Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d 14 ± 6), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with me-tritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t -tests and fold change analyses. Me-tabolites with false discovery rate adjusted P ≤ 0.10 on t -tests were used for partial least squares – discriminant analysis PLS-DA coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t -tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.


INTRODUCTION
Metritis is a highly prevalent disease in Holstein cows shortly after calving (Pinedo et al., 2020), causing severe economic losses to the dairy industry (Pérez-Báez et al., 2021).Metritis is characterized by a dysbiosis of the uterine microbiome in which pathogenic bacteria such as Fusobacterium necrophorum, Bacteroides pyogenes, and Porphyromonas levii overtake the uterine commensals (Jeon et al., 2015(Jeon et al., , 2016;;Galvão et al., 2019).Whether pathogenic bacteria proliferate and overtake the uterine commensals is determined by how efficiently the immune system respond against these pathogens.In this regard, cows that develop metritis have been shown to experience immune dysregulation around parturition, which was characterized by systemic inflammation and immune cell activation in the prepartum period (Casaro et al., 2023;Galvão et al., 2012), decreased gene expression and production of proinflammatory cytokines, and decreased polymorphonuclear cells (PMN) phagocytosis and killing activity postpartum (Hammon et al., 2006;Galvão et al., 2011Galvão et al., , 2012)).This immune dysregulation has been associated with greater prepartum body weight and body fat mobilization (Casaro et al., 2023), greater accumulation of circulating fatty acid and ketone bodies (Hammon et al., 2006), less blood calcium (Martinez et al., 2012), and greater levels of oxidative stress before the onset of metritis (Mikulková et al., 2020).
Although the associations between individual blood analytes related to lipolysis and inflammation, immune function, and metritis have been studied, the blood metabolomic changes associated with metritis development must involve more analytes than nonesterified fatty acids (Hammon et al., 2006;Ospina et al., 2010), ketone bodies (Hammon et al., 2006;Ospina et al., 2010), calcium (Martinez et al., 2012), or haptoglobin (Huzzey et al., 2009).Indeed, previous research using targeted serum metabolomics showed that most of the metabolites that differed between healthy and metritic cows from 8 weeks prepartum until metritis diagnosis were products of protein metabolism such as the amino acids valine, leucine, ornithine, serine, and glutamic acid (Hailemariam et al., 2018).
In addition to finding differences in metabolite concentrations, blood metabolomics can offer new insights into cellular mechanisms associated with lipolysis, inflammation, and immune function, which may predispose cows to metritis development.Therefore, the hypothesis of the current study was that the plasma metabolites from cows that develop metritis are associated with specific cellular pathways and processes involved in lipolysis, inflammation, and immune dysregulation.Hence, the objective of the study was to compare the peripartum plasma metabolome between cows that develop metritis and cows that do not develop metritis and use the significant metabolites to explore cellular pathways and processes.

MATERIALS AND METHODS
This case-control observational study was conducted at the University of Florida Dairy Unit from September of 2019 to March of 2020.
Because the study was part of a study evaluating immune responses, the sample size was based on previous studies evaluating immune responses in dairy cows with and without uterine disease (Galvão et al., 2010(Galvão et al., , 2012;;Martinez et al., 2012).A sample size, similar to the one used to evaluate neutrophil phagocytosis and killing ability (n = 110 cows; 55 with risk factors for metritis and 55 without risk factors for metritis; Martinez et al., 2012), was selected for this study.Following enrollment of 110 cows, 52 cows developed metritis and 58 remained healthy.This sample size allows for detection of significant differences in continuous outcomes with an effect size of 0.4, α = 0.05, and β = 0.80.Analyses of the urine or serum metabolome using principal component analysis, and partial least square discriminant analysis (PLS-DA; Dervishi et al., 2016a,b;Hailemariam et al., 2018) were able to show statistical differences including 6 cows per group; therefore, inclusion of a larger number of cows per group (n = 52) was expected to ensure sufficient power for the characterization of changes in the plasma metabolome associated with metritis in the current study.All procedures involving cows were approved by the Institutional Animal Care and Use Committee of the University of Florida; protocol number 201910623.

