BHB Concentrations Research Articles

Evaluation of Freestyle Optium Neo H and TaiDoc-TD4235 point-of-care meters for measuring blood β-hydroxybutyrate concentration in sheep.

Accurate and rapid measurement of blood β-hydroxybutyrate (β-OHB) concentrations is critical to identify hyperketonemia in sheep. The objective of this study was to test the performance of Freestyle Optium Neo H and TaiDoc-TD4235 point-of-care (POC) devices for measuring blood β-OHB concentrations in sheep. Venous blood samples from 105 sheep were evaluated with Freestyle Optium Neo H and TaiDoc-TD4235 meters. A reference approach was the laboratory measurement of serum BHB concentrations using the Randox D-3 Hydroxybutyrate reagent kit. Data were analyzed using Passing- Bablok regression and Bland-Altman analysis. The diagnostic accuracy was assessed using serum β-OHB concentration at a cut-off value of 0.8 mmol/L. Passing-Bablok regression analysis revealed an intercept of 0.066 (CI95 : 0.018-0.125) and slope of 1.242 (CI95 : 1.136-1.342) for Freestyle Optium Neo H. Intercept and slope values for the TaiDoc-TD4235 meter were 0.625 (CI95 : 0.539-0.724) and 1.265 (CI95 : 1.044-1.497), respectively. The Bland-Altman plot revealed a bias of 0.224 mmol/L and 0.737 mmol/L for Freestyle Optium Neo H and TaiDoc-TD4235 meters, respectively, compared with the reference method. Observed Total Error (TEobs ) was 56.7% for the Freestyle Optium Neo H and 168.7% for the Taidoc-TD4235. The sensitivity and specificity for the Freestyle Optium Neo H were 100% and 80.9%, respectively, at the threshold of 0.8 mmol/L. The Taidoc-TD4235 demonstrated a sensitivity of 93.3% and specificity of 8.9% at the threshold of 0.8 mmol/L. Because of the large TEobs , the Freestyle Optium Neo H and TaiDoc-TD4235 meters should not be used to quantify blood β-OHB concentrations in sheep.

Effects of early weaning on the reproductive performance of suckled Nelore cows in the subsequent breeding season.

This study aimed to evaluate the effects of early weaning (EW) on body composition, hormone concentrations and metabolites, and reproductive performance of Nelore cows in the subsequent breeding season (BS). Suckled cows that became pregnant by timed-AI (TAI) in the 2020-BS were exposed in 2021 to early weaning at 150 d (27 primiparous [PRI] and 74 multiparous [MUL]) or conventional weaning (CW) at 240 d postpartum (30 PRI and 77 MUL). Body weight (BW) and body condition score (BCS) were determined at 2020-BS, EW, CW, prepartum, and 2021-BS. Blood samples were collected at EW, CW, prepartum (54.75 ± 0.56 d prepartum), and 2021-TAI and assayed for insulin-like growth factor-1 (IGF-I), non-esterified fatty acid (NEFA), and β-hydroxybutyrate (BHB) concentrations. In 2021-BS, cows were exposed to a P4/E2-based protocol for TAI at day 0 (D0), and a second TAI was performed at D22 in females detected with luteolysis (D20) by Doppler ultrasound. The presence of corpus luteum (CL) on D10, estrous expression, and dominant follicle (DF) diameter, and blood perfusion (BP) on D2 and D0 were determined. Data were analyzed by ANOVA or logistic regression of SAS as a 2 × 2 factorial with main factors of parity (PRI or MUL) and weaning strategy (EW or CW). An interaction of parity and weaning strategy was not observed (P > 0.1), but the weight (kg) and BCS were greater (P < 0.05) in MUL cows at the five timepoints, and EW cows were heavier than CW at the moment of CW (541 vs. 493 kg; and 5.3 vs. 4.3), prepartum (551 vs. 506 kg; and 5.2 vs. 4.4) and 2021-BS (475 vs. 450 kg; and 4.5 vs. 3.7). Plasma urea concentration at 2021-BS was greater (P = 0.01) for PRI than for MUL. A parity-by-time interaction was observed (P ≤ 0.05) for concentrations of IGF-I, NEFA, and BHB. PRI cows had greater (P ≤ 0.05) concentrations of IGF-I at EW and greater (P ≤ 0.05) prepartum concentrations of NEFA and BHB than MUL cows. The proportion of cows with CL at D10 was not affected (P > 0.1) by weaning but was greater (P < 0.05) in MUL than in PRI cows (40.4 vs. 15.7%). The diameter of DF and proportion of BP on D0 were greater (P < 0.05) in EW cows than in CW cows. The pregnancy rate (P/AI, %) at the first TAI was greater (P < 0.05) in EW cows (60% vs. 45%), whereas no difference (P > 0.1) was observed at the second TAI. Cumulative P/AI (first and second TAIs) was greater (P < 0.05) in EW cows (81% vs. 63%). In conclusion, weaning at 150 d in Nelore cattle is a strategy to successfully recover the parous cow's body condition and to improve pregnancy success in the next BS, regardless of the cow's parity order.

