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Targeted direct injection/liquid chromatography coupled to tandem mass spectrometry-based metabolomics was employed to identify metabolite alterations that could differentiate subclinical mastitis (SCM) from control (CON) dairy cows at -8, -4, disease diagnosis, +4 and +8 wks relative to parturition. We identified and measured 128 metabolites in the serum. Univariate analysis revealed significant alterations of serum metabolites at all five time points studied. By applying multivariate analyses including principle component analysis and partial least squares-discriminant analysis, some of the metabolites were found to have the strongest power for discriminating the SCM from CON cows. The top five metabolites with the greatest variable importance in projection values were selected as potential biomarkers for SCM. A set of five serum metabolites including lysine, ornithine, isoleucine, LysoPC a C17:0, and leucine at -8 wks and five other metabolites including lysine, leucine, isoleucine, kynurenine, and sphingomyelin (SM) C26:0 at -4 wks prepartum were determined as predictive biomarkers for SCM, which provided highly predictive capabilities with AUC (area under the curve) at 1.00. Five metabolites including lysine, leucine, isoleucine, kynurenine, and SM C26:1 in the serum were identified as diagnostic biomarkers for SCM with the AUC of 1.00. Moreover, we observed that distinct metabolic pathways were affected in SCM cows including lysine degradation, biotin, cysteine, methionine, and glutathione metabolism, valine, leucine, and isoleucine biosynthesis and degradation, and aminoacyl-tRNA biosynthesis prior to and during the occurrence of the disease. Results of this study showed that metabolomics analyses can be used to identify susceptible cows to SCM starting from -8 and -4 wks prepartum and that blood can be used to diagnose cows with SCM.

Mastitis is an inflammation of the mammary gland initiated by pathogenic bacteria. In fact, mastitis is the second most important reason for the culling of cows from dairy herds, after infertility. In this review we focus on various forms of mastitis, including subclinical and clinical mastitis. We also stress the importance of the dry-off period as an important time when pathogenic bacteria might start their insult to the mammary gland. An important part of the review is the negative effects of mastitis on milk production and composition, as well as economic consequences for dairy farms. The two most important groups of bacteria that are involved in infection of the udder, Gram-negative and Gram-positive bacteria, are also discussed. Although all cows have both innate and adaptive immunity against most pathogens, some are more susceptible to the disease than others. That’s why we summarize the most important components of innate and adaptive immunity so that the reader understands the specific immune responses of the udder to pathogenic bacteria. One of the most important sections of this review is interrelationship of mastitis with other diseases, especially retained placenta, metritis and endometritis, ketosis, and laminitis. Is mastitis the cause or the consequence of this disease? Finally, the review concludes with treatment and preventive approaches to mastitis.

Postpartum uterine diseases are common in dairy cows and are a great concern for the dairy industry as they are associated with various consequences, including lower fertility, lower milk yield, and an overall negative impact on the host health. An infected uterus is a source of bacterial compounds and cytokines that spill into the systemic circulation, spreading inflammation to other organs. In this review article, we discuss a short overview of the anatomy of the reproductive tract of dairy cows and several infectious diseases of the uterus including metritis, endometritis, and pyometra. Additionally, we discuss the microbiome of the reproductive tract in health and during uterine diseases. As well, diagnostic criteria for metritis and endometritis and contributing factors for increased susceptibility to metritis infection are important topics of this review. To better understand how the uterus and reproductive tract respond to bacterial pathogens, a section of this review is dedicated to immunity of the reproductive tract. Both the innate and adaptive immunity systems are also discussed. We conclude the review with a factual discussion about the current treatments of uterine diseases and the new developments in the area of application of probiotics for uterine health. Mechanisms of actions of probiotics are discussed in detail and also some applications to prevent uterine infections in dairy cows are discussed.

During the periparturient period there is a significant increase in the incidence of multiple metabolic and infectious diseases in dairy cows. Dairy cows are fed high-grain diets immediately after calving to support production of large amounts of milk. Mounting evidence indicates these types of diets are associated with the release of high amounts of endotoxins in the rumen fluid. If infected, the udder and uterus additionally become important sources of endotoxins during the postpartum period. There is increasing evidence that endotoxins translocate from rumen, uterus, or udder into the systemic circulation and trigger chronic low-grade inflammatory conditions associated with multiple diseases including fatty liver, mastitis, retained placenta, metritis, laminitis, displaced abomasum, milk fever, and downer cow syndrome. Interestingly, endotoxin-related diseases are triggered by a bacterial component and not by a specific bacterium. This makes prevention of these type of diseases different from classical infectious diseases. Prevention of translocation of endotoxins into the host systemic circulation needs to take priority and this could be achieved with a new approach: mucosal vaccination. In this review article, we discuss all the aforementioned issues in detail and also report some of our trials with regards to mucosal vaccination of periparturient dairy cows.

Ketosis, characterized by high concentrations of ketone bodies in the blood, urine, and milk, affects a considerable number of cows immediately after calving. Although much is known about ketosis, dairy cows continue to be affected in every herd world-wide. Cows affected by ketosis are treated with palliative treatments after the disease is diagnosed. This is a very expensive approach and costs the dairy industry extra expenses, contributing to lower profitability of dairy herds. In this review article, we summarize the mainstream view on ketosis, classification of ketosis into three types, current diagnostic approaches to ketosis, and the economic impact of ketosis on dairy farms. Additionally, we discuss the most recent applications of the new ‘omics’ science of metabolomics in studying the etiopathology of ketosis as well as its contribution in identification of novel screening or diagnostic biomarkers of ketosis.

