Influence of Forage Type on Ruminal Bacterial Populations and Subsequent In Vitro Fiber Digestion1

Influence of Forage Type on Ruminal Bacterial Populations and Subsequent In Vitro Fiber Digestion 1 H. G. dUNG z,3 and V. H. V A R E L =

US Department of Agriculture Clay Center, NE 68933 ABSTRACT

forage was not observed due to feeding the same forage to the donor animals. Volatile fatty acid concentrations and proportions in the in vitro fermentations were related more to forage substrate than diet source. The results indicate that adaptation of the rumen population to diet forage composition occurred, but in vitro digestilibity was unrelated to fibrolytic bacterial numbers or proportions.

Adaptation of the rumen fibrolytic bacteria to legume, C3 grass, and C4 grass forages was examined in a 3 × 3 Latin square. Fistulated steers were fed alfalfa, smooth bromegrass, and switchgrass hays for 6 wk at 1.8% of body weight. Rumen samples were collected weekly after an overnight fast. Bacterial counts were conducted on rumen samples and all rumen samples were used in an in vitro fiber digestion study with three stages of maturity each for alfalfa, smooth bromegrass, and switchgrass as the substrates. Consumption of alfalfa hay resulted in the highest total viable counts of rumen bacteria but a lower proportion of fibrolytic counts than seen on the grass diets. Use of filter paper as the isolation substrate gave higher fibrolytic counts than seen with NDF of the forage fed as the isolation substrate. Fifty percent or more of the fibrolytic bacteria were Bacteriodes succinogenes, and the switchgrass diet resulted in higher proportions of this organism in the fibrolytic population than seen for alfalfa and smooth bromegrass hays. The rumen inoculum from animals fed alfalfa degraded the fiber fractions of all substrate forages best. Improved in vitro digestibility of a

INTRODUCTION

Received September 23, 1987. Accepted December 21, 1987. 1Mention of a trade name, proprietary products or specific equipment does not constitute a guarantee of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. 2 USDA-ARS Roman L. Hruska US Meat Animal Research Center. 3Present address: USDA-ARS US Dairy Forage Research Center and Department of Animal Science, University of Minnesota, St. Paul 55108. 1988 J Dairy Sci 71:1526-1535

Legumes and C3 and C4 grasses represent the major taxa of forages fed to ruminants. These taxonomic groups of forages differ in chemical and anatomical structure (1, 8, 23). The ability of rumen fibrolytic bacterial species to degrade plant cell walls is variable among bacterial species and among forage species for individual bacterial strains (20, 21). Alternate feeding of legume and Ca grass forages resulted in major shifts in rumen fibrolytic population structure in sheep with variable lengths of time required to reach stability (11). The dynamic response of the rumen microbial population to different diets has led to the suggestion that animals from which rumen inoculum is obtained for in vitro fermentation studies should be on a forage very similar to that being tested in the laboratory. This is done in the hope of obtaining a rumen population which will provide in vitro digestibilities most similar to in vivo values. Studies on the effect of forage fed to the donor animal on subsequent in vitro digestibility have indicated both benefits (4, 13) and lack of response (12, 15, 22) to similarities between diets and substrate forages. This experiment was designed to determine if the fibrolytic population in the rumen of cattle adapts to changing composition of forage cell walls in the diet. A legume, C3 grass, and C4 grass were used to provide a range of cell wall types. Bacterial populations and ability to degrade widely divergent forages in vitro were

1526

DIETARY ADAPTATION BY FIBROLYTIC BACTERIA determined. The time required for bacterial adaptation to take place was also monitored. M A T E R I A L S AND METHODS Animals, Diets, and R umen Sampling

