BHB Concentrations Research Articles

The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use β-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1–4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Mechanisms by which feeding synthetic zeolite A and dietary cation-anion difference diets affect feed intake, energy metabolism, and milk performance: Part II

The objectives of this study were to assess the effects of feeding 2 different diets, a low dietary cation-anion difference (DCAD) or a diet with synthetic zeolite A to multiparous Holstein cows during the close-up period on DMI and energy metabolism, as well as evaluate colostrum and milk production. A hundred and 21 multiparous Holstein cows, blocked by lactation number and expected parturition date were enrolled at 254 d of gestation and randomly assigned to 1 of 3 dietary treatments: control (CON; +190 mEq/kg; n = 40), negative DCAD (DCAD, -65 mEq/kg; n = 41; Ultra Chlor; Vita Plus, Lake Mills, WI, USA), or a diet containing sodium aluminum silicate zeolite (XZ; +278 mEq/kg, fed at 3.3% DM, targeting 500 g/day; n = 40; X-Zelit, Protekta Inc., Lucknow, ON, Canada/Vilofoss, Graasten, Denmark). Prepartum DMI was measured daily using Insentec Roughage Intake Control (RIC) gates (RIC System, Holofarm Group, Netherlands). All cows received the same postpartum diet. Blood and urine samples were collected daily beginning 14 d before parturition (D-14) until parturition (D0), and on 1, 2, 3, 6, 9, 12, 15, 18, 21, 35, and 49 d postpartum. Colostrum collected within 6 h of parturition, weighed, and based on samples Brix value, IgG concentrations, and nutrient composition were analyzed. Prepartum, cows fed XZ diet had decreased DMI (11.70 ± 0.26, 13.88 ± 0.26, and 13.45 ± 0.25 kg/d for XZ, CON, and DCAD respectively) and lower rumination (487 ± 8.1, 531 ± 8.3, and 527 ± 8.5 min for XZ, CON, and DCAD respectively) compared with CON and DCAD fed cows. However, rumination was not different postpartum due to treatment. No prepartum or postpartum differences were observed for glucose or BHB concentrations in blood between dietary treatments. Colostrum collected from cows fed XZ had the highest IgG concentrations (91.10 ± 2.63, 78.00 ± 2.63, and 78.90 ± 2.63 mg/mL for XZ, CON, and DCAD, respectively), but yield did not differ between dietary treatments. Additionally, cows in their third or greater lactation fed XZ had the highest milk production (51.0 ± 1.1 kg) during the first 49 d in milk. This study demonstrates that despite a decrease in DMI and rumination in cows fed XZ prepartum, blood BHB concentrations were not altered. Additionally, cows fed XZ had higher colostral IgG concentrations and 3+ lactation cows fed XZ produced the most milk. These data suggest that feeding XZ prepartum may improve colostrum quality and improve milk yield in mature cows, and does not impact energy metabolism.

Heat stress and feeding effects on the mucosa-associated and digesta microbiome and their relationship to plasma and digesta fluid metabolites in the jejunum of dairy cows

The intestinal microbiota plays a pivotal role in digestive processes and maintains gut health and intestinal homeostasis. These functions may be compromised by increased environmental heat which in turn reduces feed intake and gut integrity, while activating the intestinal immune system. It remains unknown whether high ambient temperatures, causing heat stress (HS) to dairy cows, disturb the eubiosis of the microbial community and if so, to which extent the reduction in feed intake and the impairment of circulating and intestinal metabolites account for the alterations of the jejunal microbiota. To address these questions, jejunal digesta, mucosa, and plasma samples from cows exposed to heat stress (HS: 28°C, temperature-humidity-index (THI) = 76, n = 10), control conditions (CON: 16°C, THI = 60, n = 10), or pair-feeding (PF: 16°C, THI = 60, n = 10) for 7 d were collected. Digesta fluids were examined for pH, acetate, nonesterified fatty acids (NEFA), glucose, and lactate, while plasma samples were analyzed for glucose, lactate, β-hydroxybutyrate (BHB), triglycerides, NEFA, creatinine and urea. The microbiota of digesta and mucosa samples were analyzed by 16S rRNA sequencing. The α diversity was higher in mucosa than digesta, but not affected by high ambient temperatures. However, the mucosa-associated microbiota appears more responsive to ambient heat than the digesta microbiome. The adaptive responses under HS conditions comprised an increased mucosal abundance of Bifidobacteriaceae, Succinivibrionaceae UCG-001, Clostridia and Lactobacillus. In the digesta, HS has exerted effects on microbial abundance of Colidextribacter and Lachnospiraceae UCG-008. Several correlations between plasma or intestinal metabolites and microbiota were elucidated, including Methanobacteriaceae correlating positively with plasma BHB and digesta glucose concentrations. Moreover, the reduction in feed intake during HS had non-negligible effects on microbial diversity and the abundance of certain taxa, underpinning the importance of nutrient supply on maintaining intestinal homeostasis.

