Ammoniation to Reduce the Toxicity of Endophyte-Infected Tall Fescue Seed fed to Rats

DRUG AND CHEMICAL TOXICOLOGY, 21(3), 373-385 (1998) AMMONIATION TO REDUCE THE TOXICITY OF ENDOPHYTE- INFECTED TALL FESCUE SEED FED TO RATS* A. Simeone’, M.L. Westendorf ’, R.E. Tucker’, L.P. Bush’, and G.E. Mitchell, Jr.’ Departments of ‘Animal Sciences and ’Agronomy University of Kentucky, Lexington 40546 ABSTRACT To assess the efficacy of ammoniation in the detoxification of endophyte-infected tall fescue (Festuca arundinacea Schreb.), 40 mule Harlan Sprague-Dawley rats were randomly assigned to the following four treatments for 28 d: endophyte-free (E-), endophyte-infected (E+), ammoniated (2% dry matter basis, 7 d) endophyte-free (AE-), and ammoniated endophyte-infected (AE+) tall fescue seed. Total pyrrolizidine alkaloid (N-acetyl and N-formyl loline) and ergovaline contents of endophyte-infected fescue seed were reduced 24 and 54%, respectively, by ammoniation. Endophyte-infected treatment groups had lower ( P < 0.01) daily feed intakes (DFI), daily weight gains (DWG), feed eficiencies, and primary serum hemagglutination titers to sheep red blood cell (SRBC) immunization than endophyte-free treatment groups. Performance parameters were higher ( P < 0.01) for ammoniated diets in comparison to non-ammoniated diets; however, anti-SRBC titers were not signijicantly different. When compared to the E+ diet, the AE+ diet increased ( P < 0.01) DFI (24%), DWG (41%) and feed eficiency (13%). INTRODUCTION Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season grass grown in the south central and southeastern U.S. Greater than 90% of tall fescue pastures in the U.S. are infected with the endophytic fungus Acremonium coenophialum”2. Although presence of the endophyte confers many favorable properties to the tall fescue plant (e.g. pest and drought resistance and improved gro~th)’’~”, toxicity symptoms are commonly ‘This paper (96-07-087) is published with the approval of the director of the Kentucky Agric. Exp. Sta. Correspondence to: George E. Mitchell, Jr., Tel. 606-257-2957, Fax 606-257-34 12 313 Copyright 0 1998 by Marcel Dekker. Inc. www.dekker.com D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. 374 SIMEONE ET AL. observed in cattle grazing endophyte-infected tall fescue. These symptoms, collectively referred to as fescue toxicosis. include reduced feed intake and gain, heat intolerance, increased respiration rate, decreased milk production, impaired reproductive performance, and reduced serum pr~lactin~.’.*.~.’~.’’ . Signs of fescue toxicosis can be experimentally induced in rats. Consequently, they have been utilized as models in fescue toxicosis research 12.1 3.14.1 Reports from feedlot nutritionists and veterinarians that “fescue” cattle are unthrifty, require excessive treatment for sickness and suffer high death losses have suggested that immune function may also be compromised. Lower serum complement levels and higher feedlot morbidity scores have been observed in cattle on endophyte-infected versus endophyte-free pasture^'^.'^. Using rodent models, consumption of endophyte-infected fescue has been shown to depress T cell-dependent antibody responses and T and B cell mi togene~ i s ’~ .~~ . Alkaloids produced by, or in response, to the tall fescue endophyte have been associated with the etiology of fescue toxicosis. In particular, concentrations of the pyrrolizidine alkaloids N-acetyl and N-formyl loline and the ergopeptine alkaloid ergovaline have been used as indicators of the potential toxicity of endophyte-infected tall fescue. The tall fescue toxin(s) have been shown to be susceptible to chemical and physical treatments. Growth inhibition of rats fed tall fescue seed was partially alleviated by autoclaving or by treating with either dilute NaOH or HCl”. Reductions in the concentrations of both pyrrolizidine alkaloids and ergovaline as a result of in vitro mminal fermentation have also been Due to the labile nature of the tall fescue toxin(s), chemical treatment of infected forage and (or) seed may provide a solution to the toxicity of harvested fescue. One such treatment that may potentially minimize the adverse effects of endophyte-infected fescue consumption is ammoniation. Although predominantly used to improve fiber digestibility and crude protein content of low-quality