The receiving period is a stressful event in the life of beef cattle, where cattle are subjected to transportation, comingling, potential pathogen exposure, and decreased feed intake (Duff and Galyean, 2007). Combined, these stressors ultimately contribute to decreased immune defenses, leaving cattle vulnerable to illnesses such as Bovine Respiratory Disease (BRD; Rice et al., 2007; Taylor et al., 2010). Continuous activation of the immune response creates a substantial stress scenario in vivo, further contributing over time to the suppression of cellular immunity (Nui et al., 2022). As the body attempts to remedy illness through activation of the immune response, oxidative bursts, as a product of phagocytosis by immune cells, result in a rapid increase of reactive oxygen species (ROS) which disrupt a key balance between endogenous antioxidants and pro-oxidants (Gorski et al., 2012).

Methionine is an essential but often considered a limiting amino acid in the diet of cattle due to rumen degradation and/or composition of feedstuffs (Richardson and Hatfield, 1978; Waterman et al., 2012). However, when accessibility is achieved, methionine has been shown to not only improve and regulate some metabolic processes but also mitigate oxidative stress through the activation of vital endogenous antioxidants, like glutathione (Lopes et al., 2019). Using ruminally-protected methionine, research has indicated benefits to the intestinal environment, improved milk production, and increased immune benefits in all ages as an inflammation modulator (Abdelmegeid et al., 2017; Barker et al., 2024; Motta et al., 2025). Nonetheless, the benefits of a ruminally-protected methionine supplement have not been evaluated thoroughly in beef cattle, specifically during the receiving period.

Oxidative stress occurs in the body with an imbalance between pro-oxidants and antioxidants, which favors pro-oxidants. Severe oxidative stress has been demonstrated to reduce feed efficiency, increase protein degradation, and further contribute to a reduction in meat quality (Bottje et al., 2006; Russell et al., 2016). Additionally, metabolomic analysis has risen in popularity in recent years as a way to understand metabolic differences and to classify, metabolic changes as potential biomarkers, which may further be used to define oxidative stress. Therefore, the primary objective of this study was to determine the oxidative status in the plasma of receiving beef calves following lipopolysaccharide administration to individual calves supplemented with a methionine analogue and to explore the novelty of metabolomics analyses in beef cattle. A second objective sought to explore the plasma metabolites expressed in response to LPS administration.

The role of the antioxidant defense system is to scavenge free radicals and destroy oxidants. However, a deficiency, or absolute depletion, of these defenses can contribute to oxidative stress (Benzie and Strain, 1996). The FRAP assay works by analyzing the ability of an antioxidant to donate hydrogen to an oxidant (Huang et al., 2005). In this case, it can be speculated that the decline in Fe ²⁺ beginning 2 h post-LPS administration is due to the sacrificial action of antioxidants working in the calf. As free radicals begin to increase in the body due to immune activation, the system is flooded with prooxidants. The antioxidants must then work to control propagation by donating hydrogen atoms and stopping the chain reaction (Sies, 1993).

The body is naturally equipped with endogenous antioxidants to fight off and scavenge for ROS. These natural antioxidants include glutathione, superoxide dismutase, glucose 6-phosphate dehydrogenase (G6PD), or other metal-binding proteins and vitamins (Ellah, 2010). Many of these systems work in a redox fashion, supplying each other with the means to successfully maintain ROS invasion by donation of hydrogen atoms or “attacking” free radicals. While a decline in antioxidant capacity, or ferric-reducing ability as in this study, may be seen as a negative result at first glance, it may be demonstrating the ability of this system to decrease ROS production following an acute phase response. While antioxidant capacity was slow to return to initial values, it did increase over time following LPS administration, displaying the body’s natural defense system and return to homeostasis. Yi et al. (2025) observed increased endogenous antioxidant concentrations in serum, such as superoxide dismutase, glutathione, and catalase, following supplementation of guanidinoacetic acid and methionine. Similar results were seen in liver tissues with increased superoxide dismutase, glutathione, and catalase concentrations following supplementation. While the animals in Yi et al. were not exposed to any immune challenges, methionine may increase the body’s ability to produce its own protection.

