INTRODUCTION
Metabolomics is a new biological analysis method that provides precise analysis of molecules (molecular weight of >1,000). It is typically used to investigate the activity and status of cellular and organismal metabolism on a global or network scale to delineate the end point of physiology and pathophysiology [
1–
3]. Analytical technologies that are used in metabolomics include nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS) [
4,
5]. The NMR spectroscopy has a lower metabolite coverage than GC-MS and LC-MS; however, it has a short analysis time, low cost, and is easily comparable worldwide [
6]. For this reason, research on metabolites using NMR spectroscopy as well as MS instruments (GC-MS and LC-MS) is actively being conducted.
Representative metabolomics research using serum and plasma are performed to obtain diagnostic or therapeutic biomarkers of human diseases (Alzheimer’s, cancer, etc.) [
7–
9], or to identify potential biomarkers that may improve muscle and meat quality traits (water-holding capacity, meat color, tenderness, flavor, palatability, etc.) in animals [
10]. Representative metabolomic studies using urine have been conducted for the detection of steroids biomarkers (screening for cancer, mental and central nervous system disorders, and endocrine diseases; monitoring of drug therapy; doping control, etc.) [
11].
In recent years, research on metabolomics using biofluids (rumen fluid, serum, plasma, milk, urine, feces, etc.) of ruminants has been actively conducted worldwide. Metabolomics research using ruminant serum and plasma have been conducted to investigate the relationship between negative energy balance (NEB) and early lactation in dairy cows [
12], feed efficiency in black angus steers [
13], heat stress (HS) in beef cattle breeds [
14], and in search of biomarkers of ketosis and bovine respiratory diseases (BRD) [
15,
16]. Research on metabolomics using ruminant urine has focused on urea metabolism and recycling [
17], postpartum diseases such as metritis and lameness in dairy cows [
18,
19], HS in beef cattle breeds [
14], and nitrogen efficiency in dairy cows [
20]. Such studies have helped in the diagnosis and prevention of metabolic diseases and increased the productivity of ruminants.
In Korea, metabolomics research using ruminant biofluids has been conducted for the comparison of volatile fatty acid and monosaccharide metabolite concentrations in the rumen fluid of Hanwoo cattle using proton NMR (
1H-NMR) spectroscopy, high-performance liquid chromatography, and high-performance anion-exchange chromatography [
21]. In recent years, metabolomic comparisons of various Hanwoo cattle biofluids (rumen fluid, serum, and urine) have also been conducted using
1H-NMR spectroscopy [
22]. In addition, research on the metabolite changes in the milk and feces of dairy cattle with different feeding ratios of roughage and concentrate [
23,
24], and metabolomic comparison of dairy cattle rumen fluid and milk using
1H-NMR spectroscopy have been performed [
25]. However, overall, metabolomics research using humans, foods, and monogastric animals are aplenty, while studies on metabolomics using biofluids of ruminants are nascent. Therefore, more research using ruminant biofluids should be conducted.
In this study, we measured the metabolites in dairy cattle serum and urine by using 1H-NMR spectroscopy. The metabolites were then identified and classified to construct a database of each sample, wherein the concentration of each metabolite was provided. In addition, the roles of the metabolites observed in this study were compared to those reported in a previous study. The results of this study provide useful a database for the analysis of metabolites in ruminant serum and urine in Korea.
DISCUSSION
Ketosis is a metabolic disease in lactating dairy cattle and is, typically caused by high milk production or extreme peripartum reduction in energy intake and is specifically a high risk for cows suffering from severe NEB [
31,
32]. Diseases like this negatively impact the health, reproductive, performance, milk production capacity, and milk composition of cows, leading to a decrease in dairy industry profitability [
32]. A typical method for the diagnosis of ketosis in lactating dairy cattle is to measure the BHBA concentration associated with ketone body metabolites (BHBA, acetoacetate, and acetone) in the blood (serum and plasma) and urine [
32]. Moreover, a high concentration ketone body metabolites in blood and urine have been associated with decreased feed intake and increased other periparturient diseases [
32]. In this study, ketone body metabolites were measured in serum and urine. According to Xu et al [
12], changes in the concentrations of acetone, arginine, BHBA, glucose, glycine, kynurenine, and panthothenate in the plasma may be an indicator of NEB. Further, as reported by Luke et al [
15], an increase in the BHBA concentrations in early lactation dairy cattle was positively correlated with acetate, betaine, creatine, glycine, and phosphocholine, and negatively correlated with alanine, dimethyl sulfone, glucose, lactate, and valine in the serum. All metabolites associated with NEB were measured in this study, except for kynurenine; further, the metabolites associated with increased BHBA concentration in dairy cattle during early lactation were also measured in this study, except for dimethyl sulfone and phosphocholine. Therefore, metabolites measured by serum
1H-NMR spectroscopy could potentially be used to diagnose and, perhaps, prevent ketosis, which could significantly affect dairy industry profitability.