forages, ammoniation has also been used to detoxify aflatoxin-contaminated feedstuffs23.24.25.26.27.28.29. D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. AMMONIATION TO REDUCE TOXICITY 375 The objective of this experiment was to determine the effects of ammoniating endophyte-infected tall fescue seed on (1) the alkaloid content of the seed and (2) performance and immunocompetence of animals consuming the seed using a rat model. METHODS Husbandry, and Body Weight and Feed Intake Measurements Forty male Harlan Sprague-Dawley (Harlan Sprague-Dawley Inc., Indianapolis, IN) rats (average initial weight 103 g) were stratified by weight and randomly allotted to one of four treatment groups (10 animalsltreatment) six d prior to study start. During a 28-d trial each group was fed diets containing either endophyte-free (E-), endophyte-infected (E+), ammoniated endophyte-free (AE-), or ammoniated endophyte-infected (AE+) tall fescue seed. Animals were housed in individual suspended cages (35.6 x 17.8 cm wire-mesh). All rats received rodent Chow@ (Rodent Laboratory Chow 5001, Purina Mills, Inc., St. Louis, MO) and water ad libitum for 48 h prior to the initiation of treatments. Experimental diets and water were available ad libitum for the duration of the trial. Feed intake was recorded daily and body weight was recorded every 4 d. Feed efficiency (G/F) was calculated as gram of weight gain per gram of feed consumed. Ammoniation Procedure and Preparation of Diets Endophyte-free (E-) Festuca arundinacea Fider and 50% endophyte-infected (E+) Festuca arundinacea Kentucky 3 1 tall fescue seed was ground (2 mm Wiley mill screen) and divided equally into four polyethylene bags of endophte-free seed and four polyethylene bags of endophyte-infected seed (3.4 kghag). Each bag was placed into a plastic, five-gallon container. Two containers each of E- and E+ seed were untreated and stored (20°C), while the remaining two containers of E- and E+ seed were ammoniated. Fescue seed to be ammoniated was adjusted to 35% moisture by adding the appropriate amount of water and mixing (Hobart). Anhydrous ammonia (20 g per kg seed dry matter [DM]) was applied by inserting a perforated tygon tube into the center of each container. D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. 376 SIMEONE ET AL. Ammonia dosage was calculated as the increase in the weight of the container after a slow admission of ammonia. Following ammoniation, containers were sealed and stored (20°C) for 7 d. Prior to use, all ammoniated seed was air-dried for 6 d for evaporation of moisture and volatilization of excess ammonia. Non-ammoniated and ammoniated seed were each mixed 1 : l with ground (2 mm) rodent Chow@ (93.9% DM, 24.5% crude protein [CP]). Chemical and Alkaloid Analyses Samples of the four fescue seed treatments were analyzed in duplicate for DM3’, CP (Foss-Heraeus Macron N. UIC Inc., Joliet, IL) and neutral detergent fiber (NDF) and acid detergent fiber (ADF)”. Pyrrolizidine alkaloid (N-acetyl and N-formyl loline) and ergovaline concentrations were determined by modifications of the capillary gas-liquid chromatographic method of Kennedy and and the HPLC procedure of Yates and respectively. Alkaloid analyses were run in duplicate. Preparation of Sheep Red Blood CelkF and Serum Hemagglutination Titer The procedures for sheep red blood cell (SRBC) preparation and primary serum hemagglutination titer were those described by Dewla. Sheep red blood cells were obtained from whole blood collected from a wether bled via jugular venipuncture into a sterile heparinized tube. Cells were washed twice (750 x g for 15 min) in phosphate-buffered saline (PBS, pH 7.3, Sigma, St. Louis, MO) and resuspended to a final 10% immunization volume. On d 14, rats in all groups were injected i.p. (using a tuberculin syringe and 25-gauge needle) with 0.1 mL of the 10% SRBC volume. Rats were sacrificed by decapitation at the end of the 28-d trial. Blood was collected from each rat into a non-heparinized 12 x 75 rnm polystyrene tube and allowed to clot at the time of sacrifice. Serum was obtained by centrifugation (1200 x g for 30 min) and subjected to two-fold dilutions with PBS (ranging from 1 :4 to 1:4096). Each serum/PBS dilution (200 pL) was placed within one of 12 wells of a 96-well round-bottom microtiter plate (Becton-Dickinson, Lincoln Park, NJ). Fifty