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The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Our objective was to investigate the effects of intravenous (IV) or intrauterine (IU) lipopolysaccharide (LPS) challenge at 5 or 40 d postpartum (DPP) on clinical signs, systemic and uterine inflammation, dry matter intake (DMI), and milk yield (MY). Holstein cows at 5 DPP (n = 23) or at 40 DPP (n = 24) were blocked by parity and randomly assigned to 1 of 3 treatments: (1) IV-LPS (0.0625 μg/kg BW [5 DPP] or 0.1 μg/kg BW [40 DPP] over 1h), (2) IU-LPS (100 μg [5 DPP] or 300 μg [40 DPP] in 20 mL of saline), or (3) 20 mL of saline IU (IU-SAL; same for 5 and 40 DPP). The proportion of polymorphonuclear (PMN) cells was measured by endometrial cytology at d −1, 1, 4, and 7 relative to treatment. Blood haptoglobin (Hp), serum amyloid A (SAA), and LPS-binding protein (LBP), DMI, and MY were measured from d −1 through d 7. Data were analyzed separately for each DPP group in multivariable linear regression models accounting for repeated measures. Both DPP groups showed increases in rectal temperature and heart and respiratory rates, and decrease in rumination rate following IV-LPS, but not following IU-LPS. At 5 DPP, endometrial PMN proportion was similar in IU-LPS and IU-SAL. Serum Hp was unaffected by LPS challenge, SAA was greater in IV-LPS from 12 h to 24 h after challenge, and LBP was greater in IV-LPS from 8 h to 24 h. At 40 DPP, PMN was greater in IU-LPS (37 ± 4%) than in IU-SAL (15 ± 4%) 1 d after LPS challenge. Serum Hp was greater from 24 h to 72 h after challenge in IV-LPS than in the other groups, SAA was greater in IV-LPS from 6 h to 48 h, and LBP was greater in IV-LPS from 8 h to 24 h. At both 5 and 40 DPP, treatment did not affect DMI, but MY was lesser in IV-LPS cows at 12 h and 24 h than in IU-SAL or IU-LPS. The IV-LPS challenge resulted in more pronounced changes in clinical signs and acute phase protein (APP) concentrations than IU-LPS or IU-SAL at 40 DPP, but more subtle or inconsistent changes at 5 DPP. These may be due to the different doses of LPS used at 5 and 40 DPP or possibly due to the high variation in baseline clinical signs and APP observed in all groups at 5 DPP. The IU-LPS increased uterine PMN 1 d after challenge at 40 DPP but not at 5 DPP. At each time, IU-LPS did not produce changes in clinical signs or markers of systemic inflammation. [ABSTRACT FROM AUTHOR]

ABSTRACT: Our objective was to investigate the effects of intravenous (IV) or intrauterine (IU) lipopolysaccharide (LPS) challenge at 5 or 40 d postpartum (DPP) on clinical signs, systemic and uterine inflammation, dry matter intake (DMI), and milk yield (MY). Holstein cows at 5 DPP (n = 23) or at 40 DPP (n = 24) were blocked by parity and randomly assigned to 1 of 3 treatments: (1) IV-LPS (0.0625 μg/kg BW [5 DPP] or 0.1 μg/kg BW [40 DPP] over 1h), (2) IU-LPS (100 μg [5 DPP] or 300 μg [40 DPP] in 20 mL of saline), or (3) 20 mL of saline IU (IU-SAL; same for 5 and 40 DPP). The proportion of polymorphonuclear (PMN) cells was measured by endometrial cytology at d −1, 1, 4, and 7 relative to treatment. Blood haptoglobin (Hp), serum amyloid A (SAA), and LPS-binding protein (LBP), DMI, and MY were measured from d −1 through d 7. Data were analyzed separately for each DPP group in multivariable linear regression models accounting for repeated measures. Both DPP groups showed increases in rectal temperature and heart and respiratory rates, and decrease in rumination rate following IV-LPS, but not following IU-LPS. At 5 DPP, endometrial PMN proportion was similar in IU-LPS and IU-SAL. Serum Hp was unaffected by LPS challenge, SAA was greater in IV-LPS from 12 h to 24 h after challenge, and LBP was greater in IV-LPS from 8 h to 24 h. At 40 DPP, PMN was greater in IU-LPS (37 ± 4%) than in IU-SAL (15 ± 4%) 1 d after LPS challenge. Serum Hp was greater from 24 h to 72 h after challenge in IV-LPS than in the other groups, SAA was greater in IV-LPS from 6 h to 48 h, and LBP was greater in IV-LPS from 8 h to 24 h. At both 5 and 40 DPP, treatment did not affect DMI, but MY was lesser in IV-LPS cows at 12 h and 24 h than in IU-SAL or IU-LPS. The IV-LPS challenge resulted in more pronounced changes in clinical signs and acute phase protein (APP) concentrations than IU-LPS or IU-SAL at 40 DPP, but more subtle or inconsistent changes at 5 DPP. These may be due to the different doses of LPS used at 5 and 40 DPP or possibly due to the high variation in baseline clinical signs and APP observed in all groups at 5 DPP. The IU-LPS increased uterine PMN 1 d after challenge at 40 DPP but not at 5 DPP. At each time, IU-LPS did not produce changes in clinical signs or markers of systemic inflammation.