Cows, Housing, and Feeding
A total of 128 Holstein cows (71 primigravid and 57 multigravid) were enrolled at 260 d of gestation and followed until 13 ± 1 d relative to parturition (DRP).All cows were housed in freestall barns with sand-bedded stalls pre-and postpartum.In the prepartum period, multigravid cows were fed a TMR formulated to meet or exceed the nutrient requirements for dry Holstein cows weighing 680 kg (NRC, 2001) twice daily.Postpartum, multiparous cows were fed a TMR formulated to meet or exceed the nutrient requirements for lactating Holstein cows weighing 680 kg and producing 45 kg of 3.5% fat-corrected milk (NRC, 2001) twice daily.Nulliparous cows were housed in a freestall barn with individual feeding gates (Calan Broadbent Feeding System, American Calan Inc., Northwood, NH) before 241 d of gestation and were fed a TMR once daily.After parturition, primiparous cows were moved to a postpartum pen also equipped with individual feeding gates and each cow was assigned to an individual gate until 100 DRP.Nulliparous animals were part of an experiment where they were assigned to a control, which was offered 100 g/cow/d ground corn as a top-dress to the total mixed diet, and to a treatment, which consisted of top-dress addition of calcidiol (ROVIMIX Hy-D 1.25%.12.5 mg/g calcidiol; DSM Nutritional Products, Parsippany, NJ) at a concentration of 10 mg/kg ground corn from 248 d of gestation until 100 DRP.Postpartum, all cows were milked 2 times a day at 0600 and 1800 h.The rolling herd average milk yield was approximately 11,000 kg.

Case Definition and Diagnosis
Metritis was diagnosed by examination of the uterine discharge with a Metricheck device (Metricheck, Simcro, New Zealand) at 3 ± 1, 7 ± 1, 10 ± 1 and 13 ± 1 DRP using a 5-point scale as previously described (Jeon et al., 2016): 1 = not fetid normal lochia, viscous, clear, red, or brown; 2 = cloudy, pink, red, or brown mucoid discharge with flecks of pus; 3 = not fetid, pink red or brown mucopurulent discharge with <50% pus; 4 = not fetid, pink, red or brown purulent discharge with ≥50% pus; 5 = fetid red-brownish, watery discharge.Cows with a discharge score ≤4 were classified as healthy and cows with a score of 5 in at least one examination were classified as having metritis.
Incidences of mastitis, digestive problems, respiratory disease, and antimicrobial treatments in the first 35 DRP were also recorded for individual cows, and cows with any of these diseases, cows submitted to antimicrobial treatment before metritis diagnosis, and cows diagnosed with metritis after 10 DRP were excluded from the study.A total of 13 cows were excluded.Four cows were excluded because they were treated with antimicrobials before metritis diagnosis.Three cows were excluded because of death.One cow was excluded because of uterine torsion and one cow was excluded because of peritonitis.Four cows were excluded because they were diagnosed with metritis at 13 ± 1 DRP; therefore, could not be paired to a healthy counterpart.A total of 52 cows with metritis were used for the analysis, which were paired with 52 healthy counterparts.