pL of the 10% SRBC preparation was added to each of the wells containing the serum dilutions. After 4 h at room temperature, the titer was defined as the reciprocal of the highest SRBC-agglutinating serum dilution. D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. AMMONIATION T O REDUCE TOXICITY 377 STATISTICAL ANALYSES Treatments ware analyzed as a completely randomized 2 x 2 factorial treatment arrangement using the General Linear Models procedure of SAS34. The main effects of endophyte (0 and 50% infected) and ammoniation (0 and 2%) and their interactions were tested by orthogonal contrasts. Treatment means were separated by Least Significant Difference. RESULTS AND DISCUSSION Chemical and Alkaloid Compositions As shown in Table I, the chemical compositions of endophyte-free and endophyte-infected tall fescue seed were similarly affected by ammoniation. Ammoniation increased CP (29%). lowered NDF (6%) and did not affect ADF. The increase in CP accounted for approximately 36% of the ammonia added. Concentrations of N-formyl loline (23.6%), N-acetyl loline (27.0%) and ergovaline (54.2%) were lower for ammoniated versus non-ammoniated fescue seed. Ergovaline was more susceptible to ammoniation-induced modification(s) in comparison to pyrrolizidine alkaloids (54.2 vs 24.3% reduction). The extreme instability of ergopeptines may be related to the increased susceptibility of ergovaline to ammoniation. Ergopeptine alkaloids are sensitive to acid, base and light and, as a result, can form various isomerization, hydrolysis and addition products3’. Ergopeptines are also thermally unstable and, in particular, the amide linkage between the ergolene ring and tricyclic peptide portion is temperature and pH sensitive36. There is a temperature rise during ammoniation due to the interaction of ammonia vapor with water in the material being treated that can last several days to several weeks before returning to ambient t e m p e r a t ~ r e ~ ’ * ~ ~ ~ ~ ~ * ~ . Body Weight and Feed Intake Measurements The main effects of endophyte and ammoniation and their interaction were present for D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. 378 SIMEONE ET AL. TABLE I Chemical md Alkaloid Compositions of TIU Fexue Seed ~ Trcrtment. Itanb E- E+ AE- AE+ DM(Yo0) 90.8 89.6 88.1 88.2 K DM CP 12.3 12.8 16.1 16.4 NDF 36.4 36.8 34.2 34.7 ADF 13.1 13.7 13.4 13.8 PyrrOliZidine Alkaloids, &l3 N-formyt loline NDc 3212 ND 2455 N-acetyl loline ND 900 ND 657 Total 41 12 3112 daily feed intake (DFI), daily weight gain (DWG) and feed efficiency (G/F; Table II). Rats fed endophyte-infected diets had lower DFI, DWG and feed efficiency (P c 0.01) when compared to rats fed endophyte-free diets. A similar response was observed for ammoniated versus non-ammoniated diets. Ammoniation of endophyte-infected fescue improved (Pc 0.01) DFI (24.2%). DWG (41.3%) and G/F (13.0%). Ammoniation reduced the alkaloid content and improved the chemical composition of endophyte-infected fescue seed; therefore, nutritional and (or) detoxifying effects of ammoniation may have been responsible for the increased productivity of AE+ versus E+-fed rats. Differences in NDF and CP were of the same magnitude between E- and AE- diets and between E+ and AE+ diets; however, significant differences in animal performance were only obtained between E+ and AE+ diets. This suggested that D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. TA BL E II Da ily Fe ed I nt ak e ( D FI ), Da ily W ag ht Ga in (D W G ) a nd F ee d E ffi ci en cy ( W ) En do ph yt e a nd A m m on ia tio n I nt er ac tio n T lW lb tl d Si en ifi cr nc eb It un E- E+ AE - AE + SE M E E A E x A Fi na l w eig ht , g 26 9. 9 18 2. 4 27 7. 8 21 7. 3 4. 6 In iti al w ei gh t g 10 4. 0 10 2. 1 10 2. 6 10 2. 7 2. 1 - - - 21 .5 12 .8 22 .5 15 .9 0. 36 ++ ++ ++ DW G , g ld 5. 9 2. 9 6. 3 4. 1 0. 13 ++ ++ ++ Dm , s /d 0. 27 0. 23 0. 28 0. 26 0. 08 ++ ++ + G /F d % nd op hy te -fi te ( E -), en do ph yt ai nf cc tc d ( E +) , a m m on ia te d en do ph yt e- he (A E- ), an d am m on ia tc d bS ig ni fic an ce of m ai n ef fe ct of en do ph yt e ( E ; E - a nd A E- vs E + an d AE +) , n ui n eff ect o f a m m on ia tio n ( A ; E- an d E+ vs A E- an d AE +) an d i nt er ac tio n be tw ee n cn do ph yt e a nd am m on ia tio n ( E x A) ; +P < 0. 05 , ++ P < 0. 01 . 