Response of Forage Fiber Degradation by Ruminal Microorganisms to Branched-Chain Volatile Fatty Acids, Amino Acids, and Dipeptides

J. Dairy Sci. 85:1183 1190 American Dairy Science Association, 2002. Response of Forage Fiber Degradation by Ruminal Microorganisms to Branched-Chain Volatile Fatty Acids, Amino Acids, and Dipeptides C.-M. J. Yang Applied Animal Science Department National I-Lan Institute of Technology I-Lan, Taiwan, 26015 ROC ABSTRACT This study evaluated the effect of branched-chain volatile fatty acids (VFA; isobutyric acid, isovaleric acid), amino acids (valine, leucine), and dipeptides (valinevaline, leucine-leucine) on neutral detergent fiber (NDF) degradation by rumen microorganisms in vitro. The CP (%) and in situ NDF degradation rate (%/h) for alfalfa, bermudagrass, and pangolagrass hays, and napiergrass silage were 17.2 and 7.5, 4.7 and 3.1, 8.3 and 5.3, and 9.6 and 3.4, respectively. In vitro NDF digestibility was the lowest for bermudagrass; alfalfa and napiergrass were the highest. When the incubation contained more ammonia initially, digestibilities increased, but relative differences among forages were unchanged. Adding branched-chain VFA (2 mm) to incubations increased digestibilities more than controls on 15 out of 16 occasions. The effectiveness varied with isoacids and forages used. Amino acid (2 mm) or dipeptide (1 mm) addition consistently increased digestibility over controls. Amino acids further increased digestibility over corresponding isoacids on 14 occasions. Improvement in digestibility over control by leucine appeared to be greater than that by valine. Digestibilities with dipeptides were always greater than those with isoacids, except for one case. Dipeptide addition further increased digestibility significantly over corresponding amino acids on only six occasions, while percent improvement in digestibility numerically by dipeptides occurred in 10 cases. Valine-valine seemed to exert different effect than leucine-leucine, depending on initial ammonia availability. The results indicate that dipeptides could be more effective than isoacids and amino acids in improving NDF digestion. Forages with high CP content or rapid NDF degradation rate appeared to respond to additives to smaller degrees. (Key words: branched-chain volatile fatty acids, amino acid, dipeptide, fiber degradation) Received September 5, 2001. Accepted December 19, 2001. Corresponding author: C.-M. J. Yang; e-mail: cmyang@ ilantech.edu.tw. Abbreviation key: ADL = acid detergent lignin, ALF = alfalfa hay, BCVFA = branched-chain VFA, BER = bermudagrass hay, ic 4 = isobutyric acid, ic 5 = isovaleric acid, NAP = napiergrass silage, PAN = pangolagrass hay. INTRODUCTION Forage fiber consumed by ruminants relies on microbial fermentation in the rumen to produce nutrients for absorption. The rate of fiber degradation in the rumen is positively related to DMI by ruminants (Van Soest, 1982; Mertens, 1987). Most fiber-degrading microorganisms in the rumen require branched-chain VFA (BCVFA) (Dehority et al., 1967; Van Gylswyk, 1970; Bryant, 1973). And, several studies have demonstrated that ruminal fiber digestion is enhanced by additional BCVFA (Van Gylswyk, 1970; Soofi et al., 1982; Gorosito et al., 1985). Ruminal BCVFA originates primarily from dietary true protein degradation, although microbial protein recycling also produces BCVFA (Miura et al., 1980). Ruminal microorganisms utilize BCVFA as a source of carbon skeleton to synthesize branched-chain amino acids (Allison et al., 1962a, 1962b). Amino acids often are stimulatory for rumen microbes even when ammonia and carbohydrates exceed requirements (Maeng and Baldwin, 1976; Cotta and Russell, 1982; Argyle and Baldwin, 1989), indicating a direct effect from amino acids. It is possible that fiber-degrading microorganisms could benefit more by direct provision of branched-chain amino acids rather than the corresponding BCVFA. Previous studies have shown that peptides are preferred substrates by ruminal microbes than the constituent amino acids in free form (Pittman and Bryant, 1964; Pittman et al., 1967; Chen et al., 1987b). Wright (1967) indicated that peptide carbon was more efficiently utilized than amino acid carbon. It is possible that use of peptides containing branched-chain amino acids could even be more effective in supplying branched-chain carbon skeleton than using related free amino acids or BCVFA. 1183

