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Ketosis, evidenced by hyperketonemia with elevated blood β-hydroxybutyrate (BHB) levels, is a significant metabolic disorder of dairy cattle, typically diagnosed within the first 6 weeks post-calving when high energy levels are essential to milk production. Our study aimed to identify genetic markers linked to hyperketonemia (HYK) patterns in Holstein cows during early lactation and compare these to HYK-negative cows. We screened 964 cows for HYK using a threshold of BHB ≥1.2 mmol/L during the first 2 weeks postpartum (screening period, SP). Cows that tested negative initially were retested the following week. Cows were deemed HYK-negative (CON group) if BHB levels were below 1.2 mmol/L in both tests, while those with BHB levels exceeding this threshold at any test were treated and classified as HYK-positive (HYK+). Post-treatment, HYK+ cows were monitored for two-week follow-up period (FP) and classified based on their recovery: cured (CUR; consistently low BHB), recurrent (REC; fluctuating BHB levels), severe (SEV; high initial BHB that decreased), or chronic (CHR; persistently high BHB). Using 489 cows that were genotyped, a GWAS was conducted using GCTA software, revealing significant associations of several SNPs across different HYK patterns when compared to the CON group. These SNPs were primarily linked to genes affecting milk traits and were enriched in biological pathways relevant to protein glycosylation, inflammatory response, glucose homeostasis, and fatty acid synthesis. Our findings highlight genomic regions, potential candidate genes, and biological pathways related to ketosis, underscoring potential targets for improving health management in dairy cattle. These insights could lead to better strategies for managing ketosis through genetic selection, ultimately enhancing dairy cattle welfare and productivity. Further research with a larger number of cows is recommended to validate these findings and help confirm the implicated SNPs and genes., (© 2024 The Author(s). Journal of Animal Breeding and Genetics published by John Wiley & Sons Ltd.)

This study systematically examines the patterns of milk yield (MY, kg), fat (FAT, %), and protein (PROT, %) in a diverse population of Thai multibreed dairy cattle, considering the tropical environment's impact on lactating cows. Using a dataset of 47,205 monthly test-day records from 4,440 first-lactation cows across 446 farms, we analyze variations and interrelationships through mathematical averaging and introduce the fat-to-protein ratio (FPR) to assess acidosis (FPR < 1.1) and ketosis (FPR > 1.5) risks during lactation. Pearson correlation analysis elucidated trait associations. The findings, aligned with established lactation norms, indicate peak production at 297 days in milk (DIM) for FAT (4.08%; SD = 0.96%), PROT (3.43%; SD = 0.47%), and 52 DIM for MY (18.09 kg; SD = 4.91 kg). Nadirs are observed at 72 DIM for FAT (3.27%; SD = 0.74%), 47 DIM for PROT (2.86%; SD = 0.36%), and 299 DIM for MY (9.05 kg; SD = 2.95 kg). FPR variations highlight acidosis (46.48%), normal (43.66%), and ketosis (9.86%), especially during early lactation (100 DIM). Significant negative correlations emerge between MY, FAT, and PROT (P < 0.05), while a positive correlation is identified between FAT and PROT (P < 0.01), with robust correlations during early lactation. This study contributes to understanding tailored nutritional strategies for dairy cows' holistic health and sustainability in tropical environments, guiding efficient production practices and mitigating health-related productivity impediments., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Precision livestock farming utilizing advanced diagnostic tools, including biosensors, can play a key role in the management of livestock operations to improve the productivity, health, and well-being of animals. Detection of ketosis, a metabolic disease that occurs in early lactation dairy cows due to a negative energy balance, is one potential on-farm use of biosensors. Beta-hydroxybutyrate (βHB) is an excellent biomarker for monitoring ketosis in dairy cows because βHB is one of the main ketones produced during this metabolic state. In this report, we developed a low-cost, Keto-sensor (graphene-based sensor) for the detection of βHB concentrations in less than a minute. On this device, graphene nanosheets were layered onto a screen-printed electrode (SPE), and then, a stabilized enzyme (beta-hydroxybutyrate dehydrogenase, NAD + , and glycerol) was used to functionalize the graphene surface enabled by EDC-NHS conjugation chemistry. The Keto-sensor offers an analytical sensitivity of 10 nm and a limit of detection (LoD) of 0.24 nm within a detection range of 0.01 μm-3.00 mm. Spike testing indicates that the Keto-sensor can detect βHB in serum samples from bovines with subclinical ketosis. The Keto-sensor developed in this study shows promising results for early detection of subclinical ketosis on farms.

The objectives of this study were to determine the farm-level hyperketolactia (HKL) prevalence, as diagnosed from the milk BHB concentration, on dairy farms milking with an automated milking system (AMS) and to describe the farm-level housing, management, and nutritional risk factors associated with increased farm-average milk BHB and the within-herd HKL prevalence in the first 45 DIM. Canadian AMS farms (n = 162; eastern Canada, n = 8; Quebec, n = 24; Ontario, n = 75; western Canada n = 55) were visited once between April and September 2019 to record housing and herd management practices. The first test milk data for each cow under 45 DIM were collected, along with the final test of the previous lactations for all multiparous cows, from April 1, 2019, to September 30, 2020. The first test milk BHB was then used to classify each individual cow as having or not having HKL (milk BHB ≥0.15 mmol/L) at the time of testing. Milk fat and protein content, milk BHB, and HKL prevalence were summarized by farm and lactation group (all, primiparous, and multiparous). During this same time period, formulated diets for dry and lactating cows, including ingredients and nutrient composition, and AMS milking data were collected. Data from the AMS were used to determine milking behaviors and milk production of each herd during the first 45 DIM. Multivariable regression models were used to associate herd-level housing, feeding management practices, and formulated nutrient composition with first test milk BHB concentrations and within-herd HKL levels separately for primiparous and multiparous cows. The within-herd HKL prevalence for all cows was 21.8%, with primiparous cows having a lower mean prevalence (12.2 ± 9.2%) than multiparous cows (26.6 ± 11.3%). Milk BHB concentration (0.095 ± 0.018 mmol/L) and HKL prevalence for primiparous cows were positively associated with formulated prepartum DMI and forage content of the dry cow diet; however, they were negatively associated with formulated postpartum DMI, the major ingredient in the concentrate supplemented through the AMS, and the partially mixed ration to AMS concentrate ratio. Multiparous cows' milk BHB concentration (0.12 ± 0.023 mmol/L) and HKL prevalence were positively associated with the length of the previous lactation, milk BHB at dry-off, prepartum diet nonfiber carbohydrate content, and the major forage fed on farm, while tending to be negatively associated with feed bunk space during lactation. This study is the first to determine the farm-level risk factors associated with herd-level prevalence of HKL in AMS dairy herds. The results may help to optimize management and guide diet formulation and thus promote the reduction of HKL prevalence., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The metabolic changes that occur during the early post-partum period in dairy cows can indeed lead to an imbalance in energy utilization, resulting in the production of excessive ketone bodies. This can have detrimental effects on the cow's health and milk production, leading to economic losses for dairy producers. The release of non-esterified fatty acids into the blood due to increased lipolysis is a key factor in the development of ketosis. Abdominal adiposity is a key factor on these outcomes in modern dairy cows. The redirection of energy and glucose towards lactose synthesis and milk yield leaves a deficit of gluconeogenic precursors, leading to the conversion of acetyl-CoA into ketone bodies instead of entering the Krebs cycle. These ketone bodies, including acetone, acetoacetate and β-hydroxybutyrate, accumulate in the blood and can be detected in various bodily fluids, such as urine, blood and milk, allowing for diagnostic testing. Prevention is indeed crucial in managing ketosis in dairy cattle. Supplementation of propylene glycol in the diet or the use of monensin, either in the diet or in the form of a slow-release bolus, can help prevent the occurrence of ketosis. However, avoiding high body condition (subcutaneous fat) and excessive abdominal adiposity during the dry period and parturition plus an adequate cow comfort are fundamental tasks to avoid ketosis and related disorders. These interventions aim to provide additional energy sources or enhance the cow's ability to utilize energy efficiently, thus reducing the reliance on excessive lipolysis and ketone body production., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Ketosis-a metabolic state characterized by elevated levels of ketone bodies in the blood or urine-reduces the performance and health of dairy cows and causes substantial economic losses for the dairy industry. Currently, beta-hydroxybutyric acid is the gold standard for determining ketosis in cows; however, as this method is only applicable postpartum, it is not conducive to the early intervention of ketosis in dairy cows. In this study, the sera of dry, periparturient, postpartum ketotic, and healthy cows were analyzed by both transcriptomics and metabolomics techniques. Moreover, changes of gene expression and metabolites were observed, and serum physiological and biochemical indexes were detected by ELISA. The purpose was to screen biomarkers that can be used to detect the incidence of dry or periparturient ketosis in cows. The results showed that ketotic cows had increased levels of glycolipid metabolism indexes, oxidizing factors, and inflammatory factors during dry periods and liver damage, which could be used as early biomarkers to predict the onset of ketosis. Transcriptomic results yielded 20 differentially expressed genes (DEGs) between ketotic and healthy cows during dry, peripartum, and postpartum periods. GO and KEGG enrichment analyses indicated that these DEGs were involved in amino acid metabolism, energy metabolism, and disease-related signaling pathways. The metabolomics sequencing results showed that ketotic cows mainly showed enrichment in tricarboxylic acid cycle, butyric acid metabolism, carbon metabolism, lysine degradation, fatty acid degradation, and other signaling pathways. Metabolites differed between ketotic and healthy cows in dry, pre-parturition, and post-parturition periods. Combined transcriptomics and metabolomics analyses identified significant enrichment in the glucagon signaling pathway and the lysine degradation signaling pathway in dry, periparturient, and postpartum ketotic cows. PRKAB2 and SETMAR-key DEGs of the glucagon signaling pathway and lysine degradation signaling pathway, respectively-can be used as key marker genes for determining the early onset of ketosis in dairy cows., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that could unduly influence our work, and that we have no professional or other personal interests of any nature or type with any product, service and/or company that could be perceived to influence the content of this article., (Copyright © 2024. Published by Elsevier Inc.)