ABSTRACT: Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0–20 µg/mL) for different times (0–24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3β, β-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the β-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of β-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/β-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0–20 µg/mL) for different times (0–24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3β, β-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the β-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of β-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/β-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis. [ABSTRACT FROM AUTHOR]

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

ABSTRACT: Dry matter intake (DMI, kg/d) is closely related to the magnitude of negative energy and protein balance during the transition period, and the metabolic adaptations to support lactation in dairy cows. Thus, DMI might affect the development of cytological endometritis in the early postpartum period. Difficulty to adapt to these metabolic changes is related to impaired immune function and increased occurrence of reproductive disorders. We aimed to examine the association of pre- and postpartum DMI, body weight (BW), body condition score, milk yield and milk composition, and days to first ovulation with cytological endometritis at 15 (CYT15) and 30 DIM (CYT30). A second objective was to understand the association of vaginal discharge with CYT15 and CYT30 and performance. We conducted a pooled statistical analysis of 5 studies, including data from 280 multiparous Holstein cows. Based on the cutoffs for the percentage of uterine polymorphonuclear cells (PMN), determined by taking the median value of the data set for 15 and 30 DIM, cows were categorized as follows: LOW15 (PMN % at 15 DIM ≤24%; n = 125), HIGH15 (PMN % at 15 DIM >24%; n = 125), LOW30 (PMN % at 30DIM ≤7%; n = 141); and HIGH30 (PMN % at 30DIM >7%; n = 139). Cows in HIGH15 consumed an average of 1.97 ± 0.5 kg/d less DM than cows in LOW15 during prepartum, and 3.01 ± 0.5 kg/d less DM during postpartum. Dry matter intake (as a percentage of BW) was higher for cows in LOW15 during pre- and postpartum than for cows in HIGH15. Moreover, cows in HIGH15 tended to have lower milk yield than cows in LOW15 from the third until the fifth week postpartum. Although DMI was not associated with CYT30, DMI (as a percentage of BW) was lower for cows in LOW30 pre- and postpartum than for cows in HIGH30. There was no association between CYT30 and milk yield. Cows in LOW15 had greater days to first ovulation than cows in HIGH15, while cows in LOW30 also had greater days to first ovulation than cows in HIGH30. Simple regression analyses demonstrated linear associations of increased DMI, particularly postpartum, with decreased uterine PMN percentage and lower vaginal discharge score. Additionally, increased units of vaginal discharge score and increased percentage units of uterine PMN were linearly associated with decreased milk yield. Corroborating with the notion of the ovarian function being associated with uterine inflammatory status, cows in HIGH15 and HIGH30 ovulated on average 3 d before than cows in LOW15 and LOW30, respectively. Cytological endometritis at 15 DIM was associated with lower DMI from 4 wk before calving until 4 wk postpartum and was associated with lower milk yield. The association of vaginal discharge with cytological endometritis was variable and dependent on the day of evaluation.

Dry matter intake (DMI, kg/d) is closely related to the magnitude of negative energy and protein balance during the transition period, and the metabolic adaptations to support lactation in dairy cows. Thus, DMI might affect the development of cytological endometritis in the early postpartum period. Difficulty to adapt to these metabolic changes is related to impaired immune function and increased occurrence of reproductive disorders. We aimed to examine the association of pre- and postpartum DMI, body weight (BW), body condition score, milk yield and milk composition, and days to first ovulation with cytological endometritis at 15 (CYT15) and 30 DIM (CYT30). A second objective was to understand the association of vaginal discharge with CYT15 and CYT30 and performance. We conducted a pooled statistical analysis of 5 studies, including data from 280 multiparous Holstein cows. Based on the cutoffs for the percentage of uterine polymorphonuclear cells (PMN), determined by taking the median value of the data set for 15 and 30 DIM, cows were categorized as follows: LOW15 (PMN % at 15 DIM ≤24%; n = 125), HIGH15 (PMN % at 15 DIM >24%; n = 125), LOW30 (PMN % at 30DIM ≤7%; n = 141); and HIGH30 (PMN % at 30DIM >7%; n = 139). Cows in HIGH15 consumed an average of 1.97 ± 0.5 kg/d less DM than cows in LOW15 during prepartum, and 3.01 ± 0.5 kg/d less DM during postpartum. Dry matter intake (as a percentage of BW) was higher for cows in LOW15 during pre- and postpartum than for cows in HIGH15. Moreover, cows in HIGH15 tended to have lower milk yield than cows in LOW15 from the third until the fifth week postpartum. Although DMI was not associated with CYT30, DMI (as a percentage of BW) was lower for cows in LOW30 pre- and postpartum than for cows in HIGH30. There was no association between CYT30 and milk yield. Cows in LOW15 had greater days to first ovulation than cows in HIGH15, while cows in LOW30 also had greater days to first ovulation than cows in HIGH30. Simple regression analyses demonstrated linear associations of increased DMI, particularly postpartum, with decreased uterine PMN percentage and lower vaginal discharge score. Additionally, increased units of vaginal discharge score and increased percentage units of uterine PMN were linearly associated with decreased milk yield. Corroborating with the notion of the ovarian function being associated with uterine inflammatory status, cows in HIGH15 and HIGH30 ovulated on average 3 d before than cows in LOW15 and LOW30, respectively. Cytological endometritis at 15 DIM was associated with lower DMI from 4 wk before calving until 4 wk postpartum and was associated with lower milk yield. The association of vaginal discharge with cytological endometritis was variable and dependent on the day of evaluation. [Display omitted] [ABSTRACT FROM AUTHOR]