1184 YANG The work by Gorosito et al. (1985) showed that fiber digestion by ruminal bacteria was improved by BCVFA. Furthermore, the addition of casein hydrolyzate (contained amino acids and peptides) in the presence of BCVFA further enhanced fiber digestion. These results provide evidence that fiber digesters were stimulated directly by amino acids and peptides. However, it is not certain that the stimulation was due to increased branched-chain skeleton supply from free amino acids or peptides per se. Peptides are intermediates in ruminal protein degradation (Chen et al., 1987a). Although peptides often show transitory accumulation in the rumen (Chen et al., 1987a; Broderick and Wallace, 1988), some are more persistent than others (Broderick et al., 1988; Wallace et al., 1990; Yang and Russell, 1992) and may escape ruminal breakdown to various degrees (Wallace et al., 1990; Yang and Russell, 1993). One way of microbial hydrolysis of large peptides in the rumen is through the action of microbial N-dipeptidyl peptidase (Wallace and McKain, 1989). Dipeptides are probably the predominant products of proteolysis encountered by ruminal microbes (Wallace and McKain, 1989; Yang and Russell, 1992). Dipeptides varied in rate of uptake and utilization by rumen microorganisms (Broderick et al., 1988; Yang and Russell, 1992). Earlier work (Yang and Russell, 1992) illustrated that valine and leucine dipeptides were more rapidly utilized by ruminal bacteria than most other tested dipeptides containing single valine or leucine molecule. Forage species vary in rate and extent of fiber digestion (Varga and Hoover, 1983; Andrighetto et al., 1993; Hoffman et al., 1993) and may respond differently to sources of BCVFA (Gorosito et al., 1985). Bermudagrass (BER) and pangolagrass (PAN) hays, and napiergrass silage (NAP) are the major forages fed to dairy cows in Taiwan, with alfalfa hay (ALF) as a supplemental forage. The purpose of this study was to compare effectiveness of branched-chain VFA (isobutyric acid, ic 4 ; isovaleric acid, ic 5 ), amino acids (Val, Leu) and their dipeptides (Val-Val, Leu-Leu) on NDF degradation of these forages by ruminal microorganisms in vitro. The addition of dipeptides containing mixed branched-chain amino acids (Val-Leu, Leu-Val) was also evaluated. Forage Preparation MATERIALS AND METHODS Alfalfa (Medicago sativa) and bermudagrass (Cynodon dactylon) hays were imported from the United States. Pangolagrass (Digtaria decumbens, A254) hay and napiergrass (Pennisetum purpureum) silage were produced locally, and were harvested at the height of approximately 1 and 2 m, respectively. After determina- tion of DM (55 C, 48 h), forage samples were ground (1 mm) and analyzed for NDF, ADF, and acid detergent lignin (ADL) (Van Soest et al., 1991). Ash, DM, and CP determinations were made according to AOAC (1984). Chemical composition of these forages is listed in Table 1. Animals Two Holstein steers (approximately 650 kg each) fitted with ruminal fistula were used for in situ measurements and as ruminal fluid donors. The steers were housed in box stalls and fed a low protein diet consisting of bermudagrass hay (Table 1) ad libitum and 2 kg of a commercial concentrate mix (12% CP) fed twice daily. Mineral salt block and water were provided for free access. In Situ Ruminal Degradation of Forages Ruminal NDF degradation kinetics of individual forages was determined in situ with Dacron bags (Varga and Hoover, 1983). Duplicate bags (52-µm pore size, Ankon Co., Fairport, NY) containing 8 g dried (55 C, 72 h) and ground (4 mm) forages were placed into the rumen of each steer at various intervals between 3 and 72 h. After removal of all bags from the rumen at one time, bags were soaked in ice water to stop microbial activity and to facilitate detachment of microbes from forage residues in bags (Craig et al., 1984). Tap water was then used to rinse rumen contents from the outside of the bags, which were subsequently washed in a washing machine (Cherney et al., 1990). Washed bags were dried (55 C, 72 h) in a forced air oven. Dried samples were ground (1 mm) and then analyzed for residual NDF (Van Soest et al., 1991). Percentages of disappearance of NDF at each incubation time were calculated from the proportion remaining in the bag after incubation in the rumen. Kinetics of NDF degradation was estimated according to the model by Ørskov and Mc- Donald (1979), using a nonlinear procedure of SAS (1996). The model was P = A + B(1 e ct ), where P = NDF disappearance (%) at incubation time (t), %; A = degraded fraction, %; B = degradable fraction at measurable rate, %; and c = degradation rate of B, %/h. In Vitro Experiment Rumen contents were collected and combined from the steers described above. At 3 h after feeding, ruminal contents were taken from various locations via rumen fistula and squeezed through four layers of gauze into a glass flask. This rumen fluid was strained again through eight layers gauze to further remove small feed particles.