In general, TBARS serve as an adequate marker of lipid oxidation. However, when analyzed in plasma, TBARS serves as an indicator of oxidative stress and damage, rather than just oxidized lipids (Armstrong and Browne, 1994). In times of stress, whether psychological or physiological (i.e., illness, disease, parturition, etc.), the body is depleted of its natural antioxidants, leaving reactive metabolites and lipid oxidative products, such as MDA, to increase rapidly. When compared to the results of the FRAP assay, the inverse relationship is indicative of an imbalance between pro- and antioxidants, favoring pro-oxidants. This further suggests that the opportunity for oxidative stress was greatest early in the response to LPS, but restoration of antioxidant systems, as indicated by FRAP values, allowed a return to homeostasis for TBARS values. However, it is important to recognize that reactive metabolites, contributing to the production of MDA as a secondary lipid oxidation product, may also be generated by phagocytosis (Myolonas and Kouretas, 1999). While initial assumptions may suggest severe oxidative stress 2 h post-LPS administration, a portion of the MDA produced may be a necessary response to neutralizing the LPS present following administration.

Miao et al. (2021) determined that birds kept in high-stocking-density housing had reduced activity of glutathione peroxidase and increased concentrations of MDA compared to those at normal stocking density. However, the study further recognized that with the addition of methionine, signs of oxidative stress were reduced as GSH/GSSG ratios increased. Further still, evaluation of methionine and oxidative stress in normal and intrauterine growth restricted (IUGR) piglets showed reduced MDA and cellular apoptosis with methionine supplementation (Su et al., 2017). An additional study evaluating broilers and stocking density found that methionine supplementation was similar to vitamin E, selenium, and astaxanthin as antioxidant sources in broiler diets, as it decreased MDA concentrations in muscle tissues, attenuating lipid oxidation (Magnuson et al., 2020).

While the current study did not find differences based on methionine supplementation on lipid oxidation products in plasma, it is worth noting that dietary supplementation of amino acids, like methionine, have been observed to suppress the side effects of lipid oxidation products like MDA (Sun et al., 2016). Methionine, and other sulfur-contain amino acids like cysteine, are capable of scavenging for free radicals, making them ideal possibilities in feeding regimens to reduce oxidative stress (Atmaca, 2004; Chen et al., 2013). Methionine can be converted into cysteine, which then serves as a precursor for the powerful antioxidant, glutathione (Miller, 2003). Further, MDA concentrations were reduced in both serum and liver samples of animals supplemented with a guanidinoacetic acid and methionine mix (Yi et al., 2025). Therefore, methionine supplementation may be of value when formulating a transition diet for receiving calves.

While still considered a ROS, hydrogen peroxide has less reactivity compared to other reactive species (Konno et al., 2021). Nonetheless, it has an extreme ability to pass through cell membranes, having greater potential for reacting with other compounds, such as iron and hemoglobin (Maurya, 2014). When white blood cells destroy pathogens in the body, they do so through oxidants such as nitric oxide, superoxide, and hydrogen peroxide (Maurya, 2014). Commonly referred to as the main contributors to oxidation, ROS play an important role in immune health and help the body fight toxins and disease (Maurya, 2014; Bassoy et al., 2021). This process explains the spike in ROS concentration following LPS administration and the return to basal concentrations following recovery. Production of ROS has been recognized as a result of normal cellular metabolism, arising from mitochondrial respiration, phagocytosis in an immune response, or Fenton Reactions. Furthermore, ROS are produced in a controlled manner for numerous physiological processes in the body, such as signaling of growth factors and cytokines, or even activating apoptosis (Brambilla et al., 2008). Nonetheless, when produced in excess, oxidative stress may occur, resulting in enzyme impairment, DNA damage, or lipid peroxidation (Ellah, 2010).

Methionine has often been considered a limiting amino acid in dairy cows, specifically when it comes to milk protein production, and may be added to the diet. In general, plant-based feeds are limited in methionine, and sufficient microbial breakdown of proteins in the rumen further add to the limitations of methionine without supplementation (Brake et al., 2013; Lopes et al., 2019; Costa et al., 2025). Rumen-protected methionine has been demonstrated to reduce oxidative stress in dairy cows during the transition period (Wu et al., 2013), as well as increase immune response capabilities. Further, methionine acts as a precursor for vital endogenous antioxidants, such as glutathione (Brosnan and Brosnan, 2006). In times of stress, disease, or other scenarios where insufficient redox reactions and antioxidant function occur, methionine may be vital to the diet. In the current study, calves receiving methionine appeared to show a milder reaction to LPS, based on ROS production alone. Additionally, calves receiving methionine had a second peak in ROS production sooner than control calves, suggesting a quicker recovery and increased immune response capability, as described by Wu et al. (2013). As the receiving period for beef calves often results in significant immune challenges, as well as physical and psychological distress, rumen-protected methionine may provide an immunity boost, which protects against the production of ROS in vivo.