The objective of this study was to determine urinary metabotypes of dairy cows prior to, during, and after diagnosis of subclinical mastitis (SCM). Twenty controls (CON) and 6 cows with SCM were included in the study. DI/LC-MS/MS was used to measure 186 metabolites in the urine at −8 and − 4 wks prepartum, disease diagnosis, and at +4 and + 8 wks postpartum. Results showed a total of 24 and 27 metabolites that differentiated SCM and CON cows at −8 and − 4 wks, respectively, with 5 top metabolites having an accuracy to predict SCM of 1.0, for both time point. Altered metabolites included several acylcarnitines (ACs), phosphatidylcholines (PCs), amino acids (AAs), and biogenic amines (BAs). During SCM diagnosis week there were a total of 22 metabolites that differentiated the SCM and CON cows including 13 ACs, 5 AAs, hexose, and phosphatidylethanolamine ae C44:3. The top 5 metabolites Symmetric dimethylarginine (SDMA), methylglutarylcarnitine, dodecanoylcarnitine, phosphatidylethanolamine ae C42:1, and phosphatidylethanolamine ae C42:0 showed an AUC of 1.0. Metabolite alterations continued at +4 and + 8 wks postpartum with 13 (9 ACs, 3 glycerophospholipids, and 1 BA) and 28 metabolites (14 ACs, 2 glycerophospholipids, hexose, 8 AAs, and 2 BAs) that differentiated the two groups, respectively. The top 5 most important metabolites, for both time points, showed AUCs of 1.0. Results of this study showed that typical urinary metabotypes preceded SCM event starting from −8 and − 4 wks prepartum. Metabotypes typical of SCM continued to be present during the week of SCM and at +4 and + 8 wks postpartum. These data show that the urine of dairy cows can be used with very high accuracy for screening dairy cows for susceptibility to SCM prior to entering into the dry off period and a potential new pen-side test can help dairy producers and veterinarians to make selective treatments.

The objective of this study was to evaluate whether whole raw milk originating from Holstein dairy cows affected by lameness alters its composition. A total of 20 healthy control cows and 6 cows diagnosed with lameness were selected out of 100 sampled cows in a nested case control study at 2 weeks postpartum, and whole raw milk samples were collected and analyzed with direct inject/liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. In total, 168 metabolites were identified and quantified using an in-house mass spectrometry library. A total of 35 of the identified metabolites decreased versus control cows. Only two metabolites (i.e., sn-glycero-3-phosphocholine and phosphatidylethanolamine ae C42:1) were increased in the milk of lame cows. In conclusion, milk metabotyping of lame cows revealed significant changes in multiple milk components, including amino acids, lipids, and biogenic amines. Most of the milk compounds identified as altered were lowered, suggesting deflection of nutrients from the mammary gland to the host needs for healing lameness-associated pathological processes.

Previously we observed that bacterial lipopolysaccharide (LPS) was able to instantly convert recombinant murine prion protein (moPrP) from an alpha-helical to a beta-sheet enriched state. The objectives of this study were to evaluate the effects of a single in vitro administration of recombinant moPrP alone or combined with detoxified lipopolysaccharide (D-LPS) on innate immunity and antibacterial gene expression in the colon of male FVB/N mice, under an Ussing chamber system. Results showed that moPrP alone affected the expression of genes related to both toll-like receptor (TLR)- and nod-like receptor (NLR)-signaling as well as pro- and anti-inflammatory responses. moPrP induced a strong antibacterial response with Slpi mRNA over expression (9-fold). Combination of moPrP with D-LPS on the mucosal side of the colon induced genes associated with TLR-signaling, apoptosis, and a very strong antibacterial response (35-fold Slpi expression). Administration of moPrP on the mucosal side and D-LPS on the serosal side triggered expression of 12 genes related to TLR signaling, apoptosis, and antibacterial responses, as illustrated by overexpression of Slpi by30-fold. The over expression of Slpi mRNA was further reaffirmed by ELISA and when moPrP was added to the mucosal side and D-LPS on the serosal side, an increased Slpi protein was observed. Application of combined moPrP and D-LPS on the mucosal side significantly increased the Slpi protein. Results of this study demonstrated that moPrP alone or combined with D-LPS affected the expression of various genes related to inflammation, antibacterial, and apoptotic responses.

The retained placenta is a common pathology of dairy cows. It is associated with a significant drop in the dry matter intake, milk yield, and increased susceptibility of dairy cows to metritis, mastitis, and displaced abomasum. The objective of this study was to identify metabolic alterations that precede and are associated with the disease occurrence. Blood samples were collected from 100 dairy cows at −8 and −4 weeks prior to parturition and on the day of retained placenta, and only 16 healthy cows and 6 cows affected by retained placenta were selected to measure serum polar metabolites by a targeted gas chromatography–mass spectroscopy (GC-MS) metabolomics approach. A total of 27 metabolites were identified and quantified in the serum. There were 10, 18, and 17 metabolites identified as being significantly altered during the three time periods studied. However, only nine metabolites were identified as being shared among the three time periods including five amino acids (Asp, Glu, Ser, Thr, and Tyr), one sugar (myo-inositol), phosphoric acid, and urea. The identified metabolites can be used as predictive biomarkers for the risk of retained placenta in dairy cows and might help explain the metabolic processes that occur prior to the incidence of the disease and throw light on the pathomechanisms of the disease.

