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Up to 50 % of dairy cows fail to resolve uterine involution and develop chronic clinical (CE) or subclinical endometritis (SE) 21 days after calving. Clinical endometritis is associated with purulent discharge, while SE is not associated with overt clinical signs. Along with numerous knowledge gaps related to its pathogenesis, SE does not allow for a straightforward and effective therapy. Therefore, it is crucial to unravel differences in the expression of genes among healthy, CE, and SE cows. This might contribute to the discovery of new drug candidates and, in consequence, a potentially effective treatment. In the present study, cows between 21 and 28 days postpartum (PP) were examined using vaginoscopy for the presence of vaginal discharge and endometrial cytology for the determination of the endometrial polymorphonuclear cell (PMN) percentage. Next, an endometrial biopsy sample was taken to investigate the expression of 13 selected candidate genes by qPCR. Uterine health status was assigned to healthy (absence of abnormal vaginal discharge and ≤5 % PMN, n = 13), SE (absence of abnormal vaginal discharge and >5 % PMN, n = 30), and CE (mucopurulent or purulent vaginal discharge and >5 % PMN, n = 9). At the same time, a blood sample was collected to assess serum progesterone concentration and to categorize cows as low (≤1 ng/mL) or high (>1 ng/mL) in progesterone. High expression of IL1B, IL6, IL17A, CXCL8, PTGES, PTGS1, PTGS2, and INHBA genes and low expression of FST was noted in the endometrium of CE compared to healthy cows. Increased endometrial INHBA expression was observed in both SE and CE compared to healthy cows. Interestingly, greater expression of PTGES and PRXL2B genes and lower expression of PTGS2 were characteristic of SE versus CE or healthy. Among cows with no overt clinical symptoms of uterine disease (healthy and SE), the endometrial expression of IL1 B, CXCL8, and PTGES was greater in cows with high versus low serum progesterone. Several genes were differentially expressed among healthy, SE, and CE cows indicating different pathways for the development of different uterine diseases. In conclusion, we found progesterone-independent SE markers, which suggests that low endometrial PTGS2 expression may be indicative of an inadequate immune response and thus contribute to the pathogenesis of SE., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2023. Published by Elsevier Inc.)

Due to the critical contribution of the uterine-associated microbiota in reproductive health, physiology, and performance, culture-independent methods have been increasingly employed to unravel key aspects of microbial ecology in the uterus of cattle. Nowadays, we know that bacterial diversity is crucial to maintain uterine health, however, there is still no consensus on the exact composition of a healthy uterine microbiota (or eubiosis). Generally, loss of bacterial diversity (or dysbiosis) contributes to the development of uterine infections, associated with increased relative abundances of Bacteroides, Fusobacterium, Trueperella, and Porphyromonas. Uterine infections are highly prevalent and gravely influence the profitability of cattle operations, animal welfare, and public health. Thus, understanding the dynamics of uterine microbial ecology is essential to develop effective strategies focused on preventing and mitigating the adverse effects of uterine dysbiosis as well as assisting in the process of restoring the core, healthy uterine microbiota. The aim of this review is to summarize research conducted in the microbial ecology of bovine uteri. We discuss the origin of the uterine microflora of healthy cows and the factors influencing its composition. In addition, we review the biology of specific pathogens that are known to increase in abundance during the occurrence of uterine disease. Lastly, we provide an overview of the bacterial biofilm in the bovine endometrium, and we briefly summarize the rationale for the use of probiotics to prevent uterine disease in cattle., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The anogenital distance (AGD) is considered a marker for prenatal androgen exposure and fertility in multiple species including humans. In dairy cattle, it is described as the length between the center of the anus and the clitoral base (AGDc). However, in other species, the distance from the center of the anus to the dorsal commissure of the vulva (AGDv) is also considered to be a predictor for fertility traits, as well as the anogenital ratio (AGR, defined as [AGDv/AGDc]*100). The primary aim of the present study was to assess whether AGDv and AGR can be used as an indicator for reproductive performance in dairy heifers. Additionally, the relation between AGDv and