Blood Sample Collection and Plasma Metabolome Analysis
All cows had blood collected in the prepartum period (−14 DRP), calving (first 24h after calving), and at diagnosis (day of metritis diagnosis).Blood was sampled from the jugular vein using a 20-gauge x 2.54-cm needle and 10-mL evacuated tube containing lithium heparin (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA).After collection, the blood tubes were placed on ice and transported to the laboratory within 2 h.Once in the laboratory, the blood tubes were centrifuged at 4000 g, 4 °C, for 10 min, and the plasma was stored at −80°C for further characterization of the plasma metabolome.The frozen plasma was submitted to the University of California's West Coast Metabolomics Center in Davis, CA for metabolome analysis.Samples were analyzed by blinded technicians using untargeted gas chromatography with time-of-flight mass spectrometry (GC-TOF-MS) in a single batch as previously described (Fiehn et al., 2008;Fiehn, 2016).The carrier selected was helium gas, and a column comprised of 95% dimethyl/5diphenyl polysiloxanesne was used.Column flow-rate was set at 1 mL/min, and initial oven temperature was set at 50°C followed by a 20°C increase per min up to a final temperature of 330°C, which was held constant for a period of 5 min.Injection temperature was set to begin at 50°C followed by a 12°C increase per second up to 250°C.Retention of primary metabolites was evaluated using default settings from ChromaTOF v. 2.32 and quantification was reported as peak height.Each metabolite was identified based on its mass and charge relationship.Metabolites were annotated using PubChem, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Human Metabolome Database.From the 1,014 detected metabolites, a total of 265 metabolites were annotated, and 749 were unknown.The metabolome data set and metadata are available at the NIH Common Fund's National Metabolomics Data Repository website, the Metabolomics Workbench (Sud et al., 2016), under Study ID ST002556; DOI: 10.21228/M8PF0K.

Statistical Analysis
Differences in metabolome associated with metritis were analyzed at each time point separately by univariate analyses using Metaboanalyst 5.0 (Pang et al., 2021) and RStudio 2023.03.0+386 (RStudio, PBC, Boston, MA).Data were first log-transformed and autoscaled.To analyze differences in the plasma metabolome between metritis and parity on each time point, we employed permutational analyses of variance (PER-MANOVA) based on Euclidean distances with 9,999 permutations using the Adonis2 function of the Vegan Package in RStudio.The models included the effects of metritis (metritis vs. no metritis), parity (primiparous vs. multiparous), and their interaction.Because no significant interaction (P > 0.08) was observed between metritis and parity, we used t-tests and fold change analyses to assess the differences in individual metabolites between metritis and no metritis groups at different time points.All P-values obtained from t-tests were adjusted for false discovery rate (FDR) to account for multiple testing (Benjamini and Hochberg, 1995).
To measure the importance of each significant metabolites, (PLS-DA) were performed with the metabolites with FDR adjusted P ≤ 0.10 on t-tests.Permutational analysis using 2,000 permutations were performed to each PLS-DA to evaluate the significance of the difference between cows that developed metritis and cows that did not develop metritis.Variance importance projection (VIP) scores were calculated from PLS-DA.
Metabolites with FDR adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses based on the KEGG database for Bos taurus using Metaboanalyst 5.0 (Pang et al., 2021), compound network analyses using Metscape 2 (Karnovsky et al., 2012) within the CytoScape 3.8 platform, and identification of affected cellular processes using the Ingenuity Pathway Analysis (IPA) Metabolomics platform (Krämer et al., 2014).Metabolic pathways with FDR adjusted P ≤ 0.05 and impact ≥0.10 (Liu et al., 2016(Liu et al., , 2019) ) were affected.Compound network analysis was performed to visualize the changes in metabolites associated with the different metabolic pathways.Categories of cellular processes with a Bonferroni adjusted P ≤ 0.05 were affected and further explored.Within affected categories, because Pvalues only measure enrichment but not directionality, only cellular processes with a Bonferroni adjusted P ≤ 0.05 and a predicted Z-score were considered as affected (Krämer et al., 2014).Affected cellular processes with a Bonferroni adjusted P ≤ 0.05 but without a predicted Z-score denote a nonclear pattern of direction related to the literature; thus, IPA does not make a prediction.A Chi-Square test was used to compare the proportion of primiparous cows that developed metritis in the control (n = 18/31) and calcidiol-treated groups (n = 19/33), and we detected no differences between treatments (P = 0.98).

Z-scores >0 indicate upregulated processes in cows
Statistical significance was considered at P ≤ 0.05, and a tendency was considered at 0.05 < P ≤ 0.10.

RESULTS
A total of 1,014 unique metabolites were identified by GC-TOF-MS in all plasma samples (Supplemental Table S1).Supplemental table and figures are available in the Mendeley Data Repository (DOI: 10 .17632/z9t5td2v2n .1)Prepartum t-Tests showed that 89 metabolites differed (FDR adjusted P ≤ 0.10; Supplemental Table S2) between cows that developed metritis and cows that did not, and of these, 26 were annotated and 63 were not annotated.