'S ta nd ar d er ro r o f t he m ea n (S EM ). ++ ++ ++ ~d op hy te -in fe ct ed (A E+ ) ta ll fes cu e s ee d m ix ed 1 : 1 w ith r od at C ho w@ . dG ru no fw ei gh tg ai np ag ra m of fk dc oh nn ne d( O IP ). D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. 380 SIMEONE ET AL. ammoniation-mediated improvements in performance were due primarily to reductions in alkaloid content and not to alterations in chemical composition. Ammoniation has been shown to increase fiber digestibility of low-quality forages and to inactivate aflatoxins in contaminated products by structurally altering components of the cell wall matrix (i.e. ester bonds between lignin and hemicellulose and intermolecular H bonds) and biologically active components of aflatoxins (dihydrofurofuran group and lactone-pentanone ring), r e s p e ~ t i v e l y ~ ' * ~ ~ . ~ ~ . ~ ~ ~ ~ . Such structural alterations may have been responsible for the partial detoxification of toxic fescue alkaloids by ammoniation. Ammonia-labile bonds in pyrrohidine alkaloids and ergovaline may be necessary for these compounds to maintain their toxic integrity. Serum Hemagglutination Titers Antibody responses to T cell-dependent antigens is a complex immune reaction involving B cells as well as antigen-presenting cells and T helper cells. Therefore, primary serum hemagglutination titers are useful in providing an overall assessment of immune function. Effects of endophyte and ammoniation and their interactions on serum titer response to SRBC immunization is presented in Table m. Endophyte-free treatment groups had higher (P< 0.01) antibody titers relative to endophyte-infected treatment groups (I54 vs 54). There was no difference in antibody response for rats fed either ammoniated or non-ammoniated diets; in addition, there were no interactions between endophyte and ammoniation for anti-SRBC titers. These results suggested that one or more components of the antibody response are compromised by the tall fescue endophyte and associated toxin(s). However, feed intake was substantially lower for rats given endophyte-infected versus endophyte-free diets. Therefore, nutritional influences may have contributed to the reduced serum titer response of endophyte-infected treatment groups. Immunocompetence is highly influenced by the nutritional status of the host; furthermore, protein-energy malnutrition is known to suppress T cell-dependent antibody response^^^.^'*^^. D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. AMMONIATION TO REDUCE TOXICITY 38 1 TABLE III Serum Hemaggiutinrtion Titer Endophyte Endophyt~-free (E- and AE-) Endophyte-infected (E+ md AE+) 153.6 54.4- Amadation Non-~x~onh!ed(E-andE+) 113.6 SEMb 13.9 E- 147.2 E+ 41.6 AE- 160.0 AE+ 67.2 SEM 19.7 Ammooiatd (AE- 8nd AE+) 94.4 -oPhYtC-fi= (E), =doPhY--d @+), - d o p h y t * b (As), and ammoniated endophytainfected (A€+) trll f k u c d diets mixed 1:l withrodentchow@. bStandard error of the mcllll (SEM). *Significant€y differtnt from mdophyt~-6ce at P < 0.01. In contrast to performance, ammoniation did not counteract the immunosuppressive effects of the endophyte-infected fescue. Although intake of AE+ was greater than that of E+, it was still considerably less than the intake of either of the endophyte-free diets. Therefore, malnutrition may have remained a contributory factor in the depressed antibody response. Additionally, ammoniation lowered both pyrrolizidine alkaloids and ergovaline; however, concentrations of these alkaloids may still have been sufficiently high to suppress immune function. Pyrrolizidine alkaloids have been shown to inhibit the proliferative responses of both murine splenocytes and bovine peripheral blood lymphocytes to concanavalin A and pokeweed mi tog en^^^. Although effects of ergovaline on immune function have not been examined, it may indirectly influence immune status by lowering prolactin levels. Prolactin is required for the development of a proper antibody response to T cell-dependent antigen^^^^'*'^^'^. D ru g an d Ch em ic al T ox ic ol og y D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 08 /1 6/ 13 Fo r p er so na l u se o nl y. 382 SIMEONE ET AL. In summary, dietary exposure to the tall fescue endophyte and associated toxin(s) depressed feed intake, gain and antibody response to SRBC immunization. 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