Assessment of Some Inflammatory Cytokines and Immunologic Factors in Dairy Cows with Subclinical Ketosis

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.

The effect of subclinical ketosis on activity at estrus and reproductive performance in dairy cattle

Subclinical ketosis (SCK) is highly prevalent and easily overlooked, with insidious and slow progression of hepatic injury, often characterized by an imbalance in immune homeostasis. In nonruminants, macrophage polarization plays an important regulatory role in hepatic lipid accumulation, fibrosis, and inflammatory processes. Thus, we aimed to investigate the status of hepatic macrophage polarization in SCK cows and to corroborate its association with liver injury and inflammation. Twelve Holstein dairy cows (parity 2-4) were selected, and liver biopsy and blood were collected on the second week postpartum (10-14 d DIM). On the basis of serum beta-hydroxybutyric acid (BHBA) concentrations, selected cows were categorized into healthy (n = 6; BHBA <1.0 mM) and SCK (n = 6; 1.2 mM ≤ BHBA < 3.0 mM) groups. Serum biochemical parameters were measured using an automatic biochemical analyzer, which indicated higher serum levels of BHBA and nonesterified fatty acids and an upregulation of liver injury indicators (aspartate aminotransferase [AST], alanine aminotransferase [ALT], total protein, globulin) in SCK cows compared with healthy cows. The ELISA assays revealed that SCK cows displayed systemic low-grade inflammation, as demonstrated by increased serum levels of haptoglobin, serum amyloid A, TGF-β, IFN-γ, and IL-1β. Liver biopsies revealed pathological histological alterations, hepatic inflammation, and macrophage polarization status. Oil Red staining indicated steatosis, whereas Sirius red staining demonstrated mild extracellular matrix deposition in the liver of SCK cows. The expression of inflammatory response-related proteins (TLR4, p-NFκB, p-I-κB, NLRP3, and Caspase 1) was elevated in the liver of SCK cows, with the increased mean fluorescence intensity of NFκB further confirming the activation of the inflammatory pathway. Furthermore, the levels of pro-inflammatory factors, TNF-α and IFN-γ, were elevated in the tissue homogenate. Macrophage phenotypic changes in SCK cows were further explored based on the results of liver injury and inflammation. Compared with healthy cows, the protein and mRNA abundance of the macrophage marker CD68 in the liver of SCK cows was higher, along with an increased mean fluorescence intensity of CD68. The SCK cows also exhibited reduced mRNA expression of the Kupffer cell marker CLEC4F and elevated chemokine levels (CXCL1 and CCL2). As evidenced by greater protein and mRNA abundance of macrophage M1 polarization markers (iNOS, IL-1β, CD86, IL-6, IL-12b, and CCL3), higher fluorescence intensity of iNOS and CD86, and an increased number of CD68+/CD86+-positive cells observed via immunofluorescence, the macrophage polarization phenotype in the liver of SCK cows was predominantly M1. In contrast, the protein and mRNA abundances of M2 polarization markers (CD206, IL-10, and Arg1) were lower in SCK cows, accompanied by a reduced fluorescence intensity of CD206 and a lower number of CD68+/CD206+-positive cells. Overall, the present study revealed that the number of macrophages in liver is enhanced during subclinical ketosis and is dominated by pro-inflammatory macrophages (M1 macrophages). This could partly explain the increased risk of steatosis, fibrosis, and inflammatory response processes in these cows.

Hepatic autophagy and mitophagy status in dairy cows with subclinical and clinical ketosis

Metabolic lipid alterations in subclinical ketotic dairy cows: A multisample lipidomic approach.

Simple SummaryEarlier disease detection may benefit cows by improving responses to treatment. We expected that changes in rumination before evident clinical signs of subclinical ketosis would result in the earlier identification of disease. Cows with subclinical ketosis 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 ketosis, rumination time in the healthy group was greater than that in subclinical ketosis cows from −0.96% (−17 day) to 187.79% (0 days, p < 0.001). Eating time at the beginning of the experiment in healthy cows was 48.92% and at the end of the period was −91.97% lesser compared to subclinical ketosis (p < 0.001).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.