Decreased lactose percentage in milk associated with quarter health disorder and hyperketolactia, a proxy for negative energy balance, in dairy cows

Several studies have described variations in lactose content (LC) in dairy cows during udder quarter health disorder or negative energy balance (NEB). However, their joint effects on LC have never been described. This was the aim of a longitudinal observational study performed on 5 Quebec dairy farms using automatic milking systems. Quarter milk samples were collected every 14 d from 5 to 300 DIM. Quarter health status was described by combining SCC level (SCC- or SCC+: < or ≥100,000 cells/mL) and infectious status (Patho- or Patho+: presence or absence of pathogens on a milk culture). Cows with NEB in early lactation (DIM <70) were identified using milk β-hydroxybutyrate (BHB) content: 0.15 mM = BHB-; 0.15 - 0.19 mM = BHB+; > 0.19 mM = BHB++. A total of 14,505 quarter cisternal milk samples were collected from 380 lactating cows. The quarter LC was analyzed using a mixed linear regression model with the following fixed effects: quarter health status, parity, time interval between last milking and sampling, quarter milk yield (in kg/d), DIM, and herd. A random quarter intercept with a repeated measures correlation structure and a cow random intercept were also specified. The LC of SCC+ quarters was lower (-0.17 ± 0.013% pts) compared with LC of SCC- quarters for both primiparous and multiparous cows. Over the 162 bacterial species identified, only 8 species had a prevalence greater than 4.0%, and just 5 of them were associated with a reduction in LC: Staphylococcus aureus, Staph. chromogenes, Streptococcus dysgalactiae, Staph. epidermidis, and Staph. simulans. Cows identified as BHB+ and BHB++ in early lactation had a lower LC (-0.05 ± 0.019% and -0.13 ± 0.020% pts, respectively) compared with BHB- cows. For BHB++ cows, in both parity groups the decrease in LC (-0.20 ± 0.025% pts) was higher in SCC+ quarters compared with SCC- quarters. Moreover, the additive effect of the quarter health status and NEB on milk LC was greater with larger increases in BHB. Our findings highlight the necessity to jointly take into consideration both quarter health status and milk BHB concentration when using LC as a biomarker for NEB.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

Investigating nutritional strategies during a rest period to improve health, growth, and behavioral outcomes of transported surplus dairy calves

The objective of this study was to investigate the effects of feeding surplus dairy calves a milk replacer (MR) or one of 2 different oral rehydration solutions (ORS) during a mid-transportation rest period on metabolic and clinical health indicators, growth, and behavioral outcomes after arrival to a calf-raising facility. Surplus dairy calves (n = 128) were transported in 4 cohorts from February to July 2022 for 12 h to a holding facility, rested for 8 h, then transported for an additional 6 h to a calf-raising facility. Upon arrival to the holding facility, calves were randomly assigned to 1 of 3 treatments: MR (n = 43), a high sodium ORS developed for diarrhea (ORS-D; n = 43), or a high potassium ORS developed for transportation (ORS-T; n = 42). The exact age of calves at transportation was unknown, however all calves were under 14 d of age. Calf body weight at enrollment was 43.9 ± 5.9 kg, 43.7 ± 6.5 kg, and 45.0 ± 4.5 kg for calves fed MR, ORS-D, and ORS-T, respectively. Calves were fed 2.0 L of their treatment twice, once upon arrival and once before leaving the holding facility. At unloading and reloading at the holding facility, calves were weighed and blood sampled. Calves were also health scored at unloading at the holding facility. After arrival at the calf-raising facility, calves were weighed, health scored, and blood sampled. Blood samples were collected at 24 and 48 h and body weight (BW) was recorded at 24 h, 48 h, 72 h, 5 d, 7 d, 14 d, and at 8 wks after arrival to the calf-raising facility. Calves were also health scored daily for 14 d, which included fecal consistency scoring and evaluating the presence or absence of respiratory disease. Lying time, lying bouts, and activity index were measured during transportation and from 3 d relative to transportation using accelerometers. At arrival to the calf-raiser, calves fed ORS-D had higher concentrations of NEFA and BHB than calves fed MR. Furthermore, calves fed ORS-T had higher concentrations of BHB at arrival to the calf raiser compared with calves fed MR. In the 14 d after arrival to the calf-raiser, there was evidence that calves fed ORS-T had a higher proportion of days with diarrhea and respiratory disease compared with those fed MR. During transportation, calves fed ORS-T had a lower activity index than calves fed MR, suggesting that ORS-T calves had lower overall activity. Additionally, on the day of transportation (d 0), ORS-T and ORS-D calves had a lower activity index than calves fed MR. There were no treatment effects on growth outcomes. The results of this study suggest that feeding MR rather than an ORS during a mid-transportation rest period could minimize fat mobilization and can potentially improve diarrhea and respiratory disease but does not affect growth outcomes after arrival to calf-raisers.

Continuous Ketone Monitoring via Wearable Microneedle Patch Platform.

