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.

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.

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, β-hydroxybutyric acid concentration < 1.2 mmol/L) and subclinical ketotic cows (SCK, n = 8, β-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-1β, 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.

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 β-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.

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.

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_I: BHB≥1,000 mmol/L, SCK_II: BHB≥1,100 mmol/L and SCK_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.

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.

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.

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 β-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.

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.