Prediction of blood metabolites from milk mid-infrared spectra in early-lactation cows

Dairy cows commonly experience an unbalanced energy status in early lactation, and this condition can lead to the onset of several metabolic disorders. Blood metabolic profile testing is a valid tool to monitor and detect the most common early lactation disorders, but blood sampling and analysis are time-consuming and expensive, and the procedure is invasive and stressful for the cows. Mid-infrared (MIR) spectroscopy is routinely used to analyze milk composition, being a cost-effective and nondestructive method. The present study aimed to assess the feasibility of using routine milk MIR spectra for the prediction of main blood metabolites in dairy cows, and to investigate associations between measured blood metabolites and milk traits. Twenty herds of Holstein Friesian, Brown Swiss, or Simmental cows located in Northeast Italy were visited 1 to 4 times between December 2017 and June 2018, and blood and milk samples were collected from all lactating cows within 35 d in milk. Concentrations of main blood metabolites and milk MIR spectra were recorded from 295 blood and milk samples and used to develop prediction models for blood metabolic traits through backward interval partial least squares analysis. Blood β-hydroxybutyrate (BHB), urea, and nonesterified fatty acids were the most predictable traits, with coefficients of determination of 0.63, 0.58, and 0.52, respectively. On the contrary, predictive performance for blood glucose, triglycerides, cholesterol, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase were not accurate. Associations of blood BHB and urea with their respective contents in milk were moderate to strong, whereas all other correlations were weak. Predicted blood BHB showed an improved performance in detecting cows with hyperketonemia (blood BHB ≥ 1.2 mmol/L), compared with commercial calibration equation for milk BHB. Results highlighted the opportunity of using milk MIR spectra to predict blood metabolites and thus to collect routine information on the metabolic status of early-lactation cows at a population level.     

An observational study on early-lactation metabolic profiles in Swedish organically and conventionally managed dairy cows

The aim of this observational study was to compare indicators of energy balance in early lactation in organically managed dairy cows (OMC) and conventionally managed dairy cows (CMC) under field conditions. The diets of OMC and CMC differ as a consequence of the rules and principles of organic dairy farming. The study was based on clinical examinations and blood samples from cows within the range from 2 wk prepartum to 6 wk postpartum, collected from 20 organic and 20 conventional dairy farms with 3 visits at each farm. The farms were located in a southeastern area of Sweden and ranged in size from 45 to 120 cows. The blood parameters selected to reflect energy metabolism were nonesterified fatty acids, β-hydroxybutyrate, glucose, and insulin. At clinical examination body condition score was registered. The shape of the lactation curve in early lactation was modeled to assess potential differences that could explain the blood parameter profiles. The conventionally managed cows increased their milk yield faster than OMC within the first 2 wk of lactation. Blood nonesterified fatty acid concentrations were similar between the management types, but with a tendency of lesser concentrations in OMC, primarily in early lactation. Postcalving β-hydroxybutyrate concentrations were constantly lesser in OMC during the first 6 wk of lactation. An interaction between season and insulin concentration necessitated stratification on season. During spring the profiles overlapped, but there was a significant difference in the first 4 d post-calving, when organically managed cows had greater insulin concentrations and in d 30 to 34 when conventionally managed cows had greater insulin concentrations. During fall the profiles overlapped completely and there was no significant difference at any point in time. Glucose concentrations tended to decrease slightly postcalving followed by a gradual elevation to a concentration just under the precalving concentration during the study period. Body condition scores decreased slightly over the study period. No differences were found between the management types with regard to glucose concentrations or registered body condition score. In conclusion, the OMC did not show a greater extent of mobilization of body tissue than CMC as expressed by our study variables. Hence, OMC adjusted the production amount according to feed intake.     

Milk metabolites and fatty acids as noninvasive biomarkers of metabolic status and energy balance in early-lactation cows