Ketone bodies (KBs), especially β-hydroxybutyrate (BHB), have gained tremendous attention as potential biomarkers as their presence in bodily fluids is closely associated with health and wellness. While a variety of blood fingerstick test strips are available for self-testing of BHB, there are major needs for wearable devices capable of continuously tracking changing BHB concentrations. To address these needs, we present here the first demonstration of a wearable microneedle-based continuous ketone monitoring (CKM) in human interstitial fluid (ISF) and illustrate its ability to closely follow the intake of ketone drinks. To ensure highly stable and selective continuous detection of ISF BHB, the new enzymatic microneedle BHB sensor relies on a gold-coated platinum working electrode modified with a reagent layer containing toluidine blue O (TBO) redox mediator, β-hydroxybutyrate dehydrogenase (HBD) enzyme, a nicotinamide adenine dinucleotide (NAD+) cofactor, along with carbon nanotubes (CNTs), chitosan (Chit), and a poly(vinyl chloride) (PVC) outer protective layer. The skin-worn microneedle sensing device operates with a miniaturized electrochemical analyzer connected wirelessly to a mobile electronic device for capturing, processing, and displaying the data. Cytotoxicity and skin penetration studies indicate the absence of potential harmful effects. A pilot study involving multiple human subjects evaluated continuous BHB monitoring in human ISF, against gold standard BHB meter measurements, revealing the close correlation between the two methods. Such microneedle-based CKM offers considerable promise for dynamic BHB tracking toward the management of diabetic ketoacidosis and personal nutrition and wellness.

Effects of different temperature-humidity indexes on milk traits of Holstein cows: A 10-year retrospective study

Test-day records (n = 723,091) collected between 2012 and 2021 from 43,015 Holstein cows at 157 farms located in northern Italy were used to study the effects of heat load on milk production and composition a posteriori. The data consisted of milk yield (kg/d), traditional gross composition traits, somatic cell score (SCS), differential somatic cell count (%), milk β-hydroxybutyrate (BHB, mmol/L), milk urea (mg/dL), and milk fatty acid composition (g/100 g of milk). Test-day records were then associated with their relative temperature-humidity indexes (THI) calculated using historical environmental data registered by weather stations. Indexes were created using either yearly or summer THI data. The yearly indexes included the average daily THI (adTHI) and the maximum daily THI (mdTHI) measured throughout the whole year, and the summer indexes focused on 3 mo only (June-August) and included the average daily summer THI (adTHIs), the maximum daily summer THI (mdTHIs), and the average daily THI of the hottest 4 h of the day (adTHI4h; 1200-1600 h). All indexes had significant effects on the majority of milk traits analyzed, with, in particular, adTHI and mdTHI being highly significant in explaining the variation of all traits. Milk yield started to decline at a higher THI compared with protein and fat content. The reduction in fat ceased in the elevated THI experienced during the summer months, as demonstrated by adTHIs, mdTHIs, and adTHI4h. The cows had a tendency for increased BHB concentration with elevated THI, suggesting a greater risk of negative energy balance in presence of heat stress. Furthermore, the concentration of the de novo fatty acids C14:0 and C16:0 was reduced in higher THI, reflecting altered mammary gland activity upon elevated heat load and stress. Milk SCS tended to increase with higher adTHIs, mdTHIs, and adTHI4h. The use of yearly indexes is recommended when investigating the effects of heat load on milk composition, whereas summer indexes are suggested when investigating traits influenced by extreme conditions, such as SCS and milk yield. With global temperatures expected to further rise in the upcoming decades, early and easy identification of cows or herds suffering from heat stress, such as through changes in milk composition, is crucial for timely intervention. Adapting measures to mitigate such effects of elevated THI on milk yield and composition is a necessity for the dairy industry to prevent detrimental impacts on dairy production.

Relationship of Body Condition Score at Calving with Reproductive Performance, some Blood Metabolites and some Pathological Conditions in Holstein Cows