AGDc and the correlation with other body measurements were explored. Data of 656 Holstein Friesian heifers at an age of 13.5 ± 1.08 months were analyzed. Respective means of 62.9 ± 8.20 mm (AGDv) and 107.6 ± 9.27 mm (AGDc) were recorded. The mean AGR ratio was calculated as 58.6 ± 6.75%, varying from 37.3 to 79.6%. The age of the heifers was not associated with any of the AGD measurements nor the ratio. Except for a very low correlation between heart girth and AGDc (r = 0.09, P < 0.05), both AGDs were largely uncorrelated with other body measurements. Linear regression models revealed that AGDc was not associated with any of the recorded fertility parameters. However, results revealed a negative association between AGDv and AGR and reproductive performance: heifers with a short AGDv and small AGR were younger at first AI (P ≤ 0.003) and at conception (P = 0.004). Based on ROC curve analyses, AGDv was the best indicator for pregnancy to first AI, with a threshold estimated at 65.3 mm. The pregnancy rate at first AI was 72.4% in heifers with a short AGDv (<65.3 mm, n = 413) compared to 61.7% in heifers with a long AGDv (≥65.3 mm, n = 243). Hence, short-AGDv heifers had 63% higher odds to conceive at first AI compared to their long-AGDv counterparts (P = 0.004). Additionally, an AGR threshold of 59,6% was determined: heifers with a small AGR (<59.6%) had 44% higher odds to be pregnant at first AI compared to heifers with an AGR ≥59.6%. Results of the present study suggest to consider AGDv and AGR as potential indicators for reproductive performance in dairy heifers. The latter implies that it is relevant to measure both AGDc and AGDv in future studies. The absence of correlation between body- and AGD-measurements furthermore suggests that AGD sizes are rather pre-than postnatally determined., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Isoflavones represent a class of phytoestrogens present in plants. In dairy cows, dietary isoflavones have been shown to negatively affect reproductive performance. To the best of our knowledge, no studies have yet been conducted to determine if calves are pre- or neonatally confronted with isoflavones and their metabolites. In the present study, we hypothesize that isoflavones are passed on from the dam to the offspring in utero. Twenty-three pregnant Holstein Friesian dams and their calves, originating from three commercial dairy farms in Belgium, were included. Heparin blood samples were collected during the first, second, and third trimester of gestation from all pregnant dams. Heparin blood and hair samples were obtained from the offspring within 24 h after parturition. Colostrum samples were collected from a subset of eight dams to determine the concentration of isoflavones and their metabolites. During the first and second trimester of gestation, the dams were fed either a youngstock (nulliparous dams) or a lactation (multiparous dams) diet. During the third trimester, both groups received a similar dry cow diet. Genistein and daidzein levels were unaffected by diet type, while their metabolite [equol, dihydrodaidzein (DHD), and o-desmethylangolensin (ODMA)] concentrations were significantly higher in the lactation group. Furthermore, metabolite concentrations decreased significantly during gestation. Isoflavones and their metabolites were detected in all colostrum samples. No correlation could be found between levels in colostrum and blood of pregnant dams or calves. Peripheral levels of isoflavones and their metabolites were significantly lower in newborn calves in comparison to their dams. Genistein and daidzein concentrations were found to be significantly higher in the calves' hair versus blood samples, suggesting prenatal exposure to isoflavones for an extended period of time. In contrast, no isoflavone metabolites were detected in the calves' hair samples. This is the first study to demonstrate that dairy calves are exposed to isoflavones during the developmentally most sensitive period of their lives. Results obtained pave the way for more extensive research to examine which effects isoflavones might have on developing organ systems like the reproductive system., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dairy cows diagnosed with metritis may experience a greater degree of oxidative stress (OS) and a deficit in the antioxidative capacity compared to healthy cows. We aimed to assess circulating OS markers and endometrial cell mitochondrial function, intracellular reactive oxygen species (ROS) production, and mean endometrial nuclear cell area in postpartum cows diagnosed with metritis or as healthy. From an initial pool of 121 Holstein cows, we retrospectively selected 34 cows and balanced for metritis (n = 17) or healthy (n = 17). Metritis was defined as an enlarged uterus with