Table 1. Chemical composition of forages. ISOACIDS, AMINO ACIDS, DIPEPTIDES, AND DIGESTION 1185 Alfalfa Bermudagrass Pangolagrass Napiergrass Items hay hay hay silage DM, % 89.2 92.3 88.9 16.2 OM, % DM 89.8 93.3 94.5 88.5 CP, % DM 17.2 4.7 8.3 9.6 NDF, % DM 48.6 72.5 71.2 66.6 ADF, % DM 37.8 31.5 39.4 43.7 ADL, 1 % DM 7.8 6.3 6.9 5.6 ADL/NDF, % NDF 16.1 8.7 9.7 8.4 1 ADL = Acid detergent lignin. The one-stage in vitro rumen fermentation condition was similar to that described by Goering and Van Soest (1970). Triplicate polyethylene centrifuge tubes (50 ml) were used for each treatment. Each fermentation tube used for incubation (anaerobic, 39 C, 24 h) contained 15 ml of artificial saliva (McDougall, 1948) without or with (NH 4 ) 2 SO 4 (480 mg/l), 5 ml of particle-free rumen fluid, and 250 mg of dried (55 C, 72 h), ground (1 mm) individual forage substrate. Forage substrates were presoaked with artificial saliva solution and prewarmed in a water bath at 39 C approximately 1 h before inoculation of rumen fluid. The microbial inoculum was saturated with CO 2, and maintained at 39 C during the inoculation of fermentation tubes. After the inoculation of fermentation medium (substrate plus artificial saliva) with rumen microbial inoculum, the gas phase (open space) of the fermentation tubes was gassed with CO 2 for 5 s, followed by closing the tube with a rubber stopper equipped with a Bunsen valve. All tubes were incubated in a water bath at 39 C for 24 h with occasional swirling of the contents by hands. Tubes containing no forage substrates and treatment additives served as control blanks. Treatments included adding ic 4 (2 mm), ic 5 (2 mm), Val (2 mm), Leu (2 mm), Val-Val (1 mm), Leu-Leu (1 mm), Val-Leu (1 mm), or Leu-Val (1 mm) to incubation tubes for each forage substrate, and were designed to provide equal moles of branched-chain carbon skeleton among treatments. Tubes without additives were designated as controls. The selection of additive quantity referred to the results by Gorosito et al. (1985), indicating that the optimal concentration of isoacids for fiber digestion by ruminal microorganisms was approximately 2 mm. Adding (NH 4 ) 2 SO 4 to the artificial saliva solution was designed to evaluate the influence of NH 3 availability. Additives were purchased from Sigma Chemical Company (St. Louis, MO) and were dissolved in artificial saliva as separate solutions. Isoleucine dipeptide was not available, and the related additives were excluded from treatments as well. The initial concentration of microbial mass in the incubation was 2.5 g DM/L. Analysis of NDF on blank incubations (without substrate) at beginning showed no detectable NDF, indicating no ruminal feed particle contamination. At the end of the incubation period, the contents of triplicate fermentation tubes were individually measured for ph, using a glass electrode, and NDF residue (Van Soest et al., 1991). For NDF measurement, the whole contents in fermentation tubes were decanted into 600-ml Berzelius beakers. Tubes were then washed twice with several portions of NDF solution to transfer all the remaining residues in tubes. Digestibility was calculated as the amount of NDF that disappeared during the incubation relative to the initial amount. The values were corrected for those obtained from control blanks. Statistical Analyses Data were analyzed by PROC GLM of SAS (1996). In situ kinetics measurements were analyzed as a completely randomized block design. The model employed for analysis of variance consisted of forage species and block (steer). The in vitro digestibility experiment was analyzed by forage species as a completely randomized design, with additive treatment as the sole source of variation in the model. Means reported are least squares means. Treatment effect was determined by an F ratio, with P < 0.05 being considered significant. Pooled in vitro digestibility data were also analyzed for forage species or ammonium sulfate addition effect. RESULTS Forage Chemical Composition Chemical composition of test forages is listed in Table 1. The CP content was numerically the lowest for BER, intermediate for PAN and NAP, and the highest for ALF. In contrast, the NDF content of ALF was apparently the lowest among the forages. Relative differences in ADF between ALF and the grasses were not as great magnitude as those in CP and NDF. A similar trend was observed for ADL. However, the lignification index,

