Ketotic Cows Research Articles

Comparison of prepartum blood parameters in dairy cows with postpartum ketosis and new risk prediction candidates.

Ketosis is a predominant metabolic problem and a risk factor for several postpartum diseases. This retrospective study aimed to evaluate the complete blood count (CBC), plasma biochemistry, and osteocalcin and identify significant prepartum and early postpartum values expressed in ketotic cows. In 135 Holstein Friesian cows, 210 parturitions of 114 primiparous and 96 multiparous cows were examined. According to the plasma concentrations of β-hydroxybutyrate (BHB; ≥ 1.4 mmol/L) or non-esterified fatty acids (NEFA; ≥ 0.7 mmol/L) in the postpartum period, cows were divided into healthy cows (CON) and ketotic cows (KET). Analyses of CBC and biochemistry profiles were performed from -6 to 4 weeks of parturition every 2 weeks (prepartum; BW-5, BW-3, and BW-1, postpartum; BW1 and BW3), and osteocalcin ELISA tests were performed using blood samples from -2 to 2 weeks of parturition (BW-1 and BW1). In primiparous KET (n = 114) before parturition, lower lymphocyte (Lym) in BW-5 and BW-3, lower red blood cell (RBC) in BW-5, higher mean corpuscular volume (MCV) in BW-1, and higher NEFA in BW-3 were significant compared with CON. Primiparous KET showed lower carboxylated osteocalcin (cOC) levels and a significant decrease after parturition. In multiparous KET (n = 96) before parturition, lower neutrophil (Neu) in BW-5, higher hemoglobin (HGB) in BW-5, higher MCV in BW-5 and BW-1, higher MCH in BW-5, lower total cholesterol (TC) in BW-5, higher triglyceride (TG) in BW-3, higher NEFA in BW-1, higher glucose (Glu) in BW-3, lower γ-glutamyl transferase (GGT) in BW-5, lower inorganic phosphate (iP) in BW-3, and higher body condition score (BCS) in BW-5 and BW-3 were significant compared with CON. Multiparous KET showed decreased cOC and uncarboxylated osteocalcin (ucOC) after parturition, which was lower than that in the CON group. The blood parameters expressing different values between CON and KET in prepartum or early postpartum periods are presumed to show individual nutrition and health states, liver function, and overweight status. These parameters could be valuable indicators that can be used to prevent the occurrence of ketosis and improve management practices by recognizing these differences in ketotic cows before calving.

Association between Tryptophan Metabolism and Inflammatory Biomarkers in Dairy Cows with Ketosis

Dairy cows with ketosis have high circulating beta-hydroxybutyric acid (BHBA) concentrations alongside which inflammation is concomitantly developed. Tryptophan (Trp) is an essential amino acid that participates in the regulation of the inflammatory response. However, the association between Trp metabolism and inflammation in dairy cows with ketosis remains unclear. Therefore, blood samples from healthy (n = 10) and ketotic (n = 10) primiparous dairy cows were collected at the calving date and the day of ketosis diagnosis (7 days in milk (7 DIM)). Serum levels of non-esterified fatty acids (NEFA), BHBA, haptoglobin (HP), serum amyloid A (SAA), lipopolysaccharide, and cortisol were analyzed. Tryptophan and its metabolites were quantified using liquid chromatography-tandem mass spectrometry. At 7 DIM, the concentrations of NEFA, BHBA, HP, and SAA were higher and the levels of Trp, kynurenine (KYN), indoleacetic acid, indole-3-lactic acid, and 3-indoxyl sulfate were lower in the dairy cows with ketosis compared with those in the healthy cows. However, the KYN/Trp and melatonin/Trp ratios increased in the cows with ketosis. At the calving date, the serum lipopolysaccharide levels did not differ between the healthy and ketotic cows, whereas the levels of NEFA, HP, and cortisol increased in the ketotic cows. Correlation analysis showed that Trp deficiency and elevated Trp metabolism in the dairy cows occurred during ketosis. Overall, our results suggest that abnormal Trp metabolism may contribute to the pathogenesis of ketosis.

