Effects of various compounds on in vitro ruminal fermentation and production of sulfide

Animal Feed Science and Technology 84 (2000) 69±81

Effects of various compounds on in vitro ruminal fermentation and production of sul®de$ L. Kung*, J.P. Bracht, J.Y. Tavares Delaware Agricultural Experiment Station, Department of Animal and Food Sciences, College of Agriculture and Natural Resources, University of Delaware, 531 South College Avenue, Newark 19717-1303, USA Received 29 January 1999; received in revised form 3 June 1999; accepted 21 December 1999

Abstract We evaluated the effect of several compounds on their ability to reduce the production of sul®de in in vitro ruminal fermentations, when sulfur content of the diet was 1% (dry matter basis). Excess sulfur increased (p<0.05) ruminal sul®de production, but had no effects on VFA production. The effect of 1, 10 and 25 ppm (¯uid basis) of 9,10 anthraquinone (AQ) and molybdenum in high sulfur diets was also studied. Speci®cally, AQ (10 ppm) and molybdenum (25 ppm) reduced (p<0.05) hydrogen sul®de production by 71 and 77%, respectively. The addition of AQ, but not molybdenum, decreased (p<0.05) the molar percentage of acetate and decreased methane production. The former compound also increased (p<0.05) the molar proportions of propionate, butyrate, and valerate. Bacitracin (1.25 ppm), oxytetracycline (1.25 ppm), chlortetracycline (5 ppm), lasalocid (5 ppm), bambermycin (0.3 ppm), monensin (5 ppm), and avoparcin (5 ppm) were tested relative to AQ (10 ppm) for their ability to affect sul®de production. Only chlortetracycline and oxytetracycline reduced (p<0.05) sul®de production, but they were less effective than AQ. Surprisingly, sul®de production was increased (p<0.05) by >50% when cultures were treated with monensin. Interactions between methanogens and sulfate-reducing organisms may be responsible for this ®nding. Several compounds have the ability to reduce ruminal sul®de production without negative effects on ruminal fermentation. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Rumen; Fermentation; Sul®de; Antimicrobials

$

Published as paper number 1654 in the Journal Series of the Delaware Agricultural Experiment Station. Corresponding author. Tel.: ‡1-302-831-2522; fax: ‡1-302-831-2822. E-mail address: [email protected] (L. Kung) *

0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 0 3 - 6

70

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

1. Introduction In ruminants, eructation (belching of gasses) is a normal process. However, as much as 60% of eructated gasses are inhaled and enter the respiratory tract (Bulgin et al., 1996). Inhalation of hydrogen sul®de gas from the rumen of cattle and sheep consuming feed (Gould et al., 1991; Hill and Ebbett, 1997) or water (Wagner et al., 1998) high in sulfate has been implicated as a potential cause of polioencephalomalacia (PEM) in ruminants. Polioencephalomalacia, which is characterized by cerebrocortical necrosis and can be fatal, may be caused by the inhalation of hydrogen sul®de produced in the rumen (Gould, 1998). When there is an inability to control the intake of sulfur or sulfate, selective antimicrobial therapy against sulfate-reducing bacteria may be the method of choice to prevent excess sul®de production. However, caution must be used to prevent detrimental side effects to the host animal. Recently, we (Kung et al., 1998) reported that 9,10 anthraquinone (AQ) was a potent inhibitor of hydrogen sul®de production in in vitro ruminal fermentations. However, little is known about the ability of other commonly used feed additives to prevent sul®de production from ruminal fermentations. The objective of this study was to evaluate the ability of various antimicrobial compounds to prevent excess production of sul®de in ruminal fermentations. 2. Materials and methods 2.1. Diets and culture conditions In all experiments, the control diet was a complete early market lamb pellet (unmedicated, Agway, Tully, NY), that was ground to pass through a 1-mm screen of a Wiley Mill (Arthur H. Thomas, Philadelphia, PA) and contained 0.29% sulfur (DM basis). The formulation of the pellet was proprietary, but contained wheat middlings, soyhulls, alfalfa meal, and corn meal as the major dietary ingredients. In treatments designated as being high in sulfur (HS), the control diet was supplemented with Na2SO4 to yield a ®nal concentration of 1.09% sulfur (DM basis). A representative sample of each diet was analyzed for nutrient content (AOAC, 1990) by a commercial laboratory (Cumberland Valley Analytical Services, Maugansville, MD) and the compositions of the diets are shown in Table 1. Soluble protein was analyzed via the borate-phosphate method described by Krishnamoorthy et al. (1983). Rumen ¯uid was obtained from two 400-kg steers with rumen ®stulas. Steers were limit-fed 1 kg of a commercial concentrate (18% CP), and had ad libitum access to 50:50 (DM basis) alfalfa±grass hay and corn silage mixture. Care and handling of the steers followed the standard approved management protocol (Consortium, 1989). Ruminal ¯uid and contents were collected 2±6 h after the morning allocation of concentrate and were placed in a sealed thermos while being transported to the lab for processing. Within 15 min of collection, ruminal ¯uid was ®ltered through four layers of cheese cloth and placed into a re-pipette dispenser that had been purged with anaerobic grade CO2 (<1 ppm O2). Effects of various compounds on in vitro ruminal fermentation were conducted in 60-ml serum bottles. A volume of warm (398C) mineral-buffer solution