As metabolomic analyses have risen in popularity in recent years, and the database of metabolites continues to grow every day, control samples were selected in the current study in order to identify potential markers of inflammation that may be driving differences in response to LPS during the most crucial time points (as observed in the oxidative stress parameters measured). The time points selected were based on all other oxidative stress assays within the current study. Taurocholic acid was the primary driver of differences between pre- and post-LPS samples. Taurocholic acid is a bile salt composed of taurine (synthesized from methionine) and either primary or secondary bile acids (Elliot and Hyde, 1971; Blaschka et al., 2020). Bile acids are synthesized in the liver using cholesterol (Russel, 2003). As the functional component of bile, bile acids aid in the emulsification of nutritional components (Russel, 2003). While beneficial to digestion, high concentrations may be considered cytotoxic, although human studies show bile acids can be excreted in urine (Blaschka et al., 2020). Further, bile acids are responsible for cholesterol catabolism and approximately 50% of daily cholesterol turnover (Insull, 2006). In human and animal studies (Uchida et al., 1985; Brufau et al., 2010), bile acid concentrations have the potential to alter glucose homeostasis, where insulin and glucose play a role in modulation of bile acid synthesis (Michael et al., 2000; Duran-Sandoval et al., 2004). Bile acid synthesis may also help modulate glucose absorption in the intestine as deficient mice have exhibited delayed glucose absorption (Van Dijk et al., 2009).

Lysophospholipids (LPL) are derived from phospholipids by either enzyme or ROS induction (Fuchs et al., 2012). While the results of the current study appear to disagree, LPL tends to increase under inflammatory conditions (Fuchs et al., 2012). The formation of LPL result from cellular stress and phospholipid disturbances (Pena et al., 1997). Lysophospholipids are often produced and participate in the process of inflammation, including redness, swelling, heat, pain, and loss of function (Serhan et al., 2020). Interestingly, LysoPE (18:3) was greatest in cattle at -2 h and decreased from 2 and 8 h. This may suggest inflammation was greater prior to the challenge, and LPS application did not contribute to the presence of LysoPE (18:3), although difficult to determine completely based on the time points evaluated. Nonetheless, this increase in LysoPE may be as a result of the jugular catheter placed prior to the study. Lipophosphatidic acid (LPA) and other LPL have been evaluated as potential therapeutic targets, showing significant potential in modulating wound healing, specifically of endothelial cells (Sturm et al., 1999; Lee et al., 2000). Therefore, this increase may actually be specific and unrelated to the challenge itself. In human studies, reduced concentrations of LPL are associated with sepsis or poor outcomes following bacterial and viral infections (Frej et al., 2016; Song et al., 2020). Therefore, the current study contradicts published literature, although it would appear that the lack of LysoPE metabolites during the challenge may have been beneficial.

Butyric acid (BA) is a 4-carbon fatty acid influencing morphology, growth, and gene expression in mammals (Prasad, 1980). In a study evaluating the rumen conditions of cattle exposed to heat stress, isobutyric acid concentrations were increased (Sales et al., 2021). When supplemented in the diet, BA helps regulate performance and inflammation (Weber et al., 2014). Weber et al. (2014) further recognized that BA, when combined with humic acid, decreased serum cortisol by 62% in weanling pigs administered LPS. The aforementioned study also suggested the combination of BA and humic acid in the diet of pigs can mitigate oxidative stress. Butyric acid was most prevalent in samples at 2 h and intermediate in 8 h samples, suggesting a potential protective effect occurring during the response to LPS administration.