The objective was to study the effects of week of lactation (WOL) and experimental nutrient restriction on concentrations of selected milk metabolites and fatty acids (FA), and assess their potential as biomarkers of energy status in early-lactation cows. To study WOL effects, 17 multiparous Holstein cows were phenotyped from calving until 7 WOL while allowed ad libitum intake of a lactation diet. Further, to study the effects of nutrient restriction, 8 of these cows received a diet containing 48% straw (high-straw) for 4 d starting at 24 ± 3 days in milk (mean ± SD), and 8 cows maintained on the lactation diet were sampled to serve as controls. Blood and milk samples were collected weekly for the WOL data set, and daily from d ?1 to 3 of nutrient restriction (or control) for the nutritional challenge data set. Milk β-hydroxybutyrate (BHB), isocitrate, glucose, glucose-6-phosphate (glucose-6P), galactose, glutamate, creatinine, uric acid, and N-acetyl-β-d-glucosaminidase activity (NAGase) were analyzed in p.m. and a.m. samples, and milk FA were analyzed in pooled p.m. and a.m. samples. Average energy balance (EB) per day ranged from ?27 MJ/d to neutral when cows received the lactation total mixed ration, and from ?109 to ?87 ± 7 MJ/d for high-straw (least squares means ± standard error of the mean). Plasma nonesterified FA concentration was 1.67 ± 0.13 mM and BHB was 2.96 ± 0.39 mM on the d 3 of high-straw (least squares means ± standard error of the mean). Milk concentrations of BHB, glucose, glucose-6P, glutamate, and uric acid differed significantly between p.m. and a.m. milkings. Milk isocitrate, glucose-6P, creatinine, and NAGase decreased, whereas milk glucose and galactose increased with WOL. Changes in milk BHB, isocitrate, glucose, glucose-6P, and creatinine were concordant during early lactation and in response to nutrient restriction. Milk galactose and NAGase were modulated by WOL only, whereas glutamate and uric acid concentrations responded to nutrient restriction only. The high-straw increased milk concentrations of FA potentially mobilized from adipose tissue (e.g., C18:0 and cis-9 C18:1 and sum of odd- and branched-chain FA (OBCFA) with carbon chain greater than 16; ∑ OBCFA >C16), and decreased concentrations of FA synthesized de novo by the mammary gland (e.g., sum of FA with 6 to 15 carbons; ∑ C6:0 to C15:0). Similar observations were made during early lactation. Plasma nonesterified FA concentrations had the best single linear regression with EB (R² = 0.62). Milk isocitrate, Σ C6:0 to C15:0. and cis-9 C18:1 had the best single linear regressions with EB (R² ≥ 0.44). Milk BHB, isocitrate, galactose, glutamate, and creatinine explained up to 64% of the EB variation observed in the current study using multiple linear regression. Milk concentrations of ∑ C6:0 to C15:0, C18:0, cis-9 C18:1, and ∑ OBCFA >C16 presented some of the best correlations and regressions with other indicators of metabolic status, lipomobilization, and EB, and their responses were concordant during early lactation and during experimental nutrient restriction. Metabolites and FA secreted in milk may serve as noninvasive indicators of metabolic status and EB of early-lactation cows.     

Association of postpartum hypocalcemia with early-lactation milk yield,reproductive performance,and culling in dairy cows

Periparturient hypocalcemia is frequently observed and considered as a gateway disease that is associated with various health issues. The objective of this study was to evaluate the association of hypocalcemia with early-lactation milk yield, reproductive performance, and culling across a large number of different managerial systems. A prospective cohort study was conducted based on a convenience sample of 125 dairy herds from 8 federal states of Germany between February 2015 and August 2016. A blood sample was drawn from 1,709 animals within 48 h after parturition and analyzed for serum calcium concentration. After discarding cows (n = 283) with missing data, a total of 1,426 cows were considered for final analyses. The median time from calving to sampling was 14.0 h (interquartile range = 5.0–24.9 h). For each herd, a record of the herd management software was requested 150 d after the last cow was sampled. Serum calcium concentration of each cow was associated with early-lactation milk yield (Dairy Herd Improvement Association equivalent test 1 to 3), reproductive performance [days in milk (DIM) at first artificial insemination (AI), pregnancy at first AI, time to pregnancy within 150 DIM], and culling (until 60 DIM) data. Generalized linear mixed models were used to analyze continuous or categorical data. Shared frailty models were used for time to event data. Five different thresholds were used to define hypocalcemia. Thresholds ranged from 1.8 to 2.2 mmol/L using 0.1-mmol/L increments. Clinical hypocalcemia was defined as serum calcium concentration <2.0 mmol/L in combination with clinical signs (e.g., recumbency). The effect of hypocalcemia on milk yield was conditional on parity. In primiparous cows a serum calcium concentration <2.0 mmol/L (6.4% of cows were below this threshold) had no effect on milk production, whereas there was a tendency for multiparous cows with a serum calcium concentration <2.1 mmol/L (63.2% of cows were below this threshold) to produce 0.80 kg/d more milk compared with multiparous cows at or above the threshold. Multiparous cows suffering from clinical hypocalcemia produced 2.19 kg/d less milk compared with normocalcemic cows in early lactation. Calcium status was not associated with days to first insemination. Cows with a serum calcium concentration <1.9 mmol/L (34.6% of cows below this threshold) had decreased odds (odds ratio = 0.56) of pregnancy at first AI. A serum calcium concentration <1.8 mmol/L (24.1% of cows below this threshold) had a significant effect on time to pregnancy. Compared with animals with a serum calcium concentration ≥1.8 mmol/L, the hazard of becoming pregnant within 150 DIM was reduced when cows had a serum calcium concentration <1.8 mmol/L (hazard ratio = 0.68). Cows with a serum calcium concentration <2.0 mmol/L (44.3% of cows were below this threshold) had a 1.69 times greater hazard of being culled within the first 60 DIM compared with normocalcemic animals. The present study shows that the association of hypocalcemia with milk yield was conditional on parity and serum calcium concentration measured once within 48 h after calving. Considering reproductive performance and culling in early lactation, a negative effect of postpartum hypocalcemia was demonstrated.     

