Effects of ß-hydroxybutyrate and isoproterenol on lipolysis in isolated adipocytes from periparturient dairy cows and cows with clinical ketosis.

An in vitro model was used to investigate effects of ß-hydroxybutyrate and isoproterenol (ß-adrenergic receptor agonist) on lipolysis in isolated adipocytes from late pregnant and recently calved dairy cows (n=5) and cows with clinical ketosis (n=3). Incubation with 3.0 mmol/L ß-hydroxybutyrate reduced lipolysis in isolated adipocytes. This inhibitory effect was lower in the first lactation week (47%±16%) compared with late pregnancy (71%±6.5%). Incubation with 0.3 µmol/L isoproterenol stimulated lipolysis in isolated adipocytes from periparturient dairy cows. Basal lipolysis resulted in non-esterified fatty acid to glycerol ratios in the incubation media of 2.0±0.23 in prepartum samples, 2.1±0.23 in the first lactation week and 2.2±0.09 in cows with clinical ketosis. ß-Hydroxybutyrate reduced lipolysis by 45%±9.6% in isolated adipocytes from cows with clinical ketosis, indicating that impaired feedback of ß-hydroxybutyrate may not play a role in the disease etiology.

Genetic and nongenetic variation in plasma and milk ß-hydroxybutyrate and milk acetone concentrations of early-lactation dairy cows.

This study assessed genetic variation, heritability estimates, and genetic correlations for concentrations of plasma ß-hydroxybutyrate (BHBA), milk BHBA, and milk acetone in early lactation to investigate differences between cows in susceptibility to hyperketonemia and possibilities to use test-day milk ketone bodies for genetic improvement. Blood and test-day milk samples were collected on randomly selected dairy farms in the Netherlands from cows of various parities between 5 and 60 d in milk. Plasma samples were analyzed for BHBA (reference test for hyperketonemia) and test-day milk samples were analyzed for BHBA and acetone using Fourier-transform infrared spectroscopy. The final data set consisted of plasma BHBA concentrations of 1,615 cows from 122 herds. Milk BHBA and milk acetone concentrations were determined for 1,565 cows. Genetic variation, heritability, and proportion of phenotypic variation attributable to the herd were estimated using an animal model with fixed effects for parity and season, a covariate for days in milk, and random effects for herd, animal, and error. Genetic correlations for plasma BHBA, milk BHBA, and milk acetone were estimated using bivariate analyses. The heritability estimate for plasma BHBA concentrations in early lactation was 0.17, whereas heritability estimates for milk BHBA and milk acetone were 0.16 and 0.10, respectively. This indicates that selective breeding may contribute to a lower incidence of hyperketonemia in early lactation. For the 3 traits, the proportion of variance attributable to herd was larger than the additive genetic variance, underlining the importance of on-farm feeding and management in the etiology of hyperketonemia in fresh cows. Prevention strategies for hyperketonemia can, therefore, include both feeding and management strategies at dairy farms (short-term) and genetic improvement through breeding programs (long-term). Genetic correlations between concentrations of plasma BHBA and milk BHBA (0.52) or milk acetone (0.52) were moderate. As milk ketone bodies can be routinely analyzed at test days, this may provide a practical alternative for breeding programs aimed at reducing hyperketonemia in early lactation.

Routine detection of hyperketonemia in dairy cows using Fourier transform infrared spectroscopy analysis of ß-hydroxybutyrate and acetone in milk in combination with test-day information.

The objective of this study was to assess the quality of a diagnostic model for the detection of hyperketonemia in early lactation dairy cows at test days. This diagnostic model comprised acetone and ß-hydroxybutyrate (BHBA) concentrations in milk, as determined by Fourier transform infrared (FTIR) spectroscopy, in addition to other available test-day information. Plasma BHBA concentration was determined at a regular test day in 1,678 cows between 5 and 60 d in milk, originating from 118 randomly selected farms in the Netherlands. The observed prevalence of hyperketonemia (defined as plasma BHBA ≥1,200 µmol/L) was 11.2%. The value of FTIR predictions of milk acetone and milk BHBA concentrations as single tests for hyperketonemia were found limited, given the relatively large number of false positive test-day results. Therefore, a multivariate logistic regression model with a random herd effect was constructed, using parity, season, milk fat-to-protein ratio, and FTIR predictions of milk acetone and milk BHBA as predictive variables. This diagnostic model had 82.4% sensitivity and 83.8% specificity at the optimal cutoff value (defined as maximum sum of sensitivity and specificity) for the detection of hyperketonemia at test days. Increasing the cutoff value of the model to obtain a specificity of 95% increased the predicted value of a positive test result to 56.5%. Confirmation of test-positive samples with wet chemistry analysis of milk acetone or milk BHBA concentrations (serial testing) improved the diagnostic performance of the test procedure. The presented model was considered not suitable for individual detection of cows with ketosis due to the length of the test-day interval and the low positive predictive values of the investigated test procedures. The diagnostic model is, in our opinion, valuable for herd-level monitoring of hyperketonemia, especially when the model is combined with wet chemistry analysis of milk acetone or milk BHBA concentrations. By using the diagnostic model in combination with wet chemistry milk BHBA analysis, 84% of herds were correctly classified at a 10% alarm-level prevalence. As misclassification of herds may particularly occur when only a limited number of fresh cows are sampled, we suggest using prevalence estimates over several consecutive test days to evaluate feeding and management practices in smaller dairy farms.

