The Complex Interplay of Insulin Resistance and Metabolic Inflammation in Transition Dairy Cows.

During the transition period, dairy cows exhibit heightened energy requirements to sustain fetal growth and lactogenesis. The mammary gland and the growing fetus increase their demand for glucose, leading to the mobilization of lipids to support the function of tissues that can use fatty acids as energy substrates. These physiological adaptations lead to negative energy balance, metabolic inflammation, and transient insulin resistance (IR), processes that are part of the normal homeorhetic adaptations related to parturition and subsequent lactation. Insulin resistance is characterized by a reduced biological response of insulin-sensitive tissues to normal physiological concentrations of insulin. Metabolic inflammation is characterized by a chronic, low-level inflammatory state that is strongly associated with metabolic disorders. The relationship between IR and metabolic inflammation in transitioning cows is intricate and mutually influential. On one hand, IR may play a role in the initiation of metabolic inflammation by promoting lipolysis in adipose tissue and increasing the release of free fatty acids. Metabolic inflammation, conversely, triggers inflammatory signaling pathways by pro-inflammatory cytokines, thereby leading to impaired insulin signaling. The interaction of these factors results in a harmful cycle in which IR and metabolic inflammation mutually reinforce each other. This article offers a comprehensive review of recent advancements in the research on IR, metabolic inflammation, and their intricate interrelationship. The text delves into multiple facets of physiological regulation, pathogenesis, and their consequent impacts.

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3-11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 µg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 µmol/L CA and 80 µg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1ß, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.

The importance of developmental programming in the dairy industry.

The concept of developmental programming suggests that environmental influences during pre- and early postnatal life that can have long-term effects on future health and performance. In dairy cattle, maternal body growth, age, parity and milk yield, as well as environmental factors during gestation, have the potential to create a suboptimal environment for the developing fetus. As a result, the calf's phenotype may undergo adaptations. Moreover, developmental programming can have long-term effects on subsequent birth weight, immunity and metabolism, as well as on postnatal growth, body composition, fertility, milk yield and even longevity of dairy cows. This review provides an overview of the impact of developmental programming on later health and performance in dairy cows.

Progesterone-independent endometrial mRNA expression in dairy cows with clinical or subclinical endometritis.

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.

Manual versus deep learning measurements to evaluate cumulus expansion of bovine oocytes and its relationship with embryo development in vitro.

Cumulus expansion is an important indicator of oocyte maturation and has been suggested to be indicative of greater oocyte developmental capacity. Although multiple methods have been described to assess cumulus expansion, none of them is considered a gold standard. Additionally, these methods are subjective and time-consuming. In this manuscript, the reliability of three cumulus expansion measurement methods was assessed, and a deep learning model was created to automatically perform the measurement. Cumulus expansion of 232 cumulus-oocyte complexes was evaluated by three independent observers using three methods: (1) measurement of the cumulus area, (2) measurement of three distances between the zona pellucida and outer cumulus, and (3) scoring cumulus expansion on a 5-point Likert scale. The reliability of the methods was calculated in terms of intraclass-correlation coefficients (ICC) for both inter- and intra-observer agreements. The area method resulted in the best overall inter-observer agreement with an ICC of 0.89 versus 0.54 and 0.30 for the 3-distance and scoring methods, respectively. Therefore, the area method served as the base to create a deep learning model, AI-xpansion, which reaches a human-level performance in terms of average rank, bias and variance. To evaluate the accuracy of the methods, the results of cumulus expansion calculations were linked to embryonic development. Cumulus expansion had increased significantly in oocytes that achieved successful embryo development when measured by AI-xpansion, the area- or 3-distance method, while this was not the case for the scoring method. Measuring the area is the most reliable method to manually evaluate cumulus expansion, whilst deep learning automatically performs the calculation with human-level precision and high accuracy and could therefore be a valuable prospective tool for embryologists.

The dogma of the sterile uterus revisited: does microbial seeding occur during fetal life in humans and animals?

