Identification of genomic regions and genes associated with subclinical ketosis in periparturient dairy cows.

Subclinical ketosis (SCK) is a prevalent metabolic disorder that occurs during the transition to lactation period. It is defined as a high blood concentration of ketone bodies (beta-hydroxybutyric acid f ≥ 1.2 mmol/L) within the first few weeks of lactation, and often presents without clinical signs. SCK is mainly caused by negative energy balance (NEB). The objective of this study is to identify single nucleotide polymorphisms (SNPs) associated with SCK using genome-wide association studies (GWAS), and to predict the biological functions of proximal genes using gene-set enrichment analysis (GSEA). Blood samples were collected from 112 Holstein cows between 5 and 18 days postpartum to determine the incidence of SCK. Genomic DNA extracted from both SCK and healthy cows was examined using the Illumina Bovine SNP50K BeadChip for genotyping. GWAS revealed 194 putative SNPs and 163 genes associated with those SNPs. Additionally, GSEA showed that the genes retrieved by Database for Annotation, Visualization, and Integrated Discovery (DAVID) belonged to calcium signaling, starch and sucrose, immune network, and metabolic pathways. Furthermore, the proximal genes were found to be related to germ cell and early embryo development. In summary, this study proposes several feasible SNPs and genes associated with SCK through GWAS and GSEA. These candidates can be utilized in selective breeding programs to reduce the genetic risk for SCK and subfertility in high-performance dairy cows.

Changes in adiponectin levels of subclinical ketosis cows and their effects on steroid hormone secretion and proliferation in follicular granulosa cells.

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.

Multi-Omics Reveals Disrupted Immunometabolic Homeostasis and Oxidative Stress in Adipose Tissue of Dairy Cows with Subclinical Ketosis: A Sphingolipid-Centric Perspective.

Ketosis, especially its subclinical form, is frequently observed in high-yielding dairy cows and is linked to various diseases during the transition period. Although adipose tissue plays a significant role in the development of metabolic disorders, its exact impact on the emergence of subclinical ketosis (SCK) is still poorly understood. The objectives of this study were to characterize and compare the profiling of transcriptome and lipidome of blood and adipose tissue between SCK and healthy cows and investigate the potential correlation between metabolic disorders and lipid metabolism. We obtained blood and adipose tissue samples from healthy cows (CON, n = 8, ß-hydroxybutyric acid concentration < 1.2 mmol/L) and subclinical ketotic cows (SCK, n = 8, ß-hydroxybutyric acid concentration = 1.2-3.0 mmol/L) for analyzing biochemical parameters, transcriptome, and lipidome. We found that serum levels of nonesterified fatty acids, malonaldehyde, serum amyloid A protein, IL-1ß, and IL-6 were higher in SCK cows than in CON cows. Levels of adiponectin and total antioxidant capacity were higher in serum and adipose tissue from SCK cows than in CON cows. The top enriched pathways in whole blood and adipose tissue were associated with immune and inflammatory responses and sphingolipid metabolism, respectively. The accumulation of ceramide and sphingomyelin in adipose tissue was paralleled by an increase in genes related to ceramide biosynthesis, lipolysis, and inflammation and a decrease in genes related to ceramide catabolism, lipogenesis, adiponectin production, and antioxidant enzyme systems. Increased ceramide concentrations in blood and adipose tissue correlated with reduced insulin sensitivity. The current results indicate that the lipid profile of blood and adipose tissue is altered with SCK and that certain ceramide species correlate with metabolic health. Our research suggests that disruptions in ceramide metabolism could be crucial in the progression of SCK, exacerbating conditions such as insulin resistance, increased lipolysis, inflammation, and oxidative stress, providing a potential biomarker of SCK and a novel target for nutritional manipulation and pharmacological therapy.

The rumen microbiota contributed to the development of mastitis induced by subclinical ketosis.

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.

Subclinical ketosis leads to lipid metabolism disorder by downregulating the expression of acetyl-coenzyme A acetyltransferase 2 in dairy cows.

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, ß-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.

Multi-omics analysis reveals that the metabolite profile of raw milk is associated with dairy cows' health status.

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.

Epidemiological Features of Postpartum Subclinical Ketosis in Dairy Herds in Hokkaido, Japan.

