Effects of Isoacids, Urea, and Sulfur on Ruminal Fermentation in Sheep Fed High Fiber Diets1 A. BRONDANI,2 R. TOWNS, K. CHOU, and R. M. COOK Department of Animal Science Michigan State University East lansing 48824

H. BARRADAS Instituto NacionaJ de Investigaciones Pecuarias. Centro Experimental Pecuario "La Posta" Apartado Postal 89B SucursaJ A Veracruz, Mexico


supplementation with urea, isoacids, and


The effects of isoacids, urea N, and S on ruminal fermentation of sugarcane bagasse- or com stover-based diets were studied in sheep. Acetate production was taken as a measure of the fermentation rate. For the sugarcane bagasse diet, neither urea nor S supplementation changed ruminal acetate production. When N and S were combined, acetate production was 44% higher (3.16 vs. 2.18 mol/d). Similar effects were noted for the com stover diet. Increasing the level of isoacids from .1 to .2 g/kg BW per d in the diet did not change acetate production for either diet. However, N supplementation of the sugarcane bagasse diet containing the low level of isoacids resulted in a 49% greater acetate production (2.86 vs. 1.91 mol/d). Acetate production was 90% higher (3.74 vs. 1.97 mol/d) when the diet containing the high level of isoacids was supplemented with N. The corresponding increases for com stover were 12% (2.64 to 2.95 mol/d) and 35% (2.88 to 3.87 mol/d). The results suggest j)at NH3 N provided by the basal diet was more limiting than isoacids. Once the N deficiency was corrected, isoacids became limiting. Ruminal digestion of high fiber diets low in N was improved by

(Key words: isoacids, urea, sulfur) INTRODUCTION

An important problem in ruminant nutrition is to define nutrients required by ruminal microorganisms for maximal fermentation of feedstuffs, particularly of low protein highly fibrous plant material (5). Ruminal bacteria require isoacids (isobutyrate, 2-methylbutyrate, isovalerate, and valerate), NH3' and hydrogen sulfide for growth (3, 5, 6, 9, 10). However, little information is available concerning the interaction of these three nutrients, particularly when high fiber diets are fed. Our objective was to study the effects of the interaction of isoacids, NH3, and sulfur on ruminal fermentation in sheep fed diets containing two important crop residues, com stover and sugarcane bagasse. MATERIALS AND METHODS

Received October 31, 1990. Accepted April 5, 1991. lMicbigan Agricultural Experiment Station, Publication Number 12,470. 2Present addIess: Purina llalia S.pA., Viole del Gbisollo 20, 20151, Milano, Italy. 1991 J Dairy Sci 74:2724-2727

The experimental design for two trials was a 2 3 factorial crossover (three factors each at two levels) conducted in two 4 x 4 quasi-Latin squares (7). Double blocking criteria were animals and time (nonrandom repeated measurements were obtained from each subject assigned to a sequence of treatment combinations). In each trial, eight rumen-fistulated adult Tabasco sheep were divided into two groups of four. Individuals in groups 1 and 2 weighed 27 and 36 kg, respectively, and were assigned randomly to rows of one square, corresponding to a predetermined sequence of treatment combinations (7). The three factors were isoacids, N, and S, each at two levels.



ISOACIDS AND ACETATE PRODUcnON TABLE 1. Composition and analysis of basal diets. 1 Item

Trial 1

Trial 2

Ingredient, %

Com slover Com cobs Sugarcane bagasse Sorghum grain Bone meal Trace-mineralized salt2

25.0 25.0 44.0 55.0 49.0 .5.5 .5 .5

Analysis3 CPo % 7.2 7.7 Crude fiber. % 22.0 18.2 Digestible energy, McalIkg 2.6 2.9 Total N, % 1.15 1.23 Total S, % .14.18 N:S 8.0 6.8 10M basis.

2composition: Zn, .35%; Mn, .20%; Fe, .20%; Cu. .03%; Co, .005%; 1, .007%; and NaCl, 96%. 3CalcuIated NRC values (12).

traruminal injection of 100 I1Ci of Na[1- 14C]acetate with 100 ml of a 10% solution of polyethylene glycol (molecular weight 3350). Following infusion, ruminal fluid samples were collected every 20 min for the next 3 h. Samples were strained through four layers of surgical gauze and stored frozen at -20·C. Ruminal hydrogen sulfide was detennined using a hydrogen sulfide-sensing electrode (Lazar model GS-136). Determinations of acetate production, rumina! NH3 N, and acetate were described previously (4, 6). Overall significance of treatment effects was determined by ANOVA (7) according to the model in Equation 1. Specific differences among treatment means within each two-way treatment combination were determined by Bonferroni t tests (7).