Affected categories of cellular processes in the prepartum period were amino acid metabolism (P = 0.007; Figure 2), cell death and survival (P = 0.03; Figure 2), and cellular compromise (P = 0.03; Figure 2); therefore, were further explored.Within cell death and survival, and cellular compromise categories, predicted Z-score (Z) were not produced, indicating a nonclear pattern of direction related to the literature.Within amino acid metabolism, transport (Z = −1.4; Figure 3) and efflux (Z = −2.0; Figure 3) of amino acids were downregulated in cows that developed metritis when compared with cows that did not develop metritis.

Calving
t-Tests showed that 49 metabolites differed (FDR adjusted P ≤ 0.10; Supplemental Table S2) between cows that developed metritis and cows that did not, and of these, 26 were annotated and 23 were not annotated.
Affected categories of cellular processes at calving were inflammatory response (P < 0.001; Figure 2), small molecule biochemistry (P = 0.003; Figure 2), lipid metabolism (P = 0.003; Figure 2), cell death and survival (P = 0.003; Figure 2), carbohydrate metabolism (P = 0.006; Figure 2), energy production (P = 0.01; Figure 2), cell-to-cell signaling (P = 0.01; Figure 2), vitamin and mineral metabolism (P = 0.02; Figure 2), free radical scavenging (P = 0.02; Figure 2), immune cell trafficking (P = 0.02; Figure 2), and protein synthesis (P = 0.03; Figure 2); therefore, were further explored.All enriched categories had predicted Z-scores from at least one cellular process.Concentration of lipids (Z = 1.54; Figure 3), lipolysis (Z = 1.98; Figure 3), synthesis of reactive oxygen species (ROS; Z = 1.66; Figure 3), peroxidation of lipids (Z = 1.94; Figure 3), apoptosis (Z = 1.84; Figure 3), necrosis (Z = 2.45; Figure 3), activation of leukocytes (Z = 0.70; Figure 3), quantity of calcium (Z = 1.73; Figure 3), uptake of glucose (Z = 1.95; Figure 3), synthesis of carbohydrates (Z = 0.88; Figure 3), and metabolism of carbohydrates (Z = 0.52; Figure 3), were upregulated whereas concentration of ATP (Z = −0.86; Figure 3), metabolism of protein (Z = −0.58; Figure 3), and cell viability (Z = −0.48; Figure 3) were downregulated in cows that developed metritis when compared with cows that did not develop metritis.Compound network of significant metabolic pathways associated with differences in plasma metabolites identified by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) at Prepartum (14 ± 6 d before calving; yellow circle), Calving (first 24h after calving; blue circle), and Diagnosis (day of metritis diagnosis; 7 ± 2; red circle) between dairy cows that developed metritis (n = 52) and dairy cows that did not develop metritis (n = 52; reference).Metabolic pathways significances were based on enriched pathway analysis using Metaboanalyst 5.0.Metabolic pathways with FDR adjusted P ≤ 0.05 and impact ≥0.10 were affected and are shown in the figure.Compound network analysis was performed using Metscape 2 within the CytoScape 3.8 platform to visualize the changes in metabolites associated with the different metabolic pathways.Hexagons in green correspond to metabolites that differed between cows that developed metritis and cows that did not develop metritis, while blue hexagons correspond to remaining metabolites that compose the metabolic pathway but were not different between cows that developed metritis and cows that did not develop metritis or were not recognized in the database.Large hexagons represent metabolites with greater concentration, while small hexagons represent metabolites with lower concentration in the plasma metabolome of cows that developed metritis when compared with cows that did not develop metritis.The number next to each metabolite that were identified in each metabolic pathway correspond to the impact that specific metabolite has in each pathway.The total pathway impact corresponds to the sum of the individual metabolites impact.TCA, tricarboxylic acid.Glyox, glyoxylate.Dicarb, dicarboxylate.Arg, arginine.Prol, proline.Gly, glycine.Ser, serine.Thre, threonine.IP, inositol phosphate.

