dicoumarol concentrations and forage quality of ...

DICOUMAROL CONCENTRATIONS AND FORAGE QUALITY OF SWEETCLOVER FORAGE TREATED WITH PROPIONIC ACID OR ANHYDROUS AMMONIA1 Matt A. sanderson2, D. W. ~ e ~ and e Howard r ~ casper4 North Dakota State University, Fargo 58105

ABSTRACT

Three experiments were conducted t o determine if propionic acid (PA) or anhydrous ammonia (NH,) could prevent spoiling and subsequent dicournarol formation in sweetclover [Melilotus officinalis (L.) Lam.] hay. In Exp. 1, PA at 6 , 9 or 1 2 g/kg of wet hay prevented dicoumarol formation in 417-g samples of sweetclover hay rewetted t o 400-g/kg (wet-weight basis) moisture concentration, whereas PA a t 3 gtkg did not. In Exp. 2, PA a t 1 0 or 20 g/kg did not prevent formation of toxic dicoumarol concentrations (>20 g/kg of dry matter) in 7-kg samples of 550-and 650-g/kg moisture sweetclover hay. Anhydrous ammonia, however, a t 1 0 or 30 g/kg prevented dicoumarol formation in sweetclover samples of similar size and moisture. Propionic acid and NH, were applied t o large round bales of sweetclover hay in 1981 and 1982 for Exp. 3. Dicoumarol was not detected in the 1981 bales due t o the low (170 gtkg) hay moisture. Bales a t 200- and 370-g/kg moisture concentration treated with NH, a t 1 0 and 20 glkg of wet hay, respectively, had less than 9 mg/kg of dicoumarol in 1982. Ammonia a t 1 0 g/kg on 270-g/kg moisture bales and all propionic acid treatments did not prevent occurrence of toxic dicoumarol concentrations. Ammoniation increased (P.05) t o forced-air-dried samples of the original hay, suggesting that NH, was preserving the forage a t its original digestibility. Anhydrous ammonia appears t o be a promising preservative for preventing the occurrence of dicoumarol in sweetclover hay and thereby the sweetclover bleeding disease in livestock; however, these results need to be confirmed with further field trials and animal evaluation experiments. (Key Words: Ammoniated Feeds, Dicoumarol, Organic Acids. Hay, Sweet Clover, Melilotus Officinalis.) Introduction

lactone form) to 4-hydroxycoumarin and non-enzymic condensation of two 4-hydroxycoumarin molecules with exogenous formaldehyde (Bellis et al., 1967). The glucoside of cis-o-hydroxycinnamic acid occurs naturally in relatively high concentrations (20 to 25 glkg of dry matter) in sweetclover (Smith, 1975). Sweetclover bleeding disease was first described in 1922 (Schofield, 1922) and still plagues farmers feeding sweetclover; 180 cattle were lost from January 1976 t o October 1978 by 32 ranchers in North Dakota (Benson, 1980). Application of a hay preservative may prevent dicoumarol formation in high-coumarin sweetclover hay. Propionic acid (PA) and 'Contribution from the Dept. of Agron. and Vet. ammonia (NH3) have been used Sci.. North Dakota State Univ.. Fargo, ND 58105 as successfully as preservatives of high-moisture Journal Paper No. 1363. 'Graduate Research Assistant. Present address: (324 g moisturelkg) alfalfa (Medicago sativa L.) Dept. of Agron., Iowa State Univ., Ames, IA 50011. hay (Knapp et al., 1975, 1976). Anhydrous Dept. of Agron. ammonia also has been used to improve the Dept. of Vet. Sci. Received December 10, 1984. nutritive value of low-quality roughages Accepted June 26. 1985. (Sundstrol et al., 1978). 1243

The blood anticoagulant, dicoumarol [3,3' methylene bis (4-hydroxy-coumarin)] , often occurs in spoiled, high-coumarin sweetclover [Melilotus officinalis (L.) Lam. and M. alba Desr.] hay and is responsible for "sweetclover bleeding disease" in livestock. Dicoumarol is potentially toxic at 20 to 30 mglkg of forage dry matter (Casper et al., 1983) and may cause animal death due t o internal or external hemorrhaging or both (Smith and Gorz, 1965). Dicoumarol is formed by fungal conversion of cis-o-hydroxycinnamic acid (coumarin is the

JOURNAL OF ANIMAL SCIENCE, Vol. 61, No. 5 , 1 9 8 5

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SANDERSON ET AL.