Metabolic profiling of early-lactation dairy cows using milk mid-infrared spectra

Metabolic disorders in early lactation have negative effects on dairy cow health and farm profitability. One method for monitoring the metabolic status of cows is metabolic profiling, which uses associations between the concentrations of several metabolites in serum and the presence of metabolic disorders. In this cross-sectional study, we investigated the use of mid-infrared (MIR) spectroscopy of milk for predicting the concentrations of these metabolites in serum. Between July and October 2017, serum samples were taken from 773 early-lactation Holstein Friesian cows located on 4 farms in the Gippsland region of southeastern Victoria, Australia, on the same day as milk recording. The concentrations in sera of β-hydroxybutyrate (BHB), fatty acids, urea, Ca, Mg, albumin, and globulins were measured by a commercial diagnostic laboratory. Optimal concentration ranges for each of the 7 metabolites were obtained from the literature. Animals were classified as being either affected or unaffected with metabolic disturbances based on these ranges. Milk samples were analyzed by MIR spectroscopy. The relationships between serum metabolite concentrations and MIR spectra were investigated using partial least squares regression. Partial least squares discriminant analyses (PLS-DA) were used to classify animals as being affected or not affected with metabolic disorders. Calibration equations were constructed using data from a randomly selected subset of cows (n = 579). Data from the remaining cows (n = 194) were used for validation. The coefficient of determination (R²) of serum BHB, fatty acids, and urea predictions were 0.48, 0.61, and 0.90, respectively. Predictions of Ca, Mg, albumin, and globulin concentrations were poor (0.06 ≤ R² ≤ 0.17). The PLS-DA models could predict elevated fatty acid and urea concentrations with an accuracy of approximately 77 and 94%, respectively. A second independent validation data set was assembled in March 2018, comprising blood and milk samples taken from 105 autumn-calving cows of various breeds. The accuracies of BHB and fatty acid predictions were similar to those obtained using the first validation data set. The PLS-DA results were difficult to interpret due to the low prevalence of metabolic disorders in the data set. Our results demonstrate that MIR spectroscopy of milk shows promise for predicting the concentration of BHB, fatty acids, and urea in serum; however, more data are needed to improve prediction accuracies.     

Effect of mastitis on milk perchlorate concentrations in dairy cows

Recent surveys have identified the presence of perchlorate, a natural compound and environmental contaminant, in forages and dairy milk. The ingestion of perchlorate is of concern because of its ability to competitively inhibit iodide uptake by the thyroid and to impair synthesis of thyroid hormones. A recent study established that milk perchlorate concentrations in cattle highly correlate with perchlorate intake. However, there is evidence that up to 80% of dietary perchlorate is metabolized in clinically healthy cows, thereby restricting the available transfer of ingested perchlorate into milk. The influence of mastitis on milk perchlorate levels, where there is an increase in mammary vascular permeability and an influx of blood-derived components into milk, remains unknown. The present study examined the effect of experimentally induced mastitis on milk perchlorate levels in cows receiving normal and perchlorate-supplemented diets. Over a 12-d period, cows were ruminally infused with 1 L/d of water or water containing 8 mg of perchlorate. Five days after the initiation of ruminal infusions, experimental mastitis was induced by the intramammary infusion of 100 μg of bacterial lipopolysaccharide (LPS). Contralateral quarters infused with phosphate-buffered saline served as controls. A significant reduction in milk perchlorate concentration was observed in the LPS-challenged glands of animals ruminally infused with either water or perchlorate. In control glands, milk perchlorate concentrations remained constant throughout the study. A strong negative correlation was identified between mammary vascular permeability and milk perchlorate concentrations in LPS-infused glands. These findings, in the context of a recently published study, suggest that an active transport process is operative in the establishment of a perchlorate concentration gradient across the blood-mammary gland interface, and that increases in mammary epithelial and vascular endothelial permeability lead to a net outflow of milk perchlorate. The overall finding that mastitis results in lower milk perchlorate concentrations suggests that changes in udder health do not necessitate increased screening of milk for perchlorate.     

Assessing the effect of pregnancy stage on milk composition of dairy cows using mid-infrared spectra