The study was conducted at The Great Diwaniyah cows station, located at the southern entrance of Al-Dagarah district - Al-Diwaniyah Governorate, from 6/12/2021 to 25/2/2023, to examine the relationship between body condition score (BCS) and reproductive traits, blood biomarkers, and metabolic diseases in Iraqi Holstein-Friesian cows. The 50 cows in parity2 were divided by body condition at calving into three groups (BCS1 = 2.5-3 point (17 cows), BCS2 = 3-4 point (20 cows), and BCS3 = 4 -5 point (13 cows)) based on 1-5 point scal. They were milked twice a day, morning and evening, by an automatic milking machine. The study found a substantial decrease (P≤0.01) in days open for BCS1 (77.18 days) compared to BCS2 (84.05 days) and BCS3 (88.23 days). The BCS2 group had considerably fewer days available (P≤0.01). Compared to BCS3. The study found a significant decrease (P≤0.01) in inter-calving interval for BCS1 group (354.59 days) compared to BCS2 and BCS3 groups (360.95 and 365.69 days, respectively). BCS2 group showed considerably shorter duration (P≤0.01) compared to BCS3 group. The study found no significant influence on inseminations per pregnancy or pregnancy length in the three cow groups. The study found that the BCS2 group had significantly higher glucose concentration (62.19 mg/100 ml) post-calving compared to BCS3 group (57.86 mg/100 ml), but BCS1 group did not differ significantly (59.30 mg/100 ml). The study found a substantial rise (P≤0.01) in BHB concentration (0.922 mmol/L) in the BCS3 group compared to BCS2 and BCS1 (0.743 and 0.674 mmol/L, respectively). BCS3 group showed a substantial increase (P≤0.01) in NEFA concentration (0.590 mmol/L) compared to BCS2 and BCS1 groups (0.476 and 0.461 mmol/L, respectively). During peak production, BCS2 group had a significant increase (P≤0.05) in glucose concentration (64.04 mg/100 ml) compared to BCS3 group (58.97 mg/100 ml), while BCS1 group did not differ significantly (60.97 mg/100 ml). BCS3 group showed a substantial increase (P≤0.01) in BHB concentration (0.434 mmol/L) compared to BCS2 and BCS1 groups (0.297 and 0.318 mmol/L, respectively). The study found a substantial increase (P≤0.01) in NEFA concentration in the BCS3 group (0.290 mmol/L) compared to BCS2 and BCS1 (0.205 and 0.215 mmol/L, respectively). The study found a significant (P≤0.05) increase in ketosis in Holstein cows with BCS2 and BCS3 groups, with rates of 1 (5.0%) and 2 (15.38%), respectively, compared to the BCS1 group with no injury. No significant changes were seen in placental retention, metritis, mastitis, and neonatal mortality between cows. According to the study, bodily condition during calving is linked to reproductive features, blood metabolites, and disease infection in cows.

Milk β-hydroxybutyrate metrics and its consequences for surveillance of hyperketonaemia on commercial dairy farms.

Dairy cows that are unable to adapt to a change in their metabolic status are at risk for hyperketonaemia (HK). Reported HK herd level prevalences range a lot and we hypothesized that this is partly due to differences in used tests and monitoring protocols. Insights in milk β-hydroxybutyrate (BHB) metrics can potentially explain why the reported incidences or prevalences vary between test strategies. Automated collection and repeated analyses of individual milk samples with the DeLaval Herd Navigator™ (HN) provides real-time data on milk BHB concentrations. We aimed to use that information to gain insight in BHB metrics measured in milk from 3 to 60 days in milk (DIM). Using different cut-offs (0.08, 0.10 and 0.15 mmol/L), 5 BHB metrics were determined. Furthermore, the impact of 4 arbitrary test protocols on the detected incidence of HK was assessed. We used HN data of 3,133 cows from 35 herds. The cumulative incidence of HK between 3 and 60 DIM varied between 30.5 and 76.7% for different cut-off values. We found a higher HK incidence for higher parity cows. The first elevated BHB concentrations were roughly found between one and two weeks after calving. For higher parity cows the maximum BHB concentrations were higher, the onset of HK was earlier after calving, and the number of episodes of HK was higher. It appeared that the sensitivity of a HK test protocol can be increased by increasing the testing frequency from once to twice a week. Also extending the number of days of the test window from 4-14 to 4-21 days enhances the chance to find cows experiencing HK. In conclusion, HN data provided useful insights in milk BHB metrics. The chosen cut-off value had a large effect on the reported metrics which explains why earlier reported incidences or prevalences vary such a lot. Differences in test period and sample selection also had a large impact on the observed HK incidence. We suggest to take this in consideration while evaluating whether HK is an issue on farm level and use a uniform protocol for benchmarking of HK between farms.

Combination of milk variables and on-farm data as an improved diagnostic tool for metabolic status evaluation in dairy cattle during the transition period