Transition cow nutrition and management strategies of dairy herds in the northeastern United States: Part III—Associations of management and dietary factors with analytes, health, milk yield, and reproduction

The objective was to evaluate relationships between putative periparturient management and dietary factors at the pen and herd levels with metabolic- and inflammation-related analytes, health disorders, milk yield, and reproductive performance. Multiparous and primiparous cows from 72 farms in the northeastern United States were enrolled in a prospective cohort study. Farms were visited 3 times during the prepartum and postpartum periods: during the far-off dry, close-up dry, and fresh periods. Pen measurements were taken at each visit for the pens where cows sampled were housed, and particle size was determined for the total mixed ration for the pen. A survey was used to acquire data on herd-level management variables. Blood samples were collected from the same 11 to 24 cows per farm during the close-up and fresh period visits. Whole blood was analyzed for postpartum β-hydroxybutyrate (BHB) concentrations, and plasma was analyzed for prepartum and postpartum nonesterified fatty acids (NEFA) and postpartum haptoglobin (Hp) concentrations. Health event, milk yield, and reproductive records were acquired through the records management software program used on the farm. For the pen-level analysis, primiparous and multiparous cows were analyzed separately. For the pen- and herd-level analysis, a simple linear regression was conducted on all possible explanatory variables. Variables were included in the full multivariable general linear model if P < 0.20, and a manual backward stepwise elimination process ensued until all variables had P < 0.10. Our results indicate that pen- and herd-level management factors are associated with blood biomarkers, health, milk yield, and reproductive performance. For the prepartum period, our results support increasing the proportion of particles on the 19-mm sieve of the Penn State Particle Separator, optimizing bunk space, and not overfeeding metabolizable energy (ME), to decrease the prevalence of elevated postpartum NEFA, BHB, and Hp concentrations, decrease disorder incidence, maximize milk yield, and improve pregnancy risk to first service. For the fresh period, our results generally support optimizing bunk space, avoiding commingling, increasing feeding frequency, avoiding high physically effective undigested neutral detergent fiber (NDF) after 240 h of in vitro fermentation and high total fermentable carbohydrate diets while optimizing the inclusion of forage NDF, and ensuring adequate diet ME and metabolizable protein to reduce the prevalence of elevated postpartum NEFA, BHB, and Hp concentrations, minimize disorder incidence, maximize milk yield, and improve pregnancy risk to first service. At the herd level, our results generally support not vaccinating in the calving pen, minimizing the number of prepartum and postpartum pen moves, and avoiding long stays in the calving pen after parturition to reduce the prevalence of elevated biomarker concentrations, decrease disorder incidence, increase milk yield, and improve reproductive performance.

TMET-24. MITOCHONDRIAL AND GENE EXPRESSION CHANGES IN BRAIN TUMOR CELLS GROWN IN Β-HYDROXYBUTYRATE

Abstract Malignant brain tumors are devastating diseases with poor outcomes. The search for more effective therapeutic combinations has led to investigation of the use of a therapeutic ketogenic diet (KD) as an adjuvant therapy based on the metabolic dysregulation that is a hallmark of cancer. KD sometimes results in extended survival, but like all cancer therapies the tumor eventually becomes resistant. Resistance can arise due to the “metabolic remodeling” of resistant cells due to limitations in nutrient availability and other signals from the tumor microenvironment. We are investigating the molecular and mitochondrial changes of tumor cells grown in β-hydroxybutyrate (BHB), the main ketone produced when following a therapeutic KD, to partially recapitulate the long-term effects of the KD on patients. Murine malignant glioma cells (GL261-luc2) were cultured long term in 2, 5 or 10mM BHB. The Agilent XFe96 Seahorse Analyzer was used to measure changes in cell metabolism. Epigenetic changes that occur in the mitochondria were interrogated using DNA methylation assays. Finally, we implanted BHB resistant glioma cells intracranially in mice and carried out RNAseq to determine the mechanisms by which these cells adapt. Seahorse analyses of GL261-luc2 cells grown in BHB demonstrated changes in several respiratory parameters including maximal respiration and reserve capacity. In addition, there is a statistically significant decrease in mitochondrial DNA methylation with increasing concentrations of BHB. RNAseq analysis of BHB resistant GL261 tumors showed changes in pathways related to mitochondrial translation and the electron transport chain. Our preliminary results show that the heterogeneity seen in these tumors is recapitulated in the various mitochondrial respiration parameters. Further, these may be altered as a consequence of BHB therapy. An understanding of the genetic and mitochondrial changes that occur when cells become resistant will lead to a more effective use of the KD as an adjuvant therapy.