Lameness is a very important disorder of periparturient dairy cows with implications on milk production and composition as well as with consequences on reproductive performance. The aetiology of lameness is not clear although there have been various hypotheses suggested over the years. The objective of this study was to metabotype the urine of dairy cows prior to, during and after the onset of lameness by evaluating at weeks -8, -4 pre-calving, the week of lameness diagnosis, and +4 and +8 weeks post-calving. We used a metabolomics approach to analyse urine samples collected from dairy cows around calving (6 cows with lameness v. 20 healthy control cows). A total of 153 metabolites were identified and quantified using an in-house MS library and classified into 6 groups including: 11 amino acids (AAs), 39 acylcarnitines (ACs), 3 biogenic amines (BAs), 84 glycerophospholipids, 15 sphingolipids and hexose. A total of 23, 36, 40, 23 and 49 metabolites were observed to be significantly different between the lame and healthy cows at -8 and -4 weeks pre-calving, week of lameness diagnosis as well as at +4 and +8 weeks post-calving, respectively. It should be noted that most of the identified metabolites were elevated; however, a few of them were also lower in lame cows. Overall, ACs and glycerophospholipids, specifically phosphatidylcholines (PCs), were the metabolite groups displaying the strongest differences in the urine of pre-lame and lame cows. Lysophosphatidylcholines (LysoPCs), although to a lesser extent than PCs, were altered at all time points. Alterations in urinary AA concentrations were also observed during the current study for four time points. During the pre-calving period, there was an observed elevation of arginine (-8 week), tyrosine (-8 week) and aspartate (-4 week), as well as a depression of urinary glutamate (-4 weeks). In the current study, it was additionally observed that concentrations of several sphingomyelins and one BA were altered in pre-lame and lame cows. Symmetric dimethylarginine was elevated at both -8 weeks pre-calving and the week of lameness diagnosis. Data showed that urinary fingerprinting might be a reliable methodology to be used in the future to differentiate lame cows from healthy ones.

Ketosis and subclinical ketosis are widespread among dairy cows especially after calving. Etiopathology of ketosis has been related to negative energy balance. The objective of this study was to investigate metabolite fingerprints in the urine of pre-ketotic, ketotic, and post-ketotic cows to identify potential metabolite alterations that can be used in the future to identify susceptible cows for ketosis and metabolic pathways involved in the development of disease. In this study, NMR, DI/LC-MS/MS, and GC-MS-based metabolomics were used to analyze urine samples from 6 cows diagnosed with ketosis and 20 healthy control (CON) cows at −8 and −4 weeks prepartum, the week (+1 to +3) of ketosis diagnosis, and at +4 and +8 weeks after parturition. Univariate and multivariate analyses were used to screen metabolite panels that can identify cows at their pre-ketotic stage. A total of 54, 42, 48, 16, and 31 differential metabolites between the ketotic and CON cows were identified at −8 and −4 weeks prepartum, ketosis week, and at +4, and +8 weeks postpartum, respectively. Variable importance in projection (VIP) plots ranked the most significant differential metabolites, which differentiated ketotic cows from the CON ones. Additionally, several metabolic pathways that are related to ketosis were identified. Moreover, two promising metabolite panels were identified which clearly separated ketotic from CON cows with excellent level of sensitivity and specificity. Overall, multiple urinary metabolite alterations were identified in pre-ketotic, ketotic, and post-ketotic cows. The metabolite panels identified need to be validated in the future in a larger cohort of animals.

Red osier dogwood (ROD; Cornus sericea) is an abundant native shrub plant across North America and it is rich in bioactive compounds with total phenolic concentration up to 220 g/kg dry matter (DM) depending on the season. The phenolics include anthocyanins, gallic acid, ellagic acid, quercetin, and cyanin, which have both antioxidant and antimicrobial properties. The objective of this study was to determine the effect of increasing substitution of ROD for barley silage in a high-grain diet on DM intake, ruminal pH and fermentation, digestibility in the total digestive tract, blood metabolites and acute phase response in beef heifers. Five ruminally cannulated Angus heifers (averaged body weight of 660 ± 40.8 kg; initial age of 18 months) were used in a 5 × 5 Latin square design with 21-d periods including 14 d for adaptation to new diets and 7 d for data collection. Treatments were a control diet containing 150 g/kg barley silage and 850 g/kg barley concentrate mix (DM basis), the control diet substituted with 30, 70 or 100 g/kg of ROD for equal proportions of barley silage, respectively, and the control diet supplemented with antibiotics (330 mg monensin + 110 mg tylosin/heifer/d). Apparent digestibility was determined using Cr2O3 as external marker by sampling feces from the rectum. Intake of DM quadratically (P

In this study, we aimed to identify metabolite signatures that characterize metritis prior to, during, and after the disease incidence. Blood samples were collected from 100 Holstein cows at five time points before and after parturition. Six cows that developed metritis and 20 controls were selected for metabolomics analysis in a nested case-control study. Twenty nine serum metabolites were quantified using gas chromatography–mass spectroscopy. Results showed that similar panels of metabolites differentiated pre-metritic and control cows at 8 and 4 wk prepartum. The top most important metabolites that differentiated the two groups of cows at 8 wk prepartum were oxalate, ornithine, pyroglutamic acid, d-mannose, and glutamic acid, and at 4 wk prepartum were ornithine, pyroglutamic acid, d-mannose, glutamic acid, and phosphoric acid, suggesting their potential use as risk biomarkers for metritis. Area under the curve with values of 1.0 and 0.969 at 8 and 4 wk, respectively, indicated that those panels of metabolites have a very high sensitivity and specificity to be used as risk biomarkers for metritis. Overall, results showed that specific serum metabolite signatures can be used to screen cows for susceptibility to metritis during the dry off period, and to better understand the etiopathobiology of the disease.