In brief: Opposing conclusions have been drawn regarding the presence of viable bacteria in the healthy pregnant uterus. Current evidence in humans and animals suggests that fetomaternal tissues present only traces of bacteria whose viability is still to be proven. Abstract: The debate about the pioneer colonization of the fetus is still open, being the 'in utero colonization' hypothesis versus the 'sterile womb paradigm' the two opposing sides. The seed in this field of research sprouted in human medicine in the last decade and became a central topic in other mammals as well. We aimed to review the literature on bacterial colonization of the healthy placenta, amniotic fluid, and meconium as representatives of the fetal environment. What emerges is that confirming the colonization of fetomaternal tissues by viable bacteria is challenging in humans as well as in animals. Contamination represents the major risk in this type of research as it can be related to different parts of the study design. Sampling at natural parturition or postpartum introduces risk for colonization by the vaginal microbiome of the mother or from the environment. Culture does not reveal the presence of unculturable microorganisms, and sequencing does not allow confirming bacterial viability, while also introducing the variability associated with the data analysis. Therefore, on the basis of the present review, we provide some guidelines on the best practices when performing this type of studies. What emerges from the current literature in humans and animals is that fetomaternal tissues are characterized by a very low biomass, that the viability of bacteria eventually present is still to be confirmed, while massive colonization happens at birth, priming the individual, regardless of the species.

The anogenital ratio as an indicator of reproductive performance in dairy heifers.

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.

Genesis of clinical and subclinical endometritis in dairy cows.

In brief: Clinical and subclinical endometritis are different manifestations of reproductive tract inflammatory disease in dairy cows. This review addresses the genesis of clinical and subclinical endometritis considering metabolic stress, innate immune dysfunction, and shifts in the composition of the uterine microbiota in the postpartum period. Abstract: Up to half of dairy cows may develop one or more types of reproductive tract inflammatory disease within 5 weeks after calving. Clinical endometritis (CE) results from uterine bacterial dysbiosis with increased relative abundance of pathogenic bacteria associated with luminal epithelial damage. These bacteria cause endometrial stromal cell lysis, followed by massive polymorphonuclear neutrophil (PMN) migration, and pyogenesis. CE is defined as endometrial inflammation accompanied by purulent discharge. Purulent discharge is not always accompanied by uterine inflammation (being (rarely) vaginitis or (commonly) cervicitis), hence referred to as purulent vaginal discharge (PVD). Subclinical endometritis (SCE) is an asymptomatic uterine disease defined by a threshold of PMN on cytology that is associated with worse reproductive performance; it has not been linked with bacterial dysbiosis. Current evidence suggests that SCE is a result of metabolic and inflammatory dysfunction that impairs innate immune function and the ability of endometrial PMN to undergo apoptosis, necrosis, and ultimately achieve resolution of inflammation. CE and SCE are diagnosed between 3 and 5 weeks postpartum and commonly overlap, but they are considered distinct manifestations of reproductive tract inflammatory disease. This review addresses the genesis of CE and SCE in postpartum dairy cows considering metabolic stress, innate immune dysfunction, and shifts in the composition of the uterine microbiota.

Detection of Chlamydia and Chlamydia-like organisms in bovine placental tissue.