In dairy cows, the occurrence of subclinical ketosis (SCK) is particularly high during early lactation. Previously, we documented alterations in the abundance of adiponectin (ADPN) in anestrus cows with SCK in comparison to cows in estrus. In the present study, 60 cows were divided into two groups: control (C, n = 30) and SCK (n = 30). Based on cow's estrus situation in two group at 55-60 days postpartum, 15 anestrus SCK cows and estrus cows were designated the SCK-A group and C-E group, respectively. The SCK-A group had downregulated serum and follicular fluid ADPN levels compared with the C-E group. The serum ADPN level was positively correlated with the insulin level and follicle growth rate, and there was a positive correlation between ADPN and glucose in the follicular fluid. Primary culture of dairy cow granulosa cells (GCs) was established to observe the effect of low glucose (Glu) and/or ADPN on GCs cyclins and proteins important for steroid synthesis. The results showed that the addition of 1 µg/mL ADPN alleviated the negative effects of low Glu treatment on the proliferation of GCs and the expression of steroid secretion related protein proteins. Treatment with LY294002 (PI3K inhibitor) four experimental GCs groups: control (0 µg/mL ADPN), 1 µg/mL ADPN, LY294002 inhibitor, and 1 µg/mL ADPN+LY294002. The results showed that ADPN promotes the secretion of steroid hormones by GCs through the PI3K-AKT. In summary, ADPN plays a crucial role in ameliorating postpartum anestrus in dairy cows with SCK., Competing Interests: Declaration of Competing Interest There are no conflicts of interest related to this study., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

An economic simulation was carried out over 183 milk-producing countries to estimate the global economic impacts of 12 dairy cattle diseases and health conditions: mastitis (subclinical and clinical), lameness, paratuberculosis (Johne's disease), displaced abomasum, dystocia, metritis, milk fever, ovarian cysts, retained placenta, and ketosis (subclinical and clinical). Estimates of disease impacts on milk yield, fertility, and culling were collected from the literature, standardized, meta-analyzed using a variety of methods ranging from simple averaging to random-effects models, and adjusted for comorbidities to prevent overestimation. These comorbidity-adjusted disease impacts were then combined with a set of country-level estimates for lactational incidence or prevalence or both, herd characteristics, and price estimates within a series of Monte Carlo simulations that estimated and valued the economic losses due to these diseases. It was estimated that total annual global losses are US$65 billion (B). Subclinical ketosis, clinical mastitis, and subclinical mastitis were the costliest diseases modeled, resulting in mean annual global losses of approximately US$18B, US$13B, and US$9B, respectively. Estimated global annual losses due to clinical ketosis, displaced abomasum, dystocia, lameness, metritis, milk fever, ovarian cysts, paratuberculosis, and retained placenta were estimated to be US$0.2B, US$0.6B, US$0.6B, US$6B, US$5B, US$0.6B, US$4B, US$4B, and US$3B, respectively. Without adjustment for comorbidities, when statistical associations between diseases were disregarded, mean aggregate global losses would have been overestimated by 45%. Although annual losses were greatest in India (US$12B), the United States (US$8B), and China (US$5B), depending on the measure of losses used (losses as a percentage of gross domestic product, losses per capita, losses as a percentage of gross milk revenue), the relative economic burden of these dairy cattle diseases across countries varied markedly., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86 + , but lower MFI of CD206 + and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis, a commonly observed energy metabolism disorder in dairy cows during the peripartal period, is distinguished by increased concentrations of BHB in the blood. This condition has a negative impact on milk production and quality, causing financial losses. An untargeted metabolomics approach was performed on plasma samples from cows between 5 and 7 DIM diagnosed as controls (CON; BHB <1.2 mM, n = 30), subclinically ketotic (SCK; 1.2 < BHB <3.0 mM, n = 30), or clinically ketotic (CK; BHB >3.0 mM, n = 30). Cows were selected from a commercial farm of 214 Holstein cows (average 305-d yield in the previous lactation of 35.42 ± 7.23 kg/d; parity, 2.41 ± 1.12; BCS, 3.1 ± 0.45). All plasma and milk samples (n = 90) were subjected to liquid chromatography-MS-based metabolomic analysis. Statistical analyses were performed using GraphPad Prism 8.0, MetaboAnalyst 4.0, and R version 4.1.3. Compared with the CON group, both SCK and CK groups had greater milk fat, freezing point, and fat-to-protein ratio, as well as lower milk protein, lactose, solids-not-fat, and milk density. Within 21 d after calving, compared with CON, the SCK group experienced a reduction of 2.65 kg/d in milk yield, while the CK group experienced a decrease of 7.7 kg/d. Untargeted metabolomics analysis facilitated the annotation of a total of 5,259 and 8,423 metabolites in plasma and milk. Differentially affected metabolites were screened in CON versus SCK, CON versus CK, and SCK versus CK (unpaired t-test, false discovery rate <0.05; and absolute value of log(2)-fold change >1.5). A total of 1,544 and 1,888 differentially affected metabolites were detected in plasma and milk. In plasma, glycerophospholipid metabolism, pyrimidine metabolism, tryptophan metabolism, sphingolipid metabolism, amino sugar and nucleotide sugar metabolism, phenylalanine metabolism, and steroid hormone biosynthesis were identified as important pathways. Weighted gene co-expression network analysis (WGCNA) indicated that tryptophan metabolism is a key pathway associated with the occurrence and development of ketosis. Increases in 5-hydroxytryptophan and decreases in kynurenine and 3-indoleacetic acid in SCK and CK were suggestive of an impact at the gut level. The decrease of most glycerophospholipids indicated that ketosis is associated with disordered lipid metabolism. For milk, pyrimidine metabolism, purine metabolism, pantothenate and CoA biosynthesis, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, sphingolipid metabolism, and fatty acid degradation were identified as important pathways. The WGCNA indicated that purine and pyrimidine metabolism in plasma was highly correlated with milk yield during the peripartal period. Alterations in purine and pyrimidine metabolism characterized ketosis, with lower levels of these metabolites in both milk and blood underscoring reduced efficiency in nitrogen metabolism. Our results may help to establish a foundation for future research investigating mechanisms responsible for the occurrence and development of ketosis in peripartal cows., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Dairy cows with clinical ketosis (CK) exhibit excessive adipose tissue (AT) lipolysis and systemic inflammation. Lipolysis in cows can be induced by the canonical (hormonally induced) and inflammatory lipolytic pathways. Currently, the most common treatment for CK is oral propylene glycol (PG); however, PG does not reduce lipolysis or inflammation. Niacin (NIA) can reduce the activation of canonical lipolysis, whereas cyclooxygenase inhibitors such as flunixin meglumine (FM) can limit inflammation and inhibit the inflammatory lipolytic pathway. The objective of this study was to determine the effects of including NIA and FM in the standard PG treatment for postpartum CK on AT function. Multiparous Jersey cows (n = 18; 7.1 ± 3.8 DIM) were selected from a commercial dairy. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and drop in milk yield) and high blood levels of BHB (≥1.2 mmol/L). Cows with CK were randomly assigned to one of 3 treatments: (1) PG: 310 g administered orally once per day for 5 d, (2) PG+NIA: 24 g administered orally once per day for 3 d, and (3) PG+NIA+FM: 1.1 mg/kg administered IV once per day for 3 d. Healthy control cows (HC; n = 6) matched by lactation and DIM (±2 d) were sampled. Subcutaneous AT explants were collected at d 0 and d 7 relative to enrollment. To assess AT insulin sensitivity, explants were treated with insulin (1 µL/L) during lipolysis stimulation with a β-adrenergic receptor agonist (isoproterenol, 1 µM). Lipolysis was quantified by glycerol release in the media. Lipid mobilization and inflammatory gene networks were evaluated using quantitative PCR. Protein biomarkers of lipolysis, insulin signaling, and AT inflammation, including hormone-sensitive lipase, protein kinase B (Akt), and ERK1/2, were quantified by capillary immunoassays. Flow cytometry of AT cellular components was used to characterize macrophage inflammatory phenotypes. Statistical significance was determined by a nonparametric t-test when 2 groups (HC vs. CK) were analyzed and an ANOVA test with Tukey adjustment when 3 treatment groups (PG vs. PG+NIA vs. PG+NIA+FM) were evaluated. At d 0, AT from CK cows showed higher mRNA expression of lipolytic enzymes ABHD5, LIPE, and LPL, as well as increased phosphorylation of hormone-sensitive lipase compared with HC. At d 0, insulin reduced lipolysis by 41% ± 8% in AT from HC, but CK cows were unresponsive (-2.9 ± 4%). Adipose tissue from CK cows exhibited reduced Akt phosphorylation compared with HC. Cows with CK had increased AT expression of inflammatory gene markers, including CCL2, IL8, IL10, TLR4, and TNF, along with ERK1/2 phosphorylation. Adipose tissue from CK cows showed increased macrophage infiltration compared with HC. By d 7, AT from PG+NIA+FM cows had a more robust response to insulin, as evidenced by reduced glycerol release (36.5% ± 8% compared with PG at 26.9% ± 7% and PG+NIA at 7.4% ± 8%) and enhanced phosphorylation of Akt. By d 7, PG+NIA+FM cows presented lower inflammatory markers, including ERK1/2 phosphorylation, and reduced macrophage infiltration, compared with PG and PG+NIA. These data suggest that including NIA and FM in CK treatment improves AT insulin sensitivity and reduces AT inflammation and macrophage infiltration., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis is currently regarded as a major metabolic disorder of dairy cows, reflective of the animal's efforts to adapt to energy deficit while transitioning into lactation. Currently viewed as a pathology by some, ketosis is associatively implicated in milk production losses and peripartal health complications that increase the risk of early removal of cows from the herd, thus carrying economic losses for dairy farmers and jeopardizing the sustainability of the dairy industry. Despite decades of intense research in the mitigation of ketosis and its sequelae, our ability to lessen its purported effects remains limited. Moreover, the association of ketosis to reduced milk production and peripartal disease is often erratic and likely mired by concurrent potential confounders. In this review, we discuss the potential reasons for these apparent paradoxes in the light of currently available evidence, with a focus on the limitations of observational research and the necessary steps to unambiguously identify the effects of ketosis on cow health and performance via controlled randomized experimentation. A nuanced perspective is proposed that considers the dissociation of ketosis-as a disease-from healthy hyperketonemia. Furthermore, in consideration of a growing body of evidence that highlights positive roles of ketones in the mitigation of metabolic dysfunction and chronic diseases, we consider the hypothetical functions of ketones as health-promoting metabolites and ponder on their potential usefulness to enhance dairy cow health and productivity., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objectives of this study were to assess: 1) differences in the metabolic status, systemic inflammation, daily milk yield, and daily rumination time between Holstein dairy cows with different vaginal discharge scores (VDS) in the first 7±3 DIM, and 2) effects of intrauterine dextrose infusion on metabolic status, systemic inflammation, daily milk yield and daily rumination time in dairy cows with VDS4 and VDS5. Cows (n=641) from a farm located in central Pennsylvania were screened at 7±3 DIM (study d 0) to assess vaginal discharge scores. Vaginal discharge was scored using a five-point scale (i.e., 1- clear fluid, 2- <50% white purulent fluid, 3- >50% white purulent fluid, 4- red-brownish fluid without fetid smell, and 5- fetid red-brownish watery fluid). Cows with VDS4 and VDS5 were blocked by parity and randomly assigned to one of two treatment groups: 1) CONV (VDS4 n=15; VDS5 n= 23): two injections of ceftiofur (per label; 6.6 mg/Kg) 72 h apart; and 2) DEX (VDS4 n=15; VDS5 n=22): three intrauterine infusions of a 50% dextrose solution (1 L/cow) every 24 h. Cows that presented a VDS 1, 2, and 3 were categorized as normal vaginal discharge animals (NOMVDS; n=35) and were randomly selected and matched by parity to CONV and DEX cows. Daily milk yield and rumination time for the first 150 DIM were collected from on-farm computer records. Blood samples were collected to assess haptoglobin (HP) and β-hydroxybutyrate (BHB) concentrations at study d 0, d 7, and d 14 relative to enrollment. Subclinical ketosis was defined as having a BHB concentration >1.2 mmol/dL at any of the sampling points. The data were analyzed using the MIXED and GLIMMIX procedures of SAS as a randomized complete block design. When comparing cows with different VDS (i.e., NOMVDS, VDS4, VDS5) separately, cows with VDS5 had the highest concentration of HP at enrollment compared to cows with VDS4 and NOMVDS; however, cows with VDS4 had higher concentrations of HP compared to cows with NOMVDS. Cows with VDS4 or VDS5 had a higher incidence of subclinical ketosis compared to cows with NOMVDS (p=0.005; VDS4= 62.08±9.16%; VDS5=74.44±6.74%; NOMVDS=34.36±8.53%). Similarly, daily milk yield (p<.0001; VDS4=30.17±1.32 kg/d; VDS5=27.40±1.27 kg/d; NOMVDS=35.14±1.35 kg/d) and daily rumination time (p=0.001; VDS4=490.77±19.44 min; VDS5=465±16.67 min; NOMVDS=558.29±18.80 min) was lower for cows with VDS4 and VDS5 compared to cows with NOMVDS at 7±3 days in milk. When analyzing HP concentration between treatment groups in cows with VDS4 (p=0.70), VDS5 (p=0.25), or VDS4 and VDS5 combined (p=0.31), there was no difference in HP concentration by study d 14 between treatment groups. Interestingly, when only cows with VDS4 were considered for treatment, both treatments, DEX and CONV, increased the daily milk yield to the levels of NOMVDS cows by 14 days in milk. On the other hand, when only cows with VDS5 were considered for treatment, cows treated with DEX produced, on average, 4.48 kg/d less milk in the first 150 days in milk compared to cows treated with CONV or cows that had NOMVDS. Similarly, when cows with either VDS4 or VDS5 were considered for treatment, DEX treatment also impaired milk yield. These results suggest that cows with either VDS 4 or 5 have an altered inflammatory status, and decreased milk yield and rumination compared to cows with NOMVDS. Furthermore, DEX treatment may have similar effects on daily milk yield and metabolic status compared to CONV in cows with VDS4, while DEX is not recommended for cows with VDS5., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Published by Elsevier Ltd.)