Three crossbred ruminally cannulated steers weighing 789, 728, and 607 kg were fed alfalfa (Medicago sativa), smooth bromegrass (Brornus inermis), and switchgrass (Panicum virgatum) hays in a 3 × 3 Latin square design. Animals received fresh hay daily at 0900 h and were fed hays at 1.8% of b o d y weight. Trace mineral salt and water were available ad libitum. Animals were housed in separate pens to prevent physical contact. Animals were fed a mixture of the three hays in equal proportions for 4 wk prior to initiation of the experiment. Each period of the Latin square was 6 wk. Rumen samples were collected weekly at 0800 h after a 16-h fast overnight. This sampling time was chosen to correspond to maximal fibrolytie bacterial numbers in the rumen (16). The rumen contents were sampled by collecting material from the underside of the pad and straining through four layers of cheesecloth into a prewarmed flask. The flask was sealed for transport to the laboratory. The tureen samples were strained in the laboratory through another four layers of cheesecloth, under a CO2 stream, prior to further analysis. Microbiological Analysis

The Hungate anaerobic culture method as described by Bryant (5) was used. Rumen fluid was blended under a stream o f CO2 for 1 min with a Waring blender. Serial dilutions were made in anaerobic buffer (3) to 10 - 8 . Samples (.2 ml) from the 10 - s dilution were used to inoculate four replicate roll tubes to determine numbers of total viable bacteria. The medium consisted of (g/100 ml): clarified rumen fluid, 30; glucose, cellobiose, maltose, starch, xylose, and glycerol, each .03; trypticase (BBL Microbiology Systems, Cockeysville, MD), .2; resazurin .0001; mineral $2, 5% (23); and purified agar (BBL), 1.75. Samples, .2 ml, from each of 10 - s and 10 - 6 dilutions were used to inoculate three replicate roll tubes to determine numbers of fiber degrading bacteria. This medium consisted o f (g/100 ml): incubated clarified rumen fluid (7), 15; trypticase (BBL), .2; mineral $2 (22), 5; yeast extract, .05;

1527

and purified agar (BBL), .5. The cellulose or fiber source in this medium was either filter paper (Whatman No. 1) or cell walls from alfalfa, bromegrass or switchgrass at a final concentration of .2%. Cell walls were prepared by extraction with neutral detergent (28), followed by extensive washing of insoluble residue to remove detergent. These substrates were ball milled with flint pebbles for 18 h. Sodium carbonate, cysteine hydrochloride, and sodium sulfide were added as sterile anaerobic solutions (5) to all media. The gas phase was 100% COs and incubations were at 37°C. Numbers o f colonies on the total viable count medium and the zones o f clearing with and without colonies on the fiber media were determined after 7 and 14 d, respectively. Bacteriodes succinogenes was identified by zones of clearing without colony formation, Gram stain, cell morphology, and fermentation products. In Vitro Fermentation

Alfalfa, smooth bromegrass, and switchgrass were harvested by hand at vegetative, flowering, and postflowering stages of physiological maturity. These nine forage samples were lyophlized and ground to pass a l-ram screen prior to use as substrates for fermentation with the rumen samples. All forage substrates were fermented with rumen inocula from each of the three hay diets. Digestibility of fiber fractions of these forages was estimated by 48-h in vitro fermentations. For this procedure duplicate 500-mg samples o f each substrate, for each inoculum source, were placed in 50-ml plastic culture tubes with screw caps. Each tube was inoculated with 24 ml o f buffer (18) and 6 ml of rumen fluid. Samples were incubated for 48 h at 39°C with occasional shaking. Fermented blanks for each rumen inoculum were also run in duplicate. After 48 h, samples were centrifuged at 3000 × g for 30 rain and both residual solids and supernatants were frozen until analyzed. Chemical Analysis

Hay and forage substrates were analyzed for DM by drying overnight at 100°C and CP was estimated as Kjeldahl N × 6.25 (2). All feed, forage, and in vitro fermentation residues were analyzed for fiber content by the sequential detergent system (28). Seventy-two percent Journal of Dairy Science Vol. 71, No. 6, 1988

1528

JUNG AND VAREL the three way interaction of period × animal × diet as the error term. Subplot effects and all possible interactions were tested using the residual mean squares as the error term. The F-protected least significant difference test was used to compare individual means. All computations were d o n e using SAS (25).

sulfuric acid was used to solubilize cellulose and isolate crude lignin plus ash. Total cell wall was defined as the N D F value. Hemicellulose, cellulose, and lignin were calculated by difference (hemicellulose = N D F minus A D F , cellulose = A D F minus acid d e t e r g e n t lignin, lignin = acid detergent lignin minus ash). F e r m e n t a t i o n supernatants f r o m sampling wk 6 o f each period o f the Latin square were analyzed for total V F A c o n c e n t r a t i o n and molar p r o p o r t i o n s of the individual acids (29).