This study was carried out as an observational study in order to determine the prevalence of postpartum subclinical ketosis (SCK) in dairy herds in Hokkaido, Japan. From April 2012 to March 2014, blood ß-hydroxybutyrate (BHBA) concentration was measured once within 3-88 days in milk (DIM) in 1394 apparently healthy cows from 108 farms to diagnose SCK (≥1.2 mM). In cows within 14 DIM, this was classified as SCK II, and from 15 DIM, this was classified as SCK I. Herds with a combined percentage of SCK I and SCK II of less than 10% were classified as SCK-negative herds, those with percentages of 10-25%, were classified as alert herds, and those with one of 25% or more, we classified as positive herds. The prevalence of SCK in the entire DIM was 17.6%. The prevalence of SCK II (20.2%) tended to occur more frequently than SCK I (16.5%, p = 0.094). The frequency of SCK I was higher at the fourth parity. The number of milking cows in SCK-positive herds was significantly smaller than those of the other two types of herds (p = 0.004). The frequency of SCK-positive herds in tie stalls and with component feeding was higher than for free stall or free barn and with total mixed ration (p = 0.054 and p = 0.002). This study reveals the prevalence of SCK in Hokkaido, Japan, and shows that SCK is associated with parity and the management system.

Subclinical Ketosis in Dairy Herds: Impact of Early Diagnosis and Treatment.

Clinical and subclinical ketosis (SCK) in dairy cows occurs during the lactation period frequently in many herds, causing a reduction in milk yield and alterations in milk quality with significant economic losses for farmers. SCK is defined as a preclinical stage of ketosis characterized by an elevated ketone body level without clinical signs. Often many cows develop an elevated ketone body level during the first weeks of lactation even though it never goes up to a critical point causing clinical signs. This study aimed to evaluate the prevalence of SCK in Sicily and assess the effect of a treatment with propylene glycol (PG) to control the SCK, thus, reducing the negative effect on milk quality yield. This cross-sectional study was carried out on 22 farms located south-east of Sicily and 1,588 cows in lactation. A total of 3,989 individual milk samples were collected from calving to 80 subsequently days to check the ß-hydroxybutyrate (BHB) values in order to establish the SCK status by the Fourier Transform Infrared Spectroscopy. Moreover, the contents of fat, protein, lactose, casein, urea, somatic cell count and acetone were evaluated to identify a correlation between SCK and milk quality. A total of 1,100 cows showed BHB values higher than 0.10 mmol/L. These cows were considered SCK positive, were separated from the rest of the herd, and treated with PG (400 g/head per day), all SCK cows were treated with PG and cows without SCK were not treated. The results showed a prevalence of 41.5% of SCK-positive cows during the first 9 days of lactation. The comparison among the cure rate of treated cows shows that the treatment was most effective in the first 7 days of lactation (76.5% of treated cows) than in the following days. PG positively influenced the milk quality parameters, except for the fat proportion. Moreover, the animals treated with PG showed also an increase in milk yield, supporting the economical sustainability of treatment.

Metabolic profiling of serum and urine in lactating dairy cows affected by subclinical ketosis using proton nuclear magnetic resonance spectroscopy.

Ketosis is associated with high milk yield during lactating or insufficient feed intake in lactating dairy cows. However, few studies have been conducted on the metabolomics of ketosis in Korean lactating dairy cows. The present study aimed to investigate the serum and urine metabolites profiling of lactating dairy cows through proton nuclear magnetic resonance (1H-NMR) spectroscopy and comparing those between healthy (CON) and subclinical ketosis (SCK) groups. Six lactating dairy cows were categorized into CON and SCK groups. All experimental Holstein cows were fed total mixed ration. Serum and urine samples were collected from the jugular vein of the neck and by hand sweeping the perineum, respectively. The metabolites in the serum and urine were determined using 1H-NMR spectroscopy. Identification and quantification of metabolites was performed by Chenomx NMR Suite 8.4 software. Metabolites statistical analysis was performed by Metaboanalyst version 5.0 program. In the serum, the acetoacetate level was significantly (p < 0.05) higher in the SCK group than in the CON group, and whereas acetate, galactose and pyruvate levels tended to be higher. CON group had significantly (p < 0.05) higher levels of 5-aminolevulinate and betaine. Indole-3-acetate, theophylline, p-cresol, 3-hydroxymandelate, gentisate, N-acetylglucosamine, N-nitrosodimethylamine, xanthine and pyridoxine levels were significantly (p < 0.05) higher in the urine of the SCK group than that in the CON group, which had higher levels of homogentisate, ribose, gluconate, ethylene glycol, maltose, 3-methyl-2-oxovalerate and glycocholate. Some significantly (p < 0.05) different metabolites in the serum and urine were associated with ketosis diseases, inflammation, energy balance and body weight. This study will be contributed useful a future ketosis metabolomics studies in Korea.