Changes in milk production traits (i.e., milk yield, fat, and protein contents) with the pregnancy stage are well documented. To our knowledge, the effect of pregnancy on the detailed milk composition has not been studied so far. The mid-infrared (MIR) spectrum reflects the detailed composition of a milk sample and is obtained by a nonexhaustive and widely used method for milk analysis. Therefore, this study aimed to investigate the effect of pregnancy on milk MIR spectrum in addition to milk production traits (milk yield, fat, and protein contents). A model including regression on the number of days pregnant was applied on milk production traits (milk yield, fat, and protein contents) and on 212 spectral points from the MIR spectra of 9,757 primiparous Holstein cows from Walloon herds. Effects of pregnancy stage were expressed on a relative scale (effect divided by the squared root of the phenotypic variance); this allowed comparisons between effects on milk traits and on 212 spectral points. Effect of pregnancy stage on production traits were in line with previous studies indicating that the model accounted well for the pregnancy effect. Trends of the relative effect of the pregnancy stage on the 212 spectral points were consistent with known and observed effect on milk traits. The highest effect of the pregnancy was observed in the MIR spectral region from 968 to 1,577 cm^?1. For some specific wavenumbers, the effect was higher than for fat and protein contents in the beginning of the pregnancy (from 30 to 90 or 120 d pregnant). In conclusion, the effect of early pregnancy can be observed in the detailed milk composition through the analysis of the MIR spectrum of bovine milk. Further analyses are warranted to explore deeply the use of MIR spectra of bovine milk for breeding and management of dairy cow pregnancy.     

Intravenous trimethylamine N-oxide infusion does not modify circulating markers of liver health,glucose tolerance,and milk production in early-lactation cows

In rodents and humans, the gut bacteria-derived metabolite trimethylamine N-oxide (TMAO) has been implicated in the progression of cardiovascular disease, chronic kidney disease, fatty liver, and insulin resistance; however, the effects of TMAO on dairy cattle health and milk production have not been defined. We aimed to determine whether intravenous TMAO infusion modifies measures of liver health, glucose tolerance, and milk production in early-lactation cows. Eight early-lactation Holstein cows (30.4 ± 6.41 d in milk; 2.88 ± 0.83 lactations) were enrolled in a study with a replicated 4 × 4 Latin square design. Cows were intravenously infused TMAO at 0 (control), 20, 40, or 60 g/d for 6 d. Washout periods lasted 9 d. Intravenous glucose tolerance tests (GTT) occurred on d 5. Blood was collected daily. Milk was collected on d −1, 0, 5, and 6. Urine was collected on d −1 and 6. Circulating metabolites, milk components, and TMAO concentrations in milk, urine, and plasma were quantified. Data were analyzed using a mixed model that included the fixed effects of treatment. Concentrations of TMAO in plasma, milk, and urine increased linearly with increasing dose. Dry matter intake and milk production were not modified by treatment. Daily plasma triacylglycerol, fatty acid (FA), and glucose concentrations were not modified. Serum albumin, total protein, globulin, total bilirubin, direct bilirubin, aspartate aminotransferase, γ-glutamyl transferase, and glutamate dehydrogenase concentrations were also not modified by treatment. Serum GTT glucose, FA, and insulin concentrations were not modified by treatment. Plasma total, reduced, and oxidized glutathione concentrations were also not modified by treatment. We conclude that a 6-d intravenous infusion of TMAO does not influence measures of liver health, glucose tolerance, or milk production in early-lactation dairy cows.     

Predicting energy × protein interaction on milk yield and milk composition in dairy cows

Feed management is one of the principal levers by which the production and composition of milk by dairy cows can be modulated in the short term. The response of milk yield and milk composition to variations in either energy or protein supplies is well known. However, in practice, dietary supplies of energy and protein vary simultaneously, and their interaction is still not well understood. The objective of this trial was to determine whether energy and protein interacted in their effects on milk production and milk composition and whether the response to changes in the diets depended on the parity and potential production of cows. From the results, a model was built to predict the response of milk yield and milk composition to simultaneous variations in energy and protein supplies relative to requirements of cows. Nine treatments, defined by their energy and protein supplies, were applied to 48 cows divided into 4 homogeneous groups (primiparous or multiparous × high or low milk potential) over three 4-wk periods. The control treatment was calculated to cover the predicted requirements of the group of cows in the middle of the trial and was applied to each cow. The other 8 treatments corresponded to fixed supplies of energy and protein, higher or lower than those of the control treatment. The results highlighted a significant energy × protein interaction not only on milk yield but also on protein content and yield. The response of milk yield to energy supply was zero with a negative protein balance and increased with protein supply equal to or higher than requirements. The response of milk yield to changes in the diet was greater for cows with high production potential than for those with low production potential, and the response of milk protein content was higher for primiparous cows than for multiparous cows. The model for the response of milk yield, protein yield, and protein content obtained in this trial made it possible to predict more accurately the variations in production and composition of milk relative to the potential of the cow because of changes in diet composition. In addition, the interaction obtained was in line with a response corresponding to the more limiting of 2 factors: energy or protein.     

