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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.45 No.1 pp.51-58
DOI : https://doi.org/10.5333/KGFS.2025.45.1.51

Influence of Tall Fescue Seed on the Gene Activity of Forestomach Epithelial Cells

Ki-Won Lee1, Do Hyung Kim2*
1Grassland & Forages Division, National Institute of Animal Science, RDA, Cheonan 31000, Korea
2Department of Animal Science, Gyeongbuk Provincial College, Yecheon 36830, Korea
* Corresponding author: Do Hyung Kim, Department of Animal Science, Gyeongbuk Provincial College, Yecheon 36830, Korea
Tel: +82-54-650-0343, E-mail: dhkim@gpc.ac.kr
February 14, 2025 March 14, 2025 March 17, 2025

Abstract


This study conducted to investigate potential differences in the activity of genes involved in volatile fatty acid (VFA) absorption, pH regulation, and energy metabolism in epithelial cells of forestomach administered either endophyte-infected (E+; 4.45 mg ergovaline/kg) or endophyte-free (E−) tall fescue seed. Twelve steers [body weight (BW) = 547 ± 9 kg] were fed alfalfa cubes at 1.5 × NEm and dosed 1 kg of ground tall fescue seed daily via rumen cannula for 21 days. On day 22, steers were slaughtered, and tissue samples were collected from the rumen, reticulum, omasum, and abomasum. Gene expression analysis revealed that monocarboxylate transporters (MCT), isoform 1 and MCT4 expression levels were significantly lower (p<0.05) in the rumen epithelium of steers dosed with E+ seed, while MCT2 expression remained unchanged. Similarly, sodium hydrogen exchanger (NHE), isoform 2 expression was significantly reduced (p<0.05) in the E+ seed, whereas NHE1 and NHE3 were unaffected by the seed treatment. Additionally, expression levels of down regulated in adenoma (DRA) and anion exchanger (AE), isoform 2 were lower (p<0.05) in the rumen epithelium of E+ steers, while putative anion transporter 1, sodium bicarbonate cotransporter, isoform 1, 3-hydroxy 3-methylglutaryl coenzyme A synthase, isoform 2, and putative anion transporter 1, sodium sodium potassium ATPase pump, isoform 1 expression levels were not influenced by the seed treatment. Notably, gene expression in the reticulum, omasum, and abomasum epithelia was unaffected (p>0.05) by seed exposure. These findings suggest that endophyte-infected tall fescue seed may impair ruminal VFA absorption in its dissociated state (pH > 5.8) by downregulating MCT1 and MCT4, along with suppressing NHE2, DRA, and AE2. Therefore, this mechanism may partially explain the reduced weight gain associated with fescue toxicosis in cattle.


Dsteer , VFA transporter , Gene expression , Tall fescue

초록

    Ⅰ. INTRODUCTION

    Fescue toxicosis is primarily linked to the presence of endophytic ergot alkaloids in tall fescue (Belesky et al., 1988). The endophyte synthesizes these alkaloids. A symbiotic relationship exists between the endophyte and tall fescue, where in the fungus synthesizes ergot alkaloids, such as ergovaline, which are responsible for the observed physiological effects (Zhang et al., 2012). These alkaloids induce hypoprolactinemia and vasoconstriction, leading to clinical manifestations such as dry gangrene (fescue foot) during winter (cold temperatures) and summer slump during hot temperatures (Aiken and Strickland, 2013).

    Ergovaline, a toxic ergot alkaloid produced by the endophytic fungus Epichloë coenophiala (formerly Neotyphodium coenophialum), is present in endophyte-infected tall fescue (Lolium arundinaceum) and is a primary contributor to fescue toxicosis in cattle. Previous research has demonstrated that an extract from endophyteinfected tall fescue seed alters rumen epithelial blood flow and reduces ruminal VFA flux (Foote et al., 2013). However, it remains unclear whether these changes in absorptive function are solely due to altered blood flow or if ergot alkaloids, such as ergovaline, directly affect VFA transport mechanisms at the epithelial level.

    It is hypothesized that ergot alkaloids from endophyte-infected tall fescue seed disrupt epithelial VFA transport systems in the bovine foregut, thereby reducing VFA absorption. Therefore, we conducted to determine whether gene expression related to VFA absorption, pH regulation, and energy metabolism differs between steers fed endophyte-infected and endophyte-free tall fescue seed.

    Ⅱ. MATERIALS AND METHODS

    All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC).