This study aimed to investigate the presence of Chlamydia spp. and Parachlamydia acanthamoebae in bovine placental tissue originating from abortion and non-abortion cases in Belgium. Placentas of 164 late term bovine abortions (last trimester of gestation) and 41 non-abortion (collected after calving) cases were analysed by PCR for Chlamydia spp., Chlamydia abortus, C. psittaci and P. acanthamoebae. Additionally, a subset of 101 (75 abortion and 26 non-abortion cases) of these placenta samples were also analysed by histopathology to detect possible Chlamydia-induced lesions. In 5.4% (11/205) of the cases, Chlamydia spp. were detected, and three of those cases were positive for C. psittaci. Parachlamydia acanthamoebae was detected in 36% (75/205) of the cases, being 44% (n = 72) in abortions and 7.3% (n = 3) in non-abortions cases (p < .001). None of the cases was positive for C. abortus. Purulent and/or necrotizing placentitis with or without vasculitis was observed in 18.8% (19/101) of the histopathologically analysed placenta samples. In 5.9% (6/101) of the cases, placentitis was observed along with vasculitis. In the abortion cases, 24% (18/75) of the samples showed purulent and/or necrotizing placentitis, while purulent and/or necrotizing placentitis was visible in 3.9% (1/26) of the non-abortion cases. Placental lesions of inflammation and/or necrosis were present in 44% (15/34) of the cases where P. acanthamoebae was detected, while inflammation and/or necrosis was present in 20.9% (14/67) of the negative cases (p < .05). The detection of Chlamydia spp. and especially P. acanthamoebae, in combination with correlated histological lesions such as purulent and/or necrotizing placentitis and/or vasculitis in placental tissue following abortion, suggests a potential role of this pathogen in cases of bovine abortion in Belgium. Further in-depth studies are necessary to unravel the role of these species as abortifacient agents in cattle and to include them in bovine abortion monitoring programmes.

Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.

Detection of Anaplasma phagocytophilum in fetal and placental tissue of bovine abortions and perinatal mortalities.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne zoonotic bacterium that is the aetiologic pathogen of tick-borne fever (TBF) in ruminants. In clinical bovine cases of TBF, abortion and stillbirth may be observed. However, in this regard, the pathophysiology of TBF has not yet been completely elucidated, and no clear guidelines to diagnose A. phagocytophilum-related abortions and perinatal mortalities (APM) are available. METHODS: This exploratory study aimed to investigate the presence of A. phagocytophilum in bovine cases of APM and determine whether placental or fetal spleen tissue has the greatest sensitivity for A. phagocytophilum identification. The placenta and fetal spleen of 150 late-term bovine APM cases were analysed using real-time PCR to detect A. phagocytophilum. RESULTS: A total of 2.7% of sampled placentas were positive for A. phagocytophilum, while none of the fetal spleen samples was. LIMITATIONS: No histopathology to detect associated lesions was performed. Consequently, no evidence of causality between the detection of A. phagocytophilum and APM events could be achieved. CONCLUSION: The detection of A. phagocytophilum suggests a potential role of this pathogen in bovine APM, and placental tissue seems to be the most suitable tissue for its identification.

Viability and function dynamics of circulating versus endometrial polymorphonuclear leukocytes in postpartum dairy cows with subclinical or clinical endometritis.

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.

Prenatal environment impacts telomere length in newborn dairy heifers.

Telomere length is associated with longevity and survival in multiple species. In human population-based studies, multiple prenatal factors have been described to be associated with a newborn's telomere length. In the present study, we measured relative leukocyte telomere length in 210 Holstein Friesian heifers, within the first ten days of life. The dam's age, parity, and milk production parameters, as well as environmental factors during gestation were assessed for their potential effect on telomere length. We found that for both primi- and multiparous dams, the telomere length was 1.16% shorter for each day increase in the calf's age at sampling (P = 0.017). The dam's age at parturition (P = 0.045), and the median temperature-humidity index (THI) during the third trimester of gestation (P = 0.006) were also negatively associated with the calves' TL. Investigating multiparous dams separately, only the calf's age at sampling was significantly and negatively associated with the calves' TL (P = 0.025). Results of the present study support the hypothesis that in cattle, early life telomere length is influenced by prenatal factors. Furthermore, the results suggest that selecting heifers born in winter out of young dams might contribute to increased longevity in dairy cattle.

Dairy calves are exposed to isoflavones during the developmentally most sensitive period of their life.

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.

Studying the pre-implantation uterine microbiota in cattle using transabdominal laparoscopic low-volume lavage: Aiming for zero-contamination.

Recent studies have suggested that bacteria associated with the female reproductive tract - the uterine microbiota - may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.

Circulating and endometrial cell oxidative stress in dairy cows diagnosed with metritis.

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.

Continuous activation of the IL-17F driven inflammatory pathway in acute and chronic digital dermatitis lesions in dairy cattle.