Milk composition, particularly milk fatty acids, has been extensively studied as an indicator of the metabolic status of dairy cows during early lactation. In addition to milk biomarkers, on-farm sensor data also hold potential in providing insights into the metabolic health status of cows. While numerous studies have explored the collection of a wide range of sensor data from cows, the combination of milk biomarkers and on-farm sensor data remains relatively underexplored. Therefore, this study aims to identify associations between metabolic blood variables, milk variables, and various on-farm sensor data. Second, it seeks to examine the supplementary or substitutive potential of these data sources. Therefore, data from 85 lactations on metabolic status and on-farm data were collected during 3 wk before calving up to 5 wk after calving. Blood samples were taken on d 3, 6, 9, and 21 after calving for determination of β-hydroxybutyrate (BHB), nonesterified fatty acids (NEFA), glucose, insulin-like growth factor-1 (IGF-1), insulin, and fructosamine. Milk samples were taken during the first 3 wk in lactation and analyzed by mid-infrared for fat, protein, lactose, urea, milk fatty acids, and BHB. Walking activity, feed intake, and body condition score (BCS) were monitored throughout the study. Linear mixed effect models were used to study the association between blood variables and (1) milk variables (i.e., milk models); (2) on-farm data (i.e., on-farm models) consisting of activity and dry matter intake analyzed during the dry period ([D]) and lactation ([L]) and BCS only analyzed during the dry period ([D]); and (3) the combination of both. In addition, to assess whether milk variables can clarify unexplained variation from the on-farm model and vice versa, Pearson marginal residuals from the milk and on-farm models were extracted and related to the on-farm and milk variables, respectively. The milk models had higher coefficient of determination (R2) than the on-farm models, except for IGF-1 and fructosamine. The highest marginal R2 values were found for BHB, glucose, and NEFA (0.508, 0.427, and 0.303 vs. 0.468, 0.358, and 0.225 for the milk models and on-farm models, respectively). Combining milk and on-farm data particularly increased R2 values of models assessing blood BHB, glucose, and NEFA concentrations with the fixed effects of the milk and on-farm variables mutually having marginal R2 values of 0.608, 0.566, and 0.327, respectively. Milk C18:1 was confirmed as an important milk variable in all models, but particularly for blood NEFA prediction. On-farm data were considerably more capable of describing the IGF-1 concentration than milk data (marginal R2 of 0.192 vs. 0.086), mainly due to dry matter intake before calving. The BCS [D] was the most important on-farm variable in relation to blood BHB and NEFA and could explain additional variation in blood BHB concentration compared with models solely based on milk variables. This study has shown that on-farm data combined with milk data can provide additional information concerning the metabolic health status of dairy cows. On-farm data are of interest to be further studied in predictive modeling, particularly because early warning predictions using milk data are highly challenging or even missing.

The impact of automated, constant incomplete milking on energy balance, udder health, and subsequent performance in early lactation of dairy cows

Incomplete milking (IM) is one way of mitigating the negative energy balance (NEB) that is characteristic for early lactation and may increase the risk for disease. Our objectives were to test the effects of IM in early lactation on energy balance (EB), metabolic status, udder health, and subsequent performance. To facilitate the practical application, an automated system was used to remove the milking clusters once a predefined amount of milk is withdrawn. Forty-six Holstein cows were equally allocated to either the treatment (TRT, starting on 8 d in milk) or the control group (CON; conventional cluster removal at milk flow rate <0.3 kg/min). Milk removal in the TRT group was limited to the individual cow's milk yield 1 d before IM started and held constant for 14 d. Thereafter, all cows were conventionally milked and records related to EB, performance, and udder health were continued up to 15 wk of lactation. During the 14 d of IM, on average 11.1% less milk was obtained from the TRT cows than from the CON cows. Thereafter, milk yield increased in the TRT group, eliminating the group difference throughout the remaining observation period until wk 15 of lactation. The TRT cows tended to have less dry matter intake and also water intake than the CON cows. The extent of the NEB and the circulating concentrations of fatty acids, β-hydroxybutyrate, insulin-like growth factor-1, and leptin mostly did not differ between the groups. The IM did not affect body condition. Udder health was maintained over the entire observation period in all cows. Our results demonstrate the applicability of the automated cluster removal for limiting milk withdrawal to a defined amount in early lactation. However, it remains to be determined whether the absent effect on energy metabolism was due to the relatively stable energy status of the cows or to the relatively mild IM setting used herein.

Associations between serum health biomarker concentrations and reproductive performance, accounting for milk yield, in pasture-based Holstein cows in southeastern Australia

In this single cohort study, we investigated associations between the concentrations of a suite of serum biomarkers measured in the first 30 d of lactation and subsequent reproductive performance measured as mating start date to conception intervals, in pasture-based Holstein cows. A secondary objective was to examine associations between biomarker concentrations and 305-d milk yield to assess whether any positive associations between biomarker concentration and reproductive performance were explained by reduced milk production. The data used had been collected as part of an ongoing project from 2017 to 2020 to compile a data set from a large population of lactating dairy cows. Biomarkers measured were those associated with energy balance (β-hydroxybutyrate [BHB] and nonesterified fatty acids [NEFA]), protein nutritional status (urea and albumin), immune status (globulin, albumin to globulin ratio and haptoglobin), and macromineral status (calcium and magnesium). Associations between biomarker concentrations and mating start date to conception interval were investigated using Cox proportional hazard models, using between 634 and 1,121 lactations (varying by biomarker) from 632 to 1,103 cows and 11 to 17 mating periods from 10 to 13 herds. Based on hazard ratio (HR) estimates and associated 95% confidence intervals (CI), hazard of conception on any particular day of the herds' mating periods was positively associated with the concentrations of albumin (HR = 1.09; 95% CI: 1.05-1.12), albumin to globulin ratio (HR = 2.82; 95% CI: 1.66-4.79), calcium (HR = 2.01; 95% CI: 1.18-3.43), and magnesium (HR = 2.17; 95% CI: 1.01-4.66), and negatively associated with globulin concentration (HR = 0.98; 95% CI: 0.97 to 1.00). There was also some evidence that NEFA concentration was negatively associated (HR = 0.76; 95% CI: 0.57 to 1.01), and urea concentration positively associated (HR = 1.05; 95% CI: 0.99 to 1.11), with reproductive performance, but no evidence that BHB and haptoglobin concentrations were associated with reproductive performance. Except for NEFA, presence and direction of the associations between the biomarker and milk yield were not discordant with that for reproductive performance. Also, except for NEFA, we found no substantial evidence of nonlinear relationships between biomarker concentration and either reproductive performance or milk yield. Correlations between biomarker concentrations were generally weak, indicating that multibiomarker panels may collectively predict reproductive performance better than any single biomarker. We noted substantial variation in the concentrations of all biomarkers within, and for some biomarkers, between herd-year groups. Collectively, these results indicate that there may be scope to improve biomarker concentrations through nutritional, management, and genetic interventions, and by association, reproductive performance and milk yield may also improve.