Changes in plasma fatty acids profile in hyperketonemic ewes during early lactation: a preliminary study

The transition from late pregnancy to early lactation is characterized by marked changes in energy balance of dairy ruminants. The mobilization of adipose tissue led to an increase in plasma non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB). The aim of this study was to analyze the total plasma fatty acids of healthy and hyperketonemic dairy ewes in early lactation through gas chromatography (GC) to evaluate metabolic alterations. An observational study was used with a cross-sectional experimental design. Forty-six Sarda dairy ewes were enrolled in the immediate post-partum (7 ± 3 days in milk) and divided into two groups according to serum BHB concentration: non-hyperketonemic group (n = 28; BHB < 0.86 mmol/L) and hyperketonemic group (n = 18; BHB ≥ 0.86 mmol/L). A two-way ANOVA included the effect of group and parity was used to evaluate differences in fatty acids (FA) concentrations. A total of 34 plasma FA was assessed using GC. 12 out of 34 FA showed a significant different between groups and 3 out of 34 were tended to significance. Only NEFA concentration and stearic acid were influenced by parity. The results may suggest possible links with lipid metabolism, inflammatory and immune responses in hyperketonemic group. In conclusion, GC represents a useful tool in the study of hyperketonemia and primiparous dairy ewes might show a greater risk to develop this condition.

357 The Effects of Colostrum Consumption and Feed Restriction on Performance, Energy Balance, and Behavior in Unweaned Male Holstein Calves Being Marketed and Transported

Abstract Male Holstein calves (n = 80; 42.7 ± 1.44 kg and 14.2 ± 0.81 days of age) were used to evaluate the effects of colostrum consumption and feed restriction on performance, energy balance (serum concentration of BHB and NEFA), and behavior on calves marketed and transported during 19h. Well-colostrated (WC) calves received 4L of colostrum within the first 2h after birth and 2L of colostrum in the next 3 feedings and poor-colostrated (PC) calves received 2L of colostrum within the first 2h after birth. At approximately 14 d of age, calves were subjected to an assembly center simulation (ACS, d-4 to d-1) before a 19h-transport (d0) where they were fed 4L of milk replacer (MR)/d (moderate restriction; MO) or 4L of a rehydration solution/d (severe restriction; SV). After the ACS, treatments were: WCMO, WCSV, PCMO, PCSV and a control group (WCCT) with ad-libitum access to concentrate and not transported. From d1 to d7, all calves were fed the same diet (4L/d of MR and ad-libitum access to concentrate, straw, and water). Data were analyzed using mixed models with repeated measures. There was a time by treatment interaction (P&amp;lt; 0.01) for NEFA and BHB concentrations before transport being greater for the WCSV compared with PCSV calves. After the ACS, BW was reduced (P&amp;lt; 0.01) for the WCSV and PCSV calves compared with the WCCT, WCMO and PCMO calves. After transportation, a time by treatment interaction (P&amp;lt; 0.01) was observed in concentrate intake recovery being lesser for the PCSV and PCMO; it took 4 days for those calves to recover concentrate intake to the same extent that WCCT, WCMO, and PCMO. During the ACS, PCSV calves showed greater (P=0.05) standing times compared with the remaining calves. Colostrum consumption and the degree of feed restriction affect physiological, performance, and behavior responses in calves before and after transportation.

An investigation of blood, milk, and urine test patterns for the diagnosis of ketosis in dairy cows in early lactation.

Ketosis in dairy cattle is primarily diagnosed based on the concentrations of ketone bodies in the blood, milk, or urine. Cow-side tests using these fluids are available for rapid detection of elevated concentrations of ketone bodies. Although these tests have been extensively validated, the performance of different tests has not been compared over time. Our objectives were to investigate the relationship between point-of-care diagnostic tests measuring the concentrations of β-hydroxybutyrate (BHB) in blood (BT; Precision Xtra, Abbott Laboratories), BHB in milk (MT; Keto-Test, Elanco), and acetoacetate (AcAc) in urine (UT; Ketostix, Bayer Corporation) through cases of ketosis. Holstein cows (n = 148) were screened daily for hyperketonemia (HYK; blood BHB ≥1.2 mmol/L) from 3 to 16 d in milk (DIM); moreover, milk and urine samples were collected concomitantly and tested for ketones (ketosis thresholds: 100 µmol/L milk BHB and 5 mg/dL urine AcAc). Of the animals screened (n = 148), 74% were diagnosed with HYK. When diagnosed with HYK, cows were treated with propylene glycol orally once daily for 5 d. After the day of diagnosis (d 0), hyperketonemic cows were retested with BT, MT, and UT for 3 d (d 1, 2, and 3). We assessed the diagnostic test performance and time to ketosis (survival analyses and Cox proportional hazards models) of MT and UT compared with BT. Considering all paired samples (before and after diagnosis of HYK), MT had 61% sensitivity and 91% specificity, whereas the UT had 77% sensitivity and 94% specificity compared with BT. The specificity of MT and UT increased from d 0 to d 1, decreased on d 2, and increased on d 3. The median time to diagnosis of ketosis in blood was 5 DIM (95% CI 5 to 7 DIM); moreover, MT and UT had 2 d greater median time to diagnosis of ketosis compared with the BT [7 DIM (6 to 11 d); and 7 DIM (6 to 13 d), respectively]. We concluded that the UT is a more sensitive predictor of blood BHB concentration than the MT. The UT and MT tests diagnosed ketotic cows approximately 2 d later than the BT. The possible consequences of delay in detection of ketosis in milk and urine should be investigated.