Suitability of low-dosage oxytocin treatment to induce milk ejection in dairy cows

Chronic use of high oxytocin (OT) dosages can cause a reduced response to endogenous OT. In this study the OT dosages used in the milking practice of 82 dairy cow farms were recorded. The OT dosages per cow used were high, especially when injected i.m. (23 ± 2 IU) compared with i.v. (7 ± 1 IU). In addition, the minimum OT dosages needed to obtain normal milk removal in cows with disturbed milk ejection were investigated. Seventeen cows routinely treated with OT during milking (group T) and 17 cows without previous OT treatment were used (group C). After cessation of spontaneous milk flow, both T and C groups were injected i.v. with a low dosage of OT (0.2 or 0.5 IU/cow). The time from injection until cessation of the OT-induced milk flow was recorded (response phase). The response phase and the amounts of removed milk by effect of the OT injection increased with increasing OT dosage. Values for 0.2 and 0.5 IU/cow of OT injected i.v. were (response phase and amount of milk removed) 198 ± 27 and 302 ± 18 s and 3.4 ± 0.7 kg and 6.5 ± 1.3 kg, respectively, for the C group, and 157 ± 15 and 221 ± 16 s and 3.2 ± 0.5 and 5.5 ± 1.0 kg, respectively, for the T group. Within 20 min of the OT injection, plasma concentrations returned to basal levels. The threshold OT concentration at cessation of milk flow after injection of 0.2 or 0.5 IU/cow of OT was calculated based on the OT plasma half-life. The threshold increased with increasing dosages of OT and was higher in group T (8 ± 1 and 14 ± 1 pg/mL for 0.2 and 0.5 IU/cow, respectively) than in group C (7 ± 1 and 11 ± 1 pg/mL for 0.2 and 0.5 IU/cow, respectively). In conclusion, desensitization of the udder toward OT occurs when the udder is exposed to elevated OT plasma concentrations, both short-term during the actual milking and long-term due to chronic high-dosage OT treatment. However, low-dosage OT treatments to induce normal milk removal can minimize the observed side effects.     

Changes in cisternal compartment based on stage of lactation and time since milk ejection in the udder of dairy cows

Two experiments were conducted to study changes induced by stage of lactation and milk ejection in the cisternal compartment of the udder in dairy cows. In experiment 1, 18 cows grouped according to stage of lactation were used 12 h after milking for measuring alveolar and cisternal milk volumes (by cannula) and cisternal area (by ultrasonography) in the front quarters. Cisternal milk and cisternal area were correlated (r = 0.74 to 0.82) for all stages of lactation. As lactation advanced, volumes of alveolar and cisternal milk and cisternal area decreased. Proportion of cisternal milk varied between stages (early, 33.2%; mid, 23.1%; and late, 42.6%). In experiment 2, 7 cows were used to show return of milk from cisternal to alveolar compartments when milk ejection was induced without milking. Cisternal area was measured before (0 min) and after (3, 15, 30, and 60 min) an i.v. oxytocin (OT) injection administered immediately before normal a.m. and p.m. milking times. Cisternal area increased dramatically from 0 to 3 min (98%) and decreased slowly thereafter. The 0- and 3-min data provide clear evidence of milk ejection, and their difference indicated cistern elasticity. Maximum cisternal area in each cow was similar for the 8- and 16-h milking intervals, indicating that in both cases the cistern was completely full of milk. Decrease in cisternal area after 3 min was significant at 15, 30, and 60 min. Decreased cisternal area was interpreted as the reflux of cisternal milk to the alveolar compartment. We termed this 'cisternal recoil.' In conclusion, ultrasonography was a useful method to evaluate dynamic changes in cisternal milk throughout lactation and after udder stimulation in dairy cows. Evidence exists that udder cisterns decrease when lactation advances and milk returns to the alveolar compartment when cows remain unmilked after milk ejection.     

Long-acting insulins alter milk composition and metabolism of lactating dairy cows

This study investigated the effect of 2 different types of long-acting insulin on milk production, milk composition, and metabolism in lactating dairy cows. Multiparous cows (n = 30) averaging 88 d in milk were assigned to one of 3 treatments in a completely randomized design. Treatments consisted of control (C), Humulin-N (H; Eli Lilly and Company, Indianapolis, IN), and insulin glargine (L). The H and L treatments were administered twice daily at 12-h intervals via subcutaneous injection for 10 d. Cows were milked twice daily, and milk composition was determined every other day. Mammary biopsies were conducted on d 11, and mammary proteins extracted from the biopsies were analyzed by Western blot for components of insulin and mammalian target of rapamycin signaling pathways. Treatment had no effect on dry matter intake or milk yield. Treatment with both forms of long-acting insulin increased milk protein content and tended to increase milk protein yield over the 10-d treatment period. Analysis of milk N fractions from samples collected on d 10 of treatment suggested that cows administered L tended to have higher yields of milk protein fractions than cows administered H. Milk fat content and yield tended to be increased for cows administered long-acting insulins. Lactose content and yields were decreased by treatment with long-acting insulins. Administration of long-acting insulins, particularly L, tended to shift milk fatty acid composition toward increased short- and medium-chain fatty acids and decreased long-chain fatty acids. Plasma concentrations of glucose and urea N were lower for cows administered long-acting insulins; interactions of treatment and sampling time were indicative of more pronounced effects of L than H on these metabolites. Concentrations of nonesterified fatty acids and insulin were increased in cows administered long-acting insulins. Decreased concentrations of urea N in both plasma and milk suggested more efficient use of N in cows administered long-acting insulins. Western blot analysis of mammary tissue collected by biopsy indicated that the ratios of phosphorylated protein kinase b (Akt) to total Akt and phosphorylated ribosomal protein S6 (rpS6) to total rpS6 were not affected by long-acting insulins. Modestly elevating insulin activity in lactating dairy cows using long-acting insulins altered milk composition and metabolism. Future research should explore mechanisms by which either insulin concentrations or insulin signaling pathways in the mammary gland can be altered to enhance milk fat and protein production.     