    1. Animals, treatments, and feeding

    Twelve Angus steers [initial body weight (BW) = 547 ± 9 kg] were surgically fitted with a ruminal cannula (Bar Diamond Inc., Parma, ID, USA) and blocked by initial body weight, then randomly assigned to six blocks, with two steers per block. Steers were fed alfalfa cubes once daily (07:00 h) at 1.5 × NEm based on BW. The alfalfa cube composition [on a dry matter (DM) basis] was as follows: crude protein (CP) = 16.5%; acid detergent fiber (ADF) = 37.2%; neutral detergent fiber (NDF) = 51.9%; NEm = 5.19 MJ/kg. A mineral premix (Kentucky Nutrition Service, Lawrenceburg, KY) was top-dressed onto the feed, containing: NaCl = 92%; Zn = 5500 mg/kg; Fe = 9275 mg/kg; Mn = 4790 mg/kg; Cu = 1835 mg/kg; I = 115 mg/kg; Se = 18 mg/kg; Co = 65 mg/kg. Steers were ruminally dosed (1 kg/d) with either endophyte-infected (E+, 4.45 mg ergovaline + ergovalinine/kg DM) or endophyte-free (E−) tall fescue seed throughout the 21-day treatment period. Tall fescue seed was ground using a grinder mixer with a 1.25 cm screen before being ruminally dosed immediately after feeding. The feeding and dosing schedule was staggered for each steer to ensure that only one animal was slaughtered per day and one block was processed per week. On day 22 of each experimental period, steers were slaughtered at the abattoir, and tissue samples were immediately collected for quantitative real-time PCR (qRT-PCR) analysis.

    2. Tissue harvest

    On day 22 of each experimental period, steers were slaughtered and immediately eviscerated to obtain forestomach tissues. The rumen (ventral rumen sac, where papillae are longest), reticulum, omasum, and abomasum were carefully separated, emptied of digestive contents, and thoroughly rinsed with ice-cold physiological saline to remove residual feed and debris. Following rinsing, epithelial tissues were meticulously dissected from the underlying layers on an ice-cold tray using scissors and forceps. Aliquots of epithelial tissue (1 g) were immediately homogenized in TRI reagent (Molecular Research Center, Inc., Cincinnati, OH), flash-frozen in liquid nitrogen, and stored at -80℃ until RNA analysis.

    3. RNA extraction

    Total RNA was extracted from the epithelium using the TRI reagent according to the manufacturer’s instructions (Molecular Research Center, Inc.). RNA was further purified using the RNeasy Cleanup Kit (Qiagen, Valencia, CA), which included an on-column DNase digestion step. RNA concentration was determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA) at 260 nm and 280 nm absorbances. RNA integrity was assessed using the Experion Automated Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, CA) with the RNA StdSens analysis kit, following the manufacturer’s guidelines. RNA integrity was evaluated based on the 28S/18S ribosomal RNA ratio, with quality considered sufficient if the 28S/18S ratio was >1.2 and the RNA Quality Indicator (RQI) was >8. In the current study, the mean RQI was 8.8 ± 0.08, indicating high RNA quality suitable for downstream analysis.

    4. Reverse transcription

    Complementary DNA (cDNA) was synthesized using the AccuScript First Strand cDNA Synthesis Kit (Agilent Technologies, La Jolla, CA) following the manufacturer’s instructions.

    5. Primer design

    Bovine-specific nucleotide sequences were retrieved from previously published sequences available in the National Center for Biotechnology Information (NCBI) database. Exon junction sites for each target gene were identified using the NCBI Splign tool (http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi). Primer pairs were designed using PrimerQuest software (Integrated DNA Technologies, Inc., Coralville, IA) to ensure optimal specificity and amplification efficiency. The sequences used for primer sets in this study are listed in Table 1.

    6. Quantitative real-time PCR

    Gene expression levels of monocarboxylate transporters, isoforms 1, 2, and 4 (MCT1, MCT2, MCT4, respectively), sodiumhydrogen exchangers, isoforms 1, 2, and 3 (NHE1, NHE2, NHE3, respectively), putative anion transporter 1 (PAT1), downregulated in adenoma (DRA), anion exchanger isoform 2 (AE2), sodium bicarbonate cotransporter isoform 1 (NBC1), 3-hydroxy-3-methylglutaryl-coenzyme A synthase isoform 2 (HMGCS2), and sodium-potassium ATPase pump (ATP1) were involved in VFA absorption, pH regulation, and energy metabolism in epithelial cells and evaluated using quantitative real-time PCR (qRT-PCR).