Ketone Monoester Ingestion Alters Metabolism and Simulated Rugby Performance in Professional Players

Ketone ingestion can alter metabolism but effects on exercise performance are unclear, particularly with regard to the impact on intermittent-intensity exercise and team-sport performance. Nine professional male rugby union players each completed two trials in a double-blind, randomized, crossover design. Participants ingested either 90 ± 9g carbohydrate (CHO; 9% solution) or an energy matched solution containing 20 ± 2g CHO (3% solution) and 590mg/kg body mass β-hydroxybutyrate monoester (CHO + BHB-ME) before and during a simulated rugby union-specific match-play protocol, including repeated high-intensity, sprint and power-based performance tests. Mean time to complete the sustained high-intensity performance tests was reduced by 0.33 ± 0.41s (2.1%) with CHO + BHB-ME (15.53 ± 0.52s) compared with CHO (15.86 ± 0.80s) placebo (p = .04). Mean time to complete the sprint and power-based performance tests were not different between trials. CHO + BHB-ME resulted in blood BHB concentrations that remained >2mmol/L during exercise (p < .001). Serum lactate and glycerol concentrations were lower after CHO + BHB-ME than CHO (p < .05). Coingestion of a BHB-ME with CHO can alter fuel metabolism (attenuate circulating lactate and glycerol concentrations) and may improve high-intensity running performance during a simulated rugby match-play protocol, without improving shorter duration sprint and power-based efforts.

Factors affecting true and functional productive lifespan in Italian Holstein-Friesian cows

The identification of factors affecting longevity in dairy cows is of great importance to improve their length of productive life. Cows can be replaced for poor milk production (i.e. true productive lifespan, TPL) or other reasons than production (functional productive lifespan, FPL). The objective of the present study was to investigate the influence of milk yield, fat and protein contents, insemination status (INS), somatic cell count (SCC) and β-hydroxybutyrate (BHB) concentration in milk on TPL and FPL. Data included 70,748 Italian Holstein-Friesian cows that calved for the first time between 2014 and 2018. Proportional hazards models assuming a piecewise Weibull distribution of the baseline hazard function, with or without adjustment for milk production, were used to compute relative risks of culling associated with FPL and TPL, respectively. Our study revealed that milk yield, protein and fat contents, INS, SCC and concentration of BHB in milk exhibited significant relationships with TPL and FPL. In particular, the huge difference in risk ratios between high- and low-producing cows confirms the central role of milk production in culling decision-making. In addition, farmers appear more prone to cull cows with several events of high SCC and BHB concentration in milk. HIGHLIGHTS Milk yield, fat and protein contents, insemination status, somatic cell count and ketosis affect productive lifespan in Italian Holstein cows. These factors, therefore, contribute significantly to culling decisions. The identified risk factors of culling can be exploited to improve the current management practices in Italian Holstein Friesian herds and cows’ longevity.

Method Comparison of β-Hydroxybutyrate Using a Point-of-Care Device and Dry Chemistry Analyzer in Three Sea Turtle Species.

The ketone β-hydroxybutyrate (BHB) serves as an energy source when bodily energy stores are low. Concentrations of this blood analyte are often determined by spectrophotometric quantitative assays with a dry chemistry analyzer; however, rapid assessment with point-of-care devices have the potential to improve assessment of animals in the field or in clinical settings. We measured BHB concentrations in whole blood samples from 54 leatherback (Dermochelys coriacea), 27 loggerhead (Caretta caretta), and 14 green (Chelonia mydas) sea turtles in Florida, US with a point-of-care device and validated its use with corresponding plasma samples and dry chemistry analyzer as the gold standard. Concentrations of BHB highly correlated between the two methods for all three species, with loggerheads showing the best agreement and lowest bias. Therefore, the point-of-care device used for this study (Lucidplus β-ketone monitoring system) is probably appropriate for sea turtle BHB measurements.

Brief Research Report: Expression of PD-1 and CTLA-4 in T Lymphocytes and Their Relationship With the Periparturient Period and the Endometrial Cytology of Dairy Cows During the Postpartum Period.

The present study sought to evaluate the expression of PD-1 and CTLA-4 in blood T lymphocytes during the periparturient period and their relationship with uterine health in dairy cows, as determined by endometrial cytology and serum concentrations of β-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs), which are indicators of a negative energy balance. The second objective of this study was to investigate whether the expression of PD-1 and CTLA-4 in T lymphocytes is associated with the serum acute phase-protein haptoglobin concentration during the periparturient period. To address these objectives, 26 clinically healthy dairy cows were used. Peripheral blood was collected 14 days prepartum (T-14), at calving (T0), and 30 days postpartum (T30) to measure the expression of PD-1 and CTLA-4 in blood T lymphocytes by flow cytometry. In addition, we collected blood at T0, 10 days after parturition (T10), and T30 to obtain serum and determine the serum concentrations of NEFA, BHB, and Hp. Endometrial cytology was performed at T10, 20 days after parturition (T20), and T30. In the present study, we observed higher expression of CTLA-4 and PD-1 in T lymphocytes at parturition and in the prepartum period, which could indicate a relationship between these immune checkpoints and immunological tolerance during gestation in dairy cattle. In addition, a negative association between the expression of these immune checkpoints prepartum or at parturition and endometrial cytology at T20 and T30 was observed, indicating the negative implications of these immune response regulators in susceptibility to infections. This finding was further corroborated by the relationship between the serum concentration of haptoglobin and the expression of CTLA-4 and PD-1 by T lymphocytes. However, we did not observe a relationship between the indicators of negative energy balance, evaluated by the serum concentrations of BHB and NEFA, and the expression of the immune checkpoint markers studied. Thus, our findings represent an initial step that paves the way for the development of new therapeutic alternatives directed by the host with the objective of increasing the resistance of dairy cattle to infections in this critical period of life.