1186 YANG Table 2. In situ ruminal degradation kinetics of forage NDF. Alfalfa Bermudagrass Pangolagrass Napiergrass Items 1 hay hay hay silage SEM A 1.5 b 9.8 a 0.4 b 0.3 b 0.5 B 49.8 50.5 61.3 61.6 2.3 C 7.5 a 3.1 c 5.3 b 3.4 c 0.3 UDF 48.7 a 39.7 b 38.3 b 39.1 b 1.3 a,b,c Means in the same row with different superscripts differ (P < 0.05). 1 A = Degraded fraction, %; B = degradable fraction, %; C = degradation rate of B, %/h; UDF = undegradable fraction (100 A B), %. expressed as the proportion of ADL in NDF, also revealed marked differences, which was the greatest for ALF. The degree of NDF lignification among grasses appeared relatively similar. In Situ Ruminal NDF Degradation Kinetics Except for the degradable fraction (B), forage species had a significant influence on NDF degradation kinetics parameters (Table 2). Bermudagrass hay contained a higher degraded fraction (A) than the other three forages (P < 0.05). The degradation rate (C) was the fastest with ALF and the slowest with BER and NAP, and PAN was intermediate (P < 0.05). The undegradable fraction was higher with ALF compared with the grasses (P < 0.05). In Vitro Experiment Alfalfa hay. Additive treatments had significant influences on in vitro NDF digestibility of ALF in the absence of added ammonium sulfate (Table 3). Adding ic 4 increased the digestibility over control by 19% (P < 0.05). Digestibility was further increased by Val treatment relative to ic 4 (P < 0.05). When Val-Val was used, the digestibility was the highest among the four treatments and differed significantly from Val treatment (P < 0.05). The addition of ic 5 also resulted in higher digestibility than control (P < 0.05). When amino acid Leu was used, the digestibility did not change. Digestibility with Leu-Leu was the greatest (P < 0.05) for comparisons made among control, ic 5, Leu, and Leu-Leu. Difference in digestibility between Val-Leu and Leu-Val was not significant, but the digestibility of Val-Leu was lower than Leu-Leu (P < 0.05). When the incubation medium contained more ammonia initially, digestibilities with all treatments elevated (P < 0.05) (Table 3). The digestibility of ALF control treatment remained one of the highest among test forages (P < 0.05). Under this condition, additive treatments also had significant effects. Relative significance of differences in digestibility among control, ic 4, and Val did not alter, as in the case of no additional ammonia. Similar trend was also observed when comparing control, ic 5, Leu, and Leu-Leu, except the effect with Leu was significantly higher than ic 5 (P < 0.05). There was no difference in digestibility between Val-Leu and Leu-Val treatments. But, the digestibility of both was smaller than that of Leu-Leu (P < 0.05). Bermudagrass hay. When BER was incubated in medium containing ruminal microorganisms, less than 13% of the NDF was digested in 24 h (Table 3). The addition of ic 4 to the incubation increased the extent of NDF digestion by more than 38% (P < 0.05). Replacing Val for ic 4 also increased digestibility relative to the control (P < 0.05) but did not significantly differ from ic 4 treatment. Digestibility was the highest when dipeptide Val-Val was included in the incubation and was significantly different from the other three treatments (P < 0.05). Adding ic 5 did not result in a higher NDF digestibility than the control. However, the digestibility of Leu or Leu-Leu treatment was significantly greater than that of control or ic 5 treatment (P < 0.05). When dipeptides containing mixed amino acids, Val-Leu or