Ketosis is a common metabolic disorder in the early lactation of dairy cows. It is typically diagnosed by measuring the concentration of β-hydroxybutyrate (BHB) in the blood. This study aimed to estimate the genetic parameters of blood BHB and conducted a genome-wide association study (GWAS) based on the estimated breeding value. Phenotypic data were collected from December 2019 to August 2023, comprising blood BHB concentrations in 45,617 Holstein cows during the three weeks post-calving across seven dairy farms. Genotypic data were obtained using the Neogen Geneseek Genomic Profiler (GGP) Bovine 100 K SNP Chip and GGP Bovine SNP50 v3 (Illumina Inc., San Diego, CA, USA) for genotyping. The estimated heritability and repeatability values for blood BHB levels were 0.167 and 0.175, respectively. The GWAS result detected a total of ten genome-wide significant associations with blood BHB. Significant SNPs were distributed in Bos taurus autosomes (BTA) 2, 6, 9, 11, 13, and 23, with 48 annotated candidate genes. These potential genes included those associated with insulin regulation, such as INSIG2, and those linked to fatty acid metabolism, such as HADHB , HADHA , and PANK2 . Enrichment analysis of the candidate genes for blood BHB revealed the molecular functions and biological processes involved in fatty acid and lipid metabolism in dairy cattle. The identification of novel genomic regions in this study contributes to the characterization of key genes and pathways that elucidate susceptibility to ketosis in dairy cattle.

Our main aim was to investigate the predictive value of prepartum behaviors such as total daily rumination (TDR), total daily activity (TDA) and dry matter intake (DMI) as early indicators to detect cows at risk for hyperketonemia (HYK), hypoglycemia (HYG) or high non-esterified fatty acid (NEFA) status in the first (wk1) and second week (wk2) postpartum. In a case control study, 64 Holstein cows were enrolled 3 weeks before the expected time of calving and monitored until 15 days in milk (DIM). Postpartum blood samples were taken at D3 and D6 for wk1 and at D12 and D15 for wk2 to measure beta-hydroxybutyrate, NEFA and glucose concentration. Ear-mounted accelerometers were used to measure TDR and TDA. DMI and milk yield were obtained from farm records. Relationships between the average daily rate of change in prepartum TDR (ΔTDR), TDA (ΔTDA), and DMI (ΔDMI) with postpartum HYK, HYG and NEFA status in wk1 and wk2 post-partum were evaluated using linear regression models. Models were adjusted for potential confounding variables, and covariates retained in the final models were determined by backward selection. No evidence was found to support the premise that prepartum ΔTDR, ΔTDA or ΔDMI predicted postpartum HYK, HYG or NEFA status in wk1 or in wk2. Overall, prepartum ΔTDR, ΔTDA and ΔDMI were not effective predictors of HYK, HYG or NEFA status in the first 2 weeks postpartum., (© 2024 The Authors. This is an open access article under the CC BY-NC-ND 4.0 license.)

Subclinical ketosis (SCK) in dairy cows is often misdiagnosed because it lacks clinical signs and detection indicators. However, it is highly prevalent and may transform into clinical ketosis if not treated promptly. Due to the negative energy balance, a large amount of fat is mobilized, producing NEFA that exceeds the upper limit of liver processing, which in turn leads to the disturbance of liver lipid metabolism. The silent information regulator 1 (SIRT1) is closely related to hepatic lipid metabolism disorders. Exosomes as signal transmitters, also play a role in the circulatory system. We hypothesize that the circulating exosome-mediated adenosine 5'-monophosphate (AMP)-activated protein kinase alpha (AMPKα)-SIRT1 pathway regulates lipid metabolism disorders in SCK cows. We extracted the exosomes required for the experiment from the peripheral circulating blood of non-ketotic (NK) and SCK cows. We investigated the effect of circulating exosomes on the expression levels of mRNA and protein of the AMPKα-SIRT1 pathway in non-esterified fatty acid (NEFA)-induced dairy cow primary hepatocytes using in vitro cell experiments. The results showed that circulating exosomes increased the expression levels of Lipolysis-related genes and proteins (AMPKα, SIRT1, and PGC-1α) in hepatocytes treated with 1.2 mM NEFA, and inhibited the expression of lipid synthesis-related genes and protein (SREBP-1C). The regulation of exosomes on lipid metabolism disorders caused by 1.2 mM NEFA treatment showed the same trend as for SIRT1-overexpressing adenovirus. The added exosomes could regulate NEFA-induced lipid metabolism in hepatocytes by mediating the AMPKα-SIRT1 pathway, consistent with the effect of transfected SIRT1 adenovirus., Competing Interests: Declaration of competing interest The authors declare no potential or real conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (β-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-β-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R 2 v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R 2 v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Mastitis is a serious disease which affects animal husbandry, particularly in cow breeding. The etiology of mastitis is complex and its pathological mechanism is not yet fully understood. Our previous research in clinical investigation has revealed that subclinical ketosis can increase the number of somatic cell counts (SCC) in milk, although the underlying mechanism remains unclear. Recent studies have further confirmed the significant role of mastitis., Results: In this study, we aimed to examine the SCC, rumen microbiota, and metabolites in the milkmen of cows with subclinical ketosis. Additionally, we conducted a rumen microbiota transplant into mice to investigate the potential association between rumen microbiota disturbance and mastitis induced by subclinical ketosis in dairy cows. The study has found that cows with subclinical ketosis have a higher SCC in their milk compared to healthy cows. Additionally, there were significant differences in the rumen microbiota and the level of volatile fatty acid (VFA) between cows with subclinical ketosis and healthy cows. Moreover, transplanting the rumen microbiota from subclinical ketosis and mastitis cows into mice can induce mammary inflammation and liver function damage than transplanting the rumen flora from healthy dairy cows., Conclusions: In addition to the infection of mammary gland by pathogenic microorganisms, there is also an endogenous therapeutic pathway mediated by rumen microbiota. Targeted rumen microbiota modulation may be an effective way to prevent and control mastitis in dairy cows., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