Our objective was t o investigate the effectiveness of PA and NH3 in preventing dicoumarol formation and in preserving forage quality of sweetclover hay at various moisture concentrations. Materials and Methods

Exp. 1. Propionic acid was applied at 0, 3 , 6 , 9 and 12 glkg of wet hay t o chopped (2.5 cm length) second-year-growth sweetclover hay rewetted to 400-glkg moisture. The hay had a small amount of dicoumarol(7 mglkg) present. Hay was rewetted and treated using a method similar t o that of Lacey and Lord (1977). The rewetted and treated hay was divided into three equal samples (417 g each) and each sample packed into an uncovered 2-liter plastic jar. A copper-constantan thermocouple was inserted into each sample and temperature was monitored daily with a potentiometer. The samples were incubated at room teperature (22 C) for 12 d. After incubation, the samples were dried t o a constant weight at 55 C, ground t o pass a 1-mm screen, and analyzed for dicoumarol by the method of Casper et al. (1981). This experiment was conducted twice (trials 1 and 2). Temperature data were expressed as the difference between daily sample and ambient air temperatures at the time of measurement each day and averaged across samples and trials for each treatment. Exp. 2. Two trials were conducted t o determine if PA or NH3 could prevent dicoumarol formation and improve forage quality in small samples of sweetclover forage at various moisture concentrations. In trial 1 , sweetclover was cut at the late-bud stage and allowed to dry in the field t o 550-or 650-glkg moisture concentration or forced-air-dried at 55 C to less than 100-glkg moisture. Fifteen treatments were applied to the forage, five at each moisture level. Seven-kilogram samples of 550-glkg moisture forage were treated with PA at 1 0 or 20 glkg of wet forage or NH3 at 1 0 or 30 glkg. An untreated control was included also. Two-kilogram samples of forcedair-dried hay received the same treatments. Sevenkilogram samples at 650-glkg moisture forage were treated with PA at 1 0 or 20 glkg or N H j at 10 glkg. An untreated control and an untreated control placed inside a 75 x 100 x 150-cm plastic-covered enclosure were included also. Propionic acid was sprayed onto forage spread on a plastic sheet. The PA-treated forage

was packed into 35 x 90-cm plastic, open-mesh (5 mm) bags after treatment. Gaseous NH3 was applied t o forage packed into mesh bags and placed inside plastic-covered enclosures. The same treatment procedures were used in trials 1 and 2. Each treatment was replicated three times resulting in 45 bags of forage. In the second trial, sweetclover was cut at the full-bloom stage and allowed to;dry in the field to 120-,280- or 360- glkg moisture and forced-air-dried t o less than 100-glkg moisture. Five treatments were applied t o hay samples at each moisture level for a total of 20 treatments. Each treatment was replicated three times resulting in a total of 60 bags of hay. Propionic acid was applied at 10 and 20 glkg of wet hay and NH3 was applied at 1 0 and 30 glkg t o hay at each moisture level. Untreated hay was included at each moisture level also. Sample sizes were 3 kg for the 120-, 280- and 360glkg moisture hay and 2 kg for the forced-airdried hay. All treated samples were stored inside at ambient air temperature. Each trial was ended after 28 d. All forage samples were dried t o a constant weight at 55 C, ground in a hammer mill t o pass a 1-cm screen, subsampled, and the subsample ground in a shear mill t o pass a 1-mm screen. Dry matter, dicoumarol, total N and in vitro dry matter disappearance (IVDMD) were determined on all subsarnples. Dry-matter concentration was determined by drying forage at 105 C for 24 h. Total N was determined by a micro-Kjeldahl procedure (AOAC, 1975). Subsamples were analyzed for dicoumarol by the method of Casper e t al. (1981). In vitro dry matter disappearance was determined by the Tilley and Terry (1963) method as modified by Barnes et al. (1971). Exp, 3. Field trials were conducted in 1981 and 1982 t o determine if PA or NH3 could prevent dicoumarol formation and preserve forage quality in large round bales of sweetclover hay. In 1981, second-year-growth of sweetclover was cut at the full-bloom stage and dried in the field t o 170-glkg moisture over a 2-wk period. During this time, 12.5 mm of rain fell on the hay and the windrows were turned once to facilitate drying. The desired hay moisture for this trial was between 200 t o 250 glkg; however, very hot and dry weather conditions during the last 2 d of field curing resulted in very rapid hay drying. Consequently, the desired range was missed and the hay was