11 + 0i + P(i)j + G(i)k + Al + Bm + (AB>lm + + (AChn + (BC)mn + (ABC)lmn


Composition of the basal diets for trials 1 and 2 is given in Table 1. Corn stover was chopped with a silage chopper to lengths of 1.25 em. Both sugar cane bagasse and stover were airdried. Based on results of previous experiments (6, 14), an equal weight mixture of isoacids (isobutyrate, 2-methylbutyrate, isovalerate, and valerate) was administered at .1 and .2 glkg BW per d To achieve two levels of NH3 N in the rumen (about 5 and 15 mg/dl), the basal diet was fed either alone or supplemented with urea (high N treatment) at 1.5% of OM. To achieve two levels of sulfide in the rumen (4 and 8 l1g/ml), the basal diet was fed alone or supplemented with elemental S at .2% of OM. Sulfur and N supplementation was designed to provide four different N:S ratios. Final N:S ratios were approximately 3:1, 5:1, 8:1, and 12:1. Isoacids, urea, and S were premixed weekly with part of the sorghum and then incorporated into the totally mixed diet. The daily ration was fed at 0700 h, and feed intake was recorded daily for each animal. Water was provided for ad libitum intake. Animals were adapted to a given diet for 14 d prior to measurements. In vivo production rates of acetate were measured by a single injection radioisotope technique (4, 15). On d 15, 3 h after the morning feeding, each animal received an in-


where Yijldmn = observed value for sheep j within square i under level I of N, level m of isoacids, and level n of S during period k.; 11 = overall mean; OJ = ftxed effect of square i; P(i)j = random effect of sheep j within square i; G(i)k fixed effect of period k within square i; AI = fixed effect of level I of N; B m = fixed effect of level m of isoacids; = fixed effect of level n of S; (ABhm = fixed effect of the interaction between level of N and level of isoacids; (AC)ln = fixed effect of the interaction between level of N and level of S; (BC)mn = fixed effect of the interaction between level of isoacids and level of S; (ABC)lmn = fixed effect of the interaction among level of N, level of isoacids, and level of S, in this case, nonseparable from the effect of squares; and Ejjldmn = random residual error. Significance was declared at P < .10 unless otherwise noted



Results are presented in view of the existence of two significant two-way interactions (7), i.e., the interactions between N with S and N with isoacids. The interaction of isoacids and S was not significant. Therefore, there was no specific examination of combination means Journal of Dairy Science Vol. 74, No.8. 1991



TABLE 2. Effects of N and S on feed intake and ruminal fermentation variables in sheep fed high fiber diets. Trial 2 (com stover diet)

Trial 1 (sugarcane bagasse diet) Item OM!, g/d Feed N:S NH3 N, mg/dl1 Sulfide-S, ~2 Acetate production, moUd

Low N, High N, Low N, High N, low Slow S high S high S SE

Low N, High N, Low N, High N, low S high S high S SE low S

387 8.1 6.11 2.43

430 7.0 5.28 2.97


454 12.7 12.19 2.16 l.94b

442 3.1 5.14 5.84 2.25 b

462 5.0 13.11 5.51 3.16a

16 .84 .33 .24


412 10.7 14.19 3.34 2.82b

461 3.2 6.93 5.26

442 4.7 13.11 4.81

15 .71 .28 .21

a,"Means in the same row within a trial with different superscripts differ (P < .10). 1Significant effect of N (P < .01). 2Significant effect of S (P < .01).

involving those two factors. When two factors interact, it is appropriate to examine the means of two-way combinations (averaged over other factors, if any) to determine the nature of the interaction. For the two way combinations between N and S (Table 2), DMI did not differ among treatment group in either trial. Urea increased (P < .01) ruminal NH3 N in both trials. Similarly, S supplementation resulted in higher (P < .01) levels of sulfide-S in ruminal fluid of sheep on both trials. In both trials, acetate production was not changed by either urea or S when fed separately. Supplementation of both resulted in a 44% increase in acetate production in trial 1 and a 63% increase in trial 2. Although the lack of an increase in acetate production in the group with low N and high S may be explained by the low level of ruminal NH3 N (Table 2), the results for the group with high N and low S were not expected. The calculated N:S ratio (Table 2) and the percentage of S in the basal diets (Table 2) were within the ranges commonly recommended for adult sheep (2, 3, 13). However, S intake by animals in the group with high N and low S may have been limiting. Hume and Bird (11) reported that S intake of 1.95 gld supported maximal ruminal microbial protein synthesis in sheep. In the present study, intake of total S in the group with high N and low S averaged .65 and .74 gld, respectively, for trials 1 and 2. For the group with high N and high S, average daily intake of S for sheep in trials 1 and 2 was 1.66 and 1.80 gI d, respectively. The precise level at which ruminal sulfide concentration limits microbial growth and fermentation has not been defmed Journal of Dairy Science Vol. 74, No.8, 1991