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

71

Table 1 Nutrient composition of the low-sulfur (LS) control and high-sulfur (HS) control basal diets (DM basis) Item

LS control

HS control

DM (g kgÿ1) Crude protein (g kgÿ1) Soluble protein (g kgÿ1) Acid detergent ®ber (g kgÿ1) Neutral detergent ®ber (g kgÿ1) Non-structural carbohydrates (g kgÿ1) Ca (g kgÿ1) Phosphorus (g kgÿ1) Magnesium (g kgÿ1) Potassium (g kgÿ1) Sulfur (g kgÿ1) Iron (ppm) Manganese (ppm) Zinc (ppm) Copper (ppm)

888 166 58 248 416 300 153 45 31 128 29 313 218 149 11

909 166 53 253 412 272 156 41 29 127 109 314 167 120 9

(Goering and Van Soest, 1970) without reducing solution, was added to a similar volume of strained ruminal ¯uid. In all experiments, 29.5 ml of the rumen ¯uid and buffer solution and 0.5 ml of an appropriate treatment solution was added to each serum bottle that contained 375 mg of substrate. The bottles were then purged with anaerobic grade CO2 for 10 s and sealed with a butyl-rubber stopper and crimp seal. Serum bottles were incubated in an orbital-shaking water bath (New Brunswick Scienti®c, model G76), set at a speed of 2.5 for 24 h at 408C. All treatments were incubated in triplicate and replicated on two separate days using different donor steers. 2.2. Experiment 1 In Experiment 1, we tested the effect of varying doses of molybdenum (Mo) on ruminal fermentations. Sodium molybdate dihydrate (Na2MoO4 2H2O, Mallinckrodt, Paris, KY) was dissolved in deionized water prior to inoculation into serum bottles. Treatments were (1) a control diet with low sulfur (LS), (2) a control diet with high sulfur (HS), (3) HS and 1 ppm (¯uid basis) molybdenum, (4) HS and 10 ppm molybdenum, and (5) HS and 25 ppm molybdenum. Serum bottles for the control diet received 0.25 ml of deionized water. 2.3. Experiment 2 In Experiment 2, treatments were: (1) LS, (2) HS, (3) HS and 10 ppm (¯uid basis) of 9,10 AQ (Environmental Biocontrol, Wilmington, DE), (4) HS and 5 ppm of avoparcin, (5) HS and 1.25 ppm of bacitracin, (6) HS and 0.3 ppm of bambermycin (HoechstRoussel, Somerville, NJ), (7) HS and 5 ppm of chlortetracycline, (8) HS and 5 ppm of lasalocid, (9) HS and 5 ppm of monensin (Elanco, Green®eld, IN), and (10) HS and