The objective of this meta-analytical study was to investigate selected macrominerals, microminerals, and toxic heavy metals in the bovine whole-raw-milk (WRM) samples of published data. An analytical data set was constructed from 72 different studies from 37 countries with two types of production systems: a conventional production system (CPS) and an organic production system (OPS) compared with commercially available or retail-store-available milk (COM). Results of the meta-analytical study showed differences in the concentrations of macrominerals. Concentrations of Ca, Mg, K, and P were greater in the CPS samples, whereas Na was greater in the COM samples ( P0.05). Data also demonstrated that concentrations of microminerals like Cu, I, Fe, Mn, Se, and Zn in the organic WRM were lower ( P0.05) compared with the milk from CPS. The highest concentration of Ni was reported for COM ( P0.05); however, this value was below the minimum-risk level (MRL). Concentrations of heavy metals like As and Ni were greater in CPS milk than those in organic milk ( P0.05). In addition, there were greater concentrations of Cd and Pb in the WRM from CPS versus that from the organic farms. Concentration of Al was lowest in the OPS milk versus Al in the CPS which was 6.5-fold greater than in organic milk. The amount of Hg was below the MRL of 0.01 μmol/L for all production systems. A high variability was observed in the published data regarding the country of origin. Raw milk originating from Europe and North America was characterized by concentrations of macro- and microminerals below the MRLs as compared with that from specific countries, which had some minerals above the MRLs. For example, concentrations of Pb were above the MRL in the milk samples from Brazil, Croatia, Egypt, Mexico, Nigeria, Palestine, Romania, Serbia, and Turkey. Moreover, data from this study indicate that organic dairy farms are characterized by lower concentrations of toxic heavy metals in the WRM compared with those from CPS dairy farms.

The objective of this study was to search for potential alterations in innate immunity reactants and carbohydrate and lipid metabolism in the blood of transition dairy cows before, during, and after clinical occurrence of milk fever (MF) and identify potential predictive biomarkers of disease. One hundred pregnant multiparous Holstein dairy cows were involved in the study starting from -8wks before the expected day of parturition until +8wks postpartum as part of a large retrospective longitudinal study. Health status, DMI, milk yield, and milk composition were monitored during the whole experimental period. Six healthy cows (CON) and 6 cows that showed clinical signs of MF were selected for blood analyses. Serum concentrations of lactate, non-esterified fatty acids (NEFA), β-hydroxybutyric acid (BHBA), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF), haptoglobin (Hp), and serum amyloid A (SAA) were determined. Results indicated that concentrations of serum lactate, IL-6, TNF, SAA, and Hp were greater in cows with MF than those in the CON group at different time points. Moreover, serum lactate, TNF, SAA, and Hp were greater in cows with MF starting at -8 and -4wks prior to parturition. Both principal component analysis (PCA) and partial least squares - discriminant analysis (PLS-DA) showed separated clusters between MF and CON cows at -8, -4, and disease diagnosis weeks. Overall DMI and milk production were lower in MF-affected cows. Additionally milk fat and fat:protein ratio were greater in MF. In conclusion, cows affected by MF showed alterations in some of the innate immunity reactants and metabolites related to carbohydrate metabolism several weeks prior to appearance of clinical signs of MF. Variable importance in projection plots demonstrated that TNF and SAA in the serum were the strongest discriminators between MF cows and CON ones, which might be useful as predictive biomarkers of the disease.

A targeted quantitative metabolomics approach was used to study temporal changes of serum metabolites in cows that normally released their fetal membranes and those that retained the placenta. We identified and measured serum concentrations of 128 metabolites including amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids and hexose at -8 and -4 weeks before parturition, during the week of retained placenta (RP) diagnosis, and at +4 and +8 weeks after parturition. In addition, we aimed at identifying metabolite signatures of pre-RP in the serum that might be used as predictive biomarkers for risk of developing RP in dairy cows. Results revealed major alterations in the metabolite fingerprints of pre-RP cows starting as early as -8 weeks before parturition and continuing as far as +8 weeks after calving. Biomarker candidates found in this study are mainly biomarkers of inflammation which might not be specific to RP. Therefore, the relevance of serum Lys, Orn, acetylornithine, lysophophatidylcholine LysoPC a C28:0, Asp, Leu and Ile as potential serum biomarkers for prediction of risk of RP in dairy cows will have to be tested in the future. In addition, lower concentrations of LysoPCs, Trp, and higher kynurenine in the serum during prepartum and the week of occurrence of RP suggest involvement of inflammation in the pathobiology of RP.

Bovine milk is a nutritionally rich, chemically complex biofluid consisting of hundreds of different components. While the chemical composition of cow's milk has been studied for decades, much of this information is fragmentary and very dated. In an effort to consolidate and update this information, we have applied modern, quantitative metabolomics techniques along with computer-aided literature mining to obtain the most comprehensive and up-to-date characterization of the chemical constituents in commercial cow's milk. Using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectrometry (LC-MS), and inductively coupled plasma-mass spectrometry (ICP-MS), we were able to identify and quantify 296 bovine milk metabolites or metabolite species (corresponding to 1447 unique structures) from a variety of commercial milk samples. Through our literature analysis, we also found another 676 metabolites or metabolite species (corresponding to 908 unique structures). Detailed information regarding all 2355 of the identified chemicals in bovine milk have been made freely available through a Web-accessible database called the Milk Composition Database or MCDB ( http://www.mcdb.ca/ ).