Protein and fat mobilization and associations with serum ß-hydroxybutyrate concentrations in dairy cows.

The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum ß-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirty-four cows kept under similar conditions at a university dairy farm (no experimental treatments) were monitored from 4 wk before until 8 wk after calving. Mobilization of muscle protein was investigated by analysis of plasma 3-methylhistidine concentrations (3-MH, analyzed by a recently developed HPLC tandem mass spectrometry method) and ultrasound measurements of longissimus muscle thickness. Mobilization of fat tissue was monitored by serum nonesterified fatty acid (NEFA) concentrations and ultrasound measurements of backfat thickness. Large variation was observed between cows in onset and duration of periparturient protein and fat mobilization. Plasma 3-MH concentrations and muscle thickness profiles indicated that protein mobilization started, on average, before parturition and continued until approximately wk 4 of lactation. Serum NEFA concentrations and backfat thickness profiles showed that fat mobilization occurred from parturition until the end of the study. Thus, muscle protein mobilization occurred in advance of fat mobilization in most cows from this study. We hypothesized that this might be due to a prepartum amino acid deficiency in the absence of negative energy balance. The incidence of hyperketonemia in this study was 16/34 = 47%. With the exception of 3 cows defined as having severe hyperketonemia, cows with lower 3-MH concentrations had higher serum BHBA concentrations. A possible explanation for this observation might be that higher mobilization of protein around calving might restrict ketone body production due to the higher availability of glucogenic precursors in the period of most severe negative energy balance and highest fat mobilization. The validity of this hypothesis needs to be confirmed, but data from this study indicate that further research on the role of protein mobilization in the etiology of hyperketonemia in dairy cows is needed.

Technical note: quantification of plasma 1- and 3-methylhistidine in dairy cows by high-performance liquid chromatography-tandem mass spectrometry.

To improve monitoring of protein mobilization in dairy cows, we developed and evaluated a method to quantify 1-methylhistidine and 3-methylhistidine in plasma by HPLC-tandem mass spectrometry. The analytical method described is (1) sensitive: both histidine derivates can be detected in the picomole range; (2) accurate: intra- and interassay coefficients of variation were < 5% for all standard solutions of 1-methylhistidine and 3-methylhistidine measured (31 to 500 pmol); (3) specific: 1-methylhistidine is clearly separated from 3-methyl-histidine in plasma samples from dairy cows; and (4) flexible: can be easily adapted to measure other amino acids or compounds containing a primary amine. 1-Methylhistidine is present in plasma of dairy cows at concentrations of 5.0 ± 1.7 µM, similar to concentrations of 3-methylhistidine (4.4 ± 2.4 µM). Analytical separation of both histidine metabolites is essential when plasma 3-methylhistidine is used as indicator for muscle breakdown in dairy cows. Specific quantification of the concentration of 3-methylhistidine in bovine plasma samples by HPLC tandem mass spectrometry can improve monitoring of protein mobilization in dairy cows.

Short communication: ketone body concentration in milk determined by Fourier transform infrared spectroscopy: value for the detection of hyperketonemia in dairy cows.

The objective of this study was to evaluate Fourier transform infrared (FTIR) spectrometry to measure milk ketone bodies to detect hyperketonemic cows and compare this method with milk fat to protein ratio to detect hyperketonemia. Plasma and milk samples were obtained weekly from calving to wk 9 postpartum from 69 high-producing dairy cows. The reference test for hyperketonemia was defined as plasma concentration of beta-hydroxybutyrate (BHBA) >or=1,200 micromol/L. The weekly prevalence of hyperketonemia during the first 9 wk of lactation was, on average, 7.1%. Both BHBA and acetone in milk, determined by FTIR, had a higher sensitivity (80%) to detect hyperketonemia compared with milk fat to protein ratio (66%). Specificity was similar for the 3 diagnostic tests (71, 70, and 71%). In conclusion, FTIR predictions of BHBA or acetone in milk can detect cows with hyperketonemia in early lactation with a higher accuracy compared with the use of milk fat to protein ratio. Because of the high proportion of false-positive tests, there are concerns about the practical applicability of FTIR predictions of acetone, BHBA, and fat to protein ratio in milk to detect hyperketonemic cows.