red-brown watery or thick off-white purulent discharge occurring within 21 days postpartum. Cows with no signs of clinical disease (including dystocia or retained placenta) were referred to as healthy. Blood samples for serum reactive oxygen metabolites (d-ROM), antioxidants (OXY), and oxidative status index (OSI) tests, evaluated via photometric determination of plasma thiols, were performed at 7, 14, 21, 28, and 35 days postpartum. Furthermore, from the initial pool, a random subset of 5 cows diagnosed with metritis and 6 diagnosed as healthy we collected (at the same time points as for the blood samples) endometrial cytology samples using the cytobrush technique. From the uterine samples, we evaluated the endometrial cell mitochondrial function, intracellular ROS levels, and the endometrial cell nuclear area using MitoTracker Orange, dichlorodihydrofluorescein diacetate, and Hoechst 33258, respectively. Mixed linear regression models, accounting for repeated measurements, were fitted to assess the effect of metritis versus healthy on circulating and endometrial cell OS parameters and endometrial cell size. The effect of days postpartum and its interaction with uterine health status were forced into each model. Serum concentrations of d-ROMs and OSI were greater in metritis at 7, 14, and 35 days postpartum than in healthy cows. Interestingly, the mean endometrial cell nuclear area was lower in metritis than healthy cows at 14 and 21 days postpartum. We found no differences between metritis and healthy for endometrial cell mitochondrial function and intracellular ROS production. In conclusion, cows diagnosed with metritis experienced greater systemic OS levels than healthy cows, but their OS was not higher in the uterine milieu., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at -5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at -5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 ± 0.4%; least-squares means ± standard error) and +21 d (8.5 ± 0.3%) than at +37 d (1.4 ± 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 ± 0.4 x 10 6 /mL) than postpartum (4.9 ± 0.4 x 10 6 /mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at -5 d (90.1 ± 1.5%) than at +37 d (94.1 ± 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 ± 5.1%) than at +21 d (20.9 ± 5.1%) and +37 d (11.9 ± 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 ± 6.3%) than at +37 d (54.9 ± 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 ± 3.4%) than at +37 d (13.3 ± 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Identification of factors associated with the quality and quantity of colostrum production has always been a major challenge in cattle industry. In purebred double-muscled Belgian Blue (BB) cows, parturition is mainly performed by elective caesarean section (CS; >90%). However, the CS itself may influence colostrum production characteristics. The present study aimed to evaluate the impact of maternal and newborn calf factors and the duration of the procedure of CS on the quality and quantity of colostrum production in BB cows. The dataset includes 551 records of cow-calf pairs that were presented for an elective CS at the Ghent University veterinary clinic between 2017 and 2019. The quality (measured via a colostrum densimeter) and the quantity (measured via a standard volume scale) of colostrum were measured within 30 min after the end of the CS. Fixed effects were fitted in mixed linear regression models to test for their potential association with colostrum quality (specific gravity; SG) and quantity (liters), and generalized mixed-effects models were constructed to test the associations of fixed effects with the optimal colostrum production index (yes vs no) based on an adequate supply of both colostrum quality and quantity. The fixed effects tested were parity, the gender of the calf, birth weight, duration of CS (min), and season of birth. Our results show that parity (primiparity), duration of CS (longer CS), and calving season (summer) had a significantly negative impact on colostrum production. Concluding, both colostrum quality and quantity can be influenced by intrinsic and extrinsic factors (including duration of CS), which should be considered while feeding newborn calves delivered via CS., Competing Interests: Declarations of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bovine in vitro embryo production (IVP) following Ovum Pick Up (OPU) is all too often hampered by a large time gap between the harvest of oocytes of the first and last OPU session of the day. Immediately after retrieval, oocyte maturation is initiated, resulting in oocytes maturing at different time points which necessitates laborious scheduling of the IVP process. In this study, the potential of a commercial embryo holding medium (EHM; Syngro, Bioniche