Free fatty acids promote degranulation of azurophil granules in neutrophils by inducing production of NADPH oxidase-derived reactive oxygen species in cows with subclinical ketosis.

Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2-4) and days in milk (median = 6, range = 3-15) were classified based on serum ß-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1ß, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A+/CD63high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.

Global prevalence of subclinical ketosis in dairy cows: A systematic review and meta-analysis.

The objectives of this study were to estimate the global prevalence of SCK in dairy cows, to identify optimum thresholds for BHB determining, to explore heterogeneity for some particular characteristics (continents, diagnosis techniques, cut-off, sample type, parity, breed, and housing), and to determine the SCK prevalence trend. SCK prevalence levels in dairy cows from various countries were calculated using bibliographic databases up until May 17, 2021 to screen titles and abstracts for possible eligibility. Thirty-eight studies from six continents were then included using a random-effects model to calculate the pooled SCK prevalence. Subgroup meta-analyses were performed to assess the heterogeneity of the prevalence for the above characteristics. Meta-regression and cumulative meta-analysis were used to assess the SCK prevalence trends. The results showed that the global prevalence of SCK in dairy cows was 22.7% [95% CI 21.2-24.3%]. No significant differences in SCK prevalence were observed among continents, diagnostic techniques, cut-off values-≥1.0, ≥1.2 and ≥1.4 mmol/L, sample types-milk and blood, and parities. However, the prevalence in Holsteins (19.8%) was significantly lower than other mixed breeds (23.7%). The SCK prevalence was significantly higher in indoor barns (27.8%) than in pasture and unspecified housing. In meta-regression, the SCK prevalence was not associated with study years or days in milk. The cumulative evidence suggested that the prevalence of SCK in dairy cows was associated with a wide range of risk factors. This study illustrates that the global prevalence of SCK in dairy cows is quite high; therefore, there is an urgent need to globally reduce the SCK prevalence in dairy farms.

Seasonal effect of milk yield and blood metabolites in relation to ketosis of dairy cows fed under a high ambient temperature.

BACKGROUND AND AIM: Metabolism and environment are closely related. Under high ambient temperature (HTa), dairy cows may have different energy metabolism during summer and winter. The present study was carried out to investigate the effect of HTa on the milk yield and blood concentration of beta-hydroxybutyrate (BHBA) and glucose at the herd level. MATERIALS AND METHODS: One large dairy farm in Thailand with more than 100 crossbred Holstein cows milked each month was selected. The first experiment was performed on non-lactating cows to determine the normal daily concentrations of blood BHBA and glucose. Under the HTa condition, there was no significant change in blood BHBA and glucose concentrations. The second experiment was performed using a prospective cohort clinical design to demonstrate the seasonal effect on milk yield and blood BHBA as an indication of energy metabolism at the herd level. RESULTS: The temperature and humidity index for the winter (78.1±0.5) and summer (83.4±0.7) periods differ significantly. The average milk yield during the winter period was 17.8% higher than during the summer period. The reduction of body condition score (BCS) during early lactation was significant in the winter cows. Both higher milk yield and lower BCS in the winter cows suggested a state of negative energy balance. However, there was no difference in blood BHBA and glucose concentrations between winter and summer cows. The effect of HTa on insulin signaling appeared to be a counterbalancing factor for the ketogenic status. Based on the present results, it would be interesting to further investigate the incidence of subclinical and clinical ketosis in a dairy farm under tropical conditions. CONCLUSION: The present experiment revealed that HTa during summer decreased milk yield in dairy cows fed under tropical conditions. Higher milk yield in winter caused a greater reduction of BCS and suggested a greater negative energy balance. However, there was no seasonal effect on blood BHBA and glucose concentrations.