Diagnosis
t-Tests showed that 168 metabolites differed (FDR adjusted P ≤ 0.10; Supplemental Table S2) between cows that developed metritis and cows that did not, and of these, 66 were annotated and 102 were not annotated.

DISCUSSION
Herein, peripartum plasma metabolome was compared between cows that developed metritis and cows that did not develop metritis and the significant metab- 2) between dairy cows that developed metritis (n = 52) and dairy cows that did not develop metritis (n = 52; reference).Because P-values only measure enrichment but not directionality, only cellular processes with a Bonferroni adjusted P ≤ 0.05 and a predicted Z-score are shown.Affected cellular processes with a Bonferroni adjusted P ≤ 0.05 but without a predicted Z-score denote a nonclear pattern of direction related to the literature; thus, Ingenuity Pathway Analysis does not make a prediction.Z-scores >0 indicate upregulated processes in cows with metritis and Z-scores <0 indicate downregulated processes in cows with metritis.Redundant cellular processes were excluded for ease of interpretation, but they can be found in Supplemental Table S3.ATP, Adenosine triphosphate.Ca, calcium.ROS, reactive oxygen species.Figure was created using Excel (Version 16.17.Microsoft Corporation).
olites were used to assess their importance in between group discrimination and to explore cellular pathways and processes with the hypothesis that these cellular pathways and processes would be associated with lipolysis, systemic inflammation, oxidative stress, and immune dysregulation.Cows that developed metritis had lower amino acid metabolism in the prepartum period, suggesting less amino acid intake or more amino acid utilization.At calving, cows that developed metritis had affected cellular processes associated with greater lipolysis, cell death, oxidative stress, and greater leukocyte activation.At metritis diagnosis, cows that developed metritis continued to have affected cellular processes associated with greater lipolysis, cell death, and oxidative stress.Contrary to the day of calving, cows that developed metritis had affected cellular processes associated with a reduction in immune cell activation at metritis diagnosis.In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.Nonetheless, it is important to note the observational nature of our study; therefore, further experimentational studies need to be conducted to confirm our findings.
During the prepartum period, cows that developed metritis had impacted metabolic pathways associated with amino acid metabolism, with lower concentration of L-serine, L-valine, L-leucine, D-glycerate, Lthreonine, L-ornithine, and L-citrulline.These changes were explored at a cellular level by the IPA.Cows that developed metritis had affected cellular processes associated with lower cellular transport and efflux of amino acids.Although most cows have feed intake reduction around parturition, cows that develop metritis have a more pronounced decrease in feed intake in the last weeks prepartum (Hammon et al., 2006;Pérez-Báez et al., 2019), which leads to lower protein intake; thus, a reduction in feed intake could explain the lower concentration of plasma amino acids.Additionally, cows that develop metritis have been shown to be in a proinflammatory state in the prepartum period, characterized by greater concentration of proinflammatory cytokines and immune cell activation (Casaro et al., 2023;Kasimanickam et al., 2013).In the early stages of an inflammatory response, the liver increases the uptake of amino acids for acute phase protein production (Sax et al., 1988); therefore, it is also plausible that the lower concentration of plasma amino acids may indicate the initiation of an inflammatory response.Furthermore, cows that developed metritis had greater cell death in the prepartum period (Casaro et al., 2023), which is known to induce immune cell activation (Gong et al., 2020).Herein, we observed that cows that developed metritis had enriched cellular categories associated with cell death and survival, and cellular compromise in the prepartum period, which corroborates our previous findings.Therefore, both the decrease in DMI and the inflammatory state could be driving the decrease in plasma amino acids.Contrary to our findings, previous research observed greater concentrations of valine, leucine, ornithine, and serine 8 and 4 weeks prepartum in cows that developed metritis compared with cows that remained healthy (Hailemariam et al., 2018).This discrepancy may be explained by the differences in methodology or differences in sampling time.The untargeted approach used herein is appropriate for global metabolomics profiling, whereas a targeted approach (Hailemariam et al., 2018) is more suitable for absolute quantification and validation of observed features (Martin et al., 2015).Furthermore, it is possible that differences in DMI were not present before 14 d prepartum (Huzzey et al., 2007;Pérez-Báez et al., 2019).