Objectives of the present study were to get a deeper insight into the course of the inflammatory pathways of digital dermatitis lesions in dairy cattle by investigating the gene expression patterns throughout the different clinical stages (M0 to M4.1) of the disease. Normal skin samples (M0) were used as a reference for comparing the gene expression levels in the other M-stages through RNA Seq-technology. Principal component analysis revealed a distinct gene expression pattern associated with digital dermatitis lesions in comparison to healthy skin with a further clustering of the acute M1, M2 and M4.1 stages versus the chronic M3 and M4 stages. The majority of the up-and downregulated genes in the acute and chronic stages can be placed into a common 'core' set of genes involved in inflammation, such as A2ML1, PI3, CCL11 and elafin-like protein, whereas the most downregulated genes included keratins and anti-inflammatory molecules such as SCGB1D and MGC151921. Pathway analysis indicated the activation of the pro-inflammatory IL-17 signaling pathway in all the M stages through the upregulation of IL-17F. These results indicate that digital dermatitis is associated with an excessive inflammatory immune response concomitant with a disrupted skin barrier and impaired wound repair mechanism. Importantly, despite their macroscopically healed appearance, a significant inflammatory response (Padj < 0.05) was still measurable in the M3 and M4 lesions, potentially explaining the frequent re-activation of such lesions.

Maladaptation to the transition period and consequences on fertility of dairy cows.

After parturition, dairy cows undergo a plethora of metabolic, inflammatory, and immunologic changes to adapt to the onset of lactation. These changes are mainly due to the homeorhetic shift to support milk production when nutrient demand exceeds dietary intake, resulting in a state of negative energy balance. Negative energy balance in postpartum dairy cows is characterized by upregulated adipose tissue modelling, insulin resistance, and systemic inflammation. However, half of the postpartum cows fail to adapt to these changes and develop one or more types of clinical and subclinical disease within 5 weeks after calving, and this is escorted by impaired reproductive performance in the same lactation. Maladaptation to the transition period exerts molecular and structural changes in the follicular and reproductive tract fluids, the microenvironment in which oocyte maturation, fertilization, and embryo development occur. Although the negative effects of transition diseases on fertility are well-known, the involved pathways are only partially understood. This review reconstructs the mechanism of maladaptation to lactation in the transition period, explores their key (patho)physiological effects on reproductive organs, and briefly describes potential carryover effects on fertility in the same lactation.

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

In Vitro Production of Neutrophils Extracellular Traps Is Affected by the Lactational Stage of Dairy Cows.

We aimed to research the neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) formation capacity of polymorphonuclear cells (PMN) during different lactational stages of Holstein cows. We also aimed to validate a model which could mimic infection and inflammation in vitro by adding increasing concentrations of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) to PMN suspensions isolated from nulliparous heifers and evaluate their capacity to produce NETs and ROS. In 3 replicates, we collected blood from nulliparous heifers (n = 3), cows at the end of gestation (n = 3), early postpartum (n = 3) and in mid-lactation (n = 3) in which PMN were isolated. The production of ROS in PMN were assessed using the 2',7'-Dichlorofluorescein diacetate method, while the SYTOX Orange and Quant-iT™ PicoGreen dsDNA ultra-sensitive nucleic fluorescent acid staining methods were applied in order to quantitatively analyze the formation of NETs. Statistical analyses were performed via linear regression models using the replicate as a random. ROS values of PMN harvested from peripartum cows were 1.3 times increased compared with those in nulliparous heifers (p < 0.01). Compared with nulliparous heifers, the production of NETs by PMN isolated from mid-lactation and postpartum cows was 2.1 and 2.5 times higher (p < 0.01), respectively. In 3 replicates, in vitro stimulation of PMN isolated from nulliparous heifers (n = 3) with LPS linearly increased the production of ROS and NETs (R2 = 0.96 and 0.86, respectively). Similarly, when PMN isolated from nulliparous heifers were stimulated with PMA, a linear increase in the production of ROS (R2 = 0.99) and NETs (R2 = 0.78) was observed. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic inflammation and study fundamental aspects of the production of NETs and ROS in vitro.