Identification of predictive biomarkers of disease state in transition dairy cows

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.     

Diagnostic milk biomarkers for predicting the metabolic health status of dairy cattle during early lactation

Data on metabolic profiles of blood sampled at d 3, 6, 9, and 21 in lactation from 117 lactations (99 cows) were used for unsupervised k-means clustering. Blood metabolic parameters included β-hydroxybutyrate (BHB), nonesterified fatty acids, glucose, insulin-like growth factor-1 (IGF-1) and insulin. Clustering relied on the average and range of the 5 blood parameters of all 4 sampling days. The clusters were labeled as imbalanced (n = 42) and balanced (n = 72) metabolic status based on the values of the blood parameters. Various random forest models were built to predict the metabolic cluster of cows during early lactation from the milk composition. All the models were evaluated using a leave-group-out cross-validation, meaning data from a single cow were always present in either train or test data to avoid any data leakage. Features were either milk fatty acids (MFA) determined by gas chromatography (MFA [GC]) or features that could be determined during a routine dairy herd improvement (DHI) analysis, such as concentration of fat, protein, lactose, fat/protein ratio, urea, and somatic cell count (determined and reported routinely in DHI registrations), either or not in combination with MFA and BHB determined by mid-infrared (MIR), denoted as MFA [MIR] and BHB [MIR], respectively, which are routinely analyzed but not routinely reported in DHI registrations yet. Models solely based on fat, protein, lactose, fat/protein ratio, urea and somatic cell count (i.e., DHI model) were characterized by the lowest predictive performance [area under the receiver operating characteristic curve (AUC_ROC) = 0.69]. The combination of the features of the DHI model with BHB [MIR] and MFA [MIR] powerfully increased the predictive performance (AUC_ROC = 0.81). The model based on the detailed MFA profile determined by GC analysis did not outperform (AUC_ROC = 0.81) the model using the DHI-features in combination with BHB [MIR] and MFA [MIR]. Predictions solely based on samples at d 3 were characterized by lower performance (AUC_ROC DHI + BHB [MIR] + MFA [MIR] model at d 3: 0.75; AUC_ROC MFA [GC] model at d 3: 0.73). High predictive performance was found using samples from d 9 and 21. To conclude, overall, the DHI + BHB [MIR] + MFA [MIR] model allowed to predict metabolic status during early lactation. Accordingly, these parameters show potential for routine prediction of metabolic status.     

Effects of feeding dry glycerin to early postpartum Holstein dairy cows on lactational performance and metabolic profiles

Effects of feeding a dry glycerin product (minimal 65% of food grade glycerol, dry powder) to 39 multiparous Holstein dairy cows (19 control and 20 glycerin-supplemented; lactation number = 2.2 ± 1.3 SD) on feed intake, milk yield and composition, and blood metabolic profiles were investigated. Dry glycerin was fed at 250 g/d as a top dressing (corresponding to 162.5 g of glycerol/d) to the common lactating total mixed ration from parturition to 21 d postpartum. Individual milk was sampled from 2 consecutive milkings weekly and analyzed for components. Blood was sampled from the coccygeal vein at 4, 7, 14, and 21 (±0.92, pooled SD) d in milk and analyzed for urea nitrogen, glucose, insulin, nonesterified fatty acids, and β-hydroxybutyrate. Urine was tested for the acetoacetate level weekly by using Ketostix. Average feed intake, milk yield and components, blood metabolites, and serum insulin concentrations were not affected by dry glycerin supplementation. Glycerin-supplemented cows experienced a more positive energy status (higher concentrations of plasma glucose, lower concentrations of plasma β-hydroxybutyrate, and lower concentrations of urine ketones), which was observed during the second week of lactation, suggesting that energy availability may have been improved. This glucogenic effect of dry glycerin did not result in an increase in feed intake or milk yield during the first 3 wk of lactation, likely because of the relatively less negative energy status of cows transitioning into lactation. The tendency toward higher milk yield for glycerin-supplemented cows during wk 6 of lactation (52 vs. 46 kg/d) after the supplementation period (dry glycerin was terminated at wk 3 of lactation) suggested a potential benefit of dry glycerin on subsequent milk production, perhaps through changes in metabolism, which requires further investigation.     