72

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

1.25 ppm of oxytetracycline. Anthraquinone was a 50% wt./wt. aqueous formulation that was dissolved in water and added to incubation bottles. All other compounds were solubilized in 70% ethanol before addition to incubation bottles and fermentation volumes were corrected for the addition of water and ethanol. 2.4. Experiment 3 In experiment 3, treatments were: (1) LS, (2) HS, (3) HS and 4.5 ppm (¯uid basis) of monensin, (4) HS and 1.5 ppm of tylosin, (5) HS and 4.5 ppm of monensin and 1.5 ppm of tylosin, and (6) HS 10 ppm of 9,10 AQ. Materials and methods were as previously described. 2.5. Chemical analyses Chemical analyses were as described by Kung et al. (1998). In all experiments, the total volume of gas after 24 h of incubation was measured by noting the volume of water displaced in an inverted burette and adding this to the amount of headspace volume in each incubation bottle. To measure the sul®de present in the gas phase, 1 ml of gas was extracted from each sealed bottle, via a tuberculin syringe ®tted with a 20-gage needle and analyzed following a modi®ed method described by Siegal (1965). Gas samples were slowly bubbled into alkaline deionized water (pH of 8) in a 3-ml vaccutainer tube (Becton±Dickinson, Franklin Lakes, NJ), to which N-N-dimethyl-p-phenylenediamine dihydrochloride was added and allowed to develop undisturbed at 258C for 30 min. The samples were then read on a spectrophotometer at 670 nm. In order to measure the sul®de in the liquid phase, 1 ml of the ¯uid was extracted and mixed with 0.1 ml of 6 N HCl in a vacutainer in order to protonate sul®de and release it into the gas phase. One milliliter of this gas was analyzed in the same manner as described above. When required, the presence of methane and hydrogen were determined by gas chromatography. Using a gastight syringe, 200 ml of a gas sample was injected into a Hewlett±Packard (Avondale, PA) 5880A gas chromatograph ®tted with a thermal conductivity detector and a Porapak Q column. Argon was the carrier gas with a ¯ow rate of 11.1-ml minÿ1. Initial oven settings were at 908C for 1 min followed by a rate increase of 308C minÿ1 until a ®nal temperature of 1908C was reached. This was maintained for 6 min. Analyses of gas and liquid sul®de were completed within 2 h of the sample collection. The pH of the ®nal fermentation ¯uid was determined by pH probe after sampling for gases. The fermentation ¯uid was then acidi®ed with 1.0 ml of 25% meta-phosphoric acid (containing 10 ppm isocaproic acid as an internal standard) to 5.0 ml of the fermentation ¯uid. The acidi®ed fermentation ¯uid was analyzed for ammonia via a phenolhypochlorite method as described by Okuda et al. (1965). The VFA (acetic, propionic, butyric, iso-butyric, valeric, and iso-valeric acids) were determined on a Hewlett±Packard 5890A gas chromatograph using a 530 mm macro bore Carbowax M column (Supelco, Bellfonte, PA). The chromatograph oven was programmed as follows: 708C for 1 min, 58C increase minÿ1 to 1008C, 458C increase minÿ1 to 1708C, and a ®nal holding time of 5 min. Total VFA (TVFA) concentration was calculated as the sum of all VFAs. The molar proportions of VFA were calculated by dividing the individual VFA by TVFA.

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

73

2.6. Statistical analysis All experiments were replicated on two separate days and analyzed as completely randomized designs using the general linear model procedure of SAS (1985). The interaction of day (replicate) and treatment was used as the error term. Treatment means were compared using Tukey's test when protected by a signi®cant F value (p<0.05). 3. Results In Experiment 1, we tested the effects of molybdenum on ruminal fermentation of a diet high in sulfur (Table 2). High sulfur or molybdenum had no effect on culture pH when compared to untreated cultures. Ammonia-N was also unaffected by treatment. Compared to the LS control diet, additional sulfur alone had no effect on TVFA concentration, but TVFA was lower in high-sulfur diets treated with 25 ppm of molybdenum. Similarly, high sulfur and, with few exceptions, molybdenum had no effect on the molar proportions of individual VFA. Production of methane and hydrogen were unaffected by treatment. However, sul®de production in the gas (4.3 mmol) and liquid (0.6 mmol) phases of the HS control diet was more than twice that of the LS control diet. Adding 1.0 ppm molybdenum to the cultures with high sulfur did not affect hydrogen sul®de production, but it was decreased by 12 and 77% with 10 and 25 ppm of molybdenum, respectively. In contrast, only the highest level of molybdenum caused a reduction of sul®de in the liquid phase when compared to the untreated diet with high sulfur. In experiment 2, culture pH was greater in many of the cultures treated with various antimicrobial compounds (Table 3). The compounds had minor effects on ammonia-N concentrations. Treating high-sulfur diets with AQ resulted in the greatest depression in TVFA concentration, but it was not statistically different from the depression observed with addition of monensin. Relative to the LS control diet and to the HS control diet, addition of AQ, avoparcin, lasalocid, and monensin decreased the molar percentage of acetate. However, these compounds increased the molar percentage of propionate in the cultures with the response being greatest for monensin (36%) and AQ (32%). Typical of past responses (Garcia-Lopez et al., 1996), cultures treated with monensin had a lower molar percentage of butyrate and those treated with AQ had a higher percentage of butyrate than the LS or HS control diets. Similar to Experiment 1, production of sul®de (gas and liquid phases) was greater in the HS control diet (4.35 mmol) than the LS control diet (3.54 mmol). Only ®ve compounds suppressed hydrogen sul®de production when compared to HS control (4.35 mmol of hydrogen sul®de) and the effect was greatest for AQ (0.41 mmol), followed by chlortetracycline (1.20 mmol), oxytretracycline (1.94 mmol), bambermycin (3.71 mmol), and lasalocid (3.83 mmol). In contrast, monensin markedly increased the production of sul®de (gas and liquid phase) above levels found in the HS control diet. Avoparcin and bacitracin had no effect on hydrogen sul®de production compared to the high sulfur treatment alone. The addition of tylosin (108.1 mM) by itself or in combination with monensin (111.1 mM) to a high-sulfur diet decreased TVFA concentration when compared to the