Breath Analysis for Early Detection of Rising Ketone Bodies in Postpartum Dairy Cows Classified as at Risk of Ketosis

Ketosis is a metabolic disorder associated with high production and low energy intake in dairy cows. Fat reserves are mobilized, and gluconeogenesis occurs. Traditionally, rapid tests for blood, milk or urine are used to detect increased ketone body levels in case of ketosis. Breath analysis is relatively new and relevant for the development of automatic sensor systems for early warning. This study aims to determine whether and when a postpartum rise in ketone bodies occurs in breath with elevated blood β-hydroxybutyrate (BHB) in cows at risk of ketosis. Postpartum breath, blood, urine and milk samples were taken daily until day 7, with one prepartum sample excluding milk, and ketone body concentrations were determined. Concentrations of blood BHB were 0.4–2.6 mmol/L (lab) and 0.3–3.1 mmol/L (rapid test), breath acetone was 2.3–20.0 ppm, urine acetoacetate 0–8 mmol/L and milk BHB 0–500 µmol/L. A rise in blood BHB was related to that in urine acetoacetate and milk BHB and followed by a rise in breath acetone. However, breath acetone levels of ketotic and non-ketotic cows were similar. We conclude that rising ketone bodies can be detected in blood, urine, milk and breath, but to use breath analysis as an alternative to rapid tests, longitudinal sampling is needed.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. 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 β-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 μM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 μM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

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

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

Ketosis Alters Transcriptional Adaptations of Subcutaneous White Adipose Tissue in Holstein Cows during the Transition Period.

Simple SummaryKetosis in dairy cows is one of the common nutritional metabolic diseases during the perinatal period. We conducted the present subcutaneous white adipose tissue transcriptome-based study to investigate the pathogenesis of ketosis in dairy cows. The transcriptome results showed that the expression of PCK1 and PCK2, key enzymes of the glyceroneogenesis, was simultaneously downregulated post-partum compared to pre-partum, possibly resulting in impaired lipid storage in the adipose tissue of ketotic cows. Integrating the GO enrichment analysis and published gene expression traits, the inflammatory response biological process inhibiting PCK1 and PCK2 expression in adipose tissue may be important in the development of ketosis in dairy cows. The findings of the present study provided important clues for the therapeutic and preventive targets for ketosis.Ketosis is a common nutritional, metabolic disease during the perinatal period in dairy cows characterized by elevated blood β-hydroxybutyrate (BHBA). In this study, RNA sequencing (RNA-seq) was performed to investigate adaptive changes in adipose tissue during the perinatal period of dairy cows. Blood and tailhead subcutaneous white adipose tissue (sWAT) were obtained from ketotic cows (Ket = 8, BHBA ≥ 1.4 mmol/L) and non-ketotic cows (Nket = 6, BHBA < 1.4 mmol/L) 21 d pre-partum and 10 d post-partum. Compared with pre-partum, decreased lipid synthesis due to down-regulation of PCK1 may be in a strong association with clinical ketosis. Simultaneously, PCK2 was downregulated in the Ket postnatally compared to its expression prenatally, and the expression of PCK2 was 2.7~4.2 times higher than that of PCK1, implying a more severe lipid storage impairment in the Ket. Moreover, compared to pre-partum, the upregulated differentially expressed genes post-partum in the Ket were enriched in the inflammatory response biological process. The higher expression of TNC (tenascin C) in the post-partum Ket relative to the Nket suggested that the adipose tissue of ketotic cows might also be accompanied by tissue fibrosis. Notably, pre-partum CD209 was higher in the Ket than in the Nket, which might be used as a candidate marker for the pre-partum prediction of ketosis. Combined with published gene expression traits, these results suggested that inflammation leads to a more widespread downregulation of the lipid synthesis gene network in adipose tissue in ketotic cows. Additionally, sWAT in post-partum cows with ketosis might also be accompanied by tissue fibrosis which could make the treatment of ketosis more difficult.

