk factors associated with cytological endometritis diagnosed at artificial insemination in dairy cows.

In this study, we aimed to determine risk factors associated with cytological endometritis (CYTO) diagnosed at artificial insemination (AI) in dairy cows. The CYTO risk factors were evaluated based on 1.625 AI-CYTO samples obtained from 873 Holstein-Friesian cows from in total 18 dairy herds in Flanders (Belgium). The endometrial cytology samples were obtained using the cytotape technique, which consisted of adapting a 1.5 cm piece of paper tape to a standardly loaded AI catheter, covered with a double guard sheet. The polymorphonuclear cells' (PMNs) cut-off point for CYTO at AI was set at ≥ 1%. We constructed multilevel generalized mixed effect models in order to identify the risk factors associated with the presence of CYTO at AI. The CYTO prevalence at AI was 27.8% at the animal level, while the within-herd level prevalence ranged from 10.7 to 39.7%, with an average of 28.1%. Risk factors associated with the occurrence of CYTO were parity ≥2 [odds ratio (OR) = 1.8], days in milk (DIM) at AI ≥ 124 (OR = 0.4), and warm months of the year [July (OR = 2.9), August (OR = 2.3), and September (OR = 1.4)]. In conclusion, the present study supports that multiparous cows and cows that are inseminated in the summer months have a higher risk to suffer from CYTO at insemination, while the risk for CYTO is lower when the insemination is taking place at ≥ 124 DIM.