β-hydroxybutyrate impairs the directionality of migrating neutrophils through inhibiting the autophagy-dependent degradation of Cdc42 and Rac1 in ketotic cows

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophil need further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

Feeding colostrum and transition milk facilitates digestive tract functionality recovery from feed restriction and fasting of dairy calves

The objective of this study was to evaluate the digestive tract recovery and metabolism of feeding either bovine colostrum (BC), transition milk (TM), or milk replacer (MR) after an episode of feed restriction and fasting (FRF) in dairy calves. Thirty-five Holstein male calves (22 ± 4.8 d old) were involved in a 50-d study. After 3 d of feeding 2 L of rehydration solution twice daily and 19 h of fasting (d 1 of study), calves were randomly assigned to one of the 5 feeding treatments (n = 7): calves were offered either pooled BC during 4 (C4) or 10 (C10) days, pooled TM during 4 (TM4) or 10 (TM10) days, or MR for 10 d (CTRL) at the rate of 720 g/d DM content. Then, all calves were fed the same feeding program, gradually decreasing MR from 3 L twice daily to 2 L once daily at 12.5% DM until weaning (d 42), and concentrate feed, water, and straw were offered ad libitum until d 50. Citrulline, Cr-EDTA, β-hydroxybutyrate (BHB), and nonesterified fatty acids (NEFA) in serum and complete blood count (CBC) were determined on d -3, 1, 2, 5, and 11 relative to FRF, except BHB and NEFA at d -3. Volatile fatty acids (VFA), lactoferrin (LTF), IgA, and microbiota (Firmicutes to Bacteroidetes ratio and Fecalis prausnitzii) were analyzed in feces on d 5 and 11 before the morning feeding. Health scores were recorded daily from d -3 to d 14 as well as d 23 and 30. Feed concentrate, MR, and straw intake were recorded daily, and body weight on d -3, 1, 2, 5, and 11 and weekly afterward. Calf performance, intake, serum Cr-EDTA, CBC, fecal LTF concentrations and microbiota parameters were similar among treatments throughout the study. Serum NEFA concentrations were greater in TM4, TM10 and C10 calves compared with the CTRL ones from d 2 to 11, and after the FRF, serum concentrations of BHB were lower in CTRL calves than in the other treatments, and on d 11, serum BHB concentrations in the long treatments (C10 and TM10) remained greater than those in the shorter ones (C4 and TM4) and CTRL. Serum citrulline concentrations were similar on d -3 and 1 in all treatments, but they were greater in C4, C10, TM4, and TM10 on d 2 and 5, and on d 11 they were only greater in C10 and TM10 than in CTRL calves. Fecal IgA concentrations tended to be greater in C10 than in CTRL, TM4, and TM10 calves, and in C4 and TM10 than in CTRL animals. Fecal propionate proportion was lesser in C10 than in CTRL, TM4, and TM10 calves, while butyrate was greater in C4 and C10 than in TM4 and CTRL calves. The proportion of non-normal fecal scores of C10 fed calves was greater than TM4 and TM10 calves. Results showed that TM and BC may help to recover intestinal functionality, provide gut immune protection, and increase liver fatty acid oxidation in calves after a FRF episode.

Supplementation of an organic mineral source modulated metabolic profile, insulin sensitivity, and oxidative stress in dairy calves with different birth body weights