Comparison of Enzyme Activity in Order to Describe the Metabolic Profile of Dairy Cows during Early Lactation.

Enzymatic diagnostics have practical applications in diseases of the liver, heart, pancreas, muscles, blood, and neoplastic diseases. This study aimed to compare enzyme activity to describe dairy cows’ metabolism during early lactation. Based on their general health symptoms, the cows were assigned to one of three groups: acidotic, healthy and ketotic. Samples of milk, blood and rumen fluid were collected at 12 ± 5 days postpartum. Ketotic cows were characterized by the highest malondialdehyde (MDA, 76.098 nM/mL), glutathione reductase (GluRed, 109.852 U/L), superoxide dismutase (SOD, 294.22 U/L) and gamma-glutamyltranspeptidase (GGTP, 71.175 U/L) activity. In comparing ketotic and acidotic cows, MDA, GluRed, SOD and GGTP activity were higher by a factor of almost: 1.85, 1.89, 0.79 and 2.50, respectively. Acidotic cows were characterized by the highest aspartate aminotransferase activity (AspAT, 125.914 U/L). In comparing acidotic and ketotic cows, AspAT activity was higher by a factor of almost 1.90. The use of enzymatic markers could limit the frequency of sampling for laboratory analyses and may result in a faster diagnosis of metabolic disorders. AspAT activity in blood serum seems to be a good indicator of acidosis; GGTP may participate in the pathogenesis of ketosis.

β-hydroxybutyrate impairs monocyte function via the ROS-NLR family pyrin domain-containing three inflammasome (NLRP3) pathway in ketotic cows.

Cows with ketosis display severe metabolic stress and immune dysfunction which renders them more susceptible to infections. Monocytes, one of the major subtypes of white blood cells, play an important role in innate immune defense against infections. Thus, the aim of this study was to investigate alterations in immune function, reactive oxygen species (ROS) production and activity of the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in monocytes (CD14+) of cows with clinical ketosis (CK). Twelve healthy multiparous Holstein cows [blood β-hydroxybutyrate (BHB) concentration < 1.2 mM] and 12 cows with CK (BHB > 3.0 mM) at 3 to 14 days in milk were used for blood sample collection. To determine effects of BHB on phagocytosis, ROS and protein abundance of the NLRP3 inflammasome pathway in vitro, monocytes isolated from healthy cows were treated with 3.0 mM BHB for 0, 6, 12 or 24 h. Dry matter intake (22.7 vs. 19.0 kg) was lower in cows with CK. Serum concentrations of fatty acids (0.30 vs. 0.88 mM) and BHB (0.52 vs. 3.78 mM) were greater in cows with CK, whereas concentration of glucose was lower (4.09 vs. 2.23 mM). The adhesion, migration and phagocytosis of monocytes were lower in cows with CK, but apoptosis and ROS content were greater. Protein abundance of NLRP3, cysteinyl aspartate specific proteinase 1 (caspase 1) and interleukin-1B p17 (IL1B p17) were greater in monocytes of cows with CK, while abundance of NADPH oxidase isoform 2 (NOX2) was lower. Compared with 0 h BHB, ROS content and apoptosis were greater in the monocytes challenged for 6, 12 or 24 h BHB. Compared with 0 h BHB, protein abundance of NLRP3, caspase 1, IL1B p17 and concentration of IL1B in medium were greater in the monocytes challenged for 6, 12 or 24 h BHB. However, compared with 0 h BHB, protein abundance of NOX2 and phagocytosis of monocytes were lower in the monocytes challenged for 6, 12 or 24 h BHB. Overall, the data suggested that exogenous BHB activated the ROS-NLRP3 pathway, which might be partly responsible for immune dysfunction of dairy cows with CK.