The Effect of Supplementation of Rumen-Protected Choline on Reproductive and Productive Performances of Dairy Cows.

Simple SummaryCholine is a key methyl donor synthesized endogenously in mammals, and its availability is important for various biological functions. A review of the literature presents controversial results and deals with the question of whether rumen-protected choline supplemented during the transition period significantly affects cows’ health, production, and reproduction. However, most recent research shows that in dairy cattle, choline supplementation improves milk yield, composition, and fertility.We aimed to evaluate the effects of organic herbal preparations containing rumen-protected choline (RPC) in dairy cow milk’s BHB and progesterone (P4) concentration changes, reproduction, and production performances. Cows were divided into the following two groups: The CHOL (n = 60) cow diet was supplemented with 10 g/day RPC from 20 days pre-calving to 20 days post-calving, and CONT (n = 60) were fed a conventional diet. BHB and P4 concentrations were measured at 5–64 DIM and 21–64 DIM, respectively, with DelPro 4.2. BHB was lower in the CHOL group at 5–64 DIM than CONT p > 0.05. The first post-calving P4 peak, p < 0.001, was determined earlier in the CHOL group, and the P4 profile during 21–64 DIM was similar, p > 0.05. The insemination rate was lower, and the interval between calvings was shorter. The first insemination time was earlier in the CHOL group, p < 0.05. Milk yield was higher in the CHOL group at 21–64 DIM, p > 0.05. The CHOL group had more fat in their milk at 31–60 DIM, p < 0.05. There were no significant differences in protein and SCC between the groups, p > 0.05. Based on our results, we concluded that the supplementation of RPC pre- and post-calving had statistically significant effects on the first peak of P4, and benefited the reproduction performances, milk yield, and milk fat during the early postpartum period.

Combined biotin, folic acid, and vitamin B12 supplementation given during the transition period to dairy cows: Part I. Effects on lactation performance, energy and protein metabolism, and hormones

Biotin (B8), folates (B9), and vitamin B12 (B12) are involved and interrelated in several metabolic reactions related to energy and protein metabolism. We hypothesized that a low supply of one of the latter vitamins during the transition period would impair metabolic status. The purpose of this study was to evaluate the effect of B8 supplementation on the response of lactation performance and selected energy and protein metabolites and hormones to a combined supplementation of B9 and B12 given to periparturient dairy cows, from d -21 to 21 relative to calving. A total of 32 multiparous Holstein cows housed in tie stalls were randomly assigned, according to their previous 305-d milk yield, to 8 incomplete blocks of 4 treatments: (1) a 2-mL weekly i.m. injection of saline (0.9% NaCl; B8-/B9B12-); (2) 20 mg/d of dietary B8 (unprotected from ruminal degradation) and 2-mL weekly i.m. injection of 0.9% NaCl (B8+/B9B12-); (3) 2.6 g/d of dietary B9 (unprotected) and 2-mL weekly i.m. injection of 10 mg of B12 (B8-/B9B12+); and (4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9, and weekly i.m. injection of 10 mg of B12 (B8+/B9B12+) in a 2 × 2 factorial arrangement. Milk yield and dry matter intake were obtained daily and milk components weekly. Blood samples were taken weekly from d -21 to calving and 3 times per week from calving to 21 d following parturition. Prepartum plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHB), and adiponectin were unaffected by treatments. Biotin, B9, and B12 supplements increased their respective concentrations in plasma and milk. Cows fed the B8 supplement tended to have lower dry matter intake, but only cows in B8+/B9B12- had greater plasma concentrations of NEFA compared with B8-/B9B12-. Milk and total solid yields were greater by 13.5 and 13.9%, respectively, for B8-/B9B12+ [45.5 (standard error, SE: 1.8) and 5.81 (0.22) kg/d, respectively] compared with B8-/B9B12- [40.1 (1.9) and 5.10 (0.23) kg/d, respectively], but these effects were suppressed when combined with the B8 supplement. Cows in the B8-/B9B12+ group had decreased plasma insulin and tended to have increased NEFA concentrations, but postpartum plasma concentrations of glucose, BHB, leptin, and adiponectin were not affected. These cows also mobilized more body fat reserves, as suggested by a tendency to increased plasma NEFA and more milk total solids compared with B8-/B9B12- cows. However, plasma concentrations of BHB and adiponectin were similar among treatments. This suggests that the B9 and B12 supplements enhanced efficiency of energy metabolism in early lactation cows. Folic acid and B12 supplementation increased postpartum plasma Cys and homocysteine concentrations but did not affect plasma Met concentration, suggesting an upregulation of the transsulfuration pathway. In summary, our results showed that, under the current experimental conditions, increasing B8 supply did not improve responses to the B9 and B12 supplementation.