The present study was conducted to investigate the effects of partial replacement of inorganic minerals (IM) with advanced chelated minerals (ACMS) in the starter diets of calves of different birth body weights (BBW) on metabolic indices and glucose tolerance test. Fifty-two Holstein calves were assigned to one of 4 treatments in a 2 × 2 factorial arrangement with two mineral sources (IM or ACMS) and two BBW (low or normal birth weight; LBW or NBW). Calves were weaned on day 56 and remained in the study until day 71 of the study. Blood samples for blood metabolite analysis were collected from the jugular vein on days 30, 60, and 70 of the study, and for hematology on days 36, 56, and 70. The results showed that the interaction of starter mineral sources (MS) and BBW affected the overall plasma concentration of β-hydroxybutyrate (BHB), with the highest value being obtained during treatment with LBW-ACMS. In addition, an interaction between MS and BBW was observed in plasma insulin concentrations, with LBW-IM calves having the lowest concentrations compared to the other treatments. Plasma albumin concentrations were lower in LBW calves than in NBW calves. In addition, NBW calves had greater plasma concentrations of glucose (day 60), insulin (day 30), BHB (day 30), and malondialdehyde (MDA; day 60 and the overall period) than LBW calves, while total antioxidant capacity (TAC) tended to reverse at day 70. The ACMS starter increased plasma glucose concentration at day 30 and tended to increase plasma BHB concentration in the overall period. In addition, plasma MDA concentrations were lower in calves receiving ACMS than in calves receiving IM during the overall period. NBW calves tended to have more eosinophils (day 70), basophils (day 36), and erythrocytes (RBC, day 56, and the overall period) in plasma than LBW calves. Regardless of BBW, feeding ACMS starter increased white blood cells (WBC), neutrophils, and eosinophils at day 70 and also tended to increase neutrophils during the overall period compared with the IM groups. Red blood cell concentrations were greater in calves fed ACMS than in calves fed IM during the overall period. Glucose tolerance test showed that basal glucose concentration and clearance rate (CR) of glucose tended to be lower and higher, respectively, in calves fed ACMS than in calves fed IM. ACMS supplementation increased basal concentrations and also tended to increase maximum insulin concentrations compared with IM supplementation. In addition, calves receiving ACMS starter had greater plasma insulin area under the curve (AUC) and CR compared with calves receiving IM. Feeding ACMS tended to increase the ratio CR of insulin to CR of glucose and increased the AUC of insulin to AUC of glucose compared with feeding IM. Overall, feeding ACMS resulted in a decrease in plasma MDA concentration and an increase in neutrophil concentration, suggesting that ACMS may improve immune function in these calves. A potential insulinotropic effect was noted with ACMS feeding, which was accompanied by an improvement in glucose control rate. This is likely due to higher bioavailability of chelated minerals such as Zn and should be further investigated.

Non-specific recognition of histone modifications by H3K9bhb antibody

Ketone bodies are short-chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative energy source for the brain, heart, and skeletal muscle. Beyond this metabolic role, β-hydroxybutyrate (BHB), is gaining recognition as a signaling molecule. Lysine β-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s) that likely include acetylation. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Gene co-expression network and differential expression analyses of subcutaneous white adipose tissue reveal novel insights into the pathological mechanisms underlying ketosis in dairy cows

Ketosis is a common nutritional metabolic disease during the perinatal period in dairy cows. Although various risk factors have been identified, the molecular mechanism underlying ketosis remains elusive. In this study, subcutaneous white adipose tissue (sWAT) was biopsied for transcriptome sequencing on 10 Holstein cows with type II ketosis [blood β-hydroxybutyric acid (BHB) >1.4 mmol/L; Ket group] and another 10 cows without type II ketosis (BHB ≤1.4 mmol/L; Nket group) at d 10 after calving. Serum concentrations of nonesterified fatty acids (NEFA) and BHB, as indicators of excessive fat mobilization and circulating ketone bodies, respectively, were significantly higher in the Ket group than in the Nket group. Aspartate transaminase (AST) and total bilirubin (TBIL), as indicators of liver damage, were higher in the Ket group than in the Nket group. Weighted gene co-expression network analysis (WGCNA) of the sWAT transcriptome revealed modules significantly correlated with serum BHB, NEFA, AST, TBIL, and total cholesterol. The genes in these modules were enriched in the regulation of the lipid biosynthesis process. Neurotrophic tyrosine kinase receptor type 2 (NTRK2) was identified as the key hub gene by intramodular connectivity, gene significance, and module membership. Quantitative reverse transcription PCR analyses for these samples, as well as a set of independent samples, validated the downregulation of NTRK2 expression in the sWAT of dairy cows with type II ketosis. NTRK2 encodes tyrosine protein kinase receptor B (TrkB), which is a high-affinity receptor for brain-derived neurotrophic factor, suggesting that abnormal lipid mobilization in cows with type II ketosis might be associated with impaired central nervous system regulation of adipose tissue metabolism, providing a novel insight into the pathogenesis underlying type II ketosis in dairy cows.

Effect of source and amount of rumen-protected choline on hepatic metabolism during induction of fatty liver in dairy cows