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for normalization. All reactions were performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Each sample reaction was run in triplicate on a single plate per gene. The reaction mixture (final volume 25 μL) contained 12.5 μL of SYBR Green Master Mix, 1.5 μL each of forward and reverse primers, and 2.0 μL of diluted cDNA. The qRT-PCR thermal cycling conditions consisted of an initial incubation at 50℃ for 5 min, followed by denaturation at 95℃ for 10 min and 40 cycles of 95℃ for 15 s and 60℃ for 1 min. Dissociation curves were generated to confirm the amplification of a single, specific product. The melt curve analysis was performed with an initial denaturation at 95℃ for 15 s, cooling to 60℃ for 20 s, and a final increase to 95℃ for 15 s. Relative gene expression was quantified using the relative standard curve method, with all target gene expression levels normalized to GAPDH expression. Standard curves for GAPDH were generated using a pooled cDNA sample across steers for each tissue type, serially diluted to final concentrations of 200, 100, 50, 5, 0.5, 0.1, and 0.05 ng/μL.

    7. Fescue seed

    The quantitative determination of ergot alkaloid concentrations in tall fescue seed was performed using ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). Analyses were conducted using an Acquity UPLC-TQD triple quadrupole mass detector (Waters, Inc., Milford, MA), following the method described by Foote et al. (2012).

    8. Statistical analysis

    Data from quantitative real-time PCR (qRT-PCR) were analyzed using analysis of variance (ANOVA) via the MIXED procedure of SAS (SAS Institute Inc.) for a completely randomized block design with a factorial treatment structure. The statistical model included block, steer, treatment, tissue site, and the treatment × tissue site interaction. Block and steer were considered random effects, whereas treatment, tissue site, and their interaction were treated as fixed effects. Data were analyzed for the main effects of treatment, tissue site, and their interaction (treatment × tissue site). Least squares means (LSMeans) were calculated, and differences between means were determined using the PDIFF option in SAS. To account for multiple comparisons, Tukey’s adjustment was applied, with significance set at α ≤ 0.05.

    Ⅲ. RESULTS

    The relative expression of MCT1 mRNA, normalized to GAPDH, was significantly lower (p<0.05, Table 2) in the rumen epithelium of steers administered endophyte-infected (E+) seed compared to those receiving endophyte-free (E−) seed. However, no significant differences (p>0.05) in MCT1 expression were observed in the reticulum, omasum, or abomasum between the treatment groups. Across tissue sites, MCT1 expression in the rumen was approximately 1.7-fold, 2.4-fold, and 276-fold greater than in the reticulum, omasum, and abomasum, respectively (p<0.05). The expression of MCT2 mRNA relative to GAPDH did not differ between E+ and E− treatments in any of the tissues examined. However, MCT2 expression was significantly greater in the abomasum compared to the other tissue sites (p<0.05), whereas no differences (p> 0.05) were detected among the rumen, reticulum, and omasum. For MCT4 mRNA, seed treatment influenced expression levels only in the rumen epithelium, where steers receiving E+ seed exhibited significantly lower MCT4 expression (p<0.05). In contrast, MCT4 expression in the reticulum, omasum, and abomasum remained unaffected by treatment (p>0.05).

    The expression of NHE1 mRNA did not differ between treatment groups in any of the tissues analyzed (p>0.05). Among all tissue sites, NHE1 expression was highest in the abomasum, while levels remained similar across other tissues. In contrast, NHE2 mRNA expression was significantly reduced in the rumen of steers dosed with E+ seed compared to those receiving E− seed (p<0.05). No treatment effects were observed in the reticulum, omasum, or abomasum. Across all tissues, NHE2 expression was highest in the rumen and lowest in the abomasum.

    The expression of PAT1 mRNA, normalized to GAPDH, did not differ between seed treatments in any of the tissues analyzed (p>0.05). Across tissue sites, PAT1 expression was lowest in the abomasum and reticulum, while expression levels in the rumen and omasum were similar. Expression levels of DRA and AE2 mRNA were significantly lower in the rumen epithelium of steers receiving E+ seed compared to those fed E− seed (p<0.05). DRA expression was virtually undetectable in the abomasum, whereas AE2 expression was lowest in the abomasum but present across all tissue sites.