Effects of spray-dried plasma protein product on early-lactation dairy cows

Spray-dried plasma protein (SDP) compared with blood meal (BM) may contain various functional and active components that may benefit animal health. The objective of this experiment was to investigate the effects of feeding SDP or BM on production and blood profile in dairy cows during the transition and early-lactation periods. Seventy-two Holstein cows at 14 d before calving were used in a randomized block design. During the prepartum period, cows were fed a typical late-gestation diet containing BM (100 g/cow per day; 100BM, n = 24) or SDP (100 g/cow per day; 100SDP; n = 48). After calving, cows that were fed BM prepartum were fed a typical lactation diet formulated to provide 100 g/d of BM (100BM). Half the cows that were fed 100SDP prepartum were fed a lactation diet formulated to provide 100 g/d of SDP (100SDP; n = 24), and half were fed a diet formulated to provide 400 g/d of SDP (400SDP; n = 24) on a dry matter basis where SDP replaced BM (100SDP) or BM and soybean products (400SDP). All diets were balanced for crude protein concentration and metabolizable protein supply assuming BM and SDP were equal in rumen-degradable protein and rumen-undegradable protein. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) as a randomized block design where contrasts were made for 100BM versus 100SDP for prepartum variables and 100BM versus 100SDP and 100SDP versus 400SDP for postpartum variables. Prepartum supplementation of SDP had no effect on plasma fatty acids and β-hydroxybutyrate (2 d before calving). Plasma fatty acids (255 ± 29 µEq/mL) and β-hydroxybutyrate (675 ± 70 µmol/L) at 8 and 14 d of lactation were not affected by SDP in the diet. Feeding SDP at 100 g/d compared with 100BM increased or tended to increase milk fat, protein, and lactose contents for 16 wk after parturition. Providing SDP at 400 g/d in the diet increased milk yield (42 vs. 39 kg/d), energy-corrected milk (44 vs. 41 kg/d), energy-corrected milk per kilogram of dry matter intake, and yields of milk fat (1.60 vs. 1.48 kg/d), protein (1.21 vs. 1.16 kg/d), and lactose compared with 100SDP. Body weight losses tended to be lower for 100SDP compared with 100BM without a difference between 100SDP and 400SDP. Plasma histidine concentration (d 14 of lactation) was lower for SDP compared with 100BM. In addition, plasma 1-methyl-l-histidine tended to be lower as inclusion rate of SDP increased. In conclusion, SDP at 400 g/d increased milk and milk component yields without an increase in feed intake. Studies evaluating effects of functional and active compounds in SDP on gut microbiome, gut health, and immune functions may be needed to determine mode of action.     

Prediction of metabolic status of dairy cows in early lactation using milk fatty acids and test-day variables

Early lactation metabolic imbalance is an important physiological change affecting the health, production, and reproduction of dairy cows. The aims of this study were (1) to evaluate the potential of test-day (TD) variables with or without milk fatty acids (FA) content to classify metabolically imbalanced cows and (2) to evaluate the robustness of the metabolic classification with external data. A data set was compiled from 3 experiments containing plasma β-hydroxybutyrate, nonesterified FA, glucose, insulin-like growth factor-I, FA proportions in milk fat, and TD variables collected from 244 lactations in wk 2 after calving. Based on the plasma metabolites, 3 metabolic clusters were identified using fuzzy c-means clustering and the probabilistic membership value of each cow to the 3 clusters was determined. Comparing the mean concentration of the plasma metabolites, the clusters were differentiated into metabolically imbalanced, moderately impacted, and balanced. Following this, the 2 metabolic status groups identified were imbalanced cows (n = 42), which were separated from what we refer to as “others” (n = 202) based on the membership value of each cow for the imbalanced cluster using a threshold of 0.5. The following 2 FA data sets were composed: (1) FA (groups) having high prediction accuracy by Fourier-transform infrared spectroscopy and, thus, have practical significance, and (2) FA (groups) formerly identified as associated with metabolic changes in early lactation. Metabolic status prediction models were built using FA alone or combined with TD variables as predictors of metabolic groups. Comparison was made among models and external evaluations were performed using an independent data set of 115 lactations. The area under the receiver operating characteristics curve of the models was between 75 and 91%, indicating their moderate to high accuracy as a diagnostic test for metabolic imbalance. The addition of FA groups to the TD models enhanced the accuracy of the models. Models with FA and TD variables showed high sensitivities (80–88%). Specificities of these models (73–79%) were also moderate and acceptable. The accuracy of the FA models on the external data set was high (area under the receiver operating characteristics curve between 76 and 84). The persistently good performance of models with Fourier-transform infrared spectroscopy-quantifiable FA on the external data set showed their robustness and potential for routine screening of metabolically imbalanced cows in early lactation.     