Evaluation of Statistical Process Control Techniques in Monitoring Weekly Body Condition Scores as an Early Warning System for Predicting Subclinical Ketosis in Dry Cows.

The management of body condition score (BCS) during the dry period is associated with the postpartum health outcomes of dairy cows. However, the difference between the actual BCS and the fixed ideal value is not able to accurately predict the occurrence of postpartum diseases. This study aimed to use statistical process control (SPC) technology to monitor the BCS of dry cows, to evaluate the effect of control charts on nutritional strategies, and to explore the utility of SPC in predicting the incidence of postpartum subclinical ketosis (SCK). The BCS and SCK data of 286 cows from the dry off period to 60 days postpartum were collected to set up the early warning function. Three control charts, including a control chart for the average BCS of the herds, for the BCS of each dry cow, and for individual BCS, were established. The early warning signs for postpartum SCK development were: (1) an individual BCS more than 3.5 that remained unchanged for six weeks; (2) a capability index (CPK), an SPC tool, greater than -0.52. Using these parameters, the early warning signs of SCK development were verified in 429 dry cows. The results showed that the accuracy of early warning signal was 0.64 and the precision was 0.26. The control chart showed that the average BCS of dry cows was consistently higher than the expected upper limit of BCS during the experimental period, and that the addition of new cows to the herds increased the average BCS. In summary, the application of SPC technology to monitor the BCS of dry cows was not a good tool for the prediction of postpartum SCK occurrence but was an appropriate tool for guiding positive nutrition strategies.

Metabolic Profiling of Rumen Fluid and Milk in Lactating Dairy Cattle Influenced by Subclinical Ketosis Using Proton Nuclear Magnetic Resonance Spectroscopy.

Ketosis metabolic research on lactating dairy cattle has been conducted worldwide; however, there have been very few Korean studies. Biofluids from lactating dairy cattle are necessary to study ketosis metabolic diseases. Six Holstein cows were divided into two groups (healthy (CON) and subclinical ketosis diagnosed (SCK)). Rumen fluid and milk samples were collected using a stomach tube and a pipeline milking system, respectively. Metabolites were determined using proton nuclear magnetic resonance (NMR) spectroscopy and they were identified and quantified using the Chenomx NMR Suite 8.4 software and Metaboanalyst 5.0. In the rumen fluid of the SCK group, butyrate, sucrose, 3-hydroxybutyrate, maltose, and valerate levels were significantly higher than in the CON group, which showed higher levels of N,N-dimethylformamide, acetate, glucose, and propionate were significantly higher. Milk from the SCK group showed higher levels of maleate, 3-hydroxybutyrate, acetoacetate, galactonate, and 3-hydroxykynurenine than that from the CON group, which showed higher levels of galactitol, 1,3-dihydroxyacetone, γ-glutamylphenylalanine, 5-aminolevulinate, acetate, and methylamine. Some metabolites are associated with ketosis diseases and the quality of rumen fluid and milk. This report will serve as a future reference guide for ketosis metabolomics studies in Korea.

Exploring Dry-Film FTIR Spectroscopy to Characterize Milk Composition and Subclinical Ketosis throughout a Cow's Lactation.

The use of technologies for measurements of health parameters of individual cows may ensure early detection of diseases and maximization of individual cow and herd potential. In the present study, dry-film Fourier transform infrared spectroscopy (FTIR) was evaluated for the purpose of detecting and quantifying milk components during cows' lactation. This was done in order to investigate if these systematic changes can be used to identify cows experiencing subclinical ketosis. The data included 2329 milk samples from 61 Norwegian Red dairy cows collected during the first 100 days in milk (DIM). The resulting FTIR spectra were used for explorative analyses of the milk composition. Principal component analysis (PCA) was used to search for systematic changes in the milk during the lactation. Partial least squares regression (PLSR) was used to predict the fatty acid (FA) composition of all milk samples and the models obtained were used to evaluate systematic changes in the predicted FA composition during the lactation. The results reveal that systematic changes related to both gross milk composition and fatty acid features can be seen throughout lactation. Differences in the predicted FA composition between cows with subclinical ketosis and normal cows, in particular C14:0 and C18:1cis9, showed that dietary energy deficits may be detected by deviations in distinct fatty acid features.