ß-Hydroxybutyrate Improves Mitochondrial Function After Transient Ischemia in the Mouse

ß-Hydroxybutyrate (BHB) is a ketone body formed in high amounts during lipolysis and fasting. Ketone bodies and the ketogenic diet were suggested as neuroprotective agents in neurodegenerative disease. In the present work, we induced transient ischemia in mouse brain by unilaterally occluding the middle cerebral artery for 90 min. BHB (30 mg/kg), given immediately after reperfusion, significantly improved the neurological score determined after 24 h. In isolated mitochondria from mouse brain, oxygen consumption by the complexes I, II and IV was reduced immediately after ischemia but recovered slowly over 1 week. The single acute BHB administration after reperfusion improved complex I and II activity after 24 h while no significant effects were seen at later time points. After 24 h, plasma and brain BHB concentrations were strongly increased while mitochondrial intermediates (citrate, succinate) were unchanged in brain tissue. Our data suggest that a single administration of BHB may improve mitochondrial respiration for 1–2 days but not for later time points. Endogenous BHB formation seems to complement the effects of exogenous BHB administration.

Postmortem Diagnosis of Ketoacidosis by Determining Beta-Hydroxybutyrate Levels in Three Types of Body Fluids by Two Different Methods

Background: Postmortem assessment of endogenous ketoacidosis is primarily focused on the determination of 3-beta-hydroxybutyrate (BHB). The aim of our study was to identify the most adequate body fluid and postmortem quantification method for assessing ketoacidosis status immediately prior to death. Material and method: We performed a prospective study on 53 cases of sudden death or in-hospital death that were considered forensic cases and could present a state of ketoacidosis prior to death, the autopsies being performed at a post-mortem interval of 24–72 h. BHB analysis was performed by Multi-Functional Monitoring System XPER Technology analyzer (method A—portable analyzer) for peripheral blood, and by BHB Assay MAK041 Kit (method B) for vitreous humor (VH) and cerebrospinal fluid (CSF). Results: We identified 11 ketoacidosis cases using method A and 9 ketoacidosis cases using method B. All nine cases of ketoacidosis identified using the MAK041 kit were confirmed with the portable analyzer. For the 2 cases of ketoacidosis identified only with the portable analyzer, the values obtained by method B were at the diagnostic limit. BHB concentrations determined in VH and CSF by method B were statistically significantly correlated with each other and with peripheral blood BHB concentration. Conclusion: BHB, a marker of ketoacidosis, should be determined post-mortem whenever a metabolic imbalance is suspected irrespective of known risk factors or obvious morphological substrate to help establish the thanatogenic mechanism. BHB quantification can easily be performed using a handheld automatic analyzer and a sample of peripheral blood as BHB levels in various body fluids correlate with each other.

Impaired autophagy aggravates oxidative stress in mammary gland of dairy cows with clinical ketosis

When ketosis occurs, supraphysiological levels of free fatty acids (FFA) can cause oxidative injury to the mammary gland and autophagy can regulate the cellular oxidative status. The aim of this study was to investigate the autophagy status of mammary tissue and its associations with oxidative stress in healthy and clinically ketotic dairy cows. Mammary tissue and blood samples were collected from healthy cows [n = 15, β-hydroxybutyrate (BHB) <0.6 mM] and clinically ketotic cows (n = 15, BHB >3.0 mM) at 3 to 15 (average = 7) days in milk. For in vitro study, bovine mammary epithelial cells (BMEC) isolated from healthy cows were treated with 0, 0.3, 0.6, or 1.2 mM FFA for 24 h. Furthermore, BMEC were pretreated with 100 nM rapamycin, an autophagy activator, for 4 h or 50 mM 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h, followed by treatment with or without FFA (1.2 mM) for another 24 h. Oxidation indicators and autophagy-related protein abundance were measured. Compared with healthy cows, serum concentrations of FFA, BHB, and malondialdehyde were greater in clinically ketotic cows, but milk production (kg/d), milk protein (kg/d), activities of superoxide dismutase, catalase, and glutathione peroxidase were lower. Abundances of mRNA and protein of autophagy-related gene 5 (ATG5) and 7 (ATG7) were lower, but sequestosome-1 (SQSTM1, also called p62) greater in mammary tissue of clinically ketotic cows. The mRNA abundance of microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and protein abundance of LC3-II were lower in mammary tissue of clinically ketotic cows. In vitro, exogenous FFA increased the content of malondialdehyde and reactive oxygen species, but decreased the activities of superoxide dismutase, catalase, and plasma glutathione peroxidase. Compared with the 0 mM FFA group, abundance of ATG5, ATG7, LC3-II was greater, but p62 was lower in the 0.6 mM FFA-treated cells. Similarly, abundance of ATG5, ATG7, and LC3-II was lower, but p62 greater in the 1.2 mM FFA-treated cells relative to 0 mM FFA group. Culture with rapamycin alleviated oxidative stress induced by 1.2 mM FFA, whereas 3-MA aggravated it. Overall, results indicated that a low concentration (0.6 mM) of FFA can induce oxidative stress and activate autophagy in BMEC. At higher concentrations of FFA (1.2 mM), autophagy is impaired and oxidative stress is aggravated. Autophagy is a mechanism for BMEC to counteract FFA-induced stress. As such, it could serve as a potential target for further development of novel strategies against oxidative stress.