RESULTS A N D DISCUSSION

A m i x t u r e of the three hays was fed in the 4-wk preliminary period to obtain a m i x e d p o p u l a t i o n of fibrolytic bacteria representative o f forage diets. An earlier trial in which corn silage (Zea mays) was fed in the preliminary period resulted in a very different microbial p o p u l a t i o n after 2 wk on forage diets than was seen after subsequent changes a m o n g the forage diets (Jung and Varel, unpublished data). Because changes in fibrolytic bacterial populations among different types of forages was the characteristic o f interest in this experiment, use of a forage m i x t u r e was t h o u g h t to give a m o r e appropriate starting p o p u l a t i o n than did use of a s e m i c o n c e n t r a t e feed such as corn silage.

Statistical Analysis

The e x p e r i m e n t was analyzed as a split-plot with a 3 × 3 Latin square as the main plot. The data for microbial counts and in vitro fiber digestibility included sampling w e e k as a subplot effect. Substrate forage and physiological m a t u r i t y were additional subplot effects for in vitro fiber digestibility, and isolation substrate was an additional subplot effect for the r u m e n bacterial counts. Main plot effects for period, animal, and diet o f the Latin square were tested using the mean squares for

TABLE 1. Chemical composition of forages fed to fistulated steers and used as substrates in the in vitro fermentations. Component 1 Forage

CP ~(%

CW

HC

of DM)

CEL

LIG

(% CW)

Diets Alfalfa hay Smooth bromegrass hay Switchgrass hay

18.1 11.9 7.9

50.4 70.8 74.2

27.3 42.7 46.2

56.2 49.6 48.9

17.1 7.8 5.3

Substrates and maturity 2 Alfalfa Veg Flo Pfl

22.6 23.1 14.1

30.8 32.5 59.3

30.2 26.5 23.6

54.9 60.9 57.2

14.3 15.4 19.7

Smooth bromegrass Veg Flo Pfl

14.4 9.6 6.2

53.0 64.9 66.9

48.7 43.0 41.0

47.5 51.2 51.0

4.2 6.6 8.2

Switchgrass Veg Flo Pfl

13.2 7.6 4.5

60.4 72.3 73.8

50.7 41.6 41.9

45.4 51.3 49.3

4.0 7.3 9.1

1Cell wall (CW); hemicellulose (HC); cellulose (CEL); lignin (LIG). 2Vegetative (Veg); flowering (Flo); postflowering (Pfl). Journal of Dairy Science Vol. 71, No. 6, 1988

DIETARY ADAPTATION BY FIBROLYTIC BACTERIA

1529

TABLE 2. Effects of donor animal diet and isolation substrate on rumen bacterial counts, averaged across sampling weeks. Bacterial counts a , 2 TFB Diet

TVC

CW

(xl08 .m1-1 )

FB FP

BS

CW

FP

(xl08 .m1-1 )

CW

FP

(%)

Alfalfa hay

56.28 a

.62a

1.37a

1.21 a

2.56 a

51.2a

52.4a

Smooth bromegrass hay

37.60 b

1.20 b

1.48 a

3.47 b

4.28b

54.6 a

54.1 a

Swit chgrass hay

44.97 c

1.23b

1.72 b

2.93 b

3.97 b

72.6 b

59.8b

2.42

.06

.07

.19

.19

1.8

1.3

SEM

a'b'CMeans in the same column not sharing a common superscript differ (P<.05). 1Total viable bacterial count (TVC), total fibrolytic count (TFB), percentage of total bacteria that were fibrolytic (FB), percentage of total fibrolytic bacteria that were Bacteriodes succinogenes (BS). 2 Cell wall of diet forage (CW) or filter paper (FP) used as isolation substrate.