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

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

Ketosis in Dairy Cows during Early Lactation - Detection in Pooled Blood Serum Samples

Background: Ketosis is the most important metabolic disease with prevalence from 15 to 45%. Ketosis is diagnosed using a metabolic profile. Due to the high prevalence, it is necessary to determine a large number of metabolic profiles within farm, which represents an additional cost, so the implementation of pooled serum in assessing the metabolic status of cows was examined. The aim of this study was to validate and evaluate the influence of the relative position (Z-score) of the value of pooled sample metabolic parameters within the known reference value of healthy cows in the detection of ketosis in herd during early lactation.Materials, Methods &amp; Results: The experiment has been carried out using blood samples collected by puncture of coccygeal vein from 50 ketotic and 50 healthy cows. Laboratory analysis includes determination of beta-hydroxybutyrate-BHB, non-esterified fatty acids-NEFA, cholesterol-CHOL, triglycerides-TGC, glucose-GLU, albumin-ALB, total protein-TPROT, UREA, Ca, P, total bilirubin-TBIL and aspartat aminotransferase-AST. The pooled serum was made from 10 individual samples originating from 10 different cows. A serum aliquot of 0.1 mL was taken from each sample, and a 1 mL volume of pooled serum was finally formed. Three types of serum pools were made: 1) 30 pooled sample were from ketosis; 2) 30 pooled sample were from healthy cows and 3) 60 pooled samples containing mixed sera of healthy cows and cows with ketosis were made as follows: 10 pools contain 10% to 60% of ketotic cows (1/10 to 6/10 samples). Statistical analysis includes: a) difference in metabolite concentration and Z-score in pooled sample and arithmetic mean individual sample in healthy and ketotic cow, b) correlation between Z-score of pooled sample and arithmetic mean of individual sample, c) ability of Z-score of metabolite to divide ketotic from healthy cow, d) correlation between Z-score and % of ketotic cow in pooled sample; and e) calculation of 95%CI of pooled sample Z-scores for each % of ketotic cow in pools. Z-score and all analysis were calculated for each metabolic parameter. The results of the study show that the mean values and Z-scores of the pool and the calculated average value of the individual samples participating in that pool differ significantly in healthy cows and cows in ketosis, except for TPROT and Ca. A higher value and a higher Z-score were found for BHB, NEFA, UREA, TBIL and AST, and a lower value and a lower Z-score for TGC, CHOL, GLU, ALB and P in ketotic cows compared to healthy cows. The value of the Z-score of the pooled sample and the calculated mean values of individual samples participating in the pool are highly correlated with each other (coefficient of determination over 99%). Z-score of metabolites in the pooled sample can be used to distinguish healthy from ketotic cows (ROC AUC= 0.711 to 0.989), except for TPROT and Ca. The Z-score value of the pooled sample shows a linear correlation with the percentage of ketotic cows in the pool and the reference ranges of Z-scores change significantly as a function of the percentage of ketosis cows.Discussion: Modern research on the metabolic profile in cows requires obtaining a large amount of information from as few samples as possible. The advantages of using the Z-score are reflected in the following: this score does not depend on the absolute value of the metabolite, but on the position within the known population reference value, Z-score of sample and the arithmetic mean of individual samples included in the same pool are almost identical, the Z-score of these 2 groups of results is ideally correlated, the Z-score significantly correlates with the % of ketosis samples in the pooled sample. The use of pooled sample Z-score can be a useful in a herd level assessment of metabolic status and detection of ketosis as most important metabolic disease in dairy cows.Keywords: dairy cattle, ketosis, metabolic disease, metabolic profile, pooled serum, z-score, diagnostics.

Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis

Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of β-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.