74

Treatment

pH

LS controlb HS control HS‡Mo, 1 ppm HS‡Mo, 10 ppm HS‡Mo, 25 ppm SE

6.40 6.42 6.46 6.42 6.34 0.02

a

abc ab a ab b

NH3-N (mg dlÿ1)

Total VFAa (mM)

39.2 41.0 42.8 40.9 39.5 0.8

121.0 117.2 118.3 114.5 107.2 1.9

a a a ab b

Acetate (mol (100 mol)ÿ1)

Propionate (mol (100 mol)ÿ1)

Butyrate (mol (100 mol)ÿ1)

Methane (mmol (24 h)ÿ1)

Hydrogen (mmol (24 h)ÿ1)

Sulfide, gas (mmol (24 h)ÿ1)

Sulfide, liquid (mmol (24 h)ÿ1)

62.1 62.8 63.0 62.4 62.0 0.2

19.1 18.9 18.5 18.6 18.7 0.5

13.9 13.8 13.9 14.2 14.5 0.3

494 521 570 556 572 18

14.6 11.1 14.1 11.5 12.2 1.4

2.0 4.3 4.3 3.8 1.0 0.1

0.3 0.6 0.7 0.7 0.2 <0.1

ab ab a ab b

Sum of acetic, propionic, buytric, iso-butyric, valeric, and iso-valeric acids. The low-sulfur (LS) control diet contained 29 g kgÿ1 of sulfur. c Means within a column with different letters differ (p<0.05). b

a ab b ab ab

ab b ab ab a

d c c b a

b a a a b

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

Table 2 Effect of high dietary sulfur (HS, 109 g kgÿ1) and molybdenum (Mo) on in vitro ruminal fermentation

Treatment

pH

NH3-N (mg dlÿ1)

Total VFAa (mM)

S controlb HS control HS‡Anthraquinonec HS‡Avoparcind HS‡Bacitracine HS‡Bambermycinf HS‡Chlortetracyclineg HS‡Lasalocidd HS‡Monensind HS‡Oxytetracyclinee SE

5.87efg 5.86 f 6.00 c 6.05 b 5.94 d 5.91 de 5.98 c 5.98 c 6.13 a 5.89 ef <0.01

22.7 23.6 23.1 27.9 21.6 25.3 23.1 24.1 23.3 23.4 0.9

206.8 210.2 184.3 190.5 204.0 198.3 192.7 197.0 189.6 201.3 1.2

a

b ab b a b ab b ab b a

ab a g f bc cde ef de fg bcd

Acetate (mol (100 mol)ÿ1)

Propionate (mol (100 mol)ÿ1)

Butyrate (mol 100 mol)ÿ1)

Sulfide, gas (mmol (24 h)ÿ1)

Sulfide, liquid (mmol (24 h)ÿ1)

65.4 65.8 56.1 62.9 65.2 65.2 65.0 63.3 61.2 65.4 0.1

18.3 ef 18.0 ef 24.2 b 20.9 c 18.7 e 18.5 e 17.7 g 20.3 d 24.9 a 17.9 g <0.1

13.0 cd 13.2 cd 16.5 a 13.5 bc 12.9 d 13.3 cd 13.9 b 13.1 cd 11.0 e 13.4 cd 0.10

3.54 4.35 0.41 3.98 4.21 3.71 1.20 3.83 6.70 1.94 0.10

0.45 0.59 0.07 0.83 0.77 0.75 0.29 0.80 1.23 0.38 0.03

Sum of acetic, propionic, butyric, iso-butyric, valeric, and iso-valeric acids. The low-sulfur (LS) control diet contained 29 g kgÿ1 of sulfur. c 10 ppm of the liquid. d 5 ppm of the liquid. e 1.25 ppm of the liquid. f 0.3 ppm of the liquid. g Means within a column with different letters differ (p<0.05). b

ab a e c b b b c d ab

d b g bcd bc d f cd a e

d c f b b b e b a de

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

Table 3 Effect of high dietary sulfur (HS, 109 g kgÿ1) and various compounds on ruminal fermentation

75

76

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

Table 4 Effect of high dietary sulfur (HS, 109 g kgÿ1) and 9,10 anthraquinone (AQ), monensin (MON), and tylosin (TY) on in vitro ruminal fermentation Treatment

Total VFAa (mM)

LS controlb HS control HS‡10 ppm Aqc HS‡MONd HS‡Tye HS‡MONd‡Tye SE

122.8 129.6 120.9 114.9 108.1 111.1 1.7

abf a ab ab b b

Acetate (mol (100 mol)ÿ1)