Objectives of this experiment were to study the effect of infusing utero-pathogenic bacteria to induce endometrial inflammation on productive performance in early lactation and subsequent reproduction. Although endometritis is associated with perturbed reproduction, numerous factors may contribute to the observed association. It was hypothesized that induced endometrial inflammation, resulting in localized and systemic inflammatory responses, compromises production and reproduction. Holstein cows without clinical disease and with less than 18% polymorphonuclear leukocytes (PMN) in endometrial cytology on d 31 ± 3 postpartum had their estrous cycle synchronized. Cows were blocked by parity and genomic breeding value for cow conception rate and, within block, assigned randomly to remain as untreated controls (CON; n = 37) or to receive an intrauterine infusion of 5.19 × 108 cfu Escherichia coli and 4.34 × 108 cfu Trueperella pyogenes during the luteal phase to induce endometrial inflammation (INF; n = 48). Endometrial cytology was taken on d 2 and 7 after treatment to evaluate the proportion of PMN. Rectal temperature, dry matter intake, and yields of milk and components were measured in the first 7 d after treatment. Blood serum was analyzed for concentration of haptoglobin. Leukocytes were isolated from blood on d 2 and 7 after treatment and on d 19 after artificial insemination (AI) and mRNA was quantified for a select group of genes. Cows received AI and reproduction was followed for 300 d postpartum. Bacterial infusion induced endometrial inflammation with increased proportions of PMN in the endometrial cytology on d 2 (4.4 ± 0.7 vs. 26.3 ± 2.8%) and 7 (10.9 ± 1.7 vs. 17.4 ± 2.1%) after treatment, resulting in increased mean prevalence of subclinical endometritis (>10% PMN; 23.3 ± 6.3 vs. 80.9 ± 5.1%). Rectal temperature did not differ between CON and INF, but the concentration of haptoglobin in serum tended to increase in INF compared with CON (113 ± 14 vs. 150 ± 16 µg/mL). Induced endometrial inflammation reduced yields of milk (44.9 ± 0.8 vs. 41.6 ± 0.8 kg/d), protein (1.19 ± 0.03 vs. 1.12 ± 0.03 kg/d), and lactose (2.17 ± 0.04 vs. 2.03 ± 0.04 kg/d) and tended to reduce dry matter intake (20.7 ± 0.5 vs. 19.4 ± 0.6 kg/d) in the first 7 d after treatment. Indeed, the reduction in milk yield lasted 4 wk. However, treatment did not affect yields of energy-corrected milk or fat because treatment with INF increased the concentration of fat in milk (3.54 ± 0.10 vs. 3.84 ± 0.10%). Induced endometrial inflammation reduced pregnancy per AI at all inseminations (33.4 ± 5.1 vs. 21.6 ± 3.7%) and the hazard of pregnancy (0.61; 95% CI = 0.36–1.04), which extended the median days open by 24 d. Blood leukocytes from INF cows had increased mRNA expression of the pro-inflammatory gene IL1B on d 2 and 7 after treatment, but reduced expression of the IFN-stimulated genes ISG15 and MX2 on d 19 after AI. Induced endometrial inflammation depressed production and caused long-term negative effects on reproduction in lactating dairy cows. [ABSTRACT FROM AUTHOR]

We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at −7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and ≤5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 ± 2.1%; least squares means ± standard error) and a higher proportion of apoptosis (14.4 ± 2.0%) than healthy (92.4 ± 1.3 and 6.7 ± 1.3%, respectively) or SCE (95.3 ± 2.4 and 3.8 ± 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 ± 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 ± 0.08 MFI) or CE (6.8 ± 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 ± 5.1%) than SCE (27.4 ± 7.3%) and CE (27.7 ± 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 ± 8.6%) than in healthy (18.4 ± 7.6%) cows, but did not differ from SCE cows (25.9 ± 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows. [ABSTRACT FROM AUTHOR]

ABSTRACT: Based on the clinical stage (e.g., vaginal discharge) and bacterial species, several forms of uterine diseases (UD) exist and can be classified as different traits [i.e., different stages of endometritis (EM) and metritis (MET)], which may differ in their genetic background and causal physiological mechanisms. Consequently, the present study aimed to study (1) the effect of UD on 305-d lactation and fertility, (2) the estimation of heritabilities for UD traits using pedigree- and SNP-based relationships, and (3) genome-wide associations to detect significant SNP markers and to infer candidate genes for UD traits. The data set contained herd manager and veterinarian recorded UD traits of 14,810 first-lactating genotyped Holstein cows from 63 large-scale contract herds. Binary defined UD traits (healthy or diseased) according to the clinical stage were endometritis catarrhalis (EM I), endometritis mucopurulenta (EM II), endometritis purulenta (EM III), pyometra (EM IV), endometritis (EM_SOD; superordinate diagnosis = no specific clinical stage defined), and MET. The binary defined trait UDall included all EM and MET diagnoses. The prevalence of UDall was 26.7%. The effect of UD on 305-d lactation and fertility was estimated via linear and generalized linear mixed models. We applied linear single-trait animal models and threshold models to estimate pedigree- and SNP-based heritabilities for UD traits, and bivariate linear models for genetic correlation estimations between UDall with 305-d lactation and fertility traits. A diagnosis for UDall had significant unfavorable effects on the female fertility traits calving interval, interval from calving to first service, days open, and nonreturn rate after 90 d, but was unrelated to 305-d lactation records for production traits milk yield, protein yield, and fat yield. Heritabilities for UDall and EM stages were close to zero, displaying maximal values of 0.05 for pedigree and 0.07 for SNP-based relationship matrices. For MET, pedigree- and SNP-based heritabilities were

ABSTRACT: Endometritis is a uterine disease of dairy cows causing substantial negative effects on reproductive performance and inflicting considerable economic losses. It is typically diagnosed by endometrial cytology evaluation and commonly named cytological endometritis (CEM). In most previous studies, cows were defined as CEM positive if the proportion of polymorphonuclear cells (%PMN) in their endometrial cytology was above a pre-set threshold. Thresholds were established based on CEM diagnosis in association with reproductive performance, typically analyzed by a single reproductive parameter and calculated for all cows together. Our objective was to examine whether primiparous and multiparous cows should optimally be diagnosed for CEM by different %PMN thresholds and sampling timings, using a combination of several reproductive performance parameters. Two endometrial cytobrush cytology samples were collected from Holstein-Friesian dairy cows (n = 415; 269 multiparous; 146 primiparous), at 30–40 d in milk (DIM) and 60–70 DIM, and %PMN were evaluated microscopically (blindly; Diff-Quick stain, Medi-Market). The %PMN thresholds were set at ≥1% to ≥10%, ≥15%, and ≥20%, and accordingly, for each of the thresholds, several reproductive performance parameters were compared between CEM-positive versus CEM-negative cows. Upon application of several analytic approaches, our results indicated that optimal CEM diagnosis should be performed by different criteria in primiparous and multiparous cows: in primiparous cows at 30–40 DIM, using a threshold of ≥7%PMN, and in multiparous cows at 60–70 DIM, using a threshold of ≥4%PMN. Such a diagnostic approach provides a comprehensive view of the reproductive prognosis of CEM-positive primiparous and multiparous cows, which is pertinent information for researchers, veterinarians, and farmers.