β-Hydroxybutyrate inhibits apoptosis in bovine neutrophils through activating ERK1/2 and AKT signaling pathways

Ketosis in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation and high concentrations of blood β-hydroxybutyrate (BHB). Neutrophil apoptosis plays a key role in maintaining the balance of inflammation and functional capacity of circulating neutrophils in ketotic cows. The kinases ERK1/2 and AKT, as well as their downstream Bcl-2 family-mediated mitochondrial signaling, are important apoptosis-regulating pathways in neutrophils. The objective of our study was to investigate the effects of BHB on neutrophil apoptosis and the underlying regulatory mechanisms during ketosis. Neutrophils were isolated from 5 multiparous cows (within 3 wk postpartum) with serum BHB concentrations <0.6 mM and glucose concentrations >3.5 mM. In a series of experiments, neutrophils were treated with increasing concentrations of BHB (0, 0.6, 2, and 3 mM for 10 h) and time (0, 2, 4, 6, 8, and 10 h with 2 mM). Subsequently, a 2 mM BHB dose was used to challenge neutrophils for 8 h. Apoptosis rate of neutrophils and protein abundance of cleaved caspase 3 were lower after BHB treatment. Treatment with BHB decreased protein and mRNA abundance of the pro-apoptotic genes Bax (BAX) and Bad (BAD), whereas it increased mitochondrial membrane potential (MMP) and protein and mRNA of the anti-apoptotic genes Bcl-xL (BCL2L1) and Mcl-1 (MCL1). This indicated that a mitochondrial pathway was involved in the inhibition of neutrophil apoptosis via BHB. In addition, both SCH772984 (an inhibitor of the ERK1/2 signaling pathway) and MK-2206 (an inhibitor of the AKT signaling pathway) alleviated the BHB-induced anti-apoptotic function of the Bcl-2 family and the inhibition of MMP. Overall, our data demonstrated that high concentrations of BHB inhibit apoptosis in bovine neutrophils by activating the ERK1/2 and AKT signaling pathways. These findings provide a theoretical basis for the understanding of systemic inflammation in ketotic cows.

Activation of Transcription Factor EB Is Associated With Adipose Tissue Lipolysis in Dairy Cows With Subclinical Ketosis.

Excessive lipid mobilization for adipose tissue caused by severe negative energy balance is the pathological basis for subclinical ketosis (SCK) in dairy cows. In non-ruminants, transcription factor EB (TFEB) was reported to play a role in the regulation of lipid catabolism, but its role in the control of lipolysis in the bovine is unknown. The present study aimed to determine whether the enhanced TFEB transcriptional activity contributes to lipolysis of adipose tissue in SCK cows, and to explore the possibility of establishing a therapeutic strategy by using TFEB as a target to control lipolysis. Thirty cows with similar lactation number (median = 3, range = 2–4) and days in milk (median = 6 d, range = 3–9) were selected into a healthy control (n = 15) and SCK (n = 15) group, and used for subcutaneous adipose tissue biopsies and blood sampling. Adipocytes from healthy Holstein calves were used as a model for in vitro studies involving treatment with 10 μM isoproterenol (ISO) for 0, 1, 2 and 3 h, 250 nM of the TFEB activator Torin1 for 3 h, or used for transfection with TFEB small interfering RNA for 48 h followed by treatment with 10 μM ISO for 3 h. Compared with healthy cows, adipose tissue in SCK cows showed increased lipolysis accompanied by enhanced TFEB transcriptional activity. In vitro, ISO and Torin1 treatment increased lipolysis and enhanced TFEB transcriptional activity in calf adipocytes. However, knockdown of TFEB attenuated ISO-induced lipolysis in adipocytes. Overall, these findings indicated that enhanced transcriptional activity of TFEB may contribute to lipolysis of adipose tissue in dairy cows with SCK. The regulation of TFEB activity may be an effective therapeutic strategy for controlling overt lipolysis in ketotic cows.