DICOUMAROL CONCENTRATIONS IN SWEETCLOVER

baled at 170-glkg moisture into approximately 1.52-m-long x 1.52-m-diameter, 320-kg round bales. Propionic acid was sprayed on randomly selected windrows at 20 glkg of wet hay immediately before baling. Two bales were made of PA-treated hay. Anhydrous ammonia was applied at 30 glkg of wet hay to two bales, each wrapped separately in .15-mm clear polyethylene. Gaseous NH3 was injected into the center of each bale at 454 glmin through a 1.5-m-long x 2-cm-diameter, perforated pipe sealed at one end. Four untreated bales were made also. Untreated and PA-treated bales were stored inside and the NH3-treated bales remained wrapped in plastic outside for 30 d. The bales were core-sampled after 30 d. The surface 6 cm (15% of the total bale volume) was sampled by hand and two cores of the interior were taken at 35-cm increments (64 and 21% of the total bale volume, respectively). Two random locations were sampled on the top, bottom and two horizontal, rounded sides of each bale. Corresponding section (surface and two interior cores) samples from each side of the bale were composited. Thus, 12 samples from each bale were analyzed chemically. Values from the four sides were averaged together for one observation per section. A weighted average (based on the percentage of the total bale volume that each section represented) of the three section observations was used for the analysis of variance. In 1982, second-year-growth of sweetclover was cut at the late-bud stage and allowed t o dry in the field t o 200-, 270- or 370-glkg moisture. Three treatments were applied to hay at each moisture level and each treatment was replicated three times. Hay was baled into 1.76-m-long x 1.72-m-diameter round bales weighing approximately 450 kg. Propionic acid and NH3 were applied t o bales at each moisture level and untreated controls were included also. Propionic acid was applied at 20 glkg of wet hay to 370-glkg moisture hay and at 10 glkg t o 200and 270glkg moisture hay. Propionic acid was sprayed on randomly selected windrows immediately before baling. Anhydrous ammonia was

"old-flo system patented and marketed b y USS Agrichemicals, Atlanta, GA. 6Mention of a company or brand name does not imply endorsement o f that product to the exclusion o f other products that may be suitable.

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applied at 20 glkg of wet hay t o 370glkg moisture bales and at 1 0 glkg to 200- and 270-glkg moisture bales. The higher rate of PA and NH3 on 370-glkg bales was used because of the higher moisture concentration. Bales t o be treated with NH3 were selected at random within each moisture level. Three bales were wrapped and sealed in .15-mm clear polyethylene f o r each treatment. Ammonia was applied as a liquid by passing gaseous NH3 through a ~old-flo@5,6converter. The liquid NH3 was metered at 1,135 glmin into an open-topped plastic container placed inside the plastic wrap. The liquid NH3 was allowed t o volatilize and diffusethroughout the bales. Bales were sampled with a "Penn stateH6 bale corer 5 wk after treatment. Sampling procedures were similar t o those used in 1981 except that only the horizontal, rounded sides were sampled at three random locations for each section (surface and two interior cores; 14, 6 4 and 22% of the total bale volume, respectively). Nine samples were analyzed chemically for each bale. A weighted average of the three section observations was used for the analysis of variance. Since 1982 bales were larger than 1981 bales, the lengths for the two interior cores were increased to 40 cm. In 1981 and 1982, all samples were dried at 55 C for 48 h and ground in a shear mill to pass a 1-mm screen. Samples were analyzed for dicoumarol, total N and IVDMD as described previously. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the methods of Goering and Van Soest (1970) and acid detergent insoluble nitrogen (ADIN; 1982 samples only) was determined by analyzing the ADF residue for Kjeldahl N. In 1982, three 1-kg samples of hay were taken from various parts of the field to provide forage quality data on the original hay. Statistical Analysis. Exp. 1 was analyzed as a randomized, complete-block design. Trials 1 and 2 were considered blocks. Thus, there were two replicates with three samples per treatment in each replication. Because of the unbalanced treatment design in trial 1 of Exp. 2, each moisture level was analyzed as a separate experiment. Data at each moisture level were analyzed as a completely randomized design with three replicates. Forced-air-dried forage samples and NH3-treated samples at 550- and 650-glkg moisture had no detectable dicoumarol, so these observations were omitted from the analysis of variance. The