In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, which has profound detrimental effects on subsequent reproductive performance. To date, transcriptomic studies related to this condition were either based on biopsy-derived whole-endometrium tissue or endometrial swab or cytobrush samples, thus masking effects of disease on cell type–specific gene expression. This study tested the hypothesis that different endometrial health statuses are associated with distinct transcription profiles of endometrial stromal, glandular, and luminal epithelial cells. At 44 d postpartum (DPP), endometrial biopsies were taken from dairy cows (n = 24) classified as healthy, recovered from SCE, or affected by persistent SCE, according to endometrial cytology taken at 21 and 44 DPP. Stromal, glandular, and luminal epithelial cells were isolated from the whole-tissue biopsy by laser capture microdissection, and the cell-specific transcription profiles were determined by RNA sequencing. Differential gene expression was analyzed with DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). Results demonstrated that global transcriptomic profiles and corresponding lists of differentially expressed genes between cows with different health statuses were distinct among cell types. Results also showed that although healthy and recovered cows presented similar endometrial clinically healthy phenotypes at 44 DPP, the prior presence of immune cells still affected the transcriptome of endometrial cells at this stage, delaying complete functional recovery. Recovery or persistence of inflammation was associated with gene expression patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity in a cell type–specific manner. Identifying these signatures may contribute to the development of novel diagnostic tools and therapeutic strategies. In addition, these results may help to define preventive measures or ways to stimulate recovery from endometrial inflammation, thus helping to restore the fertility of postpartum dairy cows. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Based on the clinical stage (e.g., vaginal discharge) and bacterial species, several forms of uterine diseases (UD) exist and can be classified as different traits [i.e., different stages of endometritis (EM) and metritis (MET)], which may differ in their genetic background and causal physiological mechanisms. Consequently, the present study aimed to study (1) the effect of UD on 305-d lactation and fertility, (2) the estimation of heritabilities for UD traits using pedigree- and SNP-based relationships, and (3) genome-wide associations to detect significant SNP markers and to infer candidate genes for UD traits. The data set contained herd manager and veterinarian recorded UD traits of 14,810 first-lactating genotyped Holstein cows from 63 large-scale contract herds. Binary defined UD traits (healthy or diseased) according to the clinical stage were endometritis catarrhalis (EM I), endometritis mucopurulenta (EM II), endometritis purulenta (EM III), pyometra (EM IV), endometritis (EM_SOD; superordinate diagnosis = no specific clinical stage defined), and MET. The binary defined trait UDall included all EM and MET diagnoses. The prevalence of UDall was 26.7%. The effect of UD on 305-d lactation and fertility was estimated via linear and generalized linear mixed models. We applied linear single-trait animal models and threshold models to estimate pedigree- and SNP-based heritabilities for UD traits, and bivariate linear models for genetic correlation estimations between UDall with 305-d lactation and fertility traits. A diagnosis for UDall had significant unfavorable effects on the female fertility traits calving interval, interval from calving to first service, days open, and nonreturn rate after 90 d, but was unrelated to 305-d lactation records for production traits milk yield, protein yield, and fat yield. Heritabilities for UDall and EM stages were close to zero, displaying maximal values of 0.05 for pedigree and 0.07 for SNP-based relationship matrices. For MET, pedigree- and SNP-based heritabilities were <0.001 and 0.07, respectively. Genetic correlations ranged from 0.20 to 0.31 between UDall with 305-d milk, protein, and fat yield, and from 0.17 to 0.40 with fertility traits. The GWAS revealed 5 SNP on bovine chromosomes (BTA) 1, 8, 10, 23 for UDall, 5 SNP on BTA 26 for EM I, 1 SNP on BTA 19 for EM II, 4 SNP on BTA 2, 18, 20, 25 for EM III, and 4 SNP on BTA 4, 16, 20 for EM IV above the significance threshold. For EM_SOD, we identified 15 significantly associated SNP on 4 chromosomes, and 4 significant SNP on BTA 3, 20, 22, 28 for MET. Marker associations for UD traits were annotated to 24 potential candidate genes using the ENSEMBL database. Six of these genes were previously reported to be involved in uterine defense mechanisms or in endometritis. Further detected genes contribute to immune response mechanisms during bacterial infections. Different SNP significantly influenced different UD stages, explaining the inter-individual variations in clinical severity of uterine infections. [ABSTRACT FROM AUTHOR]

Endometritis is a uterine disease of dairy cows causing substantial negative effects on reproductive performance and inflicting considerable economic losses. It is typically diagnosed by endometrial cytology evaluation and commonly named cytological endometritis (CEM). In most previous studies, cows were defined as CEM positive if the proportion of polymorphonuclear cells (%PMN) in their endometrial cytology was above a pre-set threshold. Thresholds were established based on CEM diagnosis in association with reproductive performance, typically analyzed by a single reproductive parameter and calculated for all cows together. Our objective was to examine whether primiparous and multiparous cows should optimally be diagnosed for CEM by different %PMN thresholds and sampling timings, using a combination of several reproductive performance parameters. Two endometrial cytobrush cytology samples were collected from Holstein-Friesian dairy cows (n = 415; 269 multiparous; 146 primiparous), at 30–40 d in milk (DIM) and 60–70 DIM, and %PMN were evaluated microscopically (blindly; Diff-Quick stain, Medi-Market). The %PMN thresholds were set at ≥1% to ≥10%, ≥15%, and ≥20%, and accordingly, for each of the thresholds, several reproductive performance parameters were compared between CEM-positive versus CEM-negative cows. Upon application of several analytic approaches, our results indicated that optimal CEM diagnosis should be performed by different criteria in primiparous and multiparous cows: in primiparous cows at 30–40 DIM, using a threshold of ≥7%PMN, and in multiparous cows at 60–70 DIM, using a threshold of ≥4%PMN. Such a diagnostic approach provides a comprehensive view of the reproductive prognosis of CEM-positive primiparous and multiparous cows, which is pertinent information for researchers, veterinarians, and farmers. [ABSTRACT FROM AUTHOR]