Differentiation of Subclinical Ketosis and Liver Function Test Indices in Adipose Tissues Associated With Hyperketonemia in Postpartum Dairy Cattle.

Past studies suggested that during early lactation and the transition period, higher plasma growth hormone (GH) levels in subclinical ketosis (SCK) might involve the initiation of body adipose tissues mobilization, resulting in metabolic disorders in ruminants particularly hyperketonemia. The upregulated GH mRNA expression in adipose tissue may take part in the adipolysis process in SCK-affected cows that paves a way for study further. This study aimed to characterize the plasma levels of GH, β-hydroxybutyrate acid (BHBA) and non-esterified fatty acid (NEFA) and glucose (GLu) in ketotic cows and healthy control (CON) cows; to measure the liver function test (LFT) indices in ketotic and healthy CON cows, and finally the quantitative real-time PCR (qRT-PCR) assay of candidate genes expressed in adipose tissues of ketotic and healthy CON cows during 0 to 7 week postpartum. Three experiments were conducted. Experiment-1 involved 21 Holstein cows weighing 500–600 kg with 2–5 parities. Results showed that GH, BHBA, and NEFA levels in ketotic cows were significantly higher and the GLu level significantly lower. Pearson's correlation analysis revealed a significant positive correlation of GH with BHBA, NEFA, and GLu in ketotic and healthy CON cows. In experiment-2, dynamic monitoring of LFT indices namely, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), globulin (GLOB) and albumin/globulin (A/G) were examined. The TBIL, DBIL, and GGT indices were significantly higher in ketotic cows and TP was significantly lower. In experiment-3, mRNA expression levels of GHR and peroxisome-proliferator-activated receptor alpha (PPARα) genes in adipose tissue were significantly upregulated in ketotic cows. However, the mRNA expression of insulin-like growth factor-I (IGF-1), insulin-like growth factor-I receptor (IGF-1R), and sterol regulatory element-binding protein-1c (SREBP-1c) genes in adipose tissue were downregulated in ketotic cows. Our study concluded that during postpartum, higher plasma GH levels in SCK cows might involve the initiation of body adipose tissue mobilization, resulting in hyperketonemia.

Prevalence, risk factors and metabolic predictors associated with subclinical ketosis on dairy cattle in Batna, Eastern of Algeria

Economic impact of subclinical ketosis (SCK) in dairy herds resulted from a decreased productivity, reproductive performances, and health condition. In Algeria, data availability regarding the prevalence of SCK in dairy cows were limited. The present study is a contribution in that direction and aimed to determine the prevalence of SCK between 2 and 50 days postpartum whereas investigating herd- and cow-level risk factors and the metabolic profile associated, on two bovine populations imported and native. The study was conducted with 16 typical representative local dairy cow herds (n = 100; 2–12 years old; located at Merouana, Batna Province) with data collected in a period comprised between August and February of the following year. Concentration of cows’ plasma beta-hydroxybutyrate (BHBA) was used for the diagnosis, with values equal or higher than 1.2 mmol/L chosen as the threshold for SCK detection. Other key associated zootechnical parameters such as body condition score (BCS) were monitored at the same sampling points. Logistic regression analyses were carried out to characterize association between parameters. The average SCK prevalence was 9% and resulted highest during first- and second-week postcalving, with a peak in the first week. The herd size (< 30 lactating cows) and BCS (≤ 3) were risk factors associated with SCK prevalence. At the cow-level, risks of SCK increased in thin individuals (P = 0.058; odds ratio (OR) = 5.00, 95% confidence interval (CI) = 0.95–26.32). At the herd-level, risks of SCK increased in smaller frame size herds (P = 0.047; OR = 8.76, 95% CI = 1.03–74.49). Most cases of SCK were observed in native cows compared to imported. Interestingly, linear stepwise regression showed a good relationship between BHBA and total bilirubin (P = 0.0001; R2 = 0.59). There were significant differences between healthy and ketotic cows regarding plasma concentration of bilirubin (P = 0.003), glucose (P = 0.001), alanine aminotransferase (ALT) (P = 0.008), creatinine (P = 0.007), and sodium (P = 0.02). According to receiver operating characteristic (ROC) curve analysis, the best parameter for SCK prediction is total bilirubin with more than 4.09 g/L as a cutoff point (77.78% sensitivity and 94.51% specificity). According to metabolic profile and feeding data we suggest that most cases of SCK observed in our study are type I. Further studies are required to validate other metabolic predictors for SCK and investigate adaptation of imported cows in semiarid regions in Algeria.