Analysis of t h e hays fed to t h e animals s h o w e d m a j o r d i f f e r e n c e s a m o n g t h e forages in a m o u n t o f CP, t o t a l cell wall material, a n d c o m p o s i t i o n of cell walls (Table 1). As expected, the alfalfa h a y was l o w e s t in f i b e r content but contained the greatest concent r a t i o n o f lignin. T h e ratio of hemiceUulose to cellulose was least for alfalfa h a y (.49) a n d g r e a t e s t for switchgrass h a y (.94). T h e switchgrass also c o n t a i n e d t h e m o s t cell wall m a t e r i a l b u t t h e l o w e s t p r o p o r t i o n o f lignin (Table 1). C o n s u m p t i o n o f these d i f f e r e n t forages d i d n o t r e s u l t in a n y s i g n i f i c a n t d i f f e r e n c e s in a n i m a l w e i g h t d u r i n g t h e e x p e r i m e n t d u e t o diet. T h e

level o f feeding p r o d u c e d little w e i g h t c h a n g e d u r i n g t h e t h r e e periods o f t h e e x p e r i m e n t ( 1 5 8 g / d average gain). T h e r u m e n bacterial p o p u l a t i o n s were significantly a f f e c t e d h a y d i e t (Table 2). T o t a l viable c o u n t s were g r e a t e s t ( P < . 0 5 ) o n t h e alfalfa h a y d i e t a n d least o n t h e s m o o t h b r o m e grass hay. T h e a n i m a l weighing t h e least h a d higher ( P < . 0 5 ) t o t a l viable c o u n t s o n all diets. C o u n t s o f cellulose d e g r a d i n g b a c t e r i a o n filter p a p e r were w i t h i n t h e range r e p o r t e d previously using cellulose overlay plates (16). Because 70 t o 80% o f t o t a l r u m e n m i c r o b i a l mass is associated w i t h t h e p a r t i c u l a t e f r a c t i o n (9), filtering

TABLE 3. Effect of diet fed to donor animal on subsequent in vitro fiber digestion. Values are means taken across all substrates and sampling weeks. Digestibility of fiber component I Diet

CW

HC

CEL

LIG

Alfalfa hay

54.5 a

61.1

57.1 a

23.0

Smooth bromegrass hay Switchgrass hay

49.7 b

55.6

53.3 b

22.3

51.7c

59.7

53.7 b

23.3

(%)

SEM

.2

1.2

.4

1.0

a'b'CMeans in the same column not sharing a common superscript differ (P<.05). Cell wall (CW), hemicellulose (HC), cellulose (CELL lignin (LIG). Journal of Dairy Science Vol. 71, No. 6, 1988