Longitudinal Metabolic Biomarker Profile of Hyperketonemic Cows from Dry-Off to Peak Lactation and Identification of Prognostic Classifiers.

Currently about 30% to 50% of all dairy cows are affected by a metabolic or infectious disease during the transition period. A key factor for preventive actions is the ability to precisely predict metabolic diseases at an early stage. We report the longitudinal metabolic profile of non-esterified fatty acids, beta-hydroxybutyrate (BHB), total bilirubin, and aspartate aminotransferase in hyperketonemic dairy cows. Aiming for a novel measurement regime to improve metabolic health in dairy cows, we evaluated prognostic classifiers for hyperketonemia. In the observational longitudinal study, 99 healthy adult primiparous and multiparous Simmental dairy cows were included. Every cow was monitored weekly for 14 consecutive weeks, beginning two weeks prior to the expected day of parturition until peak lactation. Cows with serum concentrations of BHB > 0.8 mmol/L were considered hyperketonemic. Biomarker profiles were fitted by the maximum likelihood method using a mixed effects natural cubic spline model. In the hyperketonemic group, the BHB profile remained significantly higher than that of the control group until the end of the study period. As a prognostic classifier, the cut-off level of 0.54 mmol/L BHB measured on the 10th day post partum had the highest area under the curve. These results provide new longitudinal insights into the metabolic biomarker progression of dairy cows and enable an early onset diagnosis of hyperketonemia.

Rumination time as an early predictor of metritis and subclinical ketosis in dairy cows at the beginning of lactation: Systematic review-meta-analysis.

Daily rumination time (RT; min/d) is recognized as an important tool for assessing the health of dairy cows, which may depend on the disease, lactation stage and individual cows. Using a systematic review-meta-analysis, this study evaluated whether the variation in RT is effective for early detection of metritis and subclinical ketosis (SCK) in dairy cows in the pre and post-partum periods (from three weeks before to three weeks after calving). The research was carried out in four electronic databases - Scopus, Science Direct, Pubmed and Web of Science. The main inclusion criteria were original research; evaluation of RT in dairy cows; and use of RT for early identification of metritis and/or SCK in post-partum dairy cows. A random effect meta-analysis (MA) was conducted for each disease (metritis and SCK) separately, with the RT means of healthy and sick groups, measured in the pre and post-partum. The effect size used was the mean difference (MD).Twenty-two trials from six studies were included in the MA, involving 1494 dairy cows. For metritis, four trials from three studies in the pre-partum period were considered as well as five trials from four studies in the post-partum. For SCK, six trials from four studies pre-partum and seven trials from five studies in the post-partum period were taken into consideration. The heterogeneity between studies for metritis was null (I2 = 0%) and low (I2 = 5.7 %) in the pre-partum and in the post-partum, respectively. The MD of RT between healthy cows and those with metritis was different in the pre (MD =0.411 min/d; P < 0.001) and in the post-partum (MD =0.279 min/d; P < 0.001). In SCK, heterogeneity was high in the pre (I2 = 69 %) and in the post-partum (I2 = 58.1 %), and the MD of RT was similar between healthy and sick cows (P> 0.05). In a meta-regression, RT from primiparous cows showed a lower predicted value for MD (0.48 min. d; P < 0.05) compared to multiparous cows, and the increment in each unit of milk production decreased the predicted MD value by 0.08 min. d (P < 0.001). Our MA demonstrates that RT is a good predictor for early detection of metritis in pre and post-partum; however, it is not an adequate predictor for SCK. Further investigations using more frequent blood sampling and the same threshold values for BHB are required to assess the adequacy of rumination time to predict SCK.