Leu- Val, were used, the digestibility was similar to that of Val-Val, but greater than that of Leu-Leu (P < 0.05). When ammonium sulfate was added to the medium to increase initial ammonia availability, digestibilities of all treatments again trended to increase (P < 0.05) (Table 3). In this case, adding ic 4 or Val increased digestibility in an ascending order (P < 0.05) in comparison to the control. However, the addition of Val-Val did not result in a higher digestibility over that of Val. The digestibility for ic 5 remained higher than the control (P < 0.05), even when ammonia was more. Adding Leu further increased digestibility (P < 0.05), but not with Leu-Leu. With Val-Leu or Leu-Val, the digestibility did not differ in between, and was similar to that of Val- Val or Leu-Leu. Pangolagrass hay. The NDF digestibility of PAN was higher (P < 0.05) than that of BER (Table 3). In this case, adding ic 4 to the incubation still increased digestibility (P < 0.05). The digestibility for Val treatment was superior to that for ic 4 (P < 0.05). A further

ISOACIDS, AMINO ACIDS, DIPEPTIDES, AND DIGESTION 1187 Table 3. Response of forage NDF degradation extent (%) by ruminal microorganisms to branched-chain VFA, amino acids, and their dipeptides in vitro (24-h incubation, Goering and Van Soest system). Alfalfa Bermudagrass Pangolagrass Napiergrass Addition hay hay hay silage Incubation without (NH 4 ) 2 SO 4 None 21.0 f 12.6 g 18.0 g 21.0 d Isobutyric acid 25.0 e (19.0) 1 17.4 def (38.1) 19.9 f (10.6) 27.6 c (31.4) Valine 29.0 d (38.1) 19.4 bce (54.0) 24.8 cde (37.8) 36.3 a (72.9) Valine-valine 31.8 bc (51.4) 23.0 a (82.5) 27.2 ab (51.1) 37.9 a (80.5) Isovaleric acid 30.4 cd (44.8) 15.2 fg (20.6) 20.6 f (14.4) 27.5 c (31.0) Leucine 31.8 bc (51.4) 19.7 bcd (56.4) 25.8 bc (43.3) 33.8 b (61.0) Leucine-leucine 34.4 a (63.8) 18.7 de (48.4) 25.7 bd (42.8) 37.7 a (79.5) Valine-leucine 31.0 bd (47.6) 22.2 ab (76.2) 27.8 a (54.4) 37.8 a (80.0) Leucine-valine 32.8 ab (56.2) 21.8 ac (73.0) 25.6 be (42.2) 37.8 a (80.0) SEM 0.6 0.7 0.5 0.6 Incubation with (NH 4 ) 2 SO 4 None 26.4 e 16.2 f 22.7 g 28.6 c Isobutyric acid 29.5 d (11.7) 21.0 d (29.6) 25.0 f (10.1) 32.4 b (13.3) Valine 34.0 c (28.8) 23.0 bc (42.0) 29.8 de (31.3) 39.7 a (38.8) Valine-valine 34.6 c (31.1) 24.5 ab (51.2) 29.8 de (31.3 39.7 a (38.8) Isovaleric acid 34.3 c (29.9) 19.3 e (19.1) 32.0 bc (41.0) 31.4 b (9.8) Leucine 36.8 b (39.4) 24.2 ac (49.4) 33.9 a (49.3) 41.9 a (46.5) Leucine-leucine 39.0 a (47.7) 25.6 a (58.0) 33.2 ab (46.2) 41.0 a (43.4) Valine-leucine 36.3 bc (37.5) 25.1 a (54.9) 30.2 de (33.0) 40.0 a (39.9) Leucine-valine 36.7 b (39.0) 25.2 a (55.6) 30.8 cd (35.7) 40.6 a (42.0) SEM 0.5 0.5 0.6 0.7 a,b,c,d,e,f,g Means within a column with different superscripts differ (P < 0.05) for incubation without or with (NH 4 ) 2 SO 4. 1 Numbers in parentheses are percent improvement over the control incubation. increase in NDF digestibility was observed when Val- Val was used comparing with Val (P < 0.05). The addition of ic 5 also increased digestibility of PAN over the control (P < 0.05). With Leu and Leu-Leu, the digestibilities were similar, and each was significantly higher than that of control or ic 5 (P < 0.05). The digestibility of Val-Leu was superior to Leu-Val (P < 0.05). When the incubation was provided with additional ammonia, digestibilities of all treatments increased (P < 0.05), as in the case with BER (Table 3). However, relative differences (P < 0.05) in digestibilities among treatments of control, ic 4, and Val were unchanged compared with the situation of no additional ammonia. Similar trend was also observed for comparison among control, ic 5, Leu, and Leu-Leu. Adding Val-Leu or Leu- Val resulted in a digestibility similar to that of Val- Val, but lower than that of Leu-Leu (P < 0.05). Napiergrass silage. The in vitro NDF digestibility of NAP grass silage was as high as that of ALF hay, and was the highest (P < 0.05) among the grasses (Table 3). Adding ic 4 to the incubation increased