Objectives were to determine the effect of supplementing increased amounts of rumen-protected choline (RPC) from sources with low (L, 28.8%) or high (H, 60.0%) concentration of choline chloride on hepatic metabolism when cows were subjected to feed restriction to develop fatty liver. It was hypothesized that increased supplementation of RPC reduces hepatic triacylglycerol and enhances glycogen concentrations. Pregnant, nonlactating multiparous Holstein cows (n = 110) at mean (± standard deviation) 232 ± 3.9 d of gestation were blocked by body condition (4.01 ± 0.52) and assigned to receive 0 (CON), 12.9 (L12.9 or H12.9), or 25.8 (L25.8 or H25.8) g/d of choline ion. Cows were fed for ad libitum intake on d 1 to 5 and restricted to 50% of the NEL required for maintenance and pregnancy from d 6 to 13. Intake of metabolizable methionine was maintained at 19 g/d during the feed restriction period by supplying rumen-protected methionine. Hepatic tissue was sampled on d 6 and 13 and analyzed for triacylglycerol, glycogen, and mRNA expression of genes involved in choline, glucose, and fatty acids metabolism, cell signaling, inflammation, autophagy, lipid droplet dynamics, lipophagy, and endoplasmic reticulum stress response. Blood was sampled and analyzed for concentrations of fatty acids, β-hydroxybutyrate (BHB), glucose, triacylglycerol, total cholesterol, and haptoglobin. Orthogonal contrasts evaluated the effect of supplementing RPC [CON vs. (1/4·L12.9 + 1/4·L25.8 + 1/4·H12.9 + 1/4·H25.8)], source of RPC [(1/2·L12.9 + 1/2·L25.8) vs. (1/2·H12.9 + 1/2·H25.8)], amount of RPC [(1/2·L12.9 + 1/2·H12.9) vs. (1/2·L25.8 + 1/2·H25.8)], and interaction between source and amount [(1/2·L12.9 + 1/2·H25.8) vs. (1/2·H12.9 + 1/2·L25.8)]. Least squares means and standard error of the means are presented in sequence as CON, L12.9, L25.8, H12.9, H25.8. Supplementation of RPC reduced hepatic triacylglycerol (9.3 vs. 6.6 vs. 5.1 vs. 6.6 vs. 6.0 ± 0.6% as-is) and increased glycogen contents (1.8 vs. 2.6 vs. 3.6 vs. 3.1 vs. 4.1 ± 0.2% as-is) on d 13 of the experiment. Feeding RPC reduced serum haptoglobin (136.6 vs. 85.6 vs. 80.6 vs. 82.8 vs. 81.2 ± 4.6 µg/mL) during the feed restriction period; however, blood concentrations of fatty acids, BHB, glucose, triacylglycerol, and total cholesterol did not differ among treatments. During feed restriction, supplementation of RPC enhanced the mRNA expression of genes related to choline metabolism (BHMT), uptake of fatty acids (CD36), and autophagy (ATG3), and reduced the expression of a transcript associated with endoplasmic reticulum stress response (ERN1). An increase in the amount of choline ion from 12.9 to 25.8 g/d enhanced the mRNA expression of genes associated with synthesis and assembly of lipoproteins (APOB100), and inflammation (TNFA), whereas it reduced the expression of genes linked to gluconeogenesis (PC), oxidation of fatty acids (ACADM, MMUT), ketogenesis (ACAT1), and synthesis of antioxidants (SOD1) on d 13 of the experiment. Feeding RPC, independent of the product used, promoted lipotropic effects that reduced hepatic lipidosis in dairy cows.

Effect of kraft pulp inclusion in calf starter on performance, health, and plasma concentration of glucagon-like peptide 2 in calves

Kraft pulp (KP), an intermediate product obtained when wood chips are converted to paper, contains highly digestible fiber. This study evaluated the effect of KP inclusion in calf starters on growth performance, health, and plasma glucagon-like peptide 2 (GLP-2) concentration in calves. Twenty-five Holstein heifer calves were raised on a high plane of nutrition program using milk replacer containing 29% crude protein and 18% fat until 49 d after birth, and were fed calf starters containing KP at 0 (CON; n = 14) or 12% (KPS; n = 11) on a dry matter basis. All calves were fed the treatment calf starters and timothy hay ad libitum. Blood was collected at 4, 14, 21, 35, 49, 70, and 91 d after birth. Dry matter intake (DMI) of milk replacer and hay was not affected by treatment, whereas calf starter DMI was lower for KPS (0.93 kg/d) than for CON (1.03 kg/d). Higher neutral detergent fiber (NDF) content in KPS (31.7%) than in the CON starter (22.1%) resulted in higher NDF intake for KPS (0.55 kg/d) than for CON (0.47 kg/d). However, the consumption of starch was lower for KPS (0.29 kg/d) than for CON (0.33 kg/d). Despite the lower starter intake for KPS, body weight and average daily gain did not differ between treatments. No significant difference was observed in the plasma concentrations of metabolites, except for β-hydroxybutyrate (BHB); BHB concentration was lower for KPS (216 μmol/L) than for CON (257 μmol/L). The area under the curve for plasma GLP-2 concentration was higher for KPS (54.1 ng/mL × d) than for CON (36.0 ng/mL × d). Additionally, the fecal score postweaning (1.19 and 1.48 for KPS and CON, respectively) and the number of days that calves developed diarrhea throughout the experimental period (2.50 d and 8.10 d for KPS and CON, respectively) were lower for KPS than for CON. These results indicate that feeding KP reduces the severity and frequency of diarrhea without adversely affecting growth performance. This could be attributed to the increased plasma GLP-2 concentration induced by higher NDF intake.