1530

JUNG

AND VAREL

the rumen samples through cheesecloth may have biased the results by removing particulates. However, the filtered samples still contained 2.5 + .4 mg of neutral detergent cell walls per ml. Craig et al. (6) found no difference in the 48-h in vitro degradation of fiber between strained rumen fluid (SRF) and SRF plus particulate associated bacteria. Therefore, influence of particle removal on compositional studies of rumen bacterial populations remains unclear. The number of fibrolytic bacteria was low and ranking of the forages was influenced by the substrate used to enumerate the fiber degraders. Use of netural detergent cell walls in the isolation procedure from the forage which the animal was fed indicated that alfalfa fed animals had fewer (P<.05) fibrolytic bacteria than seen on the grass hays, and the two grass forages did not differ (P>.05). However, use of filter paper to isolate fibrolytic bacteria resuited in greater (P<.05) numbers than seen with neutral detergent cell walls, and in this case alfalfa and smooth bromegrass hays did not differ (P<.05) in bacterial counts while the switchgrass diet counts were significantly greater. It was expected that the use of cell wall preparations to isolate fibrolytic bacteria would result in higher counts because of the greater range of possible substrates present as opposed to only cellulose in filter paper. Possibly the reason the opposite result was observed was because almost all rumen fibrolytic bacteria can degrade cellulose, and the cellulose in filter paper is more readily degradable than that in cell walls and the hemicellulose in the cell walls was less degradable than cellulose. The complex matrix of forage cell walls, which includes lignin, may have limited growth on those substrates. When the fibrolytic bacterial counts were expressed as a percentage of the total viable bacterial counts, both enumeration substrates indicated that feeding alfalfa hay resulted in a lower (P<.05) percentage of fibrolytic bacteria than seen on the grass hays (Table 2). The greater cell solubles content of alfalfa probably caused the greater TVC counts, which reduced the proportion fibrolytics. Bacteriodes succinogenes accounted for approximately 50% of all the fibrolytic bacteria for alfalfa and smooth bromegrass fed animals, whereas percentage of B. succinogenes was greater (P<.05) on the switchgrass hay diet. Journal of Dairy Science Vol. 71, No. 6, 1988

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DIETARY ADAPTATION BY FIBROLVTIC BACTERIA

1531

TABLE 5. Effect of number of weeks that a forage was fed to the donor animals on in vitro fiber digestion. Values are means taken across all diets and substrates. Sampling week

Digestibility of fiber component 1 CW

HC

CE L

LIG

55.9 a 54.9 b 55.0 b 54.1 cd 54.7bc 53.6 d .3

26.2 a 22.4 hd 20.4 c 22.9 bd 21.8bc 23.6 d .6

(%) 1 2 3 4 5 6 SEM

52.7 a 51.1 b 52.0 ac 51.7 bc 53.7 d 50.7 b .3

58.7 a 56.3 b 59.5 a 59.1 a 62.3c 57.1 b .4

a'b'c'dMeans in the same column not sharing a common superscript differ (P<.05). 1Cell wall (CW), hemicellulose (HC), cellulose (CEL), lignin (LIG).

Ruminococcus albus was t h e p r e d o m i n a n t f i b r o l y t i c o r g a n i s m for alfalfa fed sheep, w h e r e a s Butyrivibrio was d o m i n a n t o n a C4 grass (11). However, B. succinogenes has o f t e n b e e n difficult to isolate w i t h s t a n d a r d procedures (27). T h e h i g h e r values for this species r e p o r t e d in t h e c u r r e n t e x p e r i m e n t m a y b e d u e to t h e l o w e r agar c o n c e n t r a t i o n used (17). No significant effects d u e t o s a m p l i n g w e e k were f o u n d for a n y of t h e b a c t e r i a l data. T h e diff e r e n c e s o b s e r v e d a m o n g t h e diets m u s t have o c c u r r e d w i t h an a d a p t a t i o n p e r i o d o f less t h a n 1 wk. G o u w s a n d K i s t n e r (11) had f o u n d a d a p t a t i o n t i m e s o f 2 to m o r e t h a n 5 w k for cellulolytic bacterial p o p u l a t i o n s w i t h e x t r e m e variability a m o n g animals. T h e forages collected as s u b s t r a t e s for t h e in

vitro f i b e r d i g e s t i b i l i t y s t u d y varied w i d e l y in c o m p o s i t i o n (Table 1). J u s t as for t h e hays, all t h r e e forage species were o b v i o u s l y d i f f e r e n t in c h e m i c a l c o m p o s i t i o n . Increasing physiological m a t u r i t y depressed CP c o n t e n t and elevated cell wall c o n c e n t r a t i o n o f t h e forages. Physiological m a t u r i t y also altered f i b e r c o m p o s i t i o n o f all t h e s u b s t r a t e forages w i t h h e m i c e l l u l o s e conc e n t r a t i o n decreasing, cellulose c o n c e n t r a t i o n increasing a n d t h e n declining, a n d lignin cont e n t c o n s t a n t l y increasing. Diet hays were generally similar in c o m p o s i t i o n to the flowering stage o f m a t u r i t y for t h e s e t h r e e forages ( T a b l e 1). T h e i n f l u e n c e o f d i e t fed to t h e d o n o r a n i m a l o n s u b s e q u e n t 48-h in vitro fiber dig e s t i o n b y t h e r u m e n m i c r o o r g a n i s m s is s h o w n