At calving, cows that developed metritis had impacted metabolic pathways associated with amino acid (aminoacyl-tRNA biosynthesis, tryptophan, glycine, serine, threonine, and arginine metabolism), energy (inositol phosphate metabolism, pentose and glucuronate interconversions), and lipid (glycerol metabolism) metabolism, and inflammatory response (arachidonic acid metabolism).These changes were explored at a cellular level by the IPA.Cows that developed metritis had affected cellular processes associated with higher concentration of lipids, greater lipolysis, and lower metabolism of protein.The more pronounced decrease in feed intake cows that develop metritis undergo in the last days before calving leads to greater fatty acid mobilization (Hammon et al., 2006;Pérez-Báez et al., 2019); therefore, the changes associated with protein and lipid metabolism indicate a more pronounced negative nutrient balance by the day of calving in cows that developed metritis when compared with cows that did not develop metritis.Moreover, during negative nutrient balance peripheral blood leukocytes take up free fatty acids (Contreras et al., 2010), which would explain the predicted higher cellular concentration of lipids in the peripheral blood of cows that developed metritis.Cows that developed metritis also had affected cellular processes associated with lower concentration of intracellular ATP and higher synthesis of ROS.Excessive fatty acid mobilization leads to inhibition of the electron transport chain (Cocco et al., 1999).The main goal of the electron transport chain is to produce ATP (Schönfeld and Wojtczak, 2008).Furthermore, the inhibition of the electron transport chain is one of the mechanisms by which excessive fatty acid mobilization enhances the synthesis of ROS (Cocco et al., 1999;Schönfeld and Wojtczak, 2008); therefore, the reduction in intracellular ATP together with the increase in ROS synthesis suggest that the greater lipid mobilization in cows that developed metritis led to the inhibition of the electron transport chain in the mitochondria, reducing the production of ATP and increasing the synthesis of ROS.Moreover, exacerbated lipolysis enhances peroxisomal, cytosolic, and cellular membrane ROS production, and may enhance endoplasmic reticulum ROS production (Abou-Rjeileh and Contreras, 2021), which may contribute to the higher synthesis of ROS predicted by the IPA.Furthermore, cows that developed metritis had affected cellular processes associated with higher peroxidation of lipids, lower cell viability, higher cell death, and higher leukocyte activation.Reactive oxygen species react with the cell lipid membranes, causing lipid peroxidation (Reilly et al., 1991).Lipid peroxidation leads to cell damage, and if excessive, causes cell death (Reilly et al., 1991).Following cell death, damage associated molecular patterns are released to the extracellular space (Roh and Sohn, 2018), triggering the activation of immune cells and promoting an inflammatory response (Gong et al., 2020).These results suggest that the greater leukocyte activation may be a response to damage associated molecular patterns released from dying cells.Lastly, cows that developed metritis had affected cellular processes associated with higher intracellular calcium and higher carbohydrate metabolism indicated by higher uptake of glucose, and higher synthesis and metabolism of carbohydrates.Upon activation, neutrophils and macrophages require calcium for antigen processing (Jaconi et al., 1990).Furthermore, glycolysis is the major metabolic pathway in the cytosol of neutrophils, which upon activation use the absorbed glucose to produce energy for chemotaxis (Kumar and Dikshit, 2019).Therefore, the cellular processes associated with greater calcium and carbohydrate metabolism suggest a greater usage of these nutrients by the activated immune cells in cows that developed metritis.