Transition cow nutrition and management strategies of dairy herds in the northeastern United States: Part II—Associations of metabolic- and inflammation-related analytes with health, milk yield, and reproduction

The objectives were as follows: (1) establish cow-level thresholds for prepartum nonesterified fatty acids (NEFA) and postpartum NEFA, β-hydroxybutyrate (BHB), and haptoglobin (Hp) concentrations associated with negative health events; (2) evaluate cow-level associations between biomarkers and 305-d mature equivalent milk at the fourth test day (ME305) and reproductive performance; and (3) identify herd-alarm levels (proportion of cows sampled above the critical threshold) for biomarkers that are associated with herd-level changes in disorder incidence (displaced abomasum and clinical ketosis), reproductive performance, and ME305. In a prospective cohort study, 1,473 cows from 72 farms were enrolled from the northeastern United States. Blood samples were collected from the same 11 to 24 cows per herd during the late-prepartum and early-postpartum periods. Whole blood was analyzed for postpartum BHB concentrations; plasma was analyzed for prepartum and postpartum NEFA and postpartum Hp concentrations. Critical thresholds for the biomarkers associated with health events for all cows were established using a receiver operating characteristic curve analysis. Poisson, linear mixed effects, and Cox proportional hazards models investigated the association of the biomarkers with health and performance. The prepartum NEFA and Hp threshold associated with culling was ≥0.17 mmol/L and 0.45 g/L, respectively. The postpartum NEFA and BHB thresholds associated with diagnosis of metritis, displaced abomasum, or clinical ketosis were ≥0.46 mmol/L and ≥0.9 mmol/L, respectively. Multiparous cows with prepartum NEFA concentration ≥0.17 mmol/L produced 479 kg less ME305. Multiparous and primiparous cows with postpartum NEFA concentration ≥0.46 mmol/L produced 280 kg less and 446 kg more ME305, respectively. Cows with BHB concentration ≥0.9 and ≥1.1 mmol/L produced 552 kg more ME305 and had a 20% decreased risk of pregnancy within 150 d in milk, respectively; however, multiparous cows with BHB concentration ≥1.5 mmol/L produced 376 kg less ME305. Cows with Hp concentration ≥0.45 g/L produced 492 kg less ME305 and had 28% decreased risk of pregnancy within 150 DIM. Cows with Hp concentration ≥0.45 g/L had 19% decreased pregnancy risk to first service (PRFS). Herds above the herd-alarm levels for prepartum NEFA had a 6.0-percentage unit increase in disorder incidence and a 6.0-percentage unit decrease in 21-d pregnancy rate (PR) for multiparous cows, a 3.9-percentage unit increase in PR and a 5.8-percentage unit increase in the probability of pregnancy for primiparous cows. Herds above the herd-alarm levels for postpartum NEFA had a 5.8- and 4.2-percentage unit increase in disorder incidence for multiparous and primiparous cows, respectively, a 789 kg decrease in ME305 for multiparous cows, and a 6.8- and 6.3-percentage unit decrease and increase in PR and PRFS for multiparous cows, respectively. Herds above the herd-alarm levels for BHB had an 8.5-percentage unit increase in disorder incidence, a 332 and 229 kg increase in ME305 for primiparous and multiparous cows, respectively, and a 3.2-, 5.2-, and 7.0-percentage unit decrease in PR, probability of pregnancy, and PRFS, respectively. Herds above the herd-alarm levels for postpartum Hp had a 5.3-percentage unit increase in disorder incidence. At the cow level and herd level, elevated biomarker concentrations were associated with an increased disorder risk and varied performance responses.

Evaluation of the point-of-care devices KetoSureTM and StatStrip Express® blood ketone tests using β-hydroxybutyrate spiked samples

Measurement of β-hydroxybutyrate (BHB) in blood is used clinically as a measure of ketosis when diabetic ketoacidosis is expected. With the introduction of point-of-care testing (POCT) for blood-BHB, nonproductive time is reduced to a minimum in a potential critical situation; however, studies have observed inferior quality of POCT-BHB. Recently, the POCT device KetoSure (Roche) has been introduced to the clinic. In this study, we evaluated the imprecision and linearity of KetoSure and compared this to the established StatStrip Express (Nova Biomedical) based on spiked full blood samples. We found comparable imprecision for KetoSure and StatStrip Express. However, linearity was only observed in the lower part of the measuring range for both devices. In a method comparison, higher values of BHB were measured by KetoSure than by an enzymatic endpoint spectrophotometric reference method (mean bias: 21% (95% confidence interval (CI): 4%–37%)). Conversely, StatStrip Express returned lower values of BHB (mean bias: −16% (95% CI: −38%–7%), while the widely applied POCT device FreeStyle Precision Neo (Abbott) returned values equivalent with the reference method (mean bias: 5% (95% CI: −14%–24%). In samples with concentrations of BHB above the measuring range, the POCT devices could be provoked to return falsely low results. In conclusion, the quality of KetoSure is in line with other established POCT devices; however, the KetoSure measures higher concentrations than other POCT devices. As, linearity only was observed in the lower part of the measuring range and as falsely low measures could be provoked, we advise users to interpret results with precaution.