Despite the rapid growth in popularity of the Jersey breed, most research on dairy cows in the United States has been done with the Holstein breed. Postpartum uterine diseases negatively influence reproductive performance of dairy cows and limited data are available regarding predisposing factors for uterine diseases in Jersey cows. Our objectives were to determine the prevalence and risk factors for purulent vaginal discharge (PVD) and its effect on fertility of lactating Jersey cows. This was a retrospective observational study with data collected from 3,822 Jersey cows. The Metricheck device was used for PVD diagnosis, and positive cases (≥50% of pus in exudate) were further classified for severity using the following 4 categories based on the amount of pus observed: 50 to 60% pus in exudate, 60 to 90% pus in exudate, 90 to 100% pus in exudate, and 90 to 100% pus in exudate + uterine fluid detected by palpation per rectum. Univariable and multivariable regression analyzes were conducted to dissect the risk factors for PVD and severity of PVD in Jerseys cows. The major risk factors for PVD were calving-related problems, retained fetal membrane, metritis, and days in the close-up diet. A subgroup of cows (n = 740) was scored for body condition and locomotion scores and had blood sampled in the peripartum for determination of plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate. In the multivariable analysis that considered data collected for the subgroup of cows, peripartum nonesterified fatty acids, postpartum β-hydroxybutyrate, and peripartum locomotion and body condition scores were not retained in the reduced model of predictors of PVD. Not surprisingly, pregnancy per artificial insemination following the first and second services was reduced in cows diagnosed with PVD compared with cows without PVD. In addition, PVD was associated with increased odds of pregnancy loss after the first service and reduced hazard of pregnancy by 305 d in milk. The stratification of PVD severity according to the amount of pus observed and the consistency of the uterus was meaningful, as observed by the differences in reproductive outcomes between cows with diverging amounts of pus in the retrieved exudate. Risk factors for PVD in Jersey cows was similar to previously reported for Holstein cows, and a strong detrimental effect of PVD on fertility was also observed in Jersey cows. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The objective of this study was to quantify the efficacy of a second intrauterine cephapirin treatment administered 14 d after the initial one on subsequent reproductive performance of postpartum dairy cows affected by purulent vaginal discharge (PVD) or endometritis (ENDO). In total, 4,140 Holstein cows from 30 commercial herds were enrolled in a randomized clinical trial. At 36 (±7) d in milk, cows were examined using the Metricheck device to diagnose PVD. An endometrial cytology sample was also collected from each cow to perform a leukocyte esterase test for diagnosing ENDO. Diagnosis of PVD and ENDO was done cow-side. Cows diagnosed with PVD or ENDO were assigned to receive 1 of 2 treatments: (1) a single intrauterine cephapirin infusion (500 mg of cephapirin benzathin; Metricure, Merck Animal Health, Kirkland, QC, Canada) at the time of initial examination or (2) a single intrauterine infusion at the time of initial examination and a second one 14 d later. Subsequent reproductive and culling events were collected until 200 d in milk. Statistical analyses were performed using univariable and multivariable mixed logistic regression models. In cows affected by PVD, a second intrauterine cephapirin infusion increased the pregnancy risk at first insemination in comparison with cows that only received one treatment (28.0 vs. 38.8%). In cows affected by ENDO, a second treatment also increased the pregnancy risk at first insemination compared with cows that only received one treatment (30.3 vs. 39.2%). Overall, these results demonstrate that administering a second intrauterine cephapirin infusion 14 d after the initial treatment in postpartum cows affected by PVD or ENDO did improve their subsequent reproductive performance. [ABSTRACT FROM AUTHOR]

The first aim of this study was to evaluate the relationship between the concentrations of plasma insulin-like growth factor-I (IGF-I) in the transition period and the incidence of clinical endometritis postpartum. The second aim was to evaluate the relationship between the concentration of plasma IGF-I in the transition period and the estrous synchrony response and pregnancy rate to a controlled internal drug release (CIDR) protocol. A total of 402 dairy cows, 250 multiparous and 152 primiparous, were enrolled. A blood sample was taken from all cows 2 and 1 wk precalving and wk 1, 3, 4, and 5 postcalving for subsequent analysis of IGF-I. Vaginal discharge score as a measurement of uterine health was recorded in wk 3, 4, and 5 postcalving. Estrous cycles of all cows were synchronized on ≥37 d in milk (mean = 59; range 37–93) using an 8-d CIDR protocol. All cows were scanned at 32 to 35 d after insemination to confirm pregnancy. Data were analyzed using Proc Mixed and Proc Logistic models in SAS (version 9.3; SAS Institute Inc., Cary, NC). Plasma IGF-I concentrations were significantly greater in the primiparous cows than in the multiparous cows, both before and after calving, and were therefore analyzed separately. In multiparous cows plasma IGF-I concentrations postcalving were significantly lower among cows with uterine infection than in those without infection, and multiparous cows with a plasma IGF-I concentration less than 40 ng/mL 1 wk after calving were 3 times more likely to suffer from a uterine infection 4 to 5 weeks postcalving (odds ratio 2.8; 95% confidence interval 1.0–7.6). Plasma IGF-I concentrations were significantly greater after calving in the 121 primiparous cows that exhibited estrus post-CIDR protocol (mean 125 ng/mL) than in the 25 primiparous cows that did not respond (mean 95 ng/mL). Multiparous cows with greater plasma IGF-I concentrations postcalving had a significantly greater chance of conceiving (mean plasma IGF-I concentration of conceiving cows = 86 ng/mL; nonconceiving = 70 ng/mL). These results show an association between greater concentrations of plasma IGF-I postcalving and response to CIDR protocol in primiparous animals (mean plasma IGF-I concentration of responders = 116 ng/mL; nonresponders = 95 ng/mL). In contrast no association was detected between concentrations of plasma IGF-I in relation to the response to the CIDR protocol in multiparous cows. In conclusion, changes in circulating concentrations of plasma IGF-I early postpartum may help predict reproductive status of dairy cows. [ABSTRACT FROM AUTHOR]