Prevalence of Ketosis Detected by an Electronic Cow Side Test and its Impact to Days Open on Dairy Cows

The study aimed to resolve and confirm the previous results of subclinical and clinical ketosis prevalence in northwestern Croatia detected by an electronic cow side test and its impact on days open in dairy herds. Cows (N=559) 2-8 years old from 96 farms located in northwestern Croatia were included in the study. The average milk yield was 7327.80 ± 968.21 kg. The cows were classified into two separate groups: ketotic cows group KET (n=73) with BHBA blood concentration of ≥1.4 mmol/L, and negative NEG (n=486) group with serum BHBA level of &lt;1.4 mmol/L. One droplet of blood from the caudal vein was collect from randomly selected cows in the period 7 to 15 days after parturition. BHBA level determined with Precision Xceed BHBA devices. The period from parturition to first insemination (days open to the first (artificial) insemination, DOFI) was shorter in NEG than in the POS group (110.56±10.65 days vs.114.82±12.23 days, respectively) such as period from parturition to successful conception (days open to successful conception, DOSC), (139.97±15.18 days vs. 127.99±15.87 days, respectively). The prevalence of clinical and subclinical ketosis was 15.02%. Ketosis significantly prolonged days open to first artificial insemination and days open to successful conception in cows from northwestern Croatia.

Accuracy of Subclinical Ketosis Detection with Rapid Test Methods for BHBA in Blood in Commercial Dairy Farms

This study gives an overview of the performance and accuracy of devices used for the fast measurement of β-hydroxybutyrate (BHBA) in blood for the on-farm indication of subclinical ketosis. Data were collected on ten dairy farms. In each farm, blood samples were taken from ten cows on four test days (2, 4, 9 and 11), resulting in 400 samples. The reference method was the BHBA concentration in blood serum (BHBALAB). Four different devices that measure BHBA in whole blood were tested. The thresholds applied for identifying subclinical ketosis were ≥1.0, ≥1.2 and ≥1.4 mmol/L in blood serum. The BHBALAB was assigned in three classes: low—≤0.9 mmol/L; high—&gt;0.9 mmol/L; and total—all values unclassified. Due to initial negative effects on the health and performance of cows with BHBA levels ≥0.9 mmol/L, this cut-off was chosen. The Passing–Bablok regression revealed different constant as well as absolute biases for each device in the aforementioned classes. The area under the receiver operating characteristics curve indicated highly accurate results, with 94–97% accuracy levels. As an overall conclusion, the performance of the devices was good and supports their use by farmers for the detection of subclinical ketotic cows in their herds.

Reducing milking frequency from twice to once daily as an adjunct treatment for ketosis in lactating dairy cows—A randomized controlled trial