The BRD is a multifactorial disease that can significantly impact the economic prosperity and welfare of the farm industry [
16]. Diseases like this, caused by a complex of physiological and environmental stressors, precede farm admittance; for example, transportation, mixing with unfamiliar animals/herds, or exposure to viral microbial population agents can cause BRD [
33]. According to Blakebrough-Hall et al [
16], animals suffering from BRD had higher concentrations of alpha-glucose, acetone, BHBA, creatine, creatinine, ethanol, hydroxybutyrate, isobutyrate, isoleucine, isopropanol, leucine, mannose, phenylalanine, and pyruvate metabolites. In contrast, 1-methylhistidine, acetate, alanine, citrate, glucose, glutamine, glycine, glycoprotein acetyl, hydroxyisobutyrate, low-density lipoprotein (LDL), tyrosine, and valine metabolite were found in lower concentrations. In this study, BRD-associated metabolites were measured, except for alpha-glucose, citrate, ethanol, glutamine, glycoprotein acetyl, hydroxybutyrate, hydroxyisobutyrate, isobutyrate, isopropanol, LDL, phenylalanine, and tyrosine in the serum. Hence, metabolites measured in the serum in this study supplement the literature regarding the understanding of BRD-associated metabolites and could possibly be used to verify the occurrence of BRD in cattle.
The HS has become a major issue due to the acceleration of global warming. Exposure to HS, results in reduced milk production and quality in ruminants and makes them vulnerable to diseases [
34]. Such consequences could damage the livestock industry. Liao et al [
14] conducted a comparative study of metabolite changes in the blood and urine of three cattle breeds exposed to HS. In Xuanhan yellow cattle (XHC), glucose, lactate, and pyruvate associated with glycolysis and aconitate, citrate, and fumarate associated with the tricarboxylic acid (TCA) cycle were found in higher concentrations in the serum and urine [
14]. In Simmental×Xuanhan yellow crossbred cattle (SXC), asparagine, creatinine, glutamate, glutamine, ornithine, and urea associated with the amino acid metabolism, and aconitate, citrate, and fumarate associated with the TCA cycle were observed in higher concentrations. Finally, Jersey×Maiwa yak crossbred cattle (JMY), asparagine, creatinine, fumarate, glutamine, methionine, ornithine, phenylalanine, pyruvate, tyrosine, and urea associated with amino acid metabolism were found in higher concentration in the serum [
14]. Higher concentrations of aconitate, citrate, and succinate were found in the urine of the XHC and SXC breeds, higher concentrations of methylcitrate and methylmalonate (associated with TCA cycle) were found in the urine of the JMY breed [
14]. In this study, glycolysis was measured in relation to the metabolites in the serum. In addition, pyruvate and creatinine were measured as metabolites associated with amino acid metabolism, and succinate was measured as a metabolite associated with the TCA cycle in the serum. In addition, all metabolites associated with the TCA cycle were measured in this study, except for methylcitrate and methylmalonate. Hence, the metabolites measured in this study could potentially be used to verify the occurrence of HS in cattle.
Metritis is a uterine inflammation that occurs during the first three weeks post-parturition; it not only increases veterinary costs but also damages the livestock industry [
19,
35] as a result of lower reproductive efficiency, increased culling rates, and decreased milk production [
19]. According to Dervishi et al [
19], using urine from lactating dairy cattle, the candidate group of metabolites for the diagnosis of metritis consists of increased concentrations of 1,3-dihydroxyacetone, 3-aminoisobutyrate, acetylsalicylate, ascorbate, betaine, cysteine, galactose, glutamine, glycolate, guanidoacetate, hypoxanthine, lysine, N-acetylaspartate, O-phosphocholine, threonine, xylose, trans-aconitate, and π-methylhistidine metabolite and, decreased concentrations of uracil and urea metabolites. In this study, acetylsalicylate, betaine, galactose, glycolate, guanidoacetate, O-phosphocholine, urea, xylose, trans-aconitate, and π-methylhistidine metabolites were measured in the urine. Therefore, metabolites measured in the urine in this study could be used to verify the occurrence of metritis.