Use of Butaphosphan with Cyanocobalamin in High Producing Cows and Associations with Milk Yield and Dry Matter Intake

This study aimed to verify the effect of butaphosphan combined with cyanocobalamin (B+C) on dry matter intake (DMI) and milk yield in high producing dairy cows. Eighteen multiparous Holstein cows managed in a compost barn system were enrolled on a calving date and remained under observation until 28 days in milk (DIM). The B+C group administered at 2500 mg of butaphosphan and 1.25 mg of cyanocobalamin (25 mL/cow/day, n = 9), or the control group (NaCl 0.9% administered at 25 mL/cow/day, n = 9,) receiving injections at calving, and at day 3 and 7 post-partum. The DMI, feed efficiency, and DMI/% body weight were evaluated until 21 DIM and the milk yield, rumination, activity, and lying time until 28 DIM. Various metabolites were evaluated at 0, 3, 7, 14, 21, and 28 DIM. DMI did not change with treatment. Milk yield was more significant in the B+C group than in the control group, with an increase of 3.66 kg/milk/d. The maintenance of DMI and the greater milk yield in the B+C group may suggest that the use of B+C can improve feed efficiency. No treatment effect was observed for concentrations of serum glucose, NEFA, BHB, and acetone, however, albumin was higher in B+C than in control. No effect was observed on milk composition. Our results suggest that B+C improves milk yield and feed efficiency by modulating the DMI.

Assessment of milk yield and composition, early reproductive performance, and herd removal in multiparous dairy cattle based on the week of diagnosis of hyperketonemia in early lactation

The purpose of this retrospective cohort study was to evaluate the effects of the timing of hyperketonemia (HYK) diagnosis during early lactation on milk yield and composition, reproductive performance, and herd removal. Plasma β-hydroxybutyrate (BHB) was measured twice a week during the first 2 wk of lactation in 362 multiparous Holstein cows for the diagnosis of HYK. In each week, cows were diagnosed as HYK positive (HYK+) if the plasma BHB concentrations were ≥1.2 mmol/L in at least one of the tests for the week evaluated. Milk-related outcomes (first 10 monthly milk tests) included milk yield, milk fat and protein content, milk urea nitrogen (MUN), and linear score of somatic cell count. Other performance outcomes of interest included risk of pregnancy within 150 d in milk (DIM) and herd removal (i.e., culling or death) within 300 DIM. Statistical models were built separately for cows diagnosed with HYK during the first week of lactation (wk1) and for cows diagnosed during the second week of lactation (wk2). All models for wk2 were adjusted by HYK diagnosed in wk1, along with other potential confounder variables. The association between HYK in each week and milk-related outcomes was assessed using generalized estimated equation models that accounted for repeated measures. Time to pregnancy and time to herd removal were analyzed using Cox's proportional hazard regression models. Seventy-eight cows (21.5%) tested positive for HYK during wk1, 60 cows (16.6%) in wk2, and 29 cows (8.0%) in both weeks. Hyperketonemia during wk1 was associated with a milk yield reduction of 3.7 kg [95% confidence interval (CI): -6.67 to -0.76] per cow per day throughout the lactation. Meanwhile, we did not observe evidence of an association between HYK diagnosed during wk2 and milk yield. During the first 2 monthly milk tests, cows diagnosed as HYK+ in wk1 had greater fat (0.42%; 95% CI: 0.16 to 0.67) and MUN (0.75 mg/dL; 95% CI: 0.26 to 1.24) content in milk than HYK-negative (HYK-) cows. We did not detect any evidence of an association between HYK diagnosed in wk2 and these outcomes. The HYK+ cows in wk1 had a 30% [hazard ratio (HR) = 0.70; 95% CI: 0.48 to 1.01] lower risk of pregnancy within 150 DIM and 2.48 times (95% CI: 1.63 to 2.89) higher risk of herd removal within 300 DIM than HYK- cows. Conversely, no evidence of association was observed between HYK+ cows in wk2 and risk of pregnancy by 150 DIM (HR = 0.98; 95% CI: 0.64 to 1.51) or removal from the herd within 300 DIM (HR = 0.91; 95% CI: 0.52 to 1.60). Our findings indicate that HYK diagnosed during wk1 of lactation is associated with negative performance in terms of milk yield, reproduction, and herd removal. No evidence of association was found for the same outcomes when HYK was diagnosed in wk2. Our results suggest the need to consider the timing when HYK is diagnosed when investigating its association with performance outcomes.