Expression patterns of hepatic genes involved in lipid metabolism in cows with subclinical or clinical ketosis

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

BackgroundSustained lipolysis exacerbates subclinical ketosis (SCK) in dairy cows and is associated with inflammation and adipose tissue macrophage (ATM) infiltration. While ATM involvement in adipose homeostasis and inflammation in early lactation is recognized, a comprehensive exploration of ATM polarization phenotypes in SCK cows is lacking. This study aimed to characterize ATM polarization and its link to lipolysis and inflammation in SCK cows.ResultsSubcutaneous adipose tissue samples were obtained from dairy cows to analyze protein expression and gene profiles. Compared with healthy cows, SCK cows had higher serum BHBA and NEFA, smaller adipocytes, and increased expression of lipolytic enzymes (LIPE, ATGL), indicating enhanced lipolysis. Decreased levels of FASN, PPARγ, p-SMAD3, and TGFβ suggested impaired adipogenesis. Inflammatory markers (TNF-α, IFN-γ, TLR4, Caspase1) and NFκB signaling activity were elevated. ATM infiltration was supported by increased CD9, CD68, TREM2, and CXCL1 expression. Protein abundance of M1 polarization markers (iNOS, CD86 and CCL2) in ATMs were associated with greater levels of NOS2, IL1B, CD86 and CCL2 mRNA expression in SCK cows; fluorescence intensity of NOS2 and CD86 also was elevated, alongside a higher proportion of CD68+/CD86+ immunopositive cells within adipose tissue. ELISA further quantified increased concentrations of IL-1β and CCL2. Conversely, the abundance of ATM M2 polarization markers, including CD206, IL-10, KLF4, and Arg1, at both the protein and mRNA levels demonstrated a decline. Meanwhile, the proportion of CD68+/CD206+ immune response cells was relatively low in SCK cows.ConclusionsOverall, the present study indicated an augmented macrophage presence within adipose tissue during subclinical ketosis, with a predominance of pro-inflammatory macrophages (M1 ATM). This observation suggested a vicious cycle wherein macrophage infiltration and pro-inflammatory polarization coincide with enhanced lipolysis and an amplified inflammatory cascade.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-025-01252-3.

Association of rumination time and health status with milk yield and composition in early-lactation dairy cows

It is known that ionophoric antibiotics regulate ruminal fermentation, improve the utilization of feed protein, and prevent the occurrence of ketosis and steatosis in ruminants. Ionophoric antibiotics and β-acids of hops have a similar spectrum of biological activity, that is, they inhibit the vital activity of most gram-positive microorganisms of the rumen. Bacteria, like other living organisms, need vitamin E as an active antioxidant for cell membranes. The toxicity of tocopherol is very low, so adding it to the diet of ruminants in larger quantities can stimulate celluloselytic rumen bacteria and compensate for the negative effect of ionophores on fiber breakdown. Since rumen bacteria break down a significant part of dietary choline, methionine and carnitine, ruminants must receive them in a protected form, so their influence on rumen fermentation is insignificant. Three groups of cows of the Ukrainian dairy black-spotted breed with milk yields of 5 or more thousand kg during the previous lactation were formed: with signs of clinical ketosis — 4 animals; with subclinical ketosis — 5 animals and clinically healthy — 5 animals. For a month, cows with ketosis were given a treatment supplement containing crushed granules of hop cones (20 g), vitamin E (3 g), and rumen protected choline (50 g), methionine (20 g) and carnitine (1 g). Clinically healthy cows were used as control. In the blood of cows with subclinical ketosis, the additive increased the concentration of glucose and decreased the concentration of β-hydroxybutyrate, these indicators were within the normal range. In cows with symptoms of clinical ketosis, using of the feed additive also reduced the concentration of β-hydroxybutyrate (P&lt;0.01), but it was still higher than normal. In sick cows, amylolytic and lipolytic activity was lower than in healthy cows (P&lt;0.05–0.01). Celluloselytic activity was lower only in cows with clinical ketosis. The proteolytic activity of rumen content changed in the opposite way; it was higher in sick cows (P&lt;0.05–0.01). This is a consequence of the increase in the number and activity of hyper producing ammonia bacteria in the rumen, what is characteristic for ketosis. After treatment of cows with subclinical ketosis, the celluloselytic and amylolytic activities in the rumen fluid were equal to the corresponding indicators of healthy cows, and the proteolytic activity was even slightly lower (P&lt;0.05) than in the control group. Treatment of cows with clinical form of ketosis was not as effective, although the general trends remained. During subclinical and clinical ketosis, a greater amount of ammonia was found in the rumen fluid (P&lt;0.05–0.01), because of higher proteolytic activity. In both forms of ketosis, the concentration of volatile fatty acids in the rumen decreased, and the concentration of lactate increased (P&lt;0.05–0.01). After the treatment, these indicators in cows with subclinical ketosis approached the healthy animals, while the condition of cows with clinical ketosis improved, but the concentration of ammonia continued to differ from healthy animals.