The dairy industry continues to suffer severe economic losses due to the increased disease incidence cows experience during the transition period. It has long been the classical view that the major contributing factor to the development of these periparturient diseases is the considerable increase in nutritional demands for milk production. This classical view, however, fails to account for the substantial correlation between both metabolic and infectious diseases and the detrimental effects that can occur with the provision of high-energy diets to support these nutritional demands. Currently, increasing evidence implicates bacterial endotoxins in the etiopathology of most periparturient diseases. Bacterial endotoxins are components of the outer cell wall of gram-negative and gram-positive bacteria that are highly immunostimulatory and can trigger proinflammatory immune responses. The ability of endotoxins to translocate from the mucosal tissues, including the gastrointestinal tract, mammary gland, and uterus, into the systemic circulation has been observed. Once they have entered the circulation, endotoxins potentially contribute to disease either directly, through eliciting an inflammatory response, or indirectly through other factors such as the overreaction of the natural protective mechanisms of the host. Although the evidence implicating a role of endotoxins in the pathogenesis of transition diseases continues to grow, our current knowledge of the host response to mucosal endotoxin exposure and pathogenic mechanisms remain largely unknown. Developing our understanding of the connection between endotoxemia and dairy cattle disease holds significant potential for the future development of preventative measures that could benefit the productivity of the dairy industry as well as animal welfare.

The objective of this investigation was to evaluate whether intravaginal infusion of probiotics (a lactic acid bacteria cocktail) around parturition would influence metabolic status and increase milk production of transition dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of the 3 experimental groups receiving intravaginal infusion of probiotics or carrier (i.e., sterile skim milk) once a week at wk -2, -1, and +1 relative to calving as follows: 2 consecutive probiotics before parturition and 1 carrier dose after parturition (TRT1), 3 consecutive probiotics doses around parturition (TRT2), and 3 consecutive carrier doses around parturition (CTR). The probiotics were a lyophilized culture mixture composed of FUA3089 and FUA3138 and FUA3140 with a cell count of 10 to 10 cfu/dose. Blood was sampled from wk -2 to +3 and milk was sampled on the third day in milk (DIM) and from wk +1 to +5 on a weekly basis. Feed intake and milk production was monitored until wk +8. Results showed that the TRT2 group (366.12 ± 49.77 μmol/L) had a lower ( = 0.01) concentration of NEFA in the serum than the CTR group (550.85 ± 47.16 μmol/L). The concentrations of IgG in the milk were 32.71 ± 3.00 mg/mL in the TRT1 group, 17.47 ± 4.54 mg/mL in the TRT2 group, and 6.73 ± 3.43 mg/mL in the CTR group at 3 DIM ( < 0.01). Meanwhile, both the TRT1 and the TRT2 group had lower haptoglobin in the milk compared with the CTR group at 3 DIM ( < 0.01). The TRT1 group had greater milk protein content than the CTR group (2.99 ± 0.04 vs. 2.82 ± 0.04%; = 0.02), whereas the TRT2 group tended to have greater lactose content compared with the CTR group (4.53 ± 0.03 vs. 4.44 ± 0.03%; = 0.05). The effect of treatment interacted with parity with regards to milk production and feed efficiency. Multiparous cows in the TRT1 and TRT2 groups had greater milk production and feed efficiency than those in the CTR group ( < 0.01 and = 0.02, respectively). Among primiparous cows, those in the TRT2 group had greater milk production ( = 0.04) whereas those in the TRT1 group had lower feed intake ( < 0.01) than those in the CTR group. Both the TRT1 and the TRT2 groups had enhanced feed efficiency compared with the CTR group ( < 0.01). In conclusion, intravaginal infusion of lactic acid bacteria modulated concentrations of selected serum metabolites and milk components and increased milk efficiency of transition dairy cows.

(1) Background: The objective of this study was to evaluate the urine of dairy cows for presence of metabolites with the potential to be used as screening biomarkers for lameness as well as to characterize pre-lame, lame, and post-lame cows from the metabolic prospective. (2) Methods: Six lame and 20 control healthy cows were used in this nested case-control study. Urinary 1H-NMR analysis was used to identify and measure metabolites at five time points including &minus, 8 and &minus, 4 weeks prepartum, lameness diagnosis week (1&ndash, 3 weeks postpartum) as well as at +4 and +8 weeks after calving. (3) Results: A total of 90 metabolites were identified and measured in the urine. At &minus, 4 weeks, 27 prepartum metabolites were identified as altered, at both timepoints, with 19 and 5 metabolites excreted at a lower concentration, respectively. Additionally, a total of 8 and 22 metabolites were found at greater concentration in pre-lame cows at &minus, 4 weeks, respectively. Lame cows were identified to excrete, at lower concentrations, seven metabolites during a lameness event with the top five most important metabolites being Tyr, adipate, glycerate, 3-hydroxy-3-methylglutarate, and uracil. Alterations in urinary metabolites also were present at +4 and +8 weeks after calving with N-acetylaspartate, glutamine, imidazole, pantothenate, beta-alanine and trimethylamine, with the greatest VIP (variable importance in projection) score at +4 weeks, and hipurate, pantothenate 1,3-dihydroxyacetone, galactose, and Tyr, with the greatest VIP score at +8 weeks postpartum. (4) Conclusions: Overall, results showed that urine metabotyping can be used to identify cows at risk of lameness and to better characterize lameness from the metabolic prospective. However, caution should be taken in interpretation of the data presented because of the low number of replicates.