This prospective observational study aimed to investigate the association of rumen fill and motility in post-partum Holstein cows with their future reproductive performance and subclinical ketosis (SCK). The study population consisted of two independent data sets: the first (DS1) included 237 cows from 6 herds and the second one (DS2) 709 cows from 9 herds. Rumen Fill Score (RFS) was transformed into a 3 level-trait, representing very low, low and adequate dry matter intake, respectively. A binary Rumen Contraction Score (RCS) was defined as: 0: <2 contractions/2 min, impaired rumen motility and 1: ≥2 contractions/2 min, normal rumen motility. A combined binary trait based on RFS and RCS (RFCS) was also established, representing unsatisfactory and satisfactory rumen function. Three SCK traits were defined, based on 3 different thresholds, SCK&#95;I: BHB≥1,000 mmol/L, SCK&#95;II: BHB≥1,100 mmol/L and SCK&#95;III: BHB≥1,200 mmol/L. Scores were assessed and blood samples collected on day 7 (DS1) or day 8 (DS2), postpartum. Kaplan-Meier survival analysis, multivariable Cox proportional hazards models and Generalized Linear Mixed Models were performed to evaluate the association of rumen and SCK traits with reproduction. Herd, parity, calving season and several postparturient diseases were also included as potential explanatory variables. Mean days from calving to pregnancy after the 1st artificial insemination (AI) and from calving to pregnancy (all AIs) were shorter for levels of rumen traits representing adequate DMI and normal rumen motility; in most cases these differences were statistically significant in both datasets. Cows with adequate DMI and normal rumen motility (only in DS2) had greater hazard (hazard ratio [HR] = 1.84 and 1.61, for RFS and RFCS, respectively) and odds (odds ratio [OR] = 2.49 and 1.98, for RFS and RFCS, respectively) for pregnancy at 1st AI. Assessment of the association of examined rumen traits with hazard and odds for pregnancy at all AIs yielded statistically significant results in both datasets. For RFS, RCS and RFCS, HRs ranged from 1.57 to 3.31 and ORs from 1.95 to 4.83. No statistically significant associations with hazard and odds for pregnancy at 1st or all AIs were detected, for any of the 3 SCK traits, in either dataset. Overall, the combined RFCS trait constantly identified more than twice the number of cows with future reproductive problems than a positive SCK blood test., Competing Interests: Declaration of competing interest The authors declare no financial or commercial conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Describe the use of fixed-rate intravenous insulin infusions (FRIs) in cats and dogs with diabetic ketoacidosis (DKA) and determine if this is associated with faster resolution of ketosis compared to variable-rate intravenous insulin infusions (VRIs). Secondary objectives were to evaluate complication rates, length of hospitalization (LOH), and survival to discharge (STD)., Design: Randomized clinical trial (January 2019 to July 2020)., Setting: University veterinary teaching hospital and private referral hospital., Animals: Dogs and cats with DKA and venous pH <7.3, blood glucose concentration >11 mmol/L (198 mg/dL), and β-hydroxybutyrate (BHB) concentration >3 mmol/L were eligible for inclusion. Patients were randomly assigned to receive either FRI or VRI., Interventions: Neutral (regular) insulin was administered IV as an FRI or VRI. For FRI, the rate was maintained at 0.01 IU/kg/h. For VRI, the dose was adjusted according to blood glucose concentration., Measurements and Results: Sixteen cats and 20 dogs were enrolled. Population characteristics, mean insulin infusion rate, time to resolution of ketosis (BHB <0.6 mmol/L), complications, LOH, and STD were evaluated. In cats, overall resolution of ketosis was low (9/16 [56.3%]), limiting comparison of protocols. In dogs, resolution of ketosis was high (19/20 dogs [95.0%]) but the time to resolution in the FRI group was not different than that in the VRI group (P = 0.89), despite a 25% higher average insulin infusion rate in the FRI group (P = 0.04). The incidence of complications was low and did not differ between protocols. In cats, LOH and STD did not differ between protocols. All cats that died (5/16) did so within 78 hours and none had resolution of ketosis. Dogs receiving FRI had a shorter LOH (P = 0.01) but STD did not differ between protocols. Six dogs (30.0%) did not survive to hospital discharge but all had resolution of ketosis., Conclusions: FRIs can be used in veterinary species but may not hasten resolution of ketosis., (© 2023 The Authors. Journal of Veterinary Emergency and Critical Care published by Wiley Periodicals LLC on behalf of Veterinary Emergency and Critical Care Society.)

The aim of this study was to identify the c.495C > T polymorphism within exon 1 of the osteopontin gene (OPN), and to analyze its association with susceptibility to ketosis in Polish Holstein-Friesian (HF) cows. The study utilized blood samples from 977 HF cows, for the determination of β-hydroxybutyric acid (BHB) and for DNA isolation. The c.495C > T polymorphism of the bovine osteopontin gene was determined by PCR-RFLP. The CT genotype (0.50) was deemed the most common, while TT (0.08) was the rarest genotype. Cows with ketosis most often had the CC genotype, while cows with the TT genotype had the lowest incidence of ketosis. To confirm the relationship between the genotype and ketosis in cows, a weight of evidence (WoE) was generated. A very strong effect of the TT genotype on resistance to ketosis was demonstrated. The distribution of the ROC curve shows that the probability of resistance to ketosis is > 75% if cows have the TT genotype of the OPN gene (cutoff value is 0.758). Results suggest that TT genotype at the c.495C > T locus of the OPN gene might be effective way to detect the cows with risk of ketosis., (© 2023. The Author(s).)

Excessive and protracted lipolysis in adipose tissues of dairy cows is a major risk factor for clinical ketosis (CK). This metabolic disease is common in postpartum cows when lipolysis provides fatty acids as an energy substrate to offset negative energy balance. Lipolysis in cows can be induced by the canonical (hormonally induced) and inflammatory pathways. Current treatments for CK focus on improving glucose in blood (i.e., oral propylene glycol [PG], or i.v. dextrose). However, these therapies do not inhibit the canonical and inflammatory lipolytic pathways. Niacin (NIA) can reduce activation of the canonical pathway. Blocking inflammatory responses with cyclooxygenase inhibitors such as flunixin meglumine (FM) can inhibit inflammatory lipolytic activity. The objective of this study was to determine the effects of including NIA and FM in the standard PG treatment for postpartum CK on circulating concentrations of ketone bodies. A 4-group, parallel, individually randomized trial was conducted in multiparous Jersey cows (n = 80) from a commercial dairy in Michigan during a 7-mo period. Eligible cows had CK symptoms (lethargy, depressed appetite, and milk yield) and hyperketonemia (blood β-hydroxybutyrate [BHB] ≥1.2 mmol/L). Cows with CK were randomly assigned to 1 of 3 groups where the first group received 310 g of oral PG once per day for 5 d; the second group received PG for 5 d + 24 g of oral NIA once per day for 3 d (PGNIA); and the third group received PG for 5 d + NIA for 3 d + 1.1 mg/kg i.v. FM once per day for 3 d (PGNIAFM). The control group consisted of cows that were clinically healthy (HC; untreated; BHB <1.2 mmol/L, n = 27) matching for parity and DIM with all 3 groups. Animals were sampled at enrollment (d 0), and d 3, 7, and 14 to evaluate ketone bodies and circulating metabolic and inflammatory biomarkers. Effects of treatment, sampling day, and their interactions were evaluated using mixed effects models. Logistic regression was used to calculate the odds ratio (OR) of returning to normoketonemia (BHB <1.2 mmol/L). Compared with HC, enrolled CK cows exhibited higher blood concentrations of dyslipidemia markers, including nonesterified fatty acids (NEFA) and BHB, and lower glucose and insulin levels. Cows with CK also had increased levels of biomarkers of pain (substance P), inflammation, including lipopolysaccharide-binding protein, haptoglobin, and serum amyloid A, and proinflammatory cytokines IL-4, MCP-1, MIP-1α, and TNFα. Importantly, 72.2% of CK cows presented endotoxemia and had higher circulating bacterial DNA compared with HC. By d 7, the percentage of cows with normoketonemia were higher in PGNIAFM = 87.5%, compared with PG = 58.33%, and PGNIA = 62.5%. At d 7 the OR for normoketonemia in PGNIAFM cows were 1.5 (95% CI, 1.03-2.17) and 1.4 (95% CI, 0.99-1.97) relative to PG and PGNIA, respectively. At d 3, 7, and 14, PGNIAFM cows presented the lowest values of BHB (PG = 1.36; PGNIA = 1.24; PGNIAFM = 0.89 ± 0.13 mmol/L), NEFA (PG = 0.58; PGNIA = 0.59; PGNIAFM = 0.45 ± 0.02 mmol/L), and acute phase proteins. Cows in PGNIAFM also presented the highest blood glucose increment across time points and insulin by d 7. These data provide evidence that bacteremia or endotoxemia, systemic inflammation, and pain may play a crucial role in CK pathogenesis. Additionally, targeting lipolysis and inflammation with NIA and FM during CK effectively reduces dyslipidemia biomarkers, improves glycemia, and improves overall clinical recovery., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this observational cohort study was to characterize the pattern of rumination time (RT), physical activity (PA), and lying time (LT) monitored by an automated health monitoring system, based on an ear-attached sensor, immediately before, during, and after clinical diagnosis (CD) of metabolic-digestive disorders. Sensor data were collected from 820 lactating Holstein cows monitored daily from calving up to 21 DIM for detection of health disorders (HD). Cows were grouped retrospectively in the no-clinical health disorder group (NCHD; n = 616) if no HD were diagnosed, or the metabolic-digestive group (METB-DIG; n = 58) if diagnosed with clinical ketosis or indigestion only. Cows with another clinical health disorder within -7 to +7 d of CD of displaced abomasum, clinical ketosis, or indigestion were included in the metabolic-digestive plus one group (METB-DIG+1; n = 25). Daily RT, PA, and LT, and absolute and relative changes within -7 to +7 d of CD were analyzed with linear mixed models with or without repeated measures. Rumination time and PA were smaller, and LT was greater for the METB-DIG and METB-DIG+1 group than for cows in the NCHD group for most days from -7 to +7 d of CD of HD. In general, daily RT, PA, and LT differences were larger between the METB-DIG+1 and NCHD groups than between the METB-DIG and NCHD groups. In most cases, RT and PA decreased to a nadir and LT increased to a peak immediately before or after CD of HD, with a return to levels similar to the NCHD group within 7 d of CD. Absolute values and relative changes from 5 d before CD to the day of the nadir for RT and PA or peak for LT were different for cows in the METB-DIG and METB-DIG+1 group than for the NCHD group. For PA, the METB-DIG+1 group had greater changes than the METB-DIG group. For cows affected by metabolic-digestive disorders, RT, PA, and LT on the day of CD and resolution of clinical signs were different than for cows in the NCHD group, but an increase in RT and PA or a decrease in LT was observed from the day of CD to the day of resolution of clinical signs. We conclude that dairy cows diagnosed with metabolic-digestive disorders including displaced abomasum, clinical ketosis, and indigestion presented substantial alterations in the pattern of RT, PA, and LT captured by an ear-attached sensor. Thus, automated health monitoring systems based on ear-attached sensors might be used as an aid for identifying cows with metabolic-digestive disorders. Moreover, RT, PA, and LT changes after CD might be positive indicators of recovery from metabolic-digestive disorders., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis is a metabolic disease that often occurs in dairy cows postpartum and is a result of disordered lipid metabolism. Acetyl-coenzyme A (CoA) acetyltransferase 2 (ACAT2) is important for balancing cholesterol and triglyceride (TG) metabolism; however, its role in subclinical ketotic dairy cows is unclear. This study aimed to explore the potential correlation between ACAT2 and lipid metabolism disorders in subclinical ketotic cows through in vitro and in vivo experiments. In the in vivo experiment, liver tissue and blood samples were collected from healthy cows (CON, n = 6, β-hydroxybutyric acid [BHBA] concentration <1.0 mM) and subclinical ketotic cows (subclinical ketosis [SCK], n = 6, BHBA concentration = 1.2-3.0 mM) to explore the effect of ACAT2 on lipid metabolism disorders in SCK cows. For the in vitro experiment, bovine hepatocytes (BHEC) were used as the model. The effects of BHBA on ACAT2 and lipid metabolism were investigated via BHBA concentration gradient experiments. Subsequently, the relation between ACAT2 and lipid metabolism disorder was explored by transfection with siRNA of ACAT2. Transcriptomics showed an upregulation of differentially expression genes during lipid metabolism and significantly lower ACAT2 mRNA levels in the SCK group. Compared with the CON group in vivo, the SCK group showed significantly higher expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulator element binding protein 1c (SREBP1c) and significantly lower expression levels of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyl-transferase 1A (CPT1A), sterol regulatory element binding transcription factor 2 (SREBP2), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Moreover, the SCK group had a significantly higher liver TG content and significantly lower plasma total cholesterol (TC) and free cholesterol content. These results were indicative of TG and cholesterol metabolism disorders in the liver of dairy cows with SCK. Additionally, the SCK group showed an increased expression of perilipin-2 (PLIN2), decreased expression of apolipoprotein B, and decreased plasma concentration of very low-density lipoproteins (VLDL) and low-density lipoproteins cholesterol (LDL-C) by downregulating ACAT2, which indicated an accumulation of TG in liver. In vitro experiments showed that BHBA induced an increase in the TG content of BHEC, decreased content TC, increased expression of PPARγ and SREBP1c, and decreased expression of PPARα, CPT1A, SREBP2, and HMGCR. Additionally, BHBA increased the expression of PLIN2 in BHEC, decreased the expression and fluorescence intensity of ACAT2, and decreased the VLDL and LDL-C contents. Furthermore, silencing ACAT2 expression increased the TG content; decreased the TC, VLDL, and LDL-C contents; decreased the expression of HMGCR and SREBP2; and increased the expression of SREBP1c; but had no effect on the expression of PLIN2. These results suggest that ACAT2 downregulation in BHEC promotes TG accumulation and inhibits cholesterol synthesis, leading to TG and cholesterol metabolic disorders. In conclusion, ACAT2 downregulation in the SCK group inhibited cholesterol synthesis, increased TG synthesis, and reduced the contents of VLDL and LDL-C, eventually leading to disordered TG and cholesterol metabolism., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The metabolic status of dairy cows directly influences the nutritional quality and flavor of raw milk. A comprehensive comparison of non-volatile metabolites and volatile compounds in raw milk from healthy and subclinical ketosis (SCK) cows was performed using LC-MS, GC-FID, and HS-SPME/GC-MS. SCK can significantly alter the profiles of water-soluble non-volatile metabolites, lipids, and volatile compounds of raw milk. Compared with healthy cows, milk from SCK cows had higher contents of tyrosine, leucine, isoleucine, galactose-1-phosphate, carnitine, citrate, phosphatidylethanolamine species, acetone, 2-butanone, hexanal, dimethyl disulfide and lower content of creatinine, taurine, choline, α-ketoglutaric acid, fumarate, triglyceride species, ethyl butanoate, ethyl acetate, and heptanal. The percentage of polyunsaturated fatty acids in milk was lowered in SCK cows. Our results suggest that SCK can change milk metabolite profiles, disrupt the lipid composition of milk fat globule membrane, decrease the nutritional value, and increase the volatile compounds associated with off-flavors in milk., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuchao Zhao reports financial support was provided by Beijing Natural Science Foundation. Yuchao Zhao reports financial support was provided by China Postdoctoral Science Foundation. Yuchao Zhao reports financial support was provided by Beijing Postdoctoral Research Foundation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 mM NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and β-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 mM) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 mM) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B, IL6, and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Hepatic lipidosis in dairy cows is the result of a disturbed balance between the uptake of non-esterified fatty acids (NEFA), their metabolism in the hepatocytes, and the limited efflux of TG as very-low-density lipoprotein (VLDL). Lipidosis and the associated risk for ketosis represents a consequence of selecting dairy cows primarily for milk production without considering the basic physiological mechanisms of this trait. The overall risk for lipidosis and ketosis possesses a genetic background and the recently released new breeding value of the German Holstein Friesian cows now sets the path for correction of this risk and in that confirms the assumed genetic threat. Ectopic fat deposition in the liver is the result of various steps including lipolysis, uptake of fat by the liver cell, its metabolism, and finally release as very-low-density lipoprotein (VLDL). These reactions may be modulated directly or indirectly and hence, serve as basis for prophylactic measures. The pertaining methods are described in order to support an improved understanding of the pathogenesis of lipidosis and ketosis. They consist of feeding a glucogenic diet, restricted feeding during the close-up time as well as supplementation with choline, niacin, carnitine, or the reduction of milking frequency. Prophylactic measures for the prevention of ketosis are also included in this discussion., Competing Interests: Es liegt kein Interessenkonflikt vor., (Thieme. All rights reserved.)