Short communication: Association of lying behavior and subclinical ketosis in transition dairy cows

Astragalus polysaccharides restore neutrophil functions and gut microbiota homeostasis in dairy cows with subclinical ketosis.

Can rumination time and some blood biochemical parameters be used as biomarkers for the diagnosis of subclinical acidosis and subclinical ketosis?

Simple SummaryThis study investigates the impact of subclinical ketosis (SCK) and subclinical acidosis (SCA) on dairy cow rumination, eating, drinking and locomotion behavior. It focuses on cows in their second or subsequent lactation, producing an average of 12,000 kg/year milk in their previous lactation. The cows were categorized into three groups, SCK, SCA, and healthy cows, based on the milk fat–protein ratio, blood beta-hydroxybutyrate and clinical examinations by a veterinarian. The results show significant differences between the cows affected by SCK and the healthy cows. SCK leads to a reduction in the milk yield (11.78%), rumination time (17.47%), and various eating and chewing behaviors. SCA is associated with a substantial decrease in Eating Time 2 (ET2) by 36.84% and Eating Chews 2 (EC2) by 38.10% compared to the healthy cows. These findings highlight the influence of SCA on feeding behaviors and chewing activity, with implications for nutrient intake and cow health. Additionally, SCK affects locomotion parameters, leading to a 27.36% reduction in the overall activity levels, as well as reductions in the Walking Time (WT), Other Activity Time (OAT), and Activity Change (AC). Early detection and effective management of SCK are crucial for maintaining dairy cow health and productivity. In conclusion, this research underscores the need for advanced strategies to prevent and manage subclinical metabolic disorders in the dairy industry. Effective management and early detection methods are essential to mitigate the impact of SCK and SCA on dairy cow health and benefit the dairy farming sector.This study delves into the effects of subclinical ketosis (SCK) and subclinical acidosis (SCA) on various parameters related to dairy cow rumination, eating, drinking and locomotion behavior. The research hypothesized that these subclinical metabolic disorders could affect behaviors such as rumination, feeding, and locomotion. A total of 320 dairy cows, with a focus on those in their second or subsequent lactation, producing an average of 12,000 kg/year milk in their previous lactation, were examined. These cows were classified into three groups: those with SCK, those with SCA, and healthy cows. The health status of the cows was determined based on the milk fat–protein ratio, blood beta-hydroxybutyrate, and the results of clinical examinations performed by a veterinarian. The data collected during the study included parameters from the RumiWatch sensors. The results revealed significant differences between the cows affected by SCK and the healthy cows, with reductions observed in the rumination time (17.47%) and various eating and chewing behaviors. These changes indicated that SCK had a substantial impact on the cows’ behavior. In the context of SCA, the study found significant reductions in Eating Time 2 (ET2) of 36.84% when compared to the healthy cows. Additionally, Eating Chews 2 (EC2) exhibited a significant reduction in the SCA group, with an average of 312.06 units (±17.93), compared to the healthy group’s average of 504.20 units (±18.87). These findings emphasize that SCA influences feeding behaviors and chewing activity, which can have implications for nutrient intake and overall cow health. The study also highlights the considerable impact of SCK on locomotion parameters, as the cows with SCK exhibited a 27.36% reduction in the walking time levels. These cows also displayed reductions in the Walking Time (WT), Other Activity Time (OAT), and Activity Change (AC). In conclusion, this research underscores the critical need for advanced strategies to prevent and manage subclinical metabolic disorders within the dairy farming industry. The study findings have far-reaching implications for enhancing the well-being and performance of dairy cattle. Effective management practices and detection methods are essential to mitigate the impact of SCK and SCA on dairy cow health and productivity, ultimately benefiting the dairy farming sector.