Endometritis, diagnosed either by assessing the proportion of nucleated cells that are neutrophils (PMN%) following cytology of the endometrium or by assessing the degree of purulent material within the vagina (purulent vaginal discharge or PVD score), is prevalent among dairy cows. However, limited data exist as to the degree of variation among herds in the prevalence of endometritis diagnosed by these 2 methods. Thus, we undertook a study involving uterine cytological and vaginal sampling at a median of 41 d in milk of 1,807 cows from 100 seasonally breeding dairy herds in New Zealand. The optimal cut-point for PMN% was determined by receiver operator characteristic (ROC) curve analysis using conception to first artificial insemination (AI) as the outcome variable. The prevalence of disease was then calculated at the cow and herd levels, and an estimate of the effect of clustering of cow within a herd was calculated. Reproductive outcomes were collated and associations between endometritis and reproductive outcomes assessed using multivariable models. The optimal PMN% cut-point was ≥2%. The correlation of results for cows within a herd (the intraclass correlation) was 0.03, which was significant; hence, subsequent modeling accounted for this clustering. The cow-level prevalence of PMN% ≥2% was 27.0% [95% confidence interval (CI): 25.0 to 29.1%], whereas the mean within-herd prevalence of PMN% ≥2% was 27.1% (95% CI: 24.7 to 29.6%; range: 5.0 to 63.6%), and the prevalence among herds varied significantly. An elevated PMN% (≥2%) was significantly associated with a reduction in the proportion of cows conceiving to first AI (45.8 vs. 54.5%), a reduced proportion of cows submitted for AI in the first 3 wk of the seasonal breeding program (83.7 vs. 89.3%), and a lower proportion pregnant in the first 3 (44.4 vs. 55.4%) and 6 wk (67.5 vs. 76.4%) of the breeding program relative to cows with a low PMN% (i.e., <2%). A total of 24.6% of cows had a PVD score ≥2. The herd mean prevalence of PVD score ≥2 was 25.1% (95% CI: 22.5 to 27.7%; range: 5.0 to 65.0%) and varied significantly among herds. The level of agreement (kappa) between the PVD score and PMN% was low (16.8%) and nonsignificant. The effects of PVD score and PMN% on reproductive outcomes were independent. The within-herd median prevalence of endometritis based on combining both diagnostic tests and using a Bayesian latent class model was 22.9% (Bayesian 95% CI: 10.4 to 40.1%). We conclude that more than one-fifth of dairy cows have endometritis diagnosed either by PMN% or PVD in seasonal breeding herds when assessed at an average of 41 DIM, which was, on average, 30 d before the start of the seasonal breeding program. There is large and unexplained variation in prevalence of endometritis among herds. The 2 diagnostic methods were both associated with reproductive outcomes but have low levels of agreement between them and their effects appear to be independent. [ABSTRACT FROM AUTHOR]

Uterine inflammation negatively affects reproductive performance and is an important cause of infertility and subfertility in dairy cows. Several studies have investigated the use of gene expression in endometrial samples collected by biopsy or cytology to evaluate the inflammatory response of the cow uterus. This study aimed to compare the expression of the CCL5 , CXCL8 , IL6 , and IL1B genes in the bovine endometrium according to the site of sample collection [caruncular (C) or intercaruncular (IC)], the collection method (biopsy or cytology), and the category of inflammation based on endometrial cytology (zero, medium, or high) in subclinical endometritis. The reproductive tracts of dairy cows were collected from a slaughterhouse, and punch-biopsy samples of endometrial tissues were obtained from both regions (C and IC). Endometrial cells from these regions were collected with the cytobrush technique and then used for the analysis of mRNA expression by quantitative PCR. After counting polymorphonuclear cells (PMN) by endometrial cytology, 20 uteri with an ovary at stage I (d 1–4 of estrous cycle) were categorized into 3 groups. Uteri with 0% PMN (n = 10) were assigned to group zero, uteri with 5 to 15% PMN (n = 5) to group medium (12.2 ± 1.6% PMN), and uteri with >15% PMN (n = 5) to group high (53.8 ± 32.9% PMN). All data were analyzed with 2-way ANOVA with Bonferroni multiple comparison post test. The results from gene transcripts demonstrated that the region (C or IC) of the endometrial biopsy had no influence on any of the degrees of inflammatory reaction observed. However, gene expression was more elevated in the endometrium of cows with greater inflammation compared with those without inflammation (CCL5 , CXCL8 , IL6 , IL1B) and those with medium inflammation (CCL5 , IL6). Expression of the genes evaluated did not differ between the endometrium without inflammation and with medium inflammation. However, in the high inflammation group, all genes were comparatively more expressed in samples collected by cytology relative to those derived from biopsies for both anatomical regions. In conclusion, gene expression did not differ between the C and IC tissue. Samples collected from animals with greater inflammation had greater gene expression than those with zero or medium inflammation. In addition, cytology samples had greater gene expression than biopsy samples in the high inflammation group. [ABSTRACT FROM AUTHOR]

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteriainfused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows. [ABSTRACT FROM AUTHOR]