clearly (3), but 1 ~g of sulfide-SImi of ruminal fluid has been suggested to be the lower limit for optimal fermentation (8). Concentration of sulfide-S in the groups with high N and low S was greater than 2 ~glmI but may not have been high enough for maximal fermentation. These results suggest that S supplementation based solely on the N:S ratio or on the percentage of S in DM may not be adequate when high fiber diets containing low N are fed. The total amount of S that would allow maximal microbial protein synthesis should be considered first. Once that is provided, supplementing N to attain an N:S ratio of about 10 (13) should result in maximal fermentation efficiency. Increasing the level of isoacids in the diet resulted in higher (P < .01) concentrations of these acids in the rumen for both trials (Table 3). In trial I, acetate production was higher when N was supplemented along with low isoacids (P < .05). Increasing the amount of isoacids in the diet, without adding N, did not change acetate production. When both were present at the high level, acetate production was further increased (P < .05) by 30%. These results show that rates of fermentation in the rumen can be only as high as the availability of the most limiting nutrient. In trial I, ruminal NH3 N provided by the basal diet was more limiting than isoacids. Once N supply was increased by the addition of urea to the diet, isoacids became limiting. Trends in acetate production found in trial 2 were similar to trial 1 (Table 3). However, increases (P < .05) in acetate production were found only when both N and isoacids were fed



TABLE 3. Effects of isoacids (Iso) and N on feed intake and on ruminal fermentation variables in sheep fed high fiber diets. Trial 2 (com stover diet)

Trial I (sugarcane bagasse diet) Item DM!. gld NH3 N, mg/d1


Low Iso, Low Iso. High Iso. High Iso. low N high N low N high N SE

Low Iso. Low Iso. High Iso, High Iso, low N high N low N high N SE





























2.95 b


Total isoacids,


Acetate production. mol/d





1.91 c





a,b,CMeans in the same row within a trial with different superscripts differ (P < .05). (Significant effect of N (P < .01). 2Significant effect of isoacids (P < .01).

at a high level. This suggests that ruminal NH3 N provided by the basal diet in trial 2 was adequate to support microbial fermentations as efficiently as the diet that was supplemented with N. But ruminal NH3 N was not sufficiently high to pennit utilization of the higher amounts of isoacids supplied by the high isoacid treatment. Because the production of VFA from carbohydrates in the rumen is coupled with microbial growth (1), maximal microbial yield can be attained only if precursors for protein synthesis are made available to the microbiota simultaneously and in adequate quantities. This study suggests that high fiber diets low in N are utilized better when aU three factors, N, isoacids, and S, are adequate. ACKNOWLEDGMENTS

This research was supported in part by National Science Foundation Grant INT77-0636, CONACYT = Consejo Nacional de Ciencia y Tecnologia Grant 1396 and by the Michigan Agricultural Experiment Station. REFERENCES

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gestion and metabolism in Ibe ruminant. 1. W. McDonald and Al.C. Warner, ed. Univ. New England Pub!. Unit, Annidale. New South Wales, Ausl. 4 Cook, R. M. 1966. Use of 14C 10 study utilization of substrates in ruminants. J. Dairy Sci. 49:1018. 5 Cook, R. M. 1985. Isoacids, a new feed additive for dairy cows. Page 41 in Proc. Maryland Nutr. Coni. Feed Manuf. J. A. Doerr, ed. Univ. Maryland, College Park. 6 Felix, A., R. M. Cook. and J. T. Huber. 1980. Isoacids and urea as a protein supplement for lactating cows fed com silage. J. Dairy Sci. 63:1098. 7 Gill, J. L. 1978. Design and analysis of experiments in the animal and medical sciences. Vol. 1. 2. and 3. Iowa State Univ. Press, Ames. 8 Harrison, D. G., and A. B. McAllan. 1981. Factors affectiD8 microbial growth yields in the reticulorumen. Page 205 in Digestive physiology and metabolism in ruminants. Y. Ruckebush and P. Thivend, ed. A VI Publ. Co. Inc., Westport, cr. 9 Huber, J. T., and L. Kung, Jr. 1981. Protein and nonprotein nitrogen utilization in dairy cattle. J. Dairy Sci. 64:1170. 10 Hume. I. D., 1970. Synthesis of microbial protein in the rumen. II. A response to higher volatile fatty acids. Ausl. J. Agric. Res. 21:292. 11 Hume, I. D., and P. R. Bird. 1970. Synthesis of microbial protein in the rumen. IV. The influence of the level and form of dietary sulphur. Aus!. J. Agric. Res. 21:315. 12 National Resean:h Council. 1975. Nutrient requirements of sheep. 51b rev. ed. Nat!. Acad. Sci., Washington, DC. 13 Orskov, E. R. 1982. Page 33 in Protein nutrition in ruminants. Academic Press, New York, NY. 14 Quispe-Salas, M E. 1982. A study of the effects of isoacids, urea and sulfur on the rate of fermentation in the rumen. MS. Thesis, Michigan State Univ., East Lansing. 15 Rogers, J. A., and C. L. Davis. 1982. Effects of intraruminal infusions of mineral salts on volatile fatty acid production in steers fed high grain and high roughage diets. J. Dairy Sci. 65:953. Journal of Dairy Science Vol. 74, No.8, 1991

Effects of isoacids, urea, and sulfur on ruminal fermentation in sheep fed high fiber diets.

The effects of isoacids, urea N, and S on ruminal fermentation of sugarcane bagasse- or corn stover-based diets were studied in sheep. Acetate product...
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