    The expression of NBC1 mRNA relative to GAPDH was not significantly affected by seed treatment (p>0.05). However, NBC1 expression was highest in the omasum (p<0.05), with lower levels detected in other tissues. For HMGCS2 mRNA, no differences were observed between E+ and E− treatments (p> 0.05). Expression was highest in the rumen, followed by the reticulum and omasum, with near-zero expression in the abomasum. Similarly, ATP1 mRNA expression did not differ between seed treatments (p>0.05). Among tissue sites, expression was greatest in the ruminal epithelium, with lower levels detected in other regions of the forestomach.

    Ⅳ. DISCUSSION

    The clinical manifestations of fescue toxicosis, such as reduced feed intake and body weight gain, are primarily attributed to the action of ergot alkaloids, which act as serotonergic receptor agonists (Dyer, 1993;Wagner, 2008). Activation of these receptors is associated with a decrease in feed intake, likely due to increased satiety (Simansky, 1996). In addition to their effects on appetite regulation, ergot alkaloids cause vasoconstriction in the ruminal (Foote et al., 2011) and mesenteric (Egert et al., 2014) vasculature, leading to reduced blood flow and decreased VFA absorption from the reticulorumen (Foote et al., 2013). The present study aimed to further investigate the relationship between fescue toxicosis and VFA absorption by evaluating the effects of E+ seed dosing on the expression of genes involved in VFA transport.

    Plasma membranes are relatively impermeable to anions, necessitating carrier-mediated transport mechanisms to facilitate their movement across both the luminal and basolateral membranes of epithelial cells (Rechkemmer et al., 1995). Recent studies have shown that monocarboxylate transporters (MCTs) are critical for the absorption of volatile fatty acids (VFAs) in gastrointestinal tract, with evidence indicating that MCTs paly a central role in VFA absorption in both monogastric animals and ruminants. MCTs function as cotransporters of monocarboxylate anions and protons, with multiple isoforms— MCT1, MCT2, and MCT4—known to mediate VFA transport across epithelial membranes (Halestrap and Meredith, 2004).

    NHE1 plays a key role in modulating extracellular pH within the stratum granulosum, promoting the formation of undissociated VFAs, which are more readily absorbed via passive diffusion (Graham et al., 2007). In addition, NHE1 and NHE3 are involved in proton (H⁺) export from epithelial cells, accounting for 50% and 20% of total proton efflux, respectively (Etschmann et al., 2006). Under normal ruminal pH conditions, approximately 90% of VFAs exist in a dissociated state, limiting the extent of passive diffusion. Furthermore, once protonated VFAs cross the apical membrane, they rapidly dissociate due to the intracellular pH of approximately 7.4. In response to intracellular acidification, NHE2 helps maintain intracellular pH homeostasis by exporting H⁺ ions (Connor et al., 2010).

    MCTs facilitate the elimination of protons from epithelial cells by co-transporting dissociated VFAs, lactate, and ketones with H+ into the bloodstream (Graham et al., 2007). In addition to MCTs, other transporters involved in regulating intracellular pH include sodium/hydrogen exchangers (NHE), which export protons either back into the lumen or into extracellular spaces (Connor et al., 2010). In the present study, the observed reduction in MCT1 and MCT4 expression in E+ steers was associated with decreased VFA absorption, which may have, in turn, contributed to the downregulation of NHE2 in the rumen epithelium. Given that MCT1 and MCT4 are essential for VFA transport, their decreased expression could have limited VFA uptake, subsequently reducing the demand for proton export via NHE2. This finding aligns with prior research demonstrating that NHE1 is predominantly localized to the apical plasma membrane of the stratum granulosum, with its expression decreasing in deeper layers toward the bloodstream, whereas NHE2 is distributed throughout the basale, spinosum, and granulosum strata (Graham et al., 2007). Since NHE1 activity promotes proton export into the rumen, lowering local pH near the stratum granulosum, this may increase the proportion of undissociated VFAs, which could facilitate greater VFA absorption via passive diffusion (Graham et al., 2007;Connor et al., 2010). However, given the multiple factors influencing VFA absorption mechanisms, it remains uncertain whether E+ seed dosing directly alters the uptake of undissociated VFAs.pH regulation.