The objective of this prospective field study was to evaluate the effects of extending the lactation period of high-yielding dairy cows on milk production, udder health characteristics, and development of body condition. On 40 d in milk (DIM), an examination of the genital tract (transrectal palpation, sonography, vaginoscopy) was performed. Cows without signs of clinical endometritis were blocked by parity and were randomly allocated to 1 of 3 experimental groups with a voluntary waiting period of 40, 120, and 180 d, respectively (G40, n = 135; G120, n = 141; G180, n = 139). Mean daily milk and energy-corrected milk production did not differ between the 3 groups regarding the first 305 d or for the whole lactation (d 1 and up to dry off, culling, or 600 DIM). In late lactation (306 to 600 DIM), G40 had lower average productivity (23.8 kg) compared with G120 (26.5 kg), with G180 showing intermediate values (25.7 kg). The extended lactation groups showed greater persistency, as the rate of decline based on a Wilmink function was lower for G120 (c = −0.063 and −0.045 for milk and energy-corrected milk, respectively) and G180 (c = −0.061 and −0.047) compared with G40 (c = −0.071 and −0.056). We found no difference between the 3 groups regarding the evaluated udder health characteristics (somatic cell count, incidence of mastitis, and days off milk due to mastitis). More cows in G180 (7.9%) were culled due to low productivity compared with G40 (0.7%) and as a tendency compared with G120 (2.8%). Moreover, cows of G180 showed higher median body condition score at the time of dry off compared with cows of both G40 and G120 (3.50 for G180 vs. 3.25 for both G40 and G120). At the time of dry off, G180 cows also had greater backfat thickness (25.0 mm) compared with both G40 (22.2 mm) and G120 cows (21.6 mm). Based on our results, the extension of the voluntary waiting period of high-yielding cows up to 120 d has no adverse effects regarding milk production, involuntary culling, udder health, or BCS gain. [ABSTRACT FROM AUTHOR]

The objective of the study was to update a prior meta-analysis on the efficacy of the treatment of bovine endometritis PGF2α with. Clinical endometritis in cattle is defined as the presence of a purulent or mucopurulent vaginal discharge after 20 or 26 d, respectively, postpartum. Although the prevalence and impact of this disease are well known, the questions of whether and how to treat endometritis are still controversially discussed. Hence, to ensure timeliness of scientific results, we updated an existing meta-analysis published in 2013 that showed no treatment effect of PGF2α in cases of bovine endometritis. As in the prior meta-analysis, a comprehensive literature search was conducted. After applying specific inclusion and exclusion criteria, 3 trials remained that could be added to the 6 trials in the initial meta-analysis. Data for each trial were analyzed using the meta-analysis software Review Manager (version 5.3; The Nordic Cochrane Centre, Copenhagen, Denmark). Estimated effect sizes of PGF2α were calculated on calving to first service interval (CFSI) and calving to conception interval (CCI). As revealed by the initial meta-analysis, PGF2α treatment still had no effect on both reproductive parameters. Heterogeneity remained substantial for CFSI and CCI and even increased for CCI compared with the initial meta-analysis. Subgroup analysis, however, did not reveal a source of heterogeneity. In updating the meta-analysis, publication bias for trials reporting CCI became even more pronounced, suggesting that studies with higher standard error (i.e., usually smaller studies) that reported a prolonging effect on CCI after a PGF2α treatment were less likely to be published. Finally, although the number of trials published on the chosen topic and their comparability are still limited, this update of our meta-analysis now provides results that are based on approximately twice the number of included cows. The overall outcome, however, has not substantially changed and a positive effect of a PGF2α treatment in case of clinical endometritis could not be shown. Therefore, we do not recommend a treatment of bovine endometritis with PGF2α as a means to improve reproductive performance. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine minimum inhibitory concentrations (MIC) of frequently used antimicrobials for Escherichia coli and Trueperella pyogenes isolated from postpartum bovine uteri of cows with acute puerperal metritis (APM, n = 67), cows suspected to have APM (n = 37), and healthy cows (n = 37) and to evaluate possible differences in MIC according to clinical signs. Cows with APM had reddish-brown, fetid vaginal discharge and rectal temperature (RT) ≥39.5°C within 21 d in milk; cows suspected to have APM had either reddish-brown, fetid vaginal discharge or RT ≥39.5°C within 21 d in milk; and healthy cows had neither fetid discharge nor RT ≥39.5°C. Samples were collected from cows on commercial dairy herds (n = 7) using the cytobrush technique. A total of 37 T. pyogenes isolates and 85 E. coli isolates were tested. Ceftiofur, a third-generation cephalosporin that is often used to treat APM, was the focus of analysis. Trueperella pyogenes and E. coli were isolated more often from samples of cows with APM (46 and 90%, respectively) compared with samples from healthy cows (19 and 54%, respectively). Regarding cows suspected to have APM, T. pyogenes and E. coli were numerically more often isolated (30 and 70%, respectively) than in healthy cows (19 and 54%, respectively). Minimum inhibitory concentrations of ceftiofur were low. For T. pyogenes and E. coli, MIC50 (concentration that inhibited growth of 50% of isolates) were 0.25 and 0.5 µg/mL and MIC90 (concentration that inhibited growth of 90% of isolates) were 0.5 and 1 µg/mL, respectively. Although ceftiofur inhibited all T. pyogenes at the highest concentration tested (64 µg/mL), the growth of 5.9% of E. coli was not impaired. Recently, ampicillin has been suggested as an alternative treatment for APM. Although the T. pyogenes isolates exhibited low MIC in general (MIC50 ≤0.015 µg/mL and MIC90 = 0.06 µg/mL) and 81.1% of all T. pyogenes could be inhibited at the lowest ampicillin concentration tested, 11.8% of the E. coli isolates were not impaired at the highest concentration (64 µg/mL) tested in this study. The MIC50 and MIC90 of E. coli were 4 and ≥128 µg/mL, respectively. We detected no difference in the MIC distributions of ceftiofur or ampicillin among isolates from the 3 APM groups. In summary, E. coli with high MIC against ceftiofur as well as against ampicillin were found in this study. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the effect of a selective antibiotic treatment strategy based on a quick bacteriological on-farm test (Petrifilm, 3M Corp., St. Paul, MN) compared with the conventional antibiotic treatment of all cows having clinical endometritis (CE) defined by the presence of purulent vaginal discharge on both clinical cure rate and reproductive performance. The study was simultaneously conducted with dairy cows reared under a highly supplemented rotational grazing system in Argentina and in a freestall system in Slovakia. Cows having an abnormal vaginal discharge (VD, indicative of clinical endometritis) on 21 to 35 d in milk (DIM) were randomly allocated to 1 of 2 study groups: selective treatment (ST) or conventional treatment (CT). All cows in the CT group (n = 174) received a single intrauterine administration of 500 mg of cephapirin. In the ST group (n = 178), treatment decision was made according to the results of the bacteriological on-farm test. For this test, we collected intrauterine samples with the cytobrush technique and stroke the brushes onto 2 different Petrifilm plates, one for aerobic count and another for Enterobacteriaceae count, incubated the plates, and counted the number of colonies after 24 h. Positive cows (≥5 colonies in one or both plates) received a single intrauterine treatment with 500 mg of cephapirin, whereas negative cows (<5 colonies) remained untreated. Clinical cure rate was assessed by direct vaginal inspection at 14 d after treatment (VD-0). The odds for conception at first artificial insemination, artificial insemination by 80 DIM, pregnancy by 100 DIM, and for nonpregnancy by 200 DIM were estimated with mixed logistic regression models. The hazard of conception was also assessed with proportional hazard regression model. The selective antibiotic treatment strategy based on the outcome of Petrifilm test reduced the number of required treatments (57%) and maintained similar efficacy in terms of clinical cure and reproductive performance as the conventional antibiotic treatment of all endometritic cows. [ABSTRACT FROM AUTHOR]