Subclinical ketosis risk prediction in dairy cows based on prepartum metabolic indices / [Previsão do risco de cetose subclínica em vacas de leite, baseado nos índices metabólicos pré-parto]

Ketosis can seriously impair cow performance. This study detected changes in prepartum blood metabolic parameters for predicting postpartum ketosis occurrence in dairy cows. Body condition score (BCS) was assessed before and after delivery. Blood samples of 63 cows were collected from 10 days before calving to 10 days after calving to measure metabolic parameters including ß-hydroxybutyric acid (BHBA), non-esterified fatty acid (NEFA), glucose (GLU), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total protein (TP), albumin (ALB), globulin (GLO), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). There was a postpartum subclinical ketosis incidence of 42.25%. Compared with prepartum, plasma, levels of BHBA, AST, and NEFA significantly increased postpartum, and prepartum AST (R=0.57) and NEFA (R=0.45) showed a significant positive correlation with ketosis postpartum. Plasma GLU level significantly decreased postpartum and was significantly negatively correlated with ketosis (R=-0.21). Receiver operating characteristic curve analysis revealed prepartum BSC < 2.88, and prepartum plasma AST > 68.0 U/L, GLU < 3.97mmol/L, NEFA > 0.27mmol/L, and BHBA > 0.43mmol/L, indicating a high risk of subclinical ketosis postpartum. These levels can be used as risk indicators to predict the occurrence of subclinical ketosis in postpartum cows.(AU)

A cetose pode trazer sérios prejuízos ao desempenho da vaca. Este estudo detectou alterações nos parâmetros metabólicos do sangue pré-parto para prever a cetose pós-parto que ocorre em vacas leiteiras. O escore de condição corporal (ECC) foi avaliado antes e após o parto. Foram coletadas amostras de sangue de 63 vacas entre 10 dias antes e 10 dias após o parto para medir os parâmetros metabólicos, incluindo ácido ß-hidroxibutírico (BHBA), ácido graxo não esterificado (NEFA), glicose (GLU), bilirrubina total (TBIL), bilirrubina direta (DBIL), bilirrubina indireta (IBIL), proteína total (TP), albumina (ALB), globulina (GLO), alanina aminotransferase (ALT) e aspartato aminotransferase (AST). Houve uma incidência de cetose subclínica pós-parto de 42,25%. Em comparação com o pré-parto, o plasma, os níveis de BHBA, AST e NEFA aumentaram significativamente no pós-parto, e AST no pré-parto (R = 0,57) e NEFA (R = 0,45) mostraram uma correlação significativa positiva com cetose pós-parto. O nível plasmático de GLU diminuiu significativamente no pós-parto e foi negativamente correlacionado com a cetose de forma significativa (R = -0,21). A análise da curva característica de operação do receptor revelou BSC pré-parto <2,88 e AST plasmático pré-parto> 68,0 U / L, GLU <3,97mmol / L, NEFA> 0,27mmol / L e BHBA> 0,43mmol / L, indicando um alto risco de cetose subclínica pós-parto. Esses níveis podem ser usados ​​como indicadores de risco para prever a ocorrência de cetose subclínica em vacas no pós-parto.(AU)

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, ß-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.

Influence of Subclinical Ketosis in Dairy Cows on Ingestive-Related Behaviours Registered with a Real-Time System.

According to the literature, rumination time can be used as biomarker in the diagnosis of subclinical ketosis (SCK). We hypothesized that SCK in cows influences ingestive-related behaviours registered with the real-time system. The aim of the current study was to determine the influence of SCK on dairy cows' ingestive-related behaviours registered with a real-time system. Twenty Lithuanian Black and White breed dairy cows were selected based on the following criteria: First day after calving, having two or more lactations (on average 3.0 ± 0.13 lactations), and being clinically healthy. The experiment lasted 18 days. Cows were tested 24 h a day for 17.5 days. On the day of diagnosis (day 0), data were recorded for 12 h. During the experimental period, one cow was studied for a total of 420 h. For the registration of rumination behaviour, the RumiWatch system (RWS) was used. It was found that cows with SCK showed lesser average values for the following parameters: rumination time and rumination chews (1.48 and 1.68 times respectively; p < 0.001), drinking time (1.50 times; p < 0.001), chews per minute, bolus and chews per bolus (1.12, 1.45 and 1.51 times; p < 0.001). From the 15th day before the diagnosis of SCK, rumination time in health cows was greater than that in SCK cows from -0.96% (-17 day) to 187.79% (0 days, < 0.001). We estimated the greater average value of drinking time in healthy cows compared with SCK cows from 34.22% on day -17 to -121.67% on day 0 (p < 0.001). Decrease in rumination time was associated with a significant increase in the probability of risk of SCK. Further studies are needed with a larger number of cows with SCK.