The objective of experiment I was to evaluate the association between days in the close-up group (DINCU) and milk production, early lactation diseases, reproductive performance, and culling. In experiment II behavioral changes associated with DINCU were evaluated using a neck-mounted sensor (Smarttag neck, Nedap Livestock Management, Groenlo, the Netherlands). Cow-lactations of 28,813 animals from 14,155 individual cows of 2 farms in northern Germany and western Slovakia, calving between January 2015 and December 2020, were included in the study. After exclusion of cows with a gestation length <262 and >292 d and cows with >42 DINCU data from 8,794 and 19,598 nulliparous and parous cows, respectively, were available for final statistical analyses. To analyze the association between DINCU and second test-d 305-d mature-equivalent milk projection, linear mixed models were calculated. Binary data (i.e., clinical hypocalcemia, hyperketonemia, retained placenta [RP], acute puerperal metritis [APM], mastitis, left displaced abomasum [LDA], first service pregnancy risk) were analyzed using logistic regression models. To analyze the association between DINCU and culling or death during the first 300 DIM Cox proportional hazards were used. To analyze the association between DINCU and behavior 7 d before to 7 d after calving (i.e., activity, inactivity, eating, ruminating time), linear mixed models were calculated. Nulliparous cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group had a lower milk production an increased risk for hyperketonemia, RP, and APM compared with nulliparous cows with DINCU between 21 to 28 d. Parous cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group had a lower milk production, an increased risk for RP and mastitis, a reduced first service pregnancy risk, and an increased culling risk, compared with parous cows with DINCU between 21 to 28 d. Furthermore, the risk for clinical hypocalcemia and LDA was increased in parous cows with >30 DINCU compared with parous cows with <30 DINCU. The risk for APM was increased in parous cows with <10 DINCU compared with parous cows with >10 DINCU. In nulliparous cows no association was found between DINCU and the risk for left displaced abomasum and mastitis. In experiment II, cows with 7 and 35 DINCU had an impaired behavior around calving compared with cows with 14, 21, and 28 DINCU. During the last 7 d before parturition, these cows were more inactive and had a reduced eating and ruminating time. After calving, cows with 7 DINCU spent less time eating. In conclusion, cows with <10 DINCU and cows with >30 DINCU had a lower milk production, a higher risk to incur diseases and an impaired behavior, especially before calving., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Enzyme adenosine deaminase (ADA) is a marker of inflammation in domestic animals, but it is unclear whether it is a reliable marker of oxidative stress, especially in the transition period in dairy cows. This study aims to assess if ADA and redox status measurements in saliva provide the same utility to detect disease condition as that obtained from serum. Sixty-eight multiparous Holstein cows, between 2 and 3 weeks postpartum were selected. Five study groups were established: control (healthy), and cows with ketosis, mastitis, laminitis, and metritis. The parameters measured were ADA activity, total oxidants (TOS), antioxidants (TAC), and OSi ratio.Regarding redox status, no significant differences arise in both saliva and serum being the correlations negative and not significant. In saliva, ADA activity in healthy cows differs from those with pathological processes, having the lowest activities. In serum, ADA activity is similar in the healthy and ketosis cows, showing the lowest activities meanwhile animals with mastitis, laminitis, or metritis have significantly higher activities. In conclusion, the measurement of ADA activities and redox status in saliva does not give consistent results, being preferable to measure them in serum during the transition period., (© 2023. The Author(s).)