We investigated the microbiota found in the vaginas of Holstein dairy cows during the transition period and described the differences in bacterial composition and total bacterial load (TBL) associated with disease and fertility. Vaginal swabs were collected at -7, 0, 3, and 7 d relative to parturition from 111 dairy cows housed on a commercial dairy farm near Ithaca, New York. Microbiota were characterized by next-generation DNA sequencing of the bacterial 16S rRNA gene, and TBL was determined by real-time quantitative PCR. We applied repeated-measures ANOVA to evaluate the associations of uterine disease and related risk factors with the microbiota and TBL. We estimated phylum-specific bacterial load by multiplying the TBL by the relative abundance of each phylum observed in the metagenomics results. We confirmed the validity of this approach for estimating bacterial load by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical and healthy vaginal samples. Phyla associated with uterine disease and related risk factors were Proteobacteria, Fusobacteria, and Bacteroidetes. Cows with retained placenta and healthy cows had similar TBL at the day of parturition, but at d 7 postpartum, cows with retained placenta showed a significantly higher TBL, mainly driven by higher estimated loads of Fusobacteria and Bacteroidetes. Cows diagnosed with metritis had a significantly higher estimated load of Proteobacteria at d -7 and at calving and higher estimated loads of Fusobacteria in the postpartum samples. Additionally, the estimated load of Bacteroidetes at d 7 postpartum was higher for cows diagnosed with endometritis at 35 days in milk. Higher estimated loads of Fusobacteria and Bacteroidetes were also evident in cows with postpartum fever, in primiparous cows, in cows with assisted parturition, and in cows that gave birth to twins. Our findings demonstrated that microbiota composition and TBL were associated with known periparturient risk factors of uterine diseases and reproductive failure, including parity, assisted parturition, and retained fetal membranes. [ABSTRACT FROM AUTHOR]

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 45 d 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. [ABSTRACT FROM AUTHOR]

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9 mL) or Escherichia coli lipopolysaccharide (LPS; 3 µg/kg of body weight diluted in 9 mL of PBS) at 6-h intervals from 12 h before and until 9 d after ovulation during 2 cycles in a random order (ovulation = d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2 d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1β, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9 d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum. [ABSTRACT FROM AUTHOR]

This study compared physiological, health, and productive parameters in dairy cows supplemented or not with Omnigen-AF (OMN; Phibro Animal Health, Teaneck, NJ) during the transition period. Thiryeight nonlactating, multiparous, pregnant Holstein × Gir cows were ranked by body weight (BW) and body condition score (BCS), and assigned to receive (n = 19) or not (CON; n = 19) OMN at 56 g/cow daily (as-fed basis) beginning 35 d before expected date of calving. Before calving, cows were maintained in single drylot pen with ad libitum access to corn silage, and received (as-fed basis) 3 kg/cow daily of a concentrate. After calving, cows were moved to an adjacent drylot pen, milked twice daily, offered (as-fed basis) 35 kg/cow daily of corn silage, and individually received a concentrate formulated to meet their nutritional requirements after both milkings. Cows received OMN individually as top-dressing in the morning concentrate feeding. Before calving, cow BW and BCS were recorded weekly and blood samples were collected every 5 d beginning on d -35 relative to expected calving date. After calving and until 46 d in milk, BW and BCS were recorded weekly, individual milk production was recorded, and milk samples were collected daily for total solids and somatic cell count analyses. Blood was sampled daily from 0 to 7 d in milk, every other day from 9 to 21 d in milk, and every 5 d from 26 to 46 d in milk. On 30 and 46 d in milk, cows were evaluated for endometritis via cytobrush technique, based on % of polymorphonuclear (PMN) cells in 100 total cell count (PMN + endometrial cells). On 48.7 ± 1.6 d in milk, 9 cows/treatment received a lipopolysaccharide (LPS) injection (0.25 μg/ kg of BW), and blood was sampled hourly from -2 to 8 h, at 12-h intervals from 12 to 72 h, and at 24-h intervals form 96 to 120 h relative to LPS administration. No treatment differences were detected on BW, BCS, serum concentrations of cortisol, fatty acids, insulin, glucose, haptoglobin, cortisol, and insulin-like growth factor-I. Cows receiving OMN had greater milk yield (30.3 vs. 27.1 kg/d) and percentage of PMN cells in endometrial cell population (12.2 vs. 3.9%) compared with CON cows. After LPS administration, cows receiving OMN had greater mean serum haptoglobin (212 vs. 94 μg/mL), as well as greater serum concentration of tumor necrosis factor α at 1, 2, and 3 h relative to LPS injection compared with CON cows. In conclusion, OMN supplementation during the transition period enhanced innate immunity parameters and increased milk production in dairy cows. [ABSTRACT FROM AUTHOR]