Propionate (mol (100 mol)ÿ1)

Butyrate (mol (100 mol)ÿ1)

H2S (mmol (24 h)ÿ1)

Sulfide (mmol (24 h)ÿ1)

62.8 63.7 53.3 59.4 61.4 58.6 0.2

17.4 16.8 22.8 24.0 17.9 23.6 0.1

13.7 c 13.6 b 17.4 a 11.9 d 15.9 c 12.8 cd <0.1

1.41 2.26 0.19 3.05 1.84 2.37 0.08

0.60 1.33 0.13 1.69 1.02 1.63 1.7

ab a d c b c

b b a a b a

b ab c a bc ab

d bc e a c ab

a

Sum of acetic, propionic, butyric, iso-butyric, valeric, and iso-valeric acids. The low-sulfur (LS) control diet contained 29 g kgÿ1 of sulfur. c 10 ppm of the liquid. d 5 ppm of the liquid. e 1.25 ppm of the liquid. f Means within a column with different letters differ (p<0.05). b

HS control diet (129.6 mM) in Experiment 3 (Table 4). The molar proportion of acetate was decreased by 16.3% by addition of AQ, by 6.7% by monensin, 3.6% by tylosin, and by 8.0% by the combination of tylosin and monensin when compared to the HS control diet. Monensin, monensin and tylosin, and AQ had greater molar proportions of propionate than did the HS control diet. The proportion of butyrate was increased by AQ, but decreased monensin. Hydrogen sul®de and sul®de were increased with the addition of high sulfur, but decreased dramatically by addition of AQ. Tylosin caused a numerical, but not statistical decrease in production of sul®des. Addition of monensin to a highsulfur diet resulted in a 35% increase in hydrogen sul®de production when compared to the HS control diet. Hydrogen sul®de production was unaffected in HS cultures supplemented with the combination of monensin and tylosin. 4. Discussion Total VFA concentrations, the proportions of VFA, and sul®de production were somewhat variable among the different experiments, which was most likely due to the time of ruminal ¯uid collection relative to the last feeding. However, replicating each experiment on 2 days resulted in good repeatability of the results. Diets high in sulfur have resulted in outbreaks of PEM and sulfur toxicity (Gould et al., 1991; McAllister et al., 1997; Low et al., 1996). L'Estrange et al. (1972) also reported that increasing levels of sulfur caused a marked reduction in DM intake by sheep. They attributed the decrease in intake to high levels of ruminal sul®de production. The exact mechanism of high sulfate consumption and PEM is not well understood. However, the production of sul®des has deleterious effects in many biological and non-biological systems. For example, sul®de is readily absorbed through the rumen wall into the blood

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

77

stream (Bray, 1969). Once absorbed, sul®de inhibits the functions of carbonic anhydrase, dopa-oxidase, catalases, peroxidases, dehydrogenases, and dipeptidases, adversely affecting oxidative metabolism and the production of ATP (Short and Edwards, 1989). Speci®cally, sul®de is also thought to block the enzyme cytochrome c-oxidase. Sul®de also binds to hemoglobin creating sulfhemoglobin, reducing the ability of the blood to carry oxygen to tissues. In addition, sul®de also has a paralyzing effect on the carotid body and, therefore, may also inhibit normal respiration (Bulgin et al., 1996). In the environment, iron and steel structures are prone to corrosion in the presence of sul®des (Odom and Singleton, 1993). In the work place, the presence of hydrogen sul®de gas at low concentrations (50±200 ppm) is an irritant to the human respiratory tract, and at higher concentrations (200±500 ppm) it can cause hemorrhagic pulmonary edema that is often fatal (Green et al., 1991). Broad-spectrum biocides, such as hypochlorite (Odom and Singleton, 1993), methylenebis thiocyanate (Zhou and King, 1995) and gentamicin (Tanimoto et al., 1989) have been used to control sul®de production. Most, if not all, of these biocides would be impractical to use in ruminant diets because their broad antimicrobial spectrums would negatively impact ruminal fermentation. In addition, many of these compounds would be highly toxic to ruminants. Few compounds appear to be speci®c for inhibiting sulfate-reducing bacteria without adversely affecting general ruminal fermentation. For example, molybdenum (MoO4) has been proposed as an analog of sulfate which blocks the sulfate activation step that is catalyzed by ATP sulfurylase (Oremland and Capone, 1988). Taylor and Oremland (1979) showed that molybdenum speci®cally inhibits sulfate-reducing bacteria in pure culture and other investigations have shown that molybdenum inhibits sulfate-reducing bacteria in sediments (Oremland and Silverman, 1979). The ability of molybdenum to inhibit sul®de production in ruminal fermentations has been reported by others (Gawthorne and Nader, 1976). Recently, Loneragan et al. (1998) reported that sodium molybdate was capable of reducing hydrogen sul®de concentrations in the gas cap of cattle fed a high-sulfur diet, but the effect was not consistent for all cattle and liver stores of Cu decreased dramatically. Our data con®rm that molybdenum (>10 ppm of the ¯uid) can reduce hydrogen sul®de production in ruminal fermentations. At concentrations that we reported in this study (maximum of 25 ppm of the ¯uid), we observed no effect of molybdenum on rumen VFA, methane or hydrogen. This amount of molybdenum caused a depression in both, the liquid and gas sul®de and a slight decrease in total VFA, but no changes in any other culture conditions. In subsequent studies, we tested molybdenum at levels including 90 and 900 ppm of the culture ¯uid (data not shown). These levels completely inhibited sul®de production, but had no effects on VFA production. Jessie (1988) reported that molybdenum, at a concentration of 0.2 mM (32 ppm of the culture medium), inhibited pure cultures of sulfate-reducing bacteria isolated from a salt marsh, which agrees with our results. In ruminants, molybdenum is a trace mineral with a very narrow margin between the amount needed to ful®ll the animal's requirements and toxic levels. Underwood (1981) reported that, in cattle, molybdenum was toxic in the range of 20± 100 ppm on a dry matter basis. However, Huber et al. (1971) reported that lactating dairy cows showed no signs of toxicity when fed a diet containing 100 ppm of molybdenum (1.7 g/day) for 6 months, but toxicity occurred when cows were fed 200 ppm of molybdenum. Intake of 1.7 g of molybdenum in a cow weighing 625 kg with an 85-L