Inc.) to hold immature bovine oocytes was validated. We assessed the effect of holding time and temperature on (1) oocytes' maturation; (2) blastocyst development and quality at day 8 post insemination; and (3) blastocyst yield in small groups of oocytes/zygotes simulating OPU settings. Oocytes, harvested from slaughterhouse ovaries, were held for 6 h (either at 4 °C, room temperature [RT; 22-25 °C], or 38.5 °C), for 10 h (at 4 °C or RT), and for 14 h (only at RT) in 1 mL sterile glass osmometer tubes filled with EHM prior to standard maturation (22 h at 38.5 °C) and subsequent IVP. Results were compared with controls in which no prior holding was applied. Differences between the treated and control groups were assessed by generalized mixed-effects models and considered significant at P < 0.05. Generally, oocytes held up to 14 h in EHM at different temperatures remained at the germinal vesicle stage. Holding immature oocytes in EHM for 6 h at 38.5 °C and for 10 h at 4 °C significantly decreased maturation (57.1 ± 4.1% VS 80.9 ± 3.2% and 68.6 ± 3.5% VS 80.7 ± 2.9%; respectively), and development (11.0 ± 1.8% VS 36.2 ± 2.8% and 20.1 ± 3.3% VS 40.6 ± 4.6%) (P < 0.05). However, holding in EHM for both 6 and 10 h at RT, did not affect the maturation rates (83.2 ± 2.9% and 78.9 ± 3.2%) nor day 8 blastocyst rates (35.2 ± 2.7% and 40.2 ± 4.5%). Prolonging holding time to 14 h in RT decreased maturation and day 8 blastocyst yield (71.9 ± 3.5% VS 84.5 ± 2.7% and 25.7 ± 2.5% VS 39.5 ± 2.8%, respectively) (P < 0.05). Holding oocytes in EHM did not significantly affect embryonic quality as assessed by differential apoptotic staining in any of the time points. To simulate OPU-settings, small groups of 10 oocytes were held in EHM for 6 or 10 h at RT. When subsequently matured, fertilized and cultured per 8 zygotes, day 8 blastocyst rate was not affected (19.8 ± 3.5% VS 20.6 ± 3.6% and 18.8 ± 3.6% VS 18.3 ± 3.4%). In conclusion, immature bovine oocytes can be successfully conserved in EHM at RT for up to 10 h without compromising their embryonic developmental competence nor quality., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of the present study was to compare endometrial cytology with histopathology to diagnose subclinical endometritis (SCE) in dairy cows. Endometrial cytology samples were collected from Holstein-Friesian cows (n = 32) just before slaughtering. Half of them were obtained by in vivo cytobrush (IV-CB), whereas the other half by in vivo low-volume lavage (IV-LVL). After slaughtering, reproductive tracts were collected, and the endometrium was sampled at eight locations. At each location, both a ex vivo cytobrush sample (EV-CB) and a tissue sample for histopathologic examination were taken. In the histopathology slides, polymorphonuclear (PMN) cell counts were differentiated as PMN cells in direct contact with the epithelial cells of the endometrium (PMN-EP), and PMN cells present in the deeper stratum compactum (PMN-SC). Summation of both countings was referred to as PMN-total. Pearson's correlation and Cohen's kappa coefficient were used to assess the correlation and agreement between both sampling methods (in vivo cytology [IV-CB and IV-LVL] with EV-CB and PMN-total). A Poisson mixed effect model was used to analyze the PMN cells' distribution. The prevalence of SCE was 18.75% (n = 6/32) for in vivo cytology. The SCE prevalence based on EV-CB analyses and on the assessment of PMN-total was determined both at the sample (n = 256) as well as at the cow level (n = 32): EV-CB 25% (n = 64/256) and 35.5% (n = 12/32), and PMN-total 37.11% (n = 95/256) and 59.38% (n = 19/32). Correlation and agreement between IV-CB and EV-CB were r = 0.81 and k = 0.97, whereas between IV-CB and PMN-total r = 0.15 and k = 0.23, respectively. In vivo low-volume lavage correlation and agreement were r = 0.52 and k = 0.66 with EV-CB, and r = 0.45 and k = 0.44 with PMN-total. Moreover, correlation and agreement between EV-CB and PMN-total were r = 0.60 and k = 0.50, respectively. More PMN cells (P < 0.05) were detected in PMN-SC when compared to PMN-EP and EV-CB. A higher SCE prevalence was found using histopathology, rendering the latter as a more sensitive method to diagnose SCE in comparison to in vivo and ex vivo cytology. Although cytology had low and/or moderate sensitivity to diagnose SCE when compared with histopathology, its specificity is 100%, implying that all cows that were indicated to suffer from SCE using in vivo cytology were confirmed to do so by histopathologic examination. There is an uneven distribution of PMN cells throughout the endometrium, generally more PMN cells being found in the deeper stratum compactum than in contact with the superficial layers of the endometrium., (Copyright © 2016 Elsevier Inc. All rights reserved.)