Effects of postpartum uterine diseases on milk production and culling in dairy cows

The objective was to quantify the effect of postpartum uterine diseases on milk production and culling. Data from 2,178 Holstein cows in 6 herds enrolled in a randomized clinical trial were used. Milk production data from the first 4 Dairy Herd Improvement Association (DHIA) test-days and culling data from farm records were collected. Retained placenta (RP; ≥24 h after parturition) and metritis [≤20 d in milk (DIM)] were diagnosed by farm managers using standardized definitions. Farms were visited weekly and cows were examined at 35 and 56 (±3) DIM using endometrial cytology (cytobrush device), vaginal discharge scoring (Metricheck device), and measurement of cervical diameter by transrectal palpation. Diagnostic criteria for cytological endometritis (CYTO) and purulent vaginal discharge (PVD) were established based on a detrimental effect on subsequent reproduction. Statistical analyses were performed using linear mixed models, logistic regression models, and Cox proportional hazard models, accounting for the effects of experimental treatments and herd clustering. Milk production and culling were the outcomes. Primiparous and multiparous cows were modeled separately for milk production. Milk production of primiparous cows was unaffected by uterine diseases. The effect of metritis on milk production was variable over time in multiparous cows: it decreased production per cow by 3.7 kg at the first DHIA test, but was not different at later tests. Retained placenta decreased milk production by 2.6 kg/d in multiparous cows through the first 4 DHIA tests. The projected effects of metritis and RP in multiparous cows were reductions of 259 kg and 753 kg over 305 DIM, respectively; these effects were additive. Neither CYTO nor PVD affected milk production. Culling risks at 30 and 63 DIM were unaffected by RP and metritis. Culling hazard up to 300 DIM was unaffected by RP, metritis, CYTO, or PVD, whether or not pregnancy status, milk production, and displaced abomasum were accounted for. Uterine disease decreased pregnancy rate, which was a substantial risk factor for culling; however, if affected cows became pregnant they were not at greater risk of culling.     

Long-term effects of feeding monensin on milk fatty acid composition in lactating dairy cows

The objective of this study was to determine the long-term effects of feeding monensin on milk fatty acid (FA) profile in lactating dairy cows. Twenty-four lactating Holstein dairy cows (1.46 ± 0.17 parity; 620 ± 5.9 kg of live weight; 92.5 ± 2.62 d in milk) housed in a tie-stall facility were used in the study. The study was conducted as paired comparisons in a completely randomized block design with repeated measurements in a color-coded, double blind experiment. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (TMR) with a forage-to-concentrate ratio of 60:40 (control TMR; placebo premix) vs. a medicated TMR [monensin TMR; regular TMR + 24 mg of Rumensin Premix per kg of dry matter (DM)] fed ad libitum. The animals were fed and milked twice daily (feeding at 0830 and 1300 h; milking at 0500 and 1500 h). Milk samples were collected before the introduction of treatments and monthly thereafter for 6 mo and analyzed for FA composition. Monensin reduced the percentage of the short-and medium-chain saturated FA 7:0, 9:0, 15:0, and 16:0 in milk fat by 26, 35, 19, and 6%, respectively, compared with the control group. Monensin increased the percentage of the long-chain saturated FA in milk fat by 9%, total monounsaturated FA by 5%, total n-6 polyunsaturated FA (PUFA) by 19%, total n-3 PUFA by 16%, total cis-18:1 by 7%, and total conjugated linoleic acid (CLA) by 43% compared with the control group. Monensin increased the percentage of docosahexaenoic acid (22:6n-3), docosapentaenoic acid (22:5n-3), and cis-9, trans-11 CLA in milk fat by 19, 13, and 43%, respectively, compared with the control. These results suggest that monensin was at least partly effective in inhibiting the biohydrogenation of unsaturated FA in the rumen and consequently increased the percentage of n-6 and n-3 PUFA and CLA in milk, thus enhancing the nutritional properties of milk with regard to human health.     

Changes in milk urea around insemination are negatively associated with conception success in dairy cows

Dietary protein levels are a risk factor for poor reproductive performance. Conception is particularly impaired in cases of high blood or milk urea. The objective of this study was to investigate the association between conception and low milk urea or changes in milk urea around artificial insemination (AI). Data were obtained from the French Milk Control Program for a 4-yr period (2009–2012). Milk urea values between 250 and 450 mg/kg (4.3 and 7.7 mM) were considered intermediate (I), and values ≤150 mg/kg (2.6 mM) were considered low (L). Milk urea values before and after each AI were allocated into 4 classes representing the dynamics of milk urea (before-after; I-I, I-L, L-I, and L-L). Subclinical ketosis was defined using milk fat and protein contents before AI as proxies. A logistic regression with a Poisson correction and herd as a random variable was then performed on data from Holstein or all breeds of cows. The success of conception was decreased [relative risk (95% confidence interval) = 0.96 (0.94–0.99)] in low-urea cows compared with intermediate-urea cows after AI; no significant association was found for urea levels before AI. When combining data on urea before and after AI, I-L urea cows exhibited a 5 to 9% decrease in conception compared with I-I urea cows, and L-I urea cows showed no difference in conception success compared with I-I urea cows. A decreased conception success for L-L urea cows compared with I-I urea cows was observed for the analysis with cows of all breeds. This work revealed that a decrease in urea from intermediate (before AI) to low (after AI) is a risk factor for conception failure. Surveys of variation in milk urea in dairy cows close to breeding are highly recommended.