78

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

rumen would equate to a rumen concentration of 20 ppm, which is similar to the amount of molybdenum used in our study. In Experiment 2, the effects of antimicrobial compounds on ruminal VFA patterns were as expected. For example, the ionophores, monensin and lasalocid, decreased total VFA concentrations and the molar proportions of acetate, but increased the molar proportions of propionate (Chen and Wolin, 1979). The effect was greater for monensin than lasalocid. Avoparcin had similar effects on the molar percentages of VFA as lasalocid. Marounek et al. (1995) reported that avoparcin had effects on ruminal fermentation that were similar to monensin. Bambermycin, chlotetratcylcine, bacitracin, and oxytetracycline had minor effects on VFA in the current study and reduced the ®nal VFA concentration by 2.9 to 8.3%. Other studies have also concluded that bambermycin (also known as ¯avomycin) has no effect on ruminal VFA (Aitchson et al., 1989). Gram negative sulfate-reducing bacteria are primarily responsible for hydrogen sul®de production in the rumen. We previously reported that AQ was a potent inhibitor of sulfate reduction in ruminal fermentations (Kung et al., 1998) and con®rmed those ®ndings in this study. Cooling et al. (1996) reported inhibition of sul®de production using 9,10 anthraquinone due to possible uncoupling of the electron transport chain. Decreased levels of ATP result in insuf®cient energy, which is needed for subsequent activation of sulfate for further sulfate reduction. Of the compounds tested in Experiment 2, oxytetracycline, and chlortetracycline also decreased sulfate reduction. Both of these compounds have been classi®ed as broad-spectrum antibiotics, known to have activity against Gram-negative and Gram-positive bacteria (Nagaraja, 1995). The other antimicrobials tested had no effect on hydrogen sul®de production and are all generally classi®ed as having activity against Gram-positive bacteria. Unexpectedly, the cultures treated with monensin had sul®de concentrations (gas and liquid) higher than those from the HS control diet in both, experiments 2 and 3. We have since veri®ed this ®nding in several subsequent studies (unpublished data). Indirect inhibition of methanogens by monensin may have decreased competition between methanogens and sulfate-reducing bacteria, but the speci®c reason for a stimulatory effect on sul®de production by monensin is unknown. Tylosin, a compound commonly used in feedlots to reduce liver abscesses and commonly fed with monensin, numerically, but not statistically, reduced sul®de production by itself. However, when combined with the stimulatory effect of monensin, the combination of monensin and tylosin had no effect on sul®de production when compared to the HS control diet. McAllister et al. (1997) reported that the incidence of PEM cases in a feedlot in Colorado was seasonal and related to days in the feedlot. The incidence of PEM peaked between 15±30 days after cattle had entered the feedlot and peaked during summer months. Increased consumption of water high in sulfate during hot summer months, coupled with increasing adaptation to high sulfur intake by sulfate-reducing bacteria in the rumen (Cummings et al., 1995; de Oliveira et al., 1996) probably resulted in high levels of hydrogen sul®de in these cattle. Furthermore, the proportion of concentrate in the diet of incoming cattle is usually gradually increased during the ®rst 4 weeks in the feedlot. As the proportion of concentrate in the diet increased, rumen pH would decrease, resulting in a greater proportion of sul®de to be protonated since the pKa for hydrogen sul®de is 7.2. In addition, we hypothesize that another contributing factor to high levels of

