Tall fescue is the major forage used to feed cattle in pasture-based systems of the southeast and covers approximately 14 million hectares across the United States [1
]. The advantage of tall fescue is hardiness of the plants attributed to the presence of a fungal endophyte (Epichloë coenophialum
, formerly known as Neotyphodium coenophialum
and Acremonium coenophialum
) living in a mutualistic relationship with the plant [2
]. However, the endophyte produces ergot alkaloids that are toxic to animals that consume them for an extended period of time [3
]. Ergot alkaloids are able to bind biogenic amine receptors on blood vessels, resulting in vasoconstriction throughout the body [4
]. This results in a condition known as fescue toxicosis, which is commonly observed by the animal’s inability to thermoregulate [7
], poor reproductive performance [1
], and reduced average daily gain [8
], significantly reducing overall animal performance. Therefore, researchers are tasked with identifying management methods and therapeutics to alleviate these consequences to cattle producers.
Pasture management methods have been evaluated for reducing the impact of fescue toxicosis in cattle. Inter-seeding of legumes, such as red clover, to mitigate the effects of fescue toxicosis has proved beneficial in cattle grazing endophyte-infected tall fescue [9
]. Recent research has found phytoestrogenic compounds, known as isoflavones, present in red clover may be responsible for reducing the effects of fescue toxicosis. Isoflavones act as an agonist on the β-adrenergic receptors present on blood vessels to promote vasodilation [10
], reversing the effects of ergot alkaloid induced vasoconstriction. Additionally, isoflavones act as a natural antibiotic selective against hyper-ammonia-producing bacteria (HAB) and some cellulolytic and amylolytic bacteria [11
]. The reduction of ammonia levels as a result of less HAB in the rumen allows more amino acids to be absorbed and used by the ruminant, while altered celluloytic and amylolytic bacteria can influence the production of volatile fatty acids for energy. Therefore, the increase in blood flow and altered rumen fermentation may improve nutrient delivery and utilization for host metabolic processes contributing to animal growth.
The objective of the present study is to evaluate the effect of isoflavone supplementation with tall fescue seed consumption on beef steer’s rumen and serum metabolomes. Ruminal and circulating metabolites may provide insights into altered bacterial and host metabolic functions that improve steer performance on endophyte infected tall fescue with the administration of isoflavones.
The overall reductions in animal performance due to fescue toxicosis are estimated to cost the cattle industry over $
2 billion annually [13
]. Therefore, it is vital to discover management methods to reduce the impact and improve the efficiency of beef production. The objective of the current study was to use untargeted metabolomics to evaluate tall fescue seed and isoflavone consumption effects on metabolic intermediates, outputs, and pathways in the rumen and serum.
The metabolomes of the rumen and circulatory environments were first compared, independent of treatment groups, which observed distinctly unique metabolomes according to principal coordinate and abundance analyses. Highly abundant metabolites in each environment were not shared or only present in low abundances between the two environments. The metabolites identified between these different body systems are likely a result of the specific physiological functions of each system in the ruminant. The microbiome is a major contributor to rumen metabolome, as it supplies over 70% of the ruminant’s required nutrients [15
]. These microbes are highly metabolically active in order to break down feedstuffs and release metabolites to complement host metabolism of which metabolites originate from the plants and other feedstuffs consumed [16
]. Therefore, the majority of metabolites identified in the rumen are of xenobiotic origin. By evaluating the rumen metabolome, the effects of tall fescue seed and isoflavone consumption on rumen microbial metabolic processes can be inferred. The serum metabolites, however, are a result of absorbed metabolic products from the rumen and other organs. Tissues throughout the body produce intermediary metabolites from protein, carbohydrate, and lipid metabolism for energy production to perform physiological functions. These metabolites are then absorbed into the blood and can travel through the circulatory system to other tissues for further catabolic or anabolic processing. Therefore, the metabolome of the circulatory system is typically dominated by endogenous metabolites. Because of this systemic nature, metabolites in blood have been used as potential biomarkers to predict feed utilization [17
] and production parameters [18
], as well as evaluate responses to disease [19
] and stress [21
]. Evaluating the serum metabolome will determine the systemic metabolic response to tall fescue seed and isoflavone supplementation. Together, the effects on individual metabolites and metabolic pathways in rumen fluid and serum will determine how alterations in microbial and host metabolism contribute to symptoms of fescue toxicosis or the benefits isoflavones may contribute to mitigate these detrimental impacts.