This randomized controlled trial investigated the effects of temporarily reducing milking frequency (MF) on the resolution of ketosis and milk production in dairy cows in early lactation. To detect ketosis [blood β-hydroxybutyrate (BHB) ≥1.2 mmol/L], Holstein cows were screened daily from 3 to 16 d in milk using a cow-side meter. Cows diagnosed with ketosis (n = 104) were randomly assigned to twice-daily milking (TDM) or reduced to once-daily milking (ODM) for 2 wk, then returned to twice-daily milking. Both treatment groups received a 5-d treatment of an oral propylene glycol drench (PG; 300 g) beginning on the afternoon of the diagnosis; cows received additional 5-d PG treatments if they had a ketotic test result (blood BHB ≥1.2 mmol/L) at least 4 d after finishing the first PG treatment. Blood BHB tests were conducted for the first 3 d after ketosis diagnosis, and then once every 3 d for 21 d of trial (DOT). Milk and milk component data were collected weekly for 15 wk following trial enrollment. The ODM group showed rapidly and markedly decreased blood BHB concentrations (primiparous cows: 1 DOT, 0.92 ODM vs. 1.22 TDM, 15 DOT, 0.55 vs. 0.81 mmol/L; multiparous cows: 1 DOT, 1.01 vs. 1.40, 15 DOT, 0.78 vs. 1.65 mmol/L). In addition, a logistic regression model indicated that ODM cows were less likely to have blood BHB concentrations ≥1.2 mmol/L [primiparous cows: 3 DOT: ODM 1% (95% confidence interval: 0-10%) vs. TDM 43% (30-58%), 15 DOT ODM 0% (0-0.2%) vs. TDM 22% (13-36%); multiparous cows: 3 DOT: ODM 33% (24-44%) vs. TDM 59% (48-69%), 15 DOT ODM 20.9% (13-31%) vs. TDM 64% (53-74%)]. The proportion of ODM cows that required additional treatments of PG were substantially lower than the TDM group (ODM: 39%; TDM: 64%) than the TDM cows during the initial 21-d period. However, during the 2-wk treatment period, cows in the ODM group produced 26% less milk and 25% less energy-corrected milk than the TDM cows. During wk 3 to 15, when all cows were milked twice daily, ODM cows produced less milk (-14%) and energy-corrected milk (-12%) compared with the TDM group. Milk protein percentage was greater, and milk fat percentage and linear score tended to be greater in the ODM group over 15 wk. In conclusion, a 2-wk reduction of MF in ketotic cows from twice to once daily with treatment with PG resolved ketosis and decreased blood BHB concentrations more effectively than treating TDM cows with PG alone. However, the 2-wk MF reduction had immediate and long-term (up to 13 wk after cessation of MF reduction) negative effects on milk production.

Therapeutic Effects of Levocarnitine or Vitamin B Complex and E With Selenium on Glycerin-Treated Holstein Friesian Cows With Clinical Ketosis.

Currently, ketosis has no fully satisfactory resolution in dairy cows. Here, we investigated the effect of levocarnitine or vitamin B complex and E with selenium on clinically ketotic cows (β-hydroxybutyrate ≥ 3.0 mmol/L and decreased milk yield), fed glycerin. In total, 18 cases of Holstein cows with clinical ketosis during the postpartum transition period were randomly assigned to three treatments (6 cases per group): (1) levocarnitine (C+G), (2) vitamin B complex and E with selenium (VBES+G), and (3) levocarnitine and vitamin B complex and E with selenium (C+VBES+G). All groups were administered glycerin. Treatments were administered daily for 4 days. Blood sampling was performed on the onset day of ketosis (day 0), day 4, and day 6. β-Hydroxybutyrate (BHBA), milk yield (MY), and serum biochemical values were measured. Half of the animals in C+G failed to overcome clinical ketosis. VBES+G treatment ameliorated BHBA (p < 0.05), MY, and glucose on day 4. However, ketosis was exacerbated following the discontinuation of the treatment. C+VBES+G treatment improved BHBA, glucose (p < 0.05), and MY and reduced ketotic cases on days 4 and 6 with greater improvements compared to the others. In conclusion, combined treatment with levocarnitine, vitamin B complex and E with selenium, and glycerin may have the therapeutic effect on clinical ketosis.

β-Hydroxybutyrate impairs neutrophil migration distance through activation of a protein kinase C and myosin light chain 2 signaling pathway in ketotic cows

Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)-a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction-was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.