Hyperketonemia (HYK) in early lactation can have a different impact on health and productivity depending on the timing of HYK onset. While specific metabolites measured during the dry period may serve as biomarkers of HYK, the correlations between metabolites represent a challenge for the use of metabolic profiles dataset, and little has been explored on HYK. This exploratory cohort study aimed a) to characterize the correlations among metabolites measured during the late dry period in dairy cows, and b) to identify biomarkers in the late dry period associated with the onset of HYK at the first (wk1) and second (wk2) week of lactation. Individual blood samples from 440 Holstein dairy cows were collected at 21 ± 3 days before expected parturition. From each sample, 36 different metabolites were measured in serum and plasma. Hyperketonemia was diagnosed in wk1 and wk2 of lactation based on the blood concentration of beta-hydroxybutyrate (BHB > 1.2 mmol/L). Principal component analysis (PCA) was performed to reduce metabolites to a smaller number of uncorrelated components. Multivariable logistic regression models were applied to assess the associations between principal components (PC) and HYK at wk1 only (HYK+ wk1), wk2 only (HYK+ wk2), or both weeks (HYK+ wk1-2). The incidence of HYK was 16.2% in the first week, 13.0% in the second week, and 21.2% within the first two weeks of lactation. The results of PCA highlighted 10 PCs from which two were associated with HYK+ wk1 as compared with cows without HYK during the first two weeks of lactation (non-HYK); the PC a2 led by bilirubin and non-esterified fatty acids (OR = 1.29; 95%CI: 1.02-1.68), and the PC a5 led by alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) (OR = 2.77; 95%CI: 1.61-4.97). There was no evidence of an association between any PC and HYK+ wk2 (vs. non-HYK cows). Cows with elevated PC a5 (led by ALP and GGT) in the dry period were 3.18 times more likely to be HYK+ wk1 than HYK+ wk2 (OR: 3.18, 95%CI: 1.34-8.73; P = 0.013). Overall, the main hypothesis generated by our exploratory study suggests that cows with biomarkers of liver dysfunction (ALP, GGT, bilirubin) assessed by PCA at 3 weeks before calving are more likely to develop HYK during the first week of lactation compared to the second week. In addition, results suggest that cows with HYK in both of the first two weeks of lactation had an overall metabolic disbalance during the onset of the late dry period, which based on PCs, encompass biomarkers related to glucogenic and ketogenic metabolic pathways as well as liver dysfunction and fatty liver. Further research is needed to determine the underlying mechanisms associated with the different adaptations between cows that develop HYK during the first and second week of lactation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rodriguez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metabolic and oxidative stress have been characterized as risk factors during the transition period from pregnancy to lactation. Although mutual relations between both types of stress have been suggested, they rarely have been studied concomitantly. For this, a total of 99 individual transition dairy cows (117 cases, 18 cows sampled during 2 consecutive lactations) were included in this experiment. Blood samples were taken at -7, 3, 6, 9, and 21 d relative to calving and concentrations of metabolic parameters (glucose, β-hydroxybutyric acid (BHBA), nonesterified fatty acids, insulin, insulin-like growth factor 1, and fructosamine) were determined. In the blood samples of d 21, biochemical profiles related to liver function and parameters related to oxidative status were determined. First, cases were allocated to 2 different BHBA groups (ketotic vs. nonketotic, N:n = 20:33) consisting of animals with an average postpartum BHBA concentration and at least 2 out of 4 postpartum sampling points exceeding 1.2 mmol/L or remaining below 0.8 mmol/L, respectively. Second, oxidative parameters [proportion of oxidized glutathione to total glutathione in red blood cells (%)], activity of glutathione peroxidase, and of superoxide dismutase, concentrations of malondialdehyde and oxygen radical absorbance capacity were used to perform a fuzzy C-means clustering. From this, 2 groups were obtained [i.e., lower antioxidant ability (LAA 80% , n = 31) and higher antioxidant ability (HAA 80% , n = 19)], with 80% referring to the cutoff value for cluster membership. Increased concentrations of malondialdehyde, decreased superoxide dismutase activity, and impaired oxygen radical absorbance capacity were observed in the ketotic group compared with the nonketotic group, and inversely, the LAA 80% group showed increased concentrations of BHBA. In addition, the concentration of aspartate transaminase was higher in the LAA 80% group compared with the HAA 80% group. Both the ketotic and LAA 80% groups showed lower dry matter intake. However, a lower milk yield was observed in the LAA 80% group but not in the ketotic group. Only 1 out of 19 (5.3%) and 3 out of 31 (9.7%) cases from the HAA 80% and LAA 80% clusters belong to the ketotic and nonketotic group, respectively. These findings suggested that dairy cows vary in oxidative status at the beginning of the lactation, and fuzzy C-means clustering allows to classify observations with distinctive oxidative status. Dairy cows with higher antioxidant capacity in early lactation rarely develop ketosis., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Although there is evidence that ketosis negatively affects fertility, the effect of late and early ketosis on the reproductive performance of lactating cows has not been systematically investigated. The aim of this study was to evaluate the association between time and amplitude of elevated milk BHB (EMB) occurring within 42 d in milk (DIM) and subsequent reproductive performance of lactating Holstein cows. The dairy herd information data of 30,413 cows with 2 test-day milk BHB recordings during early lactation periods 1 and 2 (5-14 and 15-42 DIM, respectively) assessed as negative (<0.15 mmol/L), suspect (0.15-0.19 mmol/L), or positive (≥0.2 mmol/L) for EMB were used in this study. Based on the time and amplitude of milk BHB, cows were grouped into 7 groups: (1) healthy cows negative in both periods 1 and 2 were classified as NEG; (2) suspect in period 1 and negative in period 2: EARLY&#95;SUSP; (3) suspect in period 1 and suspect/positive in period 2: EARLY&#95;SUSP&#95;Pro; (4) positive in period 1 and negative in period 2: EARLY&#95;POS; (5) positive in period 1 and suspect/positive in period 2: EARLY&#95;POS&#95;Pro; (6) negative in period 1 and suspect in period 2: LATE&#95;SUSP; and (7) negative in period 1 and positive in period 2: LATE&#95;POS. The overall prevalence of EMB within 42 DIM was 27.4%, with the highest prevalence being EARLY&#95;SUSP (10.49%). Cows in EARLY&#95;POS and EARLY&#95;POS&#95;Pro, but not other EMB categories, had a longer interval from calving to first service compared with NEG cows. For the reproductive parameters, first service to conception interval, days open and calving interval, cows in all EMB groups except EARLY&#95;SUSP had longer intervals compared with NEG cows. These data indicate that there is a negative association between EMB within 42 d and reproductive performance after the voluntary waiting period. The intriguing findings of this study are the unaltered reproductive performance of EARLY&#95;SUSP cows, and the negative association between late EMB and reproductive performance. Hence, monitoring and prevention of ketosis during the first 6 wk of lactation is necessary to optimize reproductive performance of lactating dairy cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood β-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis ( 1 H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

This review covers the history and nomenclature of ketosis, the source and use of ketones in transition cows, and the controversial role of hyperketonemia's association with health and production outcomes in dairy cows. With the goal of assisting veterinarians with on-farm diagnostic and treatment methods, the authors present current and evolving means of direct and indirect hyperketonemia detection as well as a summary of treatment modalities and their efficacy. They encourage veterinarians to include hyperketonemia testing as part of their routine physical examinations and contemplate day in milk at hyperketonemia diagnosis when designing treatment and management strategies., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this observational study was to evaluate the association of transition cow health and estrous expression, detected by an automated activity monitoring system (Smarttag Neck, Nedap Livestock Management), with reproductive performance in lactating Holstein cows. A total of 3,750 lactating Holstein cows (1,563 primiparous cows and 2,187 multiparous cows) from a commercial dairy farm in Slovakia calving from January 2020 until July 2021 were enrolled on an ongoing basis. Activity data were recorded from d 7 until d 60 postpartum. Within this observational period, cows were classified into 3 categories: (1) no estrus event (Estrus0), (2) 1 estrus event (Estrus1), or (3) 2 or more estrus events (Estrus2+). Transition cow health was assessed by farm personnel within the first 30 d in milk (DIM) using standard operating procedures. Generalized linear mixed models were used to analyze continuous and categorical data. Cox proportional hazard models were used for time to event data. The overall prevalence of anestrus was 20.8%. Multiparous cows had a greater risk for anestrus compared with primiparous cows [odds ratio (OR) = 1.4]. Cows with stillbirth (OR = 1.76), retained placenta (OR = 2.19), puerperal metritis (OR = 1.48), or subclinical ketosis (OR = 1.51) had a greater risk for anestrus. In addition, cows calving in summer (OR = 0.82), autumn (OR = 0.38), or winter (OR = 0.56) had a higher incidence of anestrus than cows calving in spring. Estrous expression from d 7 until d 60 postpartum was associated with estrous duration (DU) and estrous intensity at first artificial insemination (AI). Cows in Estrus0 had the shortest DU at first postpartum AI (9.4 ± 0.18 h) compared with cows in Estrus1 (10.5 ± 0.13 h) and Estrus2+ (11.4 ± 0.12 h). Cows in Estrus2+ had a longer DU at first postpartum AI compared with cows in Estrus1. For Estrus0, Estrus1, and Estrus2+ cows, pregnancy per AI at first service was 42.5%, 50.9%, and 55.4%, respectively. Estrous expression from d 7 until d 60 postpartum was associated with time to first AI and time to pregnancy. Compared with Estrus0 cows, Estrus1 [hazard ratio (HR) = 1.43] and Estrus2+ cows (HR = 1.62) had an increased hazard of being inseminated within 100 DIM. Compared with Estrus2+, Estrus1 cows had a reduced hazard of being inseminated within 100 DIM (HR = 0.89). Compared with Estrus0 cows, Estrus1 (HR = 1.24) and Estrus2+ cows (HR = 1.46) had an increased hazard of becoming pregnant within 200 DIM. Median DIM to pregnancy were 121, 96, and 92 for Estrus0, Estrus1, and Estrus2+ cows, respectively. In conclusion, cows with transition cow disorders (i.e., stillbirth, retained placenta, puerperal metritis, or subclinical ketosis) had a greater chance for anestrus compared with healthy cows. Cows in Estrus0 had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrous activity from d 7 until d 60., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Accurate and rapid measurement of blood β-hydroxybutyrate (β-OHB) concentrations is critical to identify hyperketonemia in sheep., Objective: 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., Methods: 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., Results: Passing-Bablok regression analysis revealed an intercept of 0.066 (CI 95 : 0.018-0.125) and slope of 1.242 (CI 95 : 1.136-1.342) for Freestyle Optium Neo H. Intercept and slope values for the TaiDoc-TD4235 meter were 0.625 (CI 95 : 0.539-0.724) and 1.265 (CI 95 : 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 (TE obs ) 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., Conclusions: Because of the large TE obs , the Freestyle Optium Neo H and TaiDoc-TD4235 meters should not be used to quantify blood β-OHB concentrations in sheep., (© 2023 American Society for Veterinary Clinical Pathology.)