The day of metritis diagnosis cows that developed metritis had impacted metabolic pathways associated with amino acid (aminoacyl-tRNA biosynthesis, tryptophan, glycine, serine, threonine, arginine, proline, β-alanine, cysteine, and methionine metabolism), energy (TCA cycle, glyoxylate and dicarboxylate metabolism, and inositol phosphate metabolism), and pyrimidine metabolism.These changes were explored at a cellular level by the IPA.Cows that developed metritis had affected cellular processes associated with higher concentration of lipids, higher synthesis of ROS, lower concentration of ATP, lower concentration of glutathione, and lower quantity of amino acids, suggesting that the greater negative energy balance, lipid mobilization, and synthesis of ROS observed at calving are maintained at the day of metritis diagnosis.Furthermore, the greater synthesis of ROS together with the lower concentration of glutathione, which is the major cellular antioxidant (Coleman et al., 2020), suggests that cows were undergoing oxidative stress (Abuelo et al., 2019;Mikulková et al., 2020).Cows that developed metritis also had affected cellular processes associated with higher uptake, but lower efflux of amino acids, and lower metabolism of protein, which suggests that cells were using the amino acids for cellular processes other than protein synthesis.Given that cows with metritis had cellular processes associated with greater lipolysis and oxidative stress, it is likely that the amino acids were preferentially used for energy and antioxidant production (Li et al., 2007).Indeed, the metabolites that were involved in these associations were glycine, L-methionine, L-serine, L-threonine, L-alanine, and hydroxyproline, which were lower, and sarcosine, which was higher in cows that developed metritis.L-alanine is the major gluconeogenic amino acid (Aschenbach et al., 2010); thus, the greater uptake and lower efflux suggests that L-alanine was being used for de novo generation of glucose.Glycine, Lmethionine, L-serine, L-threonine, and hydroxyproline are involved in the synthesis of glutathione.Sarcosine is an N-methyl derivative of glycine, and its administration to rats induced an increase in lipid peroxidation and oxidative stress (de Andrade et al., 2017).Cows that developed metritis had affected cellular processes associated with higher oxidative stress; thus, the lower concentration of these amino acids together with the greater concentration of sarcosine suggests that the amino acids were most likely used for glutathione production.Cows that developed metritis also had affected cellular processes associated with greater cell death by necrosis and lower cell viability suggesting that the changes associated with greater cell damage observed at calving are maintained at the day of metritis diagnosis.Nonetheless, contrary to what was observed at calving, cows that developed metritis had affected cellular processes associated with lower activation of leukocytes.Although cows that develop metritis are undergoing systemic inflammation already before calving, they have lower monocyte and T cell activation in the first week postpartum (Casaro et al., 2023) indicating that the overall activation status of leukocytes is lower in cows that developed metritis.It is plausible that the prolonged inflammatory response observed due to greater oxidative stress and cell damage before calving, or already before calving (Casaro et al., 2023), could be leading to immune tolerance, which is characterized by an exhausted pool or circulating leukocytes refractory to stimulation (Rogovskii, 2020).Furthermore, cows that developed metritis had affected cellular processes associated with lower quantity of calcium, greater me- tabolism of carbohydrates, and decreased accumulation of carbohydrates.As previously mentioned, neutrophils and macrophages require calcium for antigen processing (Jaconi et al., 1990), therefore, it is plausible that the lower immune cell activation led to less uptake of calcium, or that the less availability of calcium led to lower immune cell activation.Cows that develop metritis had less serum calcium concentrations from 1 to 12 d after parturition (Martinez et al., 2012), making the latter more likely.Moreover, phagocytes depend almost exclusively on their stored glycogen for phagocytosis (Weisdorf et al., 1982), and cows that develop metritis have PMN with lower glycogen storages in the first week postpartum (Galvão et al., 2010) .Therefore, a lower accumulation together with a greater metabolism of carbohydrates suggests a reduction in immune cell glycogen storage.Results from the day of calving suggest that cows that developed metritis had a more pronounced negative nutrient balance and had greater leukocyte activation; thus, it is plausible that these cells depleted their glycogen storage upon activation at calving, leading to both less immune cell activation and lower accumulation of carbohydrates at the day of metritis diagnosis.Together, the lower quantity of calcium, higher metabolism of carbohydrates, and decreased accumulation of carbohydrates suggest another explanation for the reduction in immune cell activation in cows that developed metritis.