The objectives of this study were to identify metabolite fingerprints in the serum related to amino acid (AA), carbohydrate, and lipid metabolism in transition dairy cows at −8 and −4 wks prior to parturition, at +2 wks postpartum during lameness diagnosis as well as at +4 and +8 wks after parturition. All cases of lameness occurred at around +2 wks after parturition. Out of 100 dairy cows included in this nested case-control study only 6 pregnant multiparous (parity: 3.0 ± 0.6, Mean ± SEM) Holstein dairy cows with lameness only and 20 healthy control cows (CON) were selected for serum GC–MS metabolomics analysis. All cows selected were not injured mechanically and had similar parity (3.3 ± 0.6) and body condition score (BCS). A total of 29 metabolites were identified and quantified in the serum. Results showed that 18 and 15 metabolites differentiated pre-lame cows from CON ones at −8 and −4 wks prior to parturition. Ten metabolites were found altered at the week of lameness diagnosis. Of note: pre-lame cows were characterized by greater concentrations of several amino acids including Gly, Leu, Phe, Ser, Val, D-mannose, Myo-inositol, and phosphoric acid (PA) at −8 and −4 wks prior to lameness and at the week of lameness diagnosis. At +4 wks after parturition 11 metabolites were altered in lameness cows, and at +8 wks there were 13 metabolites that differentiated the two groups. The high accuracy of the top 6 metabolites at −8 wks prior to parturition or approximately 9–11 wks before lameness diagnosis (Glu, Orn, Phe, Ser, Val, and PA) and another 5 metabolites at −4 wks before parturition, or approximately 5–7 wks before lameness diagnosis (Leu, Orn, Phe, Ser, and D-mannose) suggest that those metabolites may serve as potential monitoring biomarkers of lameness prior to lameness diagnosis. Data also showed multiple alterations during the week of lameness as well as at +4 and +8 wks postpartum suggesting lame cows are not metabolically healthy several weeks after the incidence of lameness. Significance Lameness is one of the top three health issues of dairy cows in Canada that influences early culling of dairy cows. Despite a few efforts, there is scarcity of data regarding metabolic alterations that precede, associate, and follow lameness. We investigated whether alterations in the metabolite signatures prior, during, and after development of lameness can be used to screen cows for susceptibility to lameness, characterize lameness from the metabolic prospective, and predict the outcome of this economically important health issue of dairy cows. The results demonstrate typical metabotypes as shown by increased serum concentrations of Val, Gly, Ser, Leu, Phe, D-mannose, myo-inositol, and phosphoric acid at −8 and −4 wks prior to parturition (or −6 to −10 wks prior to occurrence of lameness) and at the week of lameness diagnosis.

The objectives of this study were: 1) to investigate ionome in three body fluids (i.e., serum, urine, and milk) of pre-milk fever (MF), MF, and post-MF dairy cows; and 2) to identify newly predictive serum/urine and diagnostic serum/urine/milk mineral biomarkers for MF. In this study, inductively coupled plasma mass spectrometry (ICP-MS) based ionomics was performed to targetedly analyze minerals in serum, urine, and milk (only at the disease wk) samples from 6 cows with MF and 20 healthy controls (CON) at -8 wks, -4 wks, disease diagnosis, +4 wks, and +8 wks relative to parturition. MetaboAnalyst 3.0 was employed for statistical analyses and biomarker analysis. Results showed that around five (out of 18) minerals were elevated in the serum of MF cows at each tested time point. Particularly, serum levels of chromium were continuously higher in both pre-MF (i.e., 3.93 vs 3.11 at -8 wks, P = 0.02; 3.79 vs 3.16 at -4 wks, P < 0.01; unit: μM) and MF cows (i.e., 3.96 vs 3.27 at the disease wk, P = 0.01) compared with CON ones. Other minerals such as aluminum, manganese, and arsenic were also elevated in the serum at early stages (i.e., at -8 and -4 wks; P < 0.05) of MF. Moreover, besides calcium (MF 37,431 vs CON 27,260 μM; P = 0.02), other two minerals (i.e., aluminum and cobalt, P < 0.05) were increased in the milk of MF cows. Moreover, four (two in serum and two in urine) predictive mineral-biomarker models for MF were developed. Overall, our ionomics data showed that MF cows had altered ionome in pre-MF, MF, and post-MF cows. MF is more complicated than hypocalcemia. ICP-MS based ionomics sheds new light on our understanding of altered mineral metabolism and identification of new biomarkers for MF in dairy cows.

The objective was to evaluate the effects of supplementing ruminally protected and non-protected active dried yeast (ADY; AB Vista, UK) on blood metabolites, acute phase protein and immune response of beef steers. Seventy-five Angus steers (initial BW 448 kg) were blocked by BW and assigned to a randomized complete block design with five treatments: control (no additives), antibiotics (ANT; 330 mg monensin + 110 mg tylosin/d), ADY (1.5 g/d), encapsulated ADY (EDY; 3 g/d) and combination of ADY and EDY (MDY). Steers were housed in individual pens and weighed every 28 d. The ADY had 1.7x10P