TABLE 6. Interaction of diet forage and in vitro cellulose and lignin fermentation of substrate forages. Values are means across physiological maturity and sampling week. Forage fiber digestibility 1 Alfalfa

Smooth bromegrass

Diet

CEL

LIG

CEL

Alfalfa hay

57.3 a

14.3

60.6 a

Smooth bromegrass hay

53.9 b

13.9

57.2b

Switchgrass hay

53.4 b

15.4

57.2 b 1.0

SEM

.7

.6

LIG

Switchgrass CEL

LIG

30.6 a

53.3 a

24.1

27.1 b

48.8b

25.9

29.4 a

50.5c

25.2

.7

1.0

.4

a,b,c Means in the same column not sharing a common superscript differ (P<.05). l Cellulose (CEL), lignin (LIG). Journal of Dairy Science Vol. 71, No. 6, 1988

<

TABLE 7,. Interaction of donor animal diet and fermentation substrate on concentration and molar proportions of volatile fatty acids. Values are means across stages of physiological maturity.

O ,q

Z

vFAd Diet

Molar proportion 1

Substrate 2

(~tmol . m l - ~)

Ac

Pr

Bu

iBu

Val

Alf Bro Swi

81.7 77.6 72.4

.687a .674b .685 a

.181a .218 b .221 b

.084a .086 ~ .078 b

.012a .009 b .007 c

.016 a .010b .008 c

iVal

Cap

Ox

Alfalfa hay 00 00

.o17a .o01b 0b

.0020a .0013b .0006 b X

Smooth bromegrass hay Alf Bro Swi

97.0 101.3 70.4

.669 a .670 ab .677b

.219 a .220 a .235 b

.076 a .089 b .076 a

.011 a .007b .005 c

.014a .o12b .007c

.009 a 0b .001 b

.0012a .0019 a .0001 b

> X

A~ Bro Swi

90.3 85.1 68.3

.688 a .676 b .685 a

.207 a .220 b .218 b

.074 a .088 b .080 c

.o10a .006 b .006 b

.013a .009 b .007 c

.o07a 0b .002 ab

.0010 .0014 .0008

r-

.002

.002

.001

.0005

.0004

.002

.0002

Switchgrass hay

SEM

4.8

abe.

.

°

' ' lv~eans m the same column and for the same diet not sharing a c o m m o n superscript differ (P<.05).

dvolatile fatty acid concentrations were greater (P<.05) for the Mfalfa and smooth bromegrass substrates than for the switchgrass. 1Acetate (Ac), propionate (Pr), butyrate (Bu), isobutyrate (iBu), valerate (Val), isovalerate (iVal), caproate (Cap). 2 Alfalfa (Alf), s m o o t h bromegrass (Bro), switchgrass (Swi).

< > t~

TABLE 8. Interaction between forage species and physiological maturity o f fermentation substrates for VFA concentration and molar proportions. Values are means across donor animal diet. Molar proportion Forage and maturity 2

VFA d

Ac

Pr

Bu

iBu

Val

iVal

Cap

(tzmol.m1-1 )

g-

>

Al~lfa Veg Flo Pfl

101.8 96.1 71.1

.663a .679 b .702 c

.209 a .203 a .196 b

.087a .078b .071 c

.011 .011 .010

.o15a .015 a .013 b

Smooth bromegrass Veg Flo Pfl

108.3 86.2 69.6

.679 a .684 a .657 b

.207 a .220 b .232 c

.092 a .079 b .092 a

.008 .007 .008

.011 a .009 b .011 a

95.5 63.1 52.5

.651 a .699 b .698 b

.250 a .217 b .208 c

.081 a .070 b .083 c

.007 .006 .005

.002

.002

.001

.0005

Switchgrass Veg FIo Pfl SEM

4.8

dconcentrations of VFA were greatest for vegetative forages and least for the postflowering stage of maturity (P<.05). ~Acetate (Ac), propionate (Pr), butyrate (Bu), isobutyrate (iBu), valerate (Val), isovalerate 0Val), caproate (Cap).