This observational study provides insights into the peripartum plasma metabolomics of cows that develop metritis; however, caution must be taken when interpreting IPA results given that their databases have low representation of ruminant experiments.

CONCLUSIONS
We observed that cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis.In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.
Casaro et al.: PERIPARTUM METABOLIC CHANGES IN COWS WITH METRITIS Casaro et al.: PERIPARTUM METABOLIC CHANGES IN COWS WITH METRITIS with metritis and Z-scores <0 indicate downregulated processes in cows with metritis.
Casaro et al.: PERIPARTUM METABOLIC CHANGES IN COWS WITH METRITIS Figure1.Compound network of significant metabolic pathways associated with differences in plasma metabolites identified by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) at Prepartum (14 ± 6 d before calving; yellow circle), Calving (first 24h after calving; blue circle), and Diagnosis (day of metritis diagnosis; 7 ± 2; red circle) between dairy cows that developed metritis (n = 52) and dairy cows that did not develop metritis (n = 52; reference).Metabolic pathways significances were based on enriched pathway analysis using Metaboanalyst 5.0.Metabolic pathways with FDR adjusted P ≤ 0.05 and impact ≥0.10 were affected and are shown in the figure.Compound network analysis was performed using Metscape 2 within the CytoScape 3.8 platform to visualize the changes in metabolites associated with the different metabolic pathways.Hexagons in green correspond to metabolites that differed between cows that developed metritis and cows that did not develop metritis, while blue hexagons correspond to remaining metabolites that compose the metabolic pathway but were not different between cows that developed metritis and cows that did not develop metritis or were not recognized in the database.Large hexagons represent metabolites with greater concentration, while small hexagons represent metabolites with lower concentration in the plasma metabolome of cows that developed metritis when compared with cows that did not develop metritis.The number next to each metabolite that were identified in each metabolic pathway correspond to the impact that specific metabolite has in each pathway.The total pathway impact corresponds to the sum of the individual metabolites impact.TCA, tricarboxylic acid.Glyox, glyoxylate.Dicarb, dicarboxylate.Arg, arginine.Prol, proline.Gly, glycine.Ser, serine.Thre, threonine.IP, inositol phosphate.
Figure2.List of cellular processes categories associated with differences in plasma metabolites identified by gas chromatography timeof-flight mass spectrometry (GC-TOF-MS) at Prepartum (14 ± 6 d before calving), Calving (First 24h after calving), and Diagnosis (day of diagnosis; 7 ± 2) between dairy cows that developed metritis (n = 52) and dairy cows that did not develop metritis (n = 52).Orange lines correspond to a Bonferroni corrected P-value of 0.05.Statistical significance was considered at P ≤ 0.05. Figure was created using Excel (Version 16.17.Microsoft Corporation).

Figure 3 .
Figure3.List of cellular processes associated with differences in plasma metabolites identified by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) at Prepartum (14 ± 6 d before calving), Calving (first 24h after calving), and Diagnosis (day of diagnosis; 7 ± 2) between dairy cows that developed metritis (n = 52) and dairy cows that did not develop metritis (n = 52; reference).Because P-values only measure enrichment but not directionality, only cellular processes with a Bonferroni adjusted P ≤ 0.05 and a predicted Z-score are shown.Affected cellular processes with a Bonferroni adjusted P ≤ 0.05 but without a predicted Z-score denote a nonclear pattern of direction related to the literature; thus, Ingenuity Pathway Analysis does not make a prediction.Z-scores >0 indicate upregulated processes in cows with metritis and Z-scores <0 indicate downregulated processes in cows with metritis.Redundant cellular processes were excluded for ease of interpretation, but they can be found in Supplemental TableS3.ATP, Adenosine triphosphate.Ca, calcium.ROS, reactive oxygen species.Figure was created using Excel (Version 16.17.Microsoft Corporation).
Casaro et al.: PERIPARTUM METABOLIC CHANGES IN COWS WITH METRITIS Casaro et al.: PERIPARTUM METABOLIC CHANGES IN COWS WITH METRITIS