    Sodium-potassium ATPase pump plays a critical role in facilitating NHE function by establishing the electrochemical gradients necessary for cellular transport and substrate exchange across epithelial membranes (Zouzoulas et al., 2005;Albrecht et al., 2008). The Na⁺-motive force generated by ATP1 is essential for sodium absorption, and its activity is indirectly linked to VFA transport via NHE-mediated proton exchange (Sehested et al., 1996), which is critical for maintaining pH balance. Since NHE and ATP1 work together to regulate intracellular pH, it was hypothesized that ATP1 expression would be lower in steers dosed with E+ seed, due to the reduced demand for proton exchange associated with VFA uptake into the rumen epithelium (Albrecht et al., 2008;Müller et al., 2002). However, this expected decrease in ATP1 expression was not observed in the current study. This finding suggests that ATP1 expression may not be as tightly regulated by NHE activity in response to E+ seed treatment as initially expected, indicating a more complex interaction between these transporters under altered physiological conditions.

    In addition to NHE, bicarbonate transport plays a crucial role in maintaining intracellular pH homeostasis. Bicarbonate export is often coupled with proton extrusion via NHE, facilitating acid-base balance within the rumen epithelium. Although the precise localization of DRA and AE2 in the rumen remains unclear, these transporters may contribute to bicarbonate secretion between the blood and the ruminal epithelium (Jacob et al., 2002;Petrovic et al., 2002). Under in vivo conditions, bicarbonate secretion by the rumen epithelium is strongly influenced by intraluminal VFA concentrations, suggesting the involvement of an anion exchanger that facilitates VFA⁻/HCO₃⁻ exchange (Gäbel et al., 1991). Among the four anion exchanger isoforms (AE1–4), AE2 has been identified as a key contributor to intracellular pH homeostasis, particularly in response to rising intracellular pH or hypertonicity (Humphreys et al., 1994, 1995;Alper et al., 2001). AE2 is predominantly expressed on the basolateral membrane of gastrointestinal epithelial cells, whereas DRA and PAT1 are localized on the apical membrane, where they function in chloride absorption and bicarbonate secretion (Connor et al., 2010). In addition to regulating acid-base balance, these transporters also participate in the import of dissociated VFAs (Bilk et al., 2005), further highlighting their role in maintaining ruminal epithelial function under varying dietary conditions.

    3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 is a mitochondrial enzyme involved in β-hydroxybutyrate (BHBA) synthesis, an essential component of ketogenesis. Previous research has suggested that HMGCS mRNA abundance is positively correlated with BHBA production, although specific isoforms were not distinguished (Lane et al., 2002). In the present study, despite the higher ruminal butyrate concentrations in E+ steers compared to E− steers, HMGCS2 mRNA abundance in the rumen and reticulum epithelium exhibited a tendency to decrease in E+ steers. Given that a significant proportion of butyrate is metabolized to BHBA, which is subsequently exported via MCTs located on the basolateral membrane (Sehested et al., 1999;Müller et al., 2002;Graham et al., 2007), the reduced expression of HMGCS2 observed in this study may indicate a potential decline in ketogenesis. This finding is consistent with the concurrent downregulation of MCT expression in the ruminal epithelium, indicating that E+ seed treatment may have contributed to both reduced butyrate transport and a subsequent decrease in ketone body synthesis.

    Ⅴ. CONCLUSIONS

    Endophyte infection in tall fescue is associated with the production of toxins, such as ergovaline, which negatively impact ruminant performance. While several physiological alterations in ruminants consuming endophyte-infected tall fescue have been identified, the precise mechanisms underlying reduced animal performance remain incompletely understood. Recent findings suggest that endophyte-infected tall fescue seed influences gene expression patterns, particularly by downregulating the expression of MCT1 and MCT4, which are associated with NHE2, DRA, and AE2. This downregulation may contribute to the reduced weight gain observed in cattle suffering from fescue toxicosis.

    Ⅵ. ACKNOWLEDGMENTS

    This study was supported by the RDA Fellowship Program of National Institute of Animal Science, and Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01592504), Rural Development Administration, Republic of Korea.

    Figure

    Table

    Primer sets for quantitative real-time PCR analysis

    Gene expression (normalized to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) by forestomach of Angus steers (n = 6 per treatment) dosed with endophyte-free tall fescue seed (E-) or endophyte-infected tall fescue seed (E+)

    MCT1, MCT2, and MCT4, Monocarboxylate transporter, isoforms 1, 2, and 4; NHE1, NHE2, and NHE3, Sodium hydrogen exchanger, isoforms 1, 2, and 3; PAT1, Putative anion transporter 1; DRA, Down regulated in adenoma; AE2, Anion exchanger 2; NBC1, Sodium bicarbonate cotransporter 1; HMGCS2, 3-hydroxy-3-methylglutaryl coenzyme a synthase 2; ATP1, Sodium potassium ATPase pump 1.
    Data are expressed as least squares means ± SEM.

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