Subclinical hyperketonemia (SCHK) is the major metabolic disease observed during the transition period in dairy goats, and is characterized by high plasma levels of nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHB). However, no prior study has comprehensively assessed metabolomic profiles of dairy goats with SCHK. Plasma samples were collected within 1 h after kidding from SCHK goats (BHB concentration >0.8 mM, n = 7) and clinically healthy goats (BHB concentration <0.8 mM, n = 7) with similar body condition score (2.75 ± 0.15, mean ± standard error of the mean) and parity (primiparous). A combination of targeted and untargeted mass spectrometric approaches was employed for analyzing the various changes in the plasma lipidome and metabolome. Statistical analyses were performed using the GraphPad Prism 8.0, SIMCA-P software (version 14.1), and R packages (version 4.1.3). Plasma aminotransferase, nonesterified fatty acids, and BHB concentrations were greater in the SCHK group, but plasma glucose concentrations were lower. A total of 156 metabolites and 466 lipids were identified. The analysis of untargeted metabolomics data by principal component analysis and orthogonal partial least squares discriminant analysis revealed a separation between SCHK and clinically healthy goats. According to the screening criteria (unpaired t-test, P < 0.05), 30 differentially altered metabolites and 115 differentially altered lipids were detected. Pathway enrichment analysis identified citrate cycle, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, and phenylalanine metabolism as significantly altered pathways. A greater concentration of plasma isocitric acid and cis-aconitic acid levels was observed in SCHK goats. In addition, AA such as lysine and isoleucine were greater, whereas alanine and phenylacetylglycine were lower in SCHK dairy goats. Dairy goats with SCHK also exhibited greater oleic acid, acylcarnitine, and phosphatidylcholine and lower choline and sphingomyelins. Acylcarnitines, oleic acid, and tridecanoic acid displayed positive correlations with several lipid species. Alanine, hippuric acid, and histidinyl-phenylalanine were negatively correlated with several lipids. Overall, altered metabolites in SCHK dairy goats indicated a more severe degree of negative energy balance. Data also indicated an imbalance in the tricarboxylic acid (TCA) cycle, lipid metabolism, and AA metabolism. The findings provide a more comprehensive understanding of the pathogenesis of SCHK in dairy goats., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Metabolic disorders as ketosis are manifestations of the animal's inability to manage the increase in energy requirement during early lactation. Generally, buffaloes show a different response to higher metabolic demands than other ruminants with a lower incidence of metabolic problems, although ketosis is one of the major diseases that may decrease the productivity in buffaloes. The aim of this study was to characterize the metabolic profile of Mediterranean buffaloes (MB) associated with 2 different levels of β-hydroxybutyrate (BHB). Sixty-two MB within 50 days in milk (DIM) were enrolled and divided into 2 groups according to serum BHB concentration: healthy group (37 MB; BHB <0.70 mmol/L; body condition score: 5.00; parity: 3.78; and DIM: 30.70) and group at risk of hyperketonemia (25 MB; BHB ≥0.70 mmol/L; body condition score: 4.50; parity: 3.76; and DIM: 33.20). The statistical analysis was conducted by one-way ANOVA and unpaired 2-sample Wilcoxon tests. Fifty-seven metabolites were identified and among them, 12 were significant or tended to be significant. These metabolites were related to different metabolic changes such as mobilization of body resources, ruminal fermentations, urea cycle, thyroid hormone synthesis, inflammation, and oxidative stress status. These findings are suggestive of metabolic changes related to subclinical ketosis status that should be further investigated to better characterize this disease in the MB., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Metabolic diseases driven by negative energy balance in dairy cattle contribute to reduced milk production, increased disease incidence, culling, and death. Cow side tests for negative energy balance markers are available but are labor-intensive. Milk sample analysis using Fourier transform infrared spectroscopy (FTIR) allows for sampling numerous cows simultaneously. FTIR prediction models have moderate accuracy for hyperketonemia diagnosis (beta-hydroxybutyrate (BHB) ≥ 1.2 mmol/L). Most research using FTIR has focused on homogenous datasets and conventional prediction models, including partial least squares, linear discriminant analysis, and ElasticNet. Our objective was to evaluate more diverse modeling options, such as deep learning, gradient boosting machine models, and model ensembles for hyperketonemia classification. We compiled a sizable, heterogeneous dataset including milk FTIR and concurrent blood samples. Blood samples were tested for blood BHB, and wavenumber data was obtained from milk FTIR analysis. Using this dataset, we trained conventional prediction models and other options listed above. We demonstrate prediction model performance is similar for convolutional neural networks and ensemble models to simpler algorithm options. Results obtained from this study indicate that deep learning and model ensembles are potential algorithm options for predicting hyperketonemia in dairy cattle. Additionally, our results indicate hyperketonemia prediction models can be developed using heterogeneous datasets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The objective of this observational study was to assess the relationship between herd-level prevalence of hyperketolactia (HPH) with management practices of the transition period and herd milk production. Dairy herds (n = 71) were selected based on their inclusion in a herd management risk assessment study (August 2014-March 2018) using a Vital 90 (Elanco) Risk Assessment tool (one assessment per farm). Data from multiple milk recording test-days (Dairy Herd Improvement, DHI; Lactanet) were included in the analysis. Tests performed within ±6 mo relative to each farm's risk assessment date were included (10 ± 2 SD tests per farm). The majority of the farms were located in Ontario (83%). For each farm DHI test, the data set included herd average milk yield (kg/cow per day), average milk fat and protein (%), average somatic cell count (cells/mL), average days in milk (DIM), number of cows tested for ketosis, number of ketosis-positive tests (milk β-hydroxybutyrate ≥0.15 mmol/L), and proportion of cows by parity groups. Overall HPH (5-21 DIM) was calculated based on data available per farm (sum of all positive tests within 5-21 DIM/sum of all cows tested within 5-21 DIM). Each farm average was obtained by considering all test-days. A logit-transformation was applied to hyperketolactia prevalence. Linear regression models (PROC GLM and MIXED of SAS, Version 9.4) were used to predict herd HPH (milk β-hydroxybutyrate ≥0.15 mmol/L within 5 to 21 DIM; the outcome of interest). Four initial models (far-off, close-up, and fresh periods, and DHI) were separately built to assess associations between their variables and HPH; a final model considered variables selected in the initial models. Univariable (liberal P < 0.25) followed by multivariable models were used to build specific models for each period of the risk assessment. Herd prevalence of hyperketolactia was 27 ± 14%, with an average herd size of 141 ± 110 cows. The final HPH model (R 2 = 24.8%) included weighted milk yield, the proportion of primiparous cows, water access in the close-up period, and access to rest areas or stall access in the fresh period. Herd prevalence of hyperketolactia was negatively associated with milk yield [odds ratio, OR = 0.96 (95% confidence interval 0.92-0.99)] and proportion of primiparous cows [OR = 0.98 (0.96-0.99)]. The odds of hyperketolactia were greater with poor water access and quality (<5 cm of linear access per cow; dirty water; only 1 water location in pen) than with ≥10.2 cm of linear access per cow; clean water; >2 water locations in pen [1.23 (1.11-2.39)] in the close-up period. The odds of hyperketolactia were greater in farms providing limited access to rest areas in the fresh period than in farms providing constant access to rest areas, without dead-ends [1.64 (1.03-2.80)]. In Canadian dairy herds, HPH in early lactation was associated with certain transition-period management practices and was negatively associated with herd productivity., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Test-day milk analysis has largely been used to study health and performance parameters in dairy cows. In this study, we estimated four health indicators of dairy cows using test-day data. Our purpose was to estimate (1) mastitis incidence rate, prevalence, and the probability of recovery; (2) the incidence proportion of ketosis; (3) the duration of inter-calving interval; and (4) the risk of a fresh cow being replaced, in a large cohort of dairy herds in the Piedmont region (Italy). We retrospectively analysed test day records of 261,121 lactating cows and 1315 herds during five years (2015-2020). Mastitis was defined by somatic cell count and ketosis by fat-to-protein ratio. Calving dates were used to calculate ICI and to estimate the removal of a fresh cow from the herd. Mixed-effect generalized linear models were used to adjust for unmeasured herd-level risk factors. The risk of mastitis increased by 120% with parity (Odds ratio [OR] = 2.20, confidence interval [CI]: 2.17 - 2.23), by 7% by months in milking (OR = 1.07, CI: 1.07 - 1.07), and even more if the cow was already affected during the same lactation (OR = 8.74, CI: 8.67 - 8.82). Lactose concentration on the previous test day was the best positive prognostic factor for mastitis recovery (OR = 1.12, CI: 1.08 - 1.17). Ketosis risk was the highest between 3rd and 4th lactations and itself increased the risk of having ICI longer than 440 days (OR = 1.12, CI: 1.02 - 1.22), and fresh-cow removal (OR = 1.75, CI: 1.58 - 1.93). Also, the removal of fresh cows was more likely when mastitis (OR = 1.31, CI: 1.19 - 1.45) or long ICI (OR = 1.34, CI: 1.22 - 1.48) occurred. For each health indicator, herd-level risk factors had an important role (18-56% of within-herd covariance). Our results indicate that milk analysis could be also useful for predicting mastitis, its cure rate, and ketosis. Cow-level risk factors are not enough to explain the risk of these issues. By studying a large population over a long period, this study provides an updated estimate of dairy cow health indicators in Piedmont (north-western Italy), useful for benchmarking dairy herds., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Case History: Serum and liver samples from 35, 2-year-old dairy heifers that had fractured one or both humeri post-calving between July and December 2019 were submitted to a diagnostic laboratory for analysis. Serum samples were analysed for albumin, β-hydroxybutyrate (BHB), creatinine, Ca, Mg, phosphate, non-esterified fatty acids (NEFA), and serum Cu concentration. Liver samples were analysed for liver Cu concentration. Data were compared to published reference intervals. Data values for heifers that prior to fracture had grazed fodder beet were also compared to values for those that had grazed pasture., Clinical Findings: Sixty-nine percent of heifers with humeral fracture had serum creatinine concentrations below the lower value of the reference range (55-130 µmol/L). In 3/32 (9%) heifers, serum NEFA concentrations were increased above the reference value indicating body fat mobilisation (≥1.2 mmol/L for peri-partum cows) and in 20/35 (57%) heifers BHB serum concentrations were above the reference value indicating subclinical ketosis (≥1.1 mmol/L for peri-partum cows). In 24/35 (69%) heifers, liver Cu concentration was low (≤ 44 µmol/kg) or marginal (45-94 µmol/kg). The concentration of Cu in serum was low (≤ 4.5 µmol/L) in 2/33 (6%) heifers and marginal (4.6-7.9 µmol/L) in 5/33 (15%) heifers. There was moderate positive correlation between the logged concentrations of Cu in paired liver and serum samples, r (31)  = 0.43; (95% CI = 0.1-0.79; p = 0.014). One heifer had a serum phosphate concentration below the lower limit of the reference range (< 1.10 mmol/L). For all heifers, the concentrations of albumin, Ca, and Mg in serum were within the reference intervals (23-38 g/L, 2.00-2.60 mmol/L, and 0.49-1.15 mmol/L respectively). Over winter, 15/35 (43%) heifers grazed predominantly pasture, 14/35 (40%) grazed fodder beet and 6/35 (17%) had a mixed diet., Clinical Relevance: In some of these heifers with humeral fractures, there was evidence for protein and/or energy malnutrition in the form of elevated NEFA and BHB concentrations and low creatinine concentrations in serum. Liver Cu concentrations were also reduced in most affected heifers. However, the absence of a control group means it is not possible to determine if these are risk factors for fracture or features common to all periparturient heifers. Clinical trials and molecular studies are needed to determine the true contribution of Cu and protein-energy metabolism to the pathogenesis of spontaneous humeral fractures in dairy heifers., Abbreviations: BHB: ß-hydroxybutyrate; NEFA: Non-esterified fatty acids.