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

79

hydrogen sul®de production could be the fact that, within the ®rst 30 days of entering the feedlot, cattle are also fed increasing amounts of monensin. There is an increased risk for development of PEM in ruminants consuming feeds or water high in sulfur or sulfate. Identi®cation of feeds high in sulfur and proper balancing of rations can reduce the chance of excess sulfur consumption in feed. Where high sulfate water is a problem, treatment by reverse osmosis conditioning can lower the sulfate content, but this is a costly proposition. Including compounds in the diet that inhibit sulfate-reducing bacteria and reduces hydrogen sul®de production may be useful. Several feed additives approved for use in the US, for example oxytetracycline and chlortetracycline, appear to have some ability to decrease hydrogen sul®de production in in vitro fermentations. The compound 9,10 anthraquinone, an experimental compound, is also a potent inhibitor of sulfate reduction and has the added bene®t of partially inhibiting methane production and improving the ratio of acetate to propionate. The experiments described in this study were tested under in vitro conditions in ruminal ¯uid that was not adapted to high sulfur diets or any of the antimicrobial compounds tested. Adaptation by ruminal microorganisms can occur and thus animal studies are needed to verify our ®ndings on an in vivo basis. Acknowledgements The authors thank C. Golt and N. Ranjit who assisted in sample analyses. J.P. Bracht and J.Y. Tavares were undergraduate Science and Engineering Scholars. J.P. Bracht used parts of this study as partial ful®llment of requirements for a Degree with Distinction.

References Aitchson, E.M., Ralph, I.M., Rowe, J.B., 1989. Evaluation of feed additives for increasing wool production from Merino sheep 1. Lasalocid, avoparcin and ¯avomycin included in lucerne-based pellets or oaten chaff fed at maintenance. Aust. J. Expt. Agric. 29, 321±325. AOAC, 1990. Of®cial Methods of Analysis (15th Edition). Association of the Of®cial Analytical Chemists, Arlington, VA. Bray, A.C., 1969. Sulphur metabolism in sheep II. The absorption of inorganic sulphate across the rumen wall of sheep. Aust. J. Agr. Res. 20, 749±756. Bulgin, M.S., Stuart, S.D., Mather, G., 1996. Elemental sulfur toxicosis in a ¯ock of sheep. JAVMA 208, 1063± 1065. Chen, M., Wolin, M.J., 1979. Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria. Appl. Environ. Microbiol. 38, 72±77. Consortium, 1989. Handbook for Agricultural Animal Care and Use in Research and Teaching. Agricultural Animal Care and Use Committee, University of Delaware, College of Agriculture and Natural Resources, Newark. Cooling III, F.B., Maloney, C.L., Nagel, E., Tabinowski, J., Odom, J.M., 1996. Inhibition of sulfate respiration by 1,8-dihydroxyanthraquinone and other anthraquinone derivatives. Appl. Environ. Microbiol. 62, 2999±3004. Cummings, B.A., Caldwell, D.R., Gould, D.H., Hamar, D.W., 1995. Identity and interactions of rumen microbes associated with dietary sulfate-induced polioencephalomalacia. Am. J. Vet. Res. 56, 1384±1389. Garcia-Lopez, P.M., Kung Jr., L., Odom, J.M., 1996. In vitro inhibition of microbial methane production by 9,10 anthraquinone. J. Anim. Sci. 74, 2276±2284.