The objectives of this study were to targetedly investigate metabolic profiles of the serum in dairy cows (both healthy controls (CON) and cows that developed subclinical mastitis (SCM) at early lactation) up to -8 wks prepartum, and evaluate the performance of new biomarkers for both early onset and progression of the disease. In this research, DI/LC-MS/MS based metabolomics was employed to identify and quantify biochemical signatures in the serum of dairy cows at -8 wks, -4 wks, disease diagnosis, +4 wks, and +8 wks relative to parturition. One hundred and twenty-eight metabolites including amino acids (21), acylcarnitines (7), biogenic amines (8), glycerophospholipids (77), sphingolipids (14), and hexose (1) were quantified in all sera. Univariate analysis showed that several metabolites (e.g., phosphatidylcholine (PC aa C30:2), hydroxysphingomyelin (SM (OH) C22:2), isoleucine, leucine, and lysine; P < 0.01) were consistently elevated in the serum of cows with SCM at five tested time points. In the supervised multivariate analysis (i.e., PLS-DA; permutation test: P < 0.05), a variable importance in projection (VIP) plot was used to rank the most significant discriminators between SCM and CON cows. Two predictive biomarker models and one diagnostic biomarker model for SCM were developed by combination of amino acids, sphingomyelin, phosphatidylcholine, and kynurenine. AUC values of three ROC curve of biomarker models were all greater than 0.995. The predictive and diagnostic models for SCM based on metabolites in serum are practical biomarker alternatives as compared to the current milk somatic cell count (SCC) assays. The data demonstrate that metabolomics may be effective for screening cows during the dry off for susceptibility to SCM and evaluating the health status of udder in dairy cows in the future. Results also help in better understanding the metabolic status and pathomechanisms of the disease.

Metritis is an uterine pathology that causes economic losses for the dairy industry. It is associated with lower reproductive efficiency, increased culling rates, decreased milk production and increased veterinary costs. To gain a more detailed view of the urine metabolome and to detect metabolite signature in cows with metritis. In addition, we aimed to identify early metabolites which can help to detect cows at risk to develop metritis in the future. We used nuclear magnetic resonance spectroscopy starting at 8 and 4 weeks prior to the expected day of parturition, during the week of diagnosis of metritis, and at 4 and 8 weeks after diagnosis of metritis in Holstein dairy cows. At 8 weeks before parturition, pre-metritic cows had a total of 30 altered metabolites. Interestingly, 28 of them increased in urine when compared with control cows (P

The objectives of this study were to test whether recombinant mouse (mo)PrP alone or in combination with LPS or under simulated endotoxemia would affect expression of genes related to host inflammatory and antimicrobial responses. To test our hypotheses colon tissues were collected from 16 male mice (FVB/N strain) and mounted in an Ussing chamber. Application of moPrP to the mucosal side of the colon affected genes related to TLR- and NLR- signaling and antimicrobial responses. When LPS was added on the mucosal side of the colon, genes related to TLR, Nlrp3 inflammasome, and iron transport proteins were over-expressed. Addition of LPS to the serosal side of the colon up-regulated genes related to TLR- and NLR-signaling, Nlrp3 inflammasome, and a chemokine. Treatment with both moPrP and LPS to the mucosal side of the colon upregulated genes associated with TLR, downstream signal transduction (DST), inflammatory response, attraction of dendritic cells to the site of inflammation, and the JNK-apoptosis pathway. Administration of moPrP to the mucosal side and LPS to the serosal side of the colon affected genes related to TLR- and NLR-signaling, DST, apoptosis, inflammatory response, cytokines, chemokines, and antimicrobial peptides. Overall this study suggests a potential role for moPrP as an endogenous 'danger signal' associated with activation of colon genes related to innate immunity and antibacterial responses.

The objectives of this study were to evaluate alterations of metabolites in the blood of dairy cows before, during, and after diagnosis of metritis and identify predictive serum metabolite biomarkers for metritis. DI/LC-MS/MS was used to analyze serum samples collected from both healthy and metritic cows during −8, −4, disease diagnosis, +4, and +8 wks relative to parturition. Results indicated that cows with metritis experienced altered concentrations of serum amino acids, glycerophospholipids, sphingolipids, acylcarnitines, and biogenic amines during the entire experimental period. Moreover, two sets of predictive biomarker models and one set of diagnostic biomarker models for metritis were developed, and all of them showed high sensitivity and specificity (e.g., high AUC values by the ROC curve evaluation), which indicate that serum metabolites identified have pretty accurate predictive, diagnostic, and prognostic abilities for metritis in transition dairy cows.

Ketosis is a prevalent metabolic disease of transition dairy cows that affects milk yield and the development of other periparturient diseases. The objective of this study was to retrospectively metabotype the serum of dairy cows affected by ketosis before clinical signs of disease, during the diagnosis of ketosis, and after the diagnosis of disease and identify potential predictive and diagnostic serum metabolite biomarkers for the risk of ketosis. Targeted metabolomics was used to identify and quantify 128 serum metabolites in healthy (CON, n = 20) and ketotic (n = 6) cows by DI/LC-MS/MS at −8 and −4 weeks prepartum, during the disease week, and at +4 and +8 weeks after parturition. Significant changes were detected in the levels of several metabolite groups including amino acids, glycerophospholipids, sphingolipids, acylcarnitines, and biogenic amines in the serum of ketotic cows during all time points studied. Results of this study support the idea that ketosis is preceded and associated and followed by alterations in multiple metabolite groups. Moreover, two sets of predictive biomarker models and one set of diagnostic biomarker model with very high sensitivity and specificity were identified. Overall, these findings throw light on the pathobiology of ketosis and some of the metabolites identified might serve as predictive biomarkers for the risk of ketosis. The data must be considered as preliminary given the lower number of ketotic cows in this study and more research with a larger cohort of cows is warranted to validate the results.