digestibility by more than 31% (P < 0.05). Addition of Val to the incubation resulted in a higher digestibility than that of ic 4 addition (P < 0.05). The effect of Val-Val was not superior to that of Val. As for ic 4,iC 5 increased digestibility relative to the control (P < 0.05). Also, as in the case of other grass hays, the digestibility was elevated over control or ic 5 when Leu or Leu-Leu was provided (P < 0.05). The digestibility with Leu-Leu was greater than that with Leu (P < 0.05). When Val-Leu or Leu-Val was used, the digestibility was similar to that of Val-Val or Leu-Leu. When ammonium sulfate was included in the medium, digestibilities for all treatments also trended to increase (P < 0.05) (Table 3), as in the case for other forages. Relative differences (P < 0.05) in digestibilities among treatments of control, ic 4, Val, and Val-Val remained unchanged. Similar trend were observed when comparisons were made among control, ic 5, Leu, and Leu-Leu treatments. However, Val-Val did not increase digestibility over Val. Digestibility with Val-Leu or Leu- Val was again similar to that of Val-Val or Leu-Leu. DISCUSSION Temperate legumes usually contain higher CP and ADL, but lower NDF and hemicellulose (NDF minus ADF) than those of temperate grasses (Canale et al., 1990; Andrighetto et al., 1993; Hoffman et al., 1993). The comparison between ALF and tropical grasses in the present study (Table 1) and other studies (Lagasse et al., 1990; Emanuele et al., 1991) also illustrated similar trend. The NDF of ALF generally contains a greater undegradable fraction than that of grass, but its degradable fraction is degraded at a faster rate (Varga and Hoover, 1983; Andrighetto et al., 1993; Hoffman et

1188 YANG al., 1993). The results obtained in this study (Table 2) agreed with earlier observations. A high degree of ligninification of ALF NDF (Table 1) was probably responsible for the undegradable fraction. When the steers were fed a low protein diet, the ph and ammonia N concentration of ruminal fluid at 3 h after feeding were 6.54 ± 0.01 and 10.9 ± 0.2 mg/dl. Ruminal acetate, propionate, and butyrate averaged 59.5, 14.3, and 6.9 mm, respectively. Isobutyrate, isovalerate, and valerate were not detected. After inoculation of rumen fluid, initial ph values of in vitro incubations without and with ammonium sulfate inclusion were 6.82 ± 0.02 and 6.94 ± 0.02, respectively. The final ph values of incubations across forages and additive treatments ranged between 6.64 and 6.86, and were not affected by treatments (data not shown). The values of ph were within the range for optimal fiber digestion in the rumen (Mackie and White, 1990). Ammonia N concentrations at the start of incubation without and with ammonium sulfate inclusion were 3.1 ± 0.2 and 6.4 ± 0.2 mg/dl, respectively. These levels were slightly below or above the concentrations, 4 to 6 mg/dl, considered adequate for ruminal fiber breakdown (Mackie and White, 1990). Under these conditions, additive treatments aimed to provide microorganisms with branchedchain carbon skeleton had significant effects on forage NDF degradation (Table 3). Van Soest (1982) indicated that forages containing larger amounts of CP generally have a greater digestibility. The results of three grasses used in the present study also a showed similar trend when comparing variations in CP contents and in vitro NDF digestibilities without additives (Tables 1 and 3). However, a comparison including ALF negates the assumption. In fact, the CP of ALF was nearly twice that of NAP, but digestibilities did not differ between these two forages. When more ammonia was provided to the incubation initially, digestibilities of control incubations increased significantly by 25.7, 28.6, 26.1, and 36.2% for ALF, BER, PAN, and NAP, respectively (Table 3). Fiber-degrading ruminal bacteria utilize primarily ammonia as a source of N for growth (Bryant, 1973). Therefore, increasing ammonia availability to microbes can enhance fiber digestion in