Z

O Ox

00 00

.0021 .O013 .0008

.0002

.0023 .0012 .0007

.009 a .o07b .006 b

.002 .001 .0002

.0006 .0005 .0004

.0004

.0022

.0002

0 0

-4 > ,4

©

a'b'CMeans in the same column and for the same diet not sharing a c o m m o n superscript differ (P<.05).

o

.013 .013 .007

Vegetative (Veg), flowering (Flo), postflowering (Pfl).

c3

1534

JUNG AND VAREL

in Table 3. Although feeding alfalfa hay resuited in the lowest percentage, and possibly numbers, of fibrolytic bacteria in the rumen, overall cell wall digestibility (CWD) was greatest (P<.05) on this diet. This improvement in CWD appears to be a function o f a greater (P<.05) cellulose digestibility (CELD) by the inoculum from the animals fed alfalfa compared with those receiving grass. Hemicellulose and lignin (LIGD) digestibility were not affected by diet. Digestibility of lignin was probably solubilization of lignin fragments rather than conversion to V F A and energy. Bezeau (4) also saw improved in vitro digestibility when alfalfa was fed. The 48-h in vitro extents of digestion for the various fiber components o f all the substrate forage species and stages of physiological maturity are given in Table 4. Smooth bromegrass was generally the highest in digestibility at all stages of maturity for all fiber components, but alfalfa and switchgrass altered their rankings depending on stage o f maturity. Switcbgrass was always similar to smooth bromegrass in fiber digestibility for vegetative tissue, whereas alfalfa was significantly lower. At the flowering stage of maturity switchgrass was lower (P<.05) for CWD, HCD, and LIGD than was alfalfa. Postflowering switchgrass and alfalfa were different (P<.05) for CWD, CELD, and LIGD; however, diet did not affect these substrate species × physiological maturity interactions. Although progressive sampling weeks were expected to result in adaptation trends in in vitro fiber digestion, the significant differences among sampling weeks were sporadic and did not suggest a consistent trend (Table 5). Most possible interactions of sampling week with the other parameters were significant for one or more of the fiber component digestibilites, but examination of the means failed to reveal any consistent effects. The interaction of diet forage and substrate species was significant for CELD and LIGD (Table 6). The effect on CELD was limited to the smooth bromegrass and switchgrass diets being the same for in vitro fermentations of alfalfa and smooth bromegrass, but switchgrass hay feeding resulted in greater (P<.05) C E L D of switchgrass than smooth bromegrass forage. Alfalfa hay as the diet source always resulted in the greatest (P<.05) CELD. Volatile fatty acid concentrations in the in vitro fermentations were similar for all three Journal of Dairy Science Vol. 71, No. 6, 1988

diets but greater (P<.05) for alfalfa and smooth bromegrass substrates than switchgrass (Table 7). The molar proportion acetate was least (P<.05) for the smooth bromegrass substrate, and the proportion propionate was least (P< • 06) for the alfalfa substrate. Other V F A proportions varied among substrate species with the branched chain V F A proportions generally related to CP content o f the substrate (Table 7). Concentration of V F A declined significantly with advancing maturity of all three substrate forages (Table 8). There was an interaction between substrate species and maturity for several individual V F A proportions. The proportions of acetate increased (P<.05) and propionate declined (P<.05) with maturity of alfalfa and switchgrass, but smooth bromegrass illustrated the opposite trend. Butyrate and valerate also showed some deviations between species × maturity comparisons (Table 8). Branched-chain amino acids and their V F A breakdown products increase in vitro digestibility (14, 19, 26). Because the alfalfa diet was highest in CP content, the concentration of branched-chain V F A was expected to be higher in this inoculum; however, only isobutyrate was greater (P< .05) for alfalfa (.069 /amol -ml _1 ) than for the grasses (0 and .025 /~mol •m1-1 for smooth bromegrass and switchgrass, respectively). Gorosito et al. (10) reported maximal response to branched-chain V F A at less than .3 /~mot-ml-:. These data indicate that higher amounts of branchedchain V F A may partially account for the improved fiber digestion on the alfalfa diet. REFERENCES