Reductions in average daily gain and delayed development of beef cattle are a major consequence of fescue toxicosis [8
]. The rumen microbiome is crucial for providing nutrients needed by the host for energy requirements and muscle development. As the microbiome has previously been shown to be affected by consuming endophyte infected tall fescue [12
], the metabolites and other products produced by the rumen microorganisms may be altered, potentially contributing to reductions in growth and feed efficiency. The metabolites produced by the rumen microorganisms are a result of the richness of the rumen microbiome. Several of these metabolites released may be related to the consumption of ergot alkaloids concentrated on the endophyte infected tall fescue seed. Many of the rumen metabolites have a relationship with purine, carbohydrate, and nucleic acid metabolism, such as hypoxanthine, xylose, and uracil, respectively [24
]; these metabolites are related to feed efficiency parameters. Clemmons et al. [26
] found that these metabolites are bio-indicators of feed efficiency in cattle showing low residual feed intake. Interestingly, they are negatively correlated to seed type in the rumen fluid of the current study; it is evident that animals are being affected by the detrimental symptoms of tall fescue toxicosis, failing to gain weight, and being less feed-efficient. Additionally, we did not observe a large number of different metabolites because of the reduction of the rumen microbiome. As a normal rumen environment, the significant presence of other metabolites, which are crucial for the ergot alkaloids metabolism and production of volatile fatty acids, was expected and improves feed efficiency and rumen microbial richness.
Vasoconstriction, induced by ergot alkaloids, occurs throughout the body, resulting in multiple observed symptoms of fescue toxicosis [4
]. Specifically, the contractility of the mesenteric vasculature surrounding the digestive tract is affected by the consumption of ergot alkaloids, potentially affecting nutrient absorption and subsequent host metabolism [5
]. However, the consumption of isoflavones promotes vasodilation to increase blood flow and mitigate fescue toxicosis effects [10
]. Ideally, oxygen and nutrient delivery to tissues is improved, thus benefiting host metabolism. Evaluating serum metabolites during induced fescue toxicosis and treatment with isoflavones may indicate the changes in metabolism systemically. The serum metabolome in the current study was greatly affected by isoflavone treatments with complete separation of animals’ global serum metabolomes between treatment groups; no metabolites differed as a result of seed type. Citrulline was identified as having the greatest influence on serum metabolomes between isoflavone treatment groups. Citrulline is an intermediary metabolite in the urea cycle, a metabolic process crucial for providing non-protein nitrogen to the ruminant [27
]. As isoflavones inhibit hyper-ammonia producing bacteria in the rumen, this reduces the amount of protein degradation, leading to decreased ammonia and nitrogen availability [11
]. Multiple metabolic pathways related to the urea cycle such as arginine biosynthesis and metabolism, pyrimidine metabolism, and nitrogen metabolism were affected in serum metabolites by isoflavone treatments. Therefore, the effect of citrulline on the serum metabolome due to isoflavone treatment may be a result of changes in the urea cycle by improving protein availability to the ruminant for muscle development.
Pantothenate was considered to be a major contributor to differences observed in the global serum metabolome among all treatment combinations, but was significantly higher in abundance in steers receiving isoflavone treatment. Previous studies have indicated different levels of pantothenate in the serum of animals differing in feed efficiency, with more feed efficient animals having greater serum levels of pantothenate [17
]. Pantothenate is a key intermediary metabolite for the formation of Coenzyme A, which is crucial for amino acid and lipid metabolism for ruminant muscle development [29
]. Additionally, the majority of metabolic pathways affected by isoflavone treatment in the current study were related to amino acid metabolism and biosynthesis. As animals experiencing fescue toxicosis often have low average daily gains [8
], the use of isoflavone supplementation may mitigate the weight gain and growth consequences of fescue toxicosis. The greater amount of available pantothenate in the serum of animals consuming isoflavones, with changes in the urea cycling of the ruminant, may improve growth and muscle development in steers affected by fescue toxicosis.
A classic symptom of fescue toxicosis is a significant reduction of prolactin secretion; this is due to the similar homology of ergot alkaloids with the neurotransmitter dopamine. Ergot alkaloids will act as an agonist by binding dopamine receptors, preventing the release of prolactin [3
]. Tyrosine is a precursor for the generation of the neurotransmitter dopamine [31
]. The current study found tyrosine metabolism to be affected by seed type in the rumen; tyrosine metabolic pathways are influenced as the signals for dopamine production are reduced during fescue toxicosis. Additionally, the study found tryptophan metabolism was affected by seed type in the serum. Tryptophan is a key amino acid in regulating protein synthesis, specifically in muscle development, of multiple species [32
]. The effects of tryptophan on muscle development are through the IGF-1 pathway [33
]. Tryptophan is a precursor to the neurotransmitter serotonin, which stimulates the production of IGF-1 [34
]. Supplementation of rumen protected tryptophan improved weight gain and feed efficiency of ruminants [35
]. The observed impact of seed type on tryptophan metabolism in the serum may indicate reduced production of serotonin and subsequent IGF-1 signaling for muscle development. Together, the impacts of fescue toxicosis on tyrosine and tryptophan metabolism were also observed previously in the plasma of steers consuming endophyte-infected or endophyte-free seed [37
]. Therefore, tall fescue seed consumption alters neurotransmitter development, leading to commonly observed symptoms of fescue toxicosis.