the rumen (Van Soest, 1982). Ruminal ammonia is apt to be limiting with low dietary CP. Under this condition, the effect of additional ammonia would be more prominent (Mackie and White, 1990). Among grasses, NAP contained the highest CP (Table 1). However, the improvement in NDF digestibility for the control by adding (NH 4 ) 2 SO 4 appeared to be the greatest, an almost 37% increase (Table 3). Although ammonia is a final product of CP breakdown in the rumen, it appears that CP content alone may not reflect potential ammonia availability. As for additive treatments, digestibilities also increased (Table 3) when ammonia was added to the medium initially. However, with a few exceptions, percent improvement by additives over control treatments dwindled compared with the situation without ammonia addition. The reduction in improvement during ammonia addition was apparently much smaller with PAN among grasses. In fact, even greater improvement was observed when ic 5, Leu, and Leu-Leu were used. A faster degradation rate among grasses (Table 2) might have been responsible, in that more energy or carbon skeleton could be produced during fermentation. This situation would be conducive to fiber degradation, especially when ammonia was more readily available. Van Kessell and Russell (1996) demonstrated that mixed ruminal bacteria only responded to amino nitrogen when carbohydrate fermentation was rapid. Some other general trends were also obvious. Reduction in improvement by additives was more prominent for NAP compared with other forages. Treatments of Val and Val- Val reduced improvement in an ascending order relative to ic 5. The extent of reduction was more drastic with Val-Val than with Leu-Leu. Fiber-degrading ruminal bacteria require BCVFA for carbon skeleton to synthesize branched-chain amino acids and long-chain fatty acids (Allison et al., 1962a, 1962b). In previous in vitro experiments, BCVFA was found to increase plant cell wall degradation by ruminal bacteria (Gorosito et al., 1985). Mixed ruminal microorganisms were used in the present study, and the results obtained were in agreement with their finding. Several in vivo studies also illustrated similar effect of BCVFA. Van Gylswyk (1970) supplemented BCVFA to sheep fed a low protein hay diet, and an improvement in cellulose digestion was found. Soofi et al. (1982) observed that mixed BCVFA improved DM digestibility of soybean stover. Gorosito et al. (1985) indicated that ic 4 and ic 5 improved digestion of isolated wheat straw cell wall to the same extent. In the present study, ic 4 or ic 5 treatment invariably resulted in significantly increased NDF digestibility over control, except with BER under ammonia limitation (Table 3). Initial ammonia did not appear to affect the significance of isoacids on digestibility. However, it was noted that, based on percent improvement over control treatments, ic 4 seemed to be superior to ic 5 in the magnitude (%) of digestion improvement with BER and NAP. The opposite trend was the case with ALF and PAN (Table 3). This implies that effectiveness of different BCVFA may vary, and may be dependent on forage species. Indeed, Gorosito et al. (1985) added mixed isoacids to in vitro incubations containing intact forages, which varied in CP content. Cell wall digestibility by ruminal bacteria was significantly

ISOACIDS, AMINO ACIDS, DIPEPTIDES, AND DIGESTION 1189 increased with alfalfa hay, orchard grass, and corn silage but not with timothy hay, reed canary grass, or bermuda grass. Isoacids are final products of dietary CP degradation. However, it can be seen from the results of previous and the present studies that isoacid adequacy is not closely related to CP content of substrates either. Several studies have indicated that amino acids often are stimulatory for rumen microbes even when ammonia and VFA exceed requirements (Maeng and Baldwin, 1976; Cotta and Russell, 1982; Argyle and Baldwin, 1989), indicating a direct effect from amino acids. In this study, the