80

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

Gawthorne, J.M., Nader, C.J., 1976. The effect of molybdenum on the conversion of sulphate to sulphide and microbial-protein-sulphur in the rumen of sheep. Br. J. Nutr. 35, 11±23. Goering, H.K., Van Soest, P.J., 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 3790, ARS-USDA, Washington, DC. Gould, D.H., 1998. Polioencephalomalacia. J. Anim. Sci. 76, 309±314. Gould, D.H., McAllister, M.M., Savage, J.C., Hamar, D.W., 1991. High sul®de concentrations in rumen ¯uid associated with nutritionally induced polioencephalomalacia in calves. Am. J. Vet. Res. 52, 1164±1169. Green, F.H.Y., Schurch, S., De Sanctis, G.T., Wallace, J.A., Cheng, S., Prior, M., 1991. Effects of hydrogen sul®de exposure on surface properties of lung surfactant. J. Appl. Physiol. 70, 1943±1949. Hill, F.I., Ebbett, P.C., 1997. Polioencephalomalacia in cattle in New Zealand fed chou mellier (Brassica oleracea). New Zealand Vet. J. 45, 37±39. Huber, J.T., Price, N.O., Engel, R.W., 1971. Response of lactating dairy cows to high levels of dietary molybdenum. J. Anim. Sci. 32, 364±367. Jessie, K.M., 1988. Inhibition of sulfate-reducing and methane-producing bacteria by MoO4 and three heavy metals. M.S. Thesis, University of Delaware. Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J., Van Soest, P.J., 1983. Nitrogen fractions in selected feeds. J. Dairy Sci. 65, 217±255. Kung, L., Jr., Hession, A.O., Bracht, J.P., 1998. Inhibition of sulfate reduction to sul®de by 9,10 anthraquinone in in vitro ruminal fermentations. J. Dairy Sci. 81, 2251±2256. L'Estrange, J.L., Upton, P.K., McAleese, D.M., 1972. Effects of dietary sulphate on voluntary feed intake and metabolism of sheep 1. A comparison between different levels of sodium sulphate and sodium chloride. Ir. J. Agric. Res. 11, 127±144. Loneragan, G.H., Wagner, J.J., Gould, D.H., Barry, F.B., Goodall, S.R., 1998. Effect of dietary molybdenum and copper on ruminal gas cap H2S levels and liver copper stores of feedlot steers, J. Dairy Sci. (Abstracts) 81 (Suppl. 1) 329. Low, J.C., Scott, P.R., Howie, F., Lewis, M., Fitzsimons, J., Spence, J.A., 1996. Sulphur-induced polioencephalomalacia in lambs. Vet. Rec. 138, 327±329. Marounek, M., Skrivanova, V., Hodrova, B., 1995. The effect of avoparcin on in vitro ruminal fermentation. Zivocisna Vyroba. 4, 165±169. McAllister, M.M., Gould, D.H., Raisbeck, M.F., Cummings, B.A., Loneragan, G.S., 1997. Evaluation of ruminal sul®de concentrations and seasonal outbreaks of polioencephalomalacia in beef cattle in a feedlot. JAVMA 211, 1275±1729. Nagaraja, T.G., 1995. Ionophores and antibiotics in ruminants. In: Wallace, R.J., Chesson, A. (Eds), Biotechnology in Animal Feeds and Animal Feeding. VCH, NY, pp. 173±204. Odom, J.M., Singleton, R., Jr., 1993. The Sulfate-Reducing Bacteria: Contemporary Perspectives. Springer, New York. Okuda, H., Fuji, S., Kawashima, Y., 1965. A direct colorimetric method for blood ammonia. Tokushima J. Exp. Med. 12, 11±14. de Oliveira, L.A., Jean-Balin, C., Corso, V.D., Benard, V.D., Durix, A., Komisarczuk-Bony, S., 1996. Effect of high sulfur diet on rumen microbial activity and rumen thiamine status in sheep receiving a semi-synthetic, thiamine-free diet. Reprod. Nutr. Dev. 36, 31±42. Oremland, R.S., Capone, D.G., 1988. Use of speci®c inhibitors in biogeochemistry and microbial ecology. Adv. Microbiol. Ecol. 10, 285±383. Oremland, R.S., Silverman, M.P., 1979. Microbial sulfate reduction measured by an automated electrical impedance technique. Geomicrobiol. J. 1, 355±372. SAS(r) User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. Short, S.B., Edwards, W.C., 1989. Sulfur (hydrogen sul®de) toxicosis in cattle. Vet. Human Toxicol. 31, 451± 453. Siegal, L.M., 1965. A microdetermination method for sul®de. Anal. Biochem. 11, 126±132. Tanimoto, Y., Tasaki, M., Okamura, K., Yamaguichi, M., Minami, K., 1989. Screening growth inhibitors of sulfate-reducing bacteria and their effects on methane formation. J. Fermen. Bioeng. 68, 353±359. Taylor, B.F., Oremland, R.S., 1979. Depletion of adenosine triphosphate in Desulfovibrio by oxyanions of Group VI elements. Curr. Microbiol. 3, 101±103.

L. Kung et al. / Animal Feed Science and Technology 84 (2000) 69±81

81

Underwood, E.J., 1981. The Mineral Nutrition of Livestock. 2nd Edition. Commonwealth Agricultural Bureaux, England, pp. 91±108. Wagner, J.J., Loneragan, G.H., Gould, D.H., Thoren, M., 1998. The effect of varying water sulfate concentration on feedyard performance and water intake of steers, J. Anim. Sci. (Abstr.) 75(Suppl. 1) 272. Zhou, X., King, V.M., 1995. A rapid bactometer method for screening of biocides against sulfate-reducing bacteria. Appl. Microbiol. Biotechnol. 43, 336±340.