Dairy cows are at a greater risk of disease due to increased energy demand during the transition period. Blood biomarkers including beta-hydroxybutyrate (BHBA) 1 and non-esterified fatty acids (NEFA) 2 are routinely used to identify animals in a state of negative energy balance (NEB) 3 . Recent research demonstrates cattle have varied response to NEB, that requires multiple blood biomarkers to characterize. This research identified five subcategories (cowtypes) of metabolic responses in transition dairy cows: Healthy, Athlete, Clever, Hyperketonemia, and Poor Metabolic Adaptation Syndrome (PMAS) 4 . The data set used in this study was collected in Germany by VIT - Vereinigte Informationssysteme Tierhaltung w.V. from 2016 to 2020. Health issues with time of diagnostic were included in the dataset. Using previously reported prediction models for blood BHB and blood NEFA and milk Fourier-transform infrared spectroscopy (FTIR) 5 data, the cowtypes in our dataset were predicted. The objective of this study is to evaluate the association of the cowtypes with the disease-free survival time in dairy cows during early post calving using an accelerated failure time regression model. Additionally, transition probabilities of the population dynamics between cowtypes are studied by means of a Markov chain model. Using Healthy cowtype as reference level, Athlete, Clever, and PMAS cowtypes were found to be significant for the disease-free survival probability (P < 0.01). Conversely, Hyperketonemia cowtype was not significant (P = 0.182). Compared to the Healthy cowtype, all other cowtypes had a negative effect on the survival probabilities, which was higher for PMAS cows. Furthermore, after computing the estimated population transition probabilities among cowtypes, the stationary distribution of the Markov chain, along with bootstrap confidence intervals were computed. The results showed 0.091 (95% CI:0.089,0.092), 0.077 (95 % CI:0.074,0.078), 0.684 (95 % CI:0.067,0.069), 0.138 (95 % CI:0.136,0.139), and 0.009 (95% CI:0.008,0.010) of probability of being in Healthy, Athlete, Clever, Hyperketonemia, and PMAS cowtype, respectively. These estimates represent the proportion of cows belonging to the different cowtypes in a herd; information which may prove useful for herd management. The application of blood biomarker predictions using milk FTIR allows us to investigate differences between predicted cowtype and movements between these states and the association with time to disease. Further research will improve our understanding of the dynamic nature of the transition period., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Data were obtained from studies in Australia, Canada, and the United States using individual cow data from 28,230 Holstein cows to evaluate associations between parity and disease. Our goal was to develop understanding of disease risks for cows of differing parity. We hypothesized that there would be increased risks of disease and changes in metabolite concentrations with increased parity. Parity ≥5 represented 2,533 cows or 9.0%, parity 4 was 9.8% (2,778), parity 3 as 19.0% (5,355), parity 2 as 28.1% (7,925), and parity 1 was 34.1% (9,639) of the sample. Of these cows, 15.5% were in Australia, 14.7% in Canada, and 69.8% in the United States. Lactational incidence (LI) risk of clinical hypocalcemia increased with parity from 0.1% for parity 1 to 13% for parity ≥5 cows. The marked increase suggests profound differences in metabolism with increased parity. The LI of clinical mastitis was 17.4%. The odds of mastitis increased with parity to 2.5 times greater in parity ≥5 than in parity 1. The LI of lameness increased with parity; specifically, the odds of lameness was 5.6 times greater for parity ≥5 than parity 1. Dystocia incidence was 8.7% and greatest for parity 1 cows. The LI of retained placenta was 7.4% and increased with parity, with the odds for parity ≥5 2.3 times greater than for parity 1. The LI of metritis was 10% and of endometritis 14%, with the greatest odds in parity 1. The LI of clinical ketosis was 3.3% with a marked increase in odds with parity. The prevalence of subclinical ketosis was 26.8% with only cows in parity 1 having lower odds than other parities. Parity ≥5 cows had greater odds (odds ratio = 1.7) of respiratory disease than parity 1 cows, which were lesser than other parities. Metabolite concentrations were evaluated in 5,154 Holstein cows in the precalving, calving, and immediate postcalving data sets. Metabolic measures near peak lactation provided 1,906 observations. Concentrations of β-hydroxybutyrate (BHB) and nonesterified fatty acids increased with parity on d 1 to 3 of lactation and at peak lactation. On d 1 to 3 after calving differences in glucose, nonesterified fatty acids, and BHB indicated a greater reliance on mobilized lipid to export energy to peripheral tissues as BHB for greater parity cows. Differences in concentrations among parity groups were marked at times, for example >0.20 mM in Ca for parity 1 and 2 to parity ≥5 and >0.33 mM for all older parities compared with parity 1 for P on the day of calving. The marked increase suggests profound differences in metabolism with increased parity are probably influenced, in part, by increased production. We found marked differences in concentrations of metabolites with parity that are consistent with reduced reproduction, health, and body condition for higher parity cows. These unfavorable differences in metabolism in Ca, P, glucose, and cholesterol concentrations for higher parity cows also complement the often-substantial differences in disease risk with parity and suggest a need to carefully consider the parity structure in study design. Managers and advisors will need to consider methods to reduce risk of health disorders tailored to cows of different ages., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis is a metabolic disease of dairy cows in the perinatal period, β-hydroxybutyrate (β-HB) is the main component of ketosis. High levels of β-HB can trigger oxidative stress and inflammatory response in dairy cows, leading to decreased milk yield and multiple postpartum diseases. Forsythin (FOR), the major constituent of the herbal medicine Forsythia, has anti-inflammatory, anti-oxidant, and antiviral effects. FOR was demonstrated to have an antioxidant effect on PC12 cells. However, the effects of FOR on β-HB-stimulated bovine macrophages (BMs) has not been reported. Thus, the aim of the present study was to investigate the effects of FOR on β-HB-stimulated BMs. Firstly, the CCK8 test confirmed that FOR (50, 100, 200 μg/mL) has no effect on BMs activity, and we selected these concentrations for subsequent experiments. Secondly, through detecting the oxidation indexes ROS, MDA and antioxidant indexes CAT and SOD, we confirmed the antioxidant effect of FOR on BMs. Next, qRT-PCR confirmed that FOR dramatically reduced the mRNA levels of IL-1β and IL-6. Furthermore, the western blotting confirmed that FOR observably down-regulated β-HB-stimulated phosphorylation of p38, ERK and Akt and up-regulated expression of Nrf2, and HO-1. Above results suggested that FOR plays antioxidant effects on β-HB-induced BMs through p38, ERK and PI3K/Akt, Nrf2 and HO-1 signaling pathways. Therefore, we speculated that FOR may be a potential medicine to alleviate β-HB-induced inflammatory response and provide a preliminary reference for the research and development of FOR., Competing Interests: Declaration of Competing Interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Ketosis is a common metabolic disorder during the post-partum transition period of dairy cattle. How the method of reproduction, parturition time, and calf birth weight affect the occurrence of ketosis on dairy herds remains elusive., Objectives: This study investigated factors associated with the severity of ketosis., Methods: We divided 186 Holstein cows into three classifications based on the highest β-hydroxybutyrate (BHBA) concentration during the post-partum transition period, namely non-ketosis (<1.2 mmol/L, n = 94), subclinical ketosis (1.2-2.9 mmol/L, n = 58), and clinical ketosis (≥3.0 mmol/L, n = 34). We evaluated characteristics of cows associated with the severity of ketosis., Results: Ketosis was not associated with the method of reproduction, parturition time, pregnancy wastage, premature delivery, retained placenta, and type of calf. Cows calving in spring and especially summer were at higher risk of severe ketosis (p < 0.01). Cows with increased body condition score (BCS) at parturition, age, lactation number, and calving interval were more likely to develop severe ketosis (p < 0.05). Cows with clinical ketosis produced most milk (29.9 ± 1.0 kg) from days four to six, whereas cows without ketosis produced the least (21.3 ± 0.8 kg) (p < 0.001). Heavier calf birth weight resulted in high risk of severe ketosis (p < 0.01), due to increased milk yield during the early lactation., Conclusions: The severity of ketosis is associated with the calving season, BCS at parturition, age, lactation number, calving interval, milk yield in the early lactation period, and calf birth weight. Nonetheless, it was not associated with the method of reproduction, parturition time, pregnancy wastage, premature delivery, retained placenta, and type of calf. This study is the first to investigate the associations between ketosis and calf birth weight. Our findings could help predict cows at risk of ketosis and take precautions., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

This study sheds light on the genetic complexity and interplay of production, body size, and metabolic health in dairy cattle. Phenotypes for body size-related traits from conformation classification (130,166 animals) and production (101,562 animals) of primiparous German Holstein cows were available. Additionally, 21,992, 16,641, and 7,096 animals were from herds with recordings of the metabolic diseases ketosis, displaced abomasum, and milk fever in first, second, and third lactation. Moreover, all animals were genotyped. Heritabilities of traits and genetic correlations between all traits were estimated and GWAS were performed. Heritability was between 0.240 and 0.333 for production and between 0.149 and 0.368 for body size traits. Metabolic diseases were lowly heritable, with estimates ranging from 0.011 to 0.029 in primiparous cows, from 0.008 to 0.031 in second lactation, and from 0.037 to 0.052 in third lactation. Production was found to have negative genetic correlations with body condition score (BCS; -0.279 to -0.343) and udder depth (-0.348 to -0.419). Positive correlations were observed for production and body depth (0.138-0.228), dairy character (DCH) (0.334-0.422), and stature (STAT) (0.084-0.158). In first parity cows, metabolic disease traits were unfavorably correlated with production, with genetic correlations varying from 0.111 to 0.224, implying that higher yielding cows have more metabolic problems. Genetic correlations of disease traits in second and third lactation with production in primiparous cows were low to moderate and in most cases unfavorable. While BCS was negatively correlated with metabolic diseases (-0.255 to -0.470), positive correlations were found between disease traits and DCH (0.269-0.469) as well as STAT (0.172-0.242). Thus, the results indicate that larger and sharper animals with low BCS are more susceptible to metabolic disorders. Genome-wide association studies revealed several significantly associated SNPs for production and conformation traits, confirming previous findings from literature. Moreover, for production and conformation traits, shared significant signals on Bos taurus autosome (BTA) 5 (88.36 Mb) and BTA 6 (86.40 to 87.27 Mb) were found, implying pleiotropy. Additionally, significant SNPs were observed for metabolic diseases on BTA 3, 10, 14, 17, and 26 in first lactation and on BTA 2, 6, 8, 17, and 23 in third lactation. Overall, this study provides important insights into the genetic basis and interrelations of relevant traits in today's Holstein cattle breeding programs, and findings may help to improve selection decisions., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The body condition assessment methods Body Condition Scoring (BCS) and backfat thickness measurement are important methods to assess the energy status of a dairy cow in the dry period and early lactation. An extensive search of the scientific literature (online and analogue) was carried out for the presented review. Various keywords filtered important articles, which were entered into a literature management program and whose content was assigned to various categories, on which the review article is based. The BCS is the tool of choice for acquiring a rapid and precise overview of the dairy herd. The backfat thickness (RFD) may be used for more detailed investigations. The aim of aformentioned studies is to attain an optimal energy balance of the dairy cow during early lactation considering energy intake and energy output as well as to minimize diseases such as fat mobilization syndrome and ketosis, and in consequence to allow for improved animal health and optimal performance., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)