The objectives of this study were to determine alterations in the serum metabolites related to amino acid (AA), carbohydrate, and lipid metabolism in transition dairy cows before diagnosis of subclinical mastitis (SCM), during, and after diagnosis of disease. A subclinical mastitis case was determined as a cow having somatic cell count (SCC)200 000/mL of milk for two or more consecutive reports. Blood samples were collected from 100 Holstein dairy cows at five time points at -8 and -4 weeks before parturition, at the week of SCM diagnosis, and +4 and +8 weeks after parturition. Twenty healthy control cows (CON) and six cows that were diagnosed with SCM were selected for serum analysis with GC-MS. At -8 weeks a total of 13 metabolites were significantly altered in SCM cows. In addition, at the week of SCM diagnosis 17 metabolites were altered in these cows. Four weeks after parturition 10 metabolites were altered in SCM cows and at +8 weeks 11 metabolites were found to be different between the two groups. Valine (Val), serine (Ser), tyrosine (Tyr), and phenylalanine (Phe) had very good predictive abilities for SCM and could be used at -8 weeks and -4 weeks before calving. Combination of Val, isoleucine (Ile), Ser, and proline (Pro) can be used as diagnostic biomarkers of SCM during early stages of lactation at +4 to +8 weeks after parturition. In conclusion, SCM is preceded and followed by alteration in AA metabolism.

In dairy cows, periparturient disease states, such as metritis, mastitis, and laminitis, are leading to increasingly significant economic losses for the dairy industry. Treatments for these pathologies are often expensive, ineffective, or not cost-efficient, leading to production losses, high veterinary bills, or early culling of the cows. Early diagnosis or detection of these conditions before they manifest themselves could lower their incidence, level of morbidity, and the associated economic losses. In an effort to identify predictive biomarkers for postpartum or periparturient disease states in dairy cows, we undertook a cross-sectional and longitudinal metabolomics study to look at plasma metabolite levels of dairy cows during the transition period, before and after becoming ill with postpartum diseases. Specifically we employed a targeted quantitative metabolomics approach that uses direct flow injection mass spectrometry to track the metabolite changes in 120 different plasma metabolites. Blood plasma samples were collected from 12 dairy cows at 4 time points during the transition period (-4 and -1 wk before and 1 and 4 wk after parturition). Out of the 12 cows studied, 6 developed multiple periparturient disorders in the postcalving period, whereas the other 6 remained healthy during the entire experimental period. Multivariate data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation between healthy controls and diseased cows at all 4 time points. This analysis allowed us to identify several metabolites most responsible for separating the 2 groups, especially before parturition and the start of any postpartum disease. Three metabolites, carnitine, propionyl carnitine, and lysophosphatidylcholine acyl C14:0, were significantly elevated in diseased cows as compared with healthy controls as early as 4 wk before parturition, whereas 2 metabolites, phosphatidylcholine acyl-alkyl C42:4 and phosphatidylcholine diacyl C42:6, could be used to discriminate healthy controls from diseased cows 1 wk before parturition. A 3-metabolite plasma biomarker profile was developed that could predict which cows would develop periparturient diseases, up to 4 wk before clinical symptoms appearing, with a sensitivity of 87% and a specificity of 85%. This is the first report showing that periparturient diseases can be predicted in dairy cattle before their development using a multimetabolite biomarker model. Further research is warranted to validate these potential predictive biomarkers.

The objective of this study was to identify urinary metabolite signatures that can be used for monitoring and diagnosing of subclinical mastitis in Holstein dairy cows. Six multiparous pregnant Holstein cows with subclinical mastitis (SCM) and 20 healthy controls (CON) were selected out of 100 cows in the study. DI/LC-MS/MS-based urinary analyses were conducted on samples collected at -8, -4, SCM diagnosis, and at +4 and +8 wks around the expected day of calving. Data indicated that 14 amino acids and derivatives were found in the urine with Gln and SDMA consistently increased (P

The study evaluated the effects of repeated oral exposure to LPS and lipoteichoic acid (LTA) on immune responses of dairy cows. Thirty pregnant Holstein cows were randomly assigned to two treatment groups. Cows received orally either 2 ml of 0.85% sterile saline solution (control group), or 2 ml of sterile saline solution containing three doses of LPS from Escherichia coli 0111 : B4 along with a flat dose of LTA from Bacillus subtilis. Blood and saliva samples were collected and analyzed for serum amyloid A (SAA); LPS-binding protein (LBP); anti-LPS plasma IgA, IgG and IgM; TNF-α; and IL-1. Results showed greater concentrations of IgA in the saliva of treated cows compared with the controls ( P 0.10). Overall, the results of this study indicate that repeated oral administration with LPS and LTA stimulates innate and humoral immune responses in periparturient dairy cows.

Milk fever, a metabolic disease of dairy cattle, is associated with perturbations of calcium homeostasis, the pathogenesis of which is not yet completely understood.The aim of this study was to investigate plasma concentrations of calcitonin gene-related peptide and selected minerals and metabolites in periparturient cows with and without milk fever.Plasma concentrations of calcitonin gene-related peptide, as well as calcium, phosphate, magnesium, iron, glucose, lactate, and cortisol, were determined in multiple plasma samples from Jersey cows with and without spontaneous milk fever.Cows affected by milk fever (n = 5) had lower concentrations of calcitonin gene-related peptide (P = .038) and inorganic phosphate (P.001) in plasma than did the controls (n = 5). Also, these cows tended to have lower calcium concentrations (P = .071). Magnesium, iron, lactate, glucose, and cortisol concentrations were comparable between both groups of cows (P.10). Around the day of calving, plasma concentrations of lactate, glucose, and cortisol increased and the concentration of iron decreased in all cows (P ≤ .01).Despite the limited number of cows evaluated, this report is the first to indicate lowered concentrations of calcitonin gene-related peptide as part of the metabolic changes during milk fever in cows. Further work with a larger cohort of animals is warranted to understand the precise role of calcitonin gene-related peptide and the potential associations with disturbances in plasma minerals typically observed during milk fever.