effect of adding amino acids on the increase in NDF degradation was almost invariably superior to that of their BCVFA counterparts, except for ALF with Leu and for BER with Val (Table 3). Again, initial ammonia appeared to have little influence on the significance by amino acids. Comparisons between amino acids revealed that percent improvement over control by Leu appeared to be greater than that by Val, except with NAP when initial ammonia was less. Many ruminal bacteria utilize peptide at a faster rate than the constituent amino acids presented in free form (Pittman and Bryant, 1964; Pittman et al., 1967; Chen et al., 1987b). In the present study, dipeptide addition significantly increased NDF digestibility over correspondent free amino acids on five occasions with lower initial ammonia (Table 3). However, there was only one case was the digestibility greater with dipeptide than with amino acid at the higher ammonia level. In this case, initial ammonia appeared to modulate the significant effect of dipeptides. Nevertheless, judgment based on numerical improvement (%) over controls, dipeptides were apparently superior to amino acids on 10 out of 16 occasions. Based on the same criteria, Val-Val appeared to exert a greater extent of improvement (%) than Leu-Leu when ammonia was less, except with ALF (Table 3). This trend appeared to reverse during increased ammonia provision. The latter finding could be explained by previous observations indicating that mixed ruminal bacteria in rumen fluid utilized Leu-Leu at a faster rate than Val-Val (Yang and Russell, 1992). Within individual forages, the digestibilities with Val-Leu and Leu-Val did not differ significantly from each other for almost all cases, and appeared to oscillate between those of Val-Val and Leu-Leu (Table 3). It appeared that dipeptides containing mixed amino acids, regardless of the position of amino acid in the molecule, were similar in efficacy. However, it was noted that the digestibility for Val-Leu was similar to that of Val-Val. On the contrary, the digestibility of Leu-Val was much closer to that of Leu-Leu. In this case, N-terminus amino acid in dipeptides with mixed amino acids seemed to be influential. Comparison of response based on averaged percent improvement over controls across individual additives for each forage revealed that PAN, which was moderate in CP content (Table 1) and fast in NDF degradation (Table 2), was almost always the least responsive to additives among grasses. Alfalfa, the highest in CP and the fastest in degradation rate, ranked second to the last (46.5%) among all forages in terms of averaged percent improvement when ammonia was less initially or the last (33.1%) when ammonia was more. It can be seen that although percent improvement by individual additives among different forage species was not consistent, it appeared that forage with high CP content or fast NDF degradation rate would respond to additives to smaller degrees. It was noteworthy that ALF seemed to respond to carbon-5 related additives better than carbon-4 additives (Table 3). Overall across forages, isoacids significantly increased NDF digestibility over control 15 out of 16 times (Table 3). On all occasions, amino acids or dipeptides increased digestibility over controls. Amino acids further increased digestibility over the corresponding isoacid treatments occurred on 14 occasions. Digestibilities with dipeptides containing a single amino acid were always greater than those with isoacids, except for one case. Dipeptides further increased digestibility over amino acid treatments on six out of 16 occasions. However, percent improvement in digestibility by dipeptides over amino acid treatments occurred in 10 out of 16 cases. The results from this study indicate that dipeptides Val-Val and Leu-Leu could be more effective than corresponding isoacids and free amino acids in improving forage NDF digestion by ruminal microorganisms. 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