Pathway analyses further highlight the body’s defenses against oxidative stress. Prior studies have recognized the function of arginine (Arg) against oxidative stress (Southern and Baker, 1983; Suschek et al., 2003; Lin et al., 2008), while Zheng et al. (2017) recognized Arg synthesis decreased in piglets experiencing oxidative stress. Arginine itself has been recognized as an important amino acid in the protection and development of the small intestine of pigs, specifically by improving villus morphology in the jejunum (Zheng et al., 2017). Arginine also had a protective effect on intestinal integrity, via attenuation of LPS-induced inflammation and inhibition of tight-junction protein downregulation (Lan et al., 2020). The study further suggested that arginine on its own aids in enhancing disease resistance by inhibiting numerous other pathways related to immune stimulation and proinflammatory conditions. Data also suggests that arginine, as an essential amino acid, is beneficial for gastrointestinal, liver, and immune functions, as well as enhancing insulin sensitivity, amongst other functions (Wu et al., 2008). This pathway may be prevalent in the data of the current study because of the role of arginine in aiding the body in defense against immune stimulation, especially with LPS. Arginine appears to play an important role in health and immune defense.

Interestingly, Arg is synthesized from proline, as well as glutamine and glutamate, making the Arg and proline metabolism pathway another key element in the pathways driving differences in the current data set. Proline is a particularly fascinating amino acid, as it is the only proteogenic secondary amino acid, and is metabolized by a family of enzymes that respond to metabolic stress and signaling (Phang, 1985; Phang et al., 2008, 2010). Proline may also be interconvertible with glutamate and Arg, where the conversion between Arg and proline is an important step in the urea cycle (Adams, 1970; Phang et al., 2010). In contrast to previous research, a new theory has emerged suggesting the proline metabolic pathway is mobilized under conditions of stress (Phang et al., 2010). It is further suggested that proline oxidase plays a vital role in cellular energy maintenance for survival, where glucose deprivation contributed to an inverse increase in proline oxidase (Pandhare et al., 2009). These data in the current study, as well as others, can be considered further, where arginine was the only amino acid affected by methionine supplementation when evaluating amino acid concentrations in plasma. On their own, methionine and arginine have strong capabilities in preventing oxidative stress through the production of glutathione and exerting anti-inflammatory properties, respectively (Dai et al., 2020).

In an in vitro study evaluating β-alanine (βA) metabolism, the non-essential amino acid increased cellular oxygen consumption, thus improving oxidative metabolism (Schnuck et al., 2016). In human applications, βA is often supplemented for athletes to prolong exercise duration (Carpentier et al., 2015). Further, supplementation of βA in the diets of weaned piglets has been shown to alleviate the inflammatory response generated during stress (Chen et al., 2022). Lactate, previously considered a waste product of metabolism, may also be used in endocrine signaling (Brooks, 1985; 2009). Lactate and its associated H⁺ ions tend to further inhibit glycolysis of immune cells and suppress glucose uptake and intracellular ATP levels in monocytes (Caslin et al., 2019). Therefore, this metabolic pathway may ease the potential damages elicited by the immune response during an endotoxin challenge.

Histidine is one of the body’s essential amino acids and is a gluconeogenic amino acid (Brosnan and Brosnan, 2020). Histidine may be metabolized into histamine via decarboxylation (Brosnan and Brosnan, 2020). Research in rats recognized that increases in serum histidine contributed to increases in histamine synthesis, specifically in the brain (Prell et al., 1996). It was later determined that, in the brain, histamine derived from histidine controls various functions, including stress response (Yoshikawa et al., 2019). While it is also well-recognized that metal ions, like iron, promote the production of free radicals, histidine may help attenuate their effects (Smolik et al., 2010). Therefore, histidine metabolism may be another key effort in the body’s attempts to manage not only immune stress, but also oxidative stress. The pathways present within the current study may aide in elucidating the events which occur to maintain immune responsiveness, prevent oxidative stress, and ultimately, return the body to homeostasis following LPS administration.

In summary, these data suggest that oxidative stress indicators from an activated immune response may be mitigated with the addition of supplemental methionine, suggesting expedited recovery of stressed animals during the receiving period. While a complete elimination of oxidative stress markers from the body is not feasible, some within the current study, specifically ROS, show marked reductions in concentrations with supplementation. Further, while prior work on methionine supplementation showed no effect on lipid oxidation products or antioxidant reducing activity, the impact on ROS could be sufficient to slow the effect of immune dysfunction on other plasma indicators. Metabolomic data further demonstrate a clear distinction between pre- and post-LPS administration metabolites present in vivo, where protective measures may naturally be activated. Ultimately, these data demonstrate increased oxidative stress products in vivo following an immune challenge, as well as their subsequent recovery between 24 to 48 h. These data will be important in further predicting and understanding the potential damage to the growth and development of cattle during the receiving period.