1 Akin, D. E., L. L. Rigsby, and R. H. Brown. 1984. Ultrastructure of cell wall degradation in Panicum species differing in digestibility. Crop. Sci. 24:156. 2 Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. Assoc. Offic. Anal. Chem., Washington, DC. 3 Betian, H. G., B. A. Linehan, M. P. Bryant, and L. V. Holdeman. 1977. Isolation of cellulolytic Bacteriodes sp. from human feces. Appl. Environ. Microbiol. 33 : 1009. 4 Bezeau, L. M. 1965. Effect of source of inoculum on digestibility of substrate in in vitro digestion trials. J. Anim. Sci. 24:823. 5 Bryant, M. P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324. 6 Craig, W. M., B. J. Hong, G. A. Broderick, and R. J. Bula. 1984. In vitro inoculum enriched with particle associated microorganisms for determining

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19 Mir, P. S., Z. Mir, and J. A. Robertson. 1986. Effect of branched-chain amino acids or fatty acid supplementation or in vitro digestibility of barley straw or alfalfa hay. Can. J. Anita. Sci. 66:151. 20 Morris, E. J. 1984. Degradation of the intact plant cell wall o f subtropical and tropical herbage by rumen bacteria. Pages 3 7 8 - 3 9 6 Herbivore nutrition in the subtropics and tropics. F.M.C. Gilchrist and R. I. Mackie, ed. Sci. Press, Craighall, So. Aft. 21 Morris, E. J., and N. O. Van Gylswyk. 1980. Comparison o f the action of rumen bacteria on cell walls from Eragrostis tel. J. Agric. Sci. 95:313. 22 Nik-Khah, A., and D. E. Tribe. 1977. A note on the effect o f diet on the inoculum used in digestibility determination in vitro. A n i m Prod. 25:103. 23 Nordkvist, E., and P. Aman. 1986. Changes during growth in anatomical and chemical composition and in vitro degradability o f lucerne. J. Sci. Food Agric. 37:1. 24- Salanitro, J. P., I. G. Fairchilds, and Y. D. Zgornicki. 1974. Isolation, culture characteristics, and identification of anaerobic bacteria from the chicken cecum. App1. Microbiol. 27:678. 25 Statistical Analysis System Inc., 1982. SAS User's guide: statistics. Cary, NC. 26 Soofi, R., G. C. Fahey, Jr., L. L. Berger, and F. C. Hinds. 1982. Effect of branched chain volatile fatty acids, trypticase, urea, and starch on in vitro dry matter disappearance of soybean stover. J. Dairy Sci. 65:1748. 27 Van Gylswyk, N. O., and H. M. Schwartz. 1984. Microbial ecology o f the rumen o f animals fed high-fibre diets. Pages 3 5 9 - 3 7 7 in Herbivore nutrition in the subtropics and tropics. F.M.C. Gilchrist and R. I. Mackie, ed. Sci. Press, Craighall, So. Afr. 28 Van Soest, P. J., and J. B. Robertson. 1980. Systems of analysis for evaluating fibrous feeds. W. J. Pigden, C. C. Balch, and M. Graham, ed. Pages 4 9 - 6 0 in Standardization o f analytical methodology for feeds. Publ. IDRC-134e, Int. Dev. Res Ctr., Ottawa, Ont., Can. 29 Varel, V. H., and A. G. Hashimoto. 1981. Effects of dietary monensin or chlortetracycline on methane production from cattle waste. Appl. Environ. Microbiol. 41: 29.

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