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SANDERSON ET AL.

sources of variation, degrees of freedom and mean squares are presented in table 1. Trial 2 in Exp. 2 and Exp. 3 were analyzed as completely randomized designs with a split-plot arrangement of treatments. Moisture levels comprised the whole plots and subplots were the 'preservative treatments. The sources of variation, degrees of freedom and denominators of F-tests for the split-plot statistical model are presented in tables 1 and 2. The least significant difference (LSD) test following a significant F test was used t o separate means (Steel and Torrie, 1980). It is recognized that the replications for the NH3-treated hay in trial 2 of Exp. 3 were nested within treatments. Therefore, a separate analysis of variance was conducted on the data from the NH3-treated bales. An F test was used t o compare the error mean square for each variable measured from the NH3 data with the error mean squares of the combined control and PA-treatment data to determine if the errors were homogeneous and could be pooled

(Steel and Torrie, 1980). The error terms were not different (P> .lo) and were pooled. Trial 1 of Exp. 3 was analyzed by one-way analysis of variance for a completely randomized design. A large round bale was considered the experimental unit. Propionic acid and NH3 treatments were replicated twice and the control treatment four times. Results and Discussion

Exp. 1. Propionic acid at 3 glkg did not prevent heating (figure 1) and dicoumarol formation in sweetclover hay rewetted to 400glkg moisture concentrations. Hay from this treatment contained 44 mg dicoumarollkg DM and was visibly moldy at the end of the experiment. Untreated hay contained 48 mglkg of dicoumarol and heated rapidly (figure 1). Hay treated with PA at 6 and 9 glkg did not heat more than 2 C above ambient air temperature. Hay treated with PA at 12 glkg (data not shown) had a temperature pattern similar t o

TABLE 1. DEGREES OF FREEDOM (DF) AND MEAN SQUARES FOR EXP. 2, TRIALS 1 AND 2 Source

df

Nitrogen

IVDMDa

Dicoumarol

(df$

Trial 1 550-g/kg moisture forage Treatment Error 650-g/kg moisture forage Treatment Error Forced-air-dried forage Treatment Error

4 10

71.7.. 4.7

12,696.1.. 478.3

30.5 18.7

(2) (6)

4 10

77.4'. 1.6

31,461.9'. 421.5

8,721.2.. 1,074.6

(3) (8)

4 10

13.8 5.3

1,177.7 1,581.9

3 8 4 12 32

88.1 * * 3.0 229.8.. 16.0 7.6

Trial 2 Moisture level (M)C Replication [MI d Treatment ( T F T X Me Residual

37,128.5.. 491.4 6,105.6. 1,821.2 1,777.5

a~~~~~ = in vitro dry matter disappearance. b ~ u m b e r sin parentheses are degrees of freedom reflecting number of samples with positive dicoumarol analysis. Forced-air-dried and NH,-treated samples at 550- and 650-g/kg moisture had no detectable dicoumarol and thus were omitted from analyses.

enom om in at or of F test = Replication d~eplicationwithin moisture level. e ~ e n o m i n a t o of r F test = Residual. *P