Grass und Forage Science (1989) Volume 44, 65-75

Particle size reduction of the leaves of a tropical and a temperategrass by cattle. II. Relation of anatomical structure to the process ofleaf breakdown through chewing and digestion

J. R. WILSON, D. E. AKINt,M. N. McLEOD AND D, J. MINSONCSIRO. Division of Tropical Crops andPastures, Queensland, Australia. iUSDA RussellAgricultural Research Centre, Athens. Georgia.USA.

Abstract

To investigate links between the anatomy of grassleaves and their resistance to breakdown in therumen, leaf blades of the tropical grass, greenpanic {Panicum maximum var, trichoglume), andthe temperate grass, Italian ryegrass {Loliummultifforum), were chopped into 50 mm lengthsand fed fresh to cattle. Particle size reduction ofthe chewed feed was studied immediately aftereating and after digestion in nylon bags in therumen for 6, 12, 24, 48, 96 h and 3 weeks. Thelengths of fibre elements, separated into groups ofdifferent diameters, were measured on samplesdigested for 96 h. The process of tissue break-down was studied using light and electron micros-copy.

Green panic leaves had twice the cross-sectio-nal area of thick-walled tissues, a higher vascularbundle frequency per unit leaf width, and less, butmore densely packed mesophyll, than did theryegrass leaves. Despite the contribution of thesecharacteristics to greater leaf rigidity, green panicwas broken down to a greater degree by chewingthan was ryegrass.

During digestion, width reduction of thechewed leaf particles was faster in ryegrass than ingreen panic because of two anatomical features:(i) the straight-walled intercostal cells of theepidermis in ryegrass were easily separated allow-

Correspondence: Dr J, R, Wilson, CSIRO Division of TropicalCrops and Pastures, 306 Carmody Road, Si, Lucia, Queens-land, Australia, 4067.

ing the epidermis to split, whereas the sinuouswalls in green panic were resistant to splitting,and (ii) the epidermis of ryegrass was linked to thevascular bundles by thin-walled mesophyll cellsand was shed when these were digested, whereasin green panic the linkage was via thick-walledbundle sheath cells causing the epidermis toremain attached for much longer. Ryegrass leafwas reduced to isolated fibres within 24 h diges-tion; this process took >48 h in green panic.These fibres all had a high resistance to lengthreduction by digestion irrespective of their anato-mical or species origin. Even after 3 weeks in therumen there was little digestive disruption to thelongitudinal walls of these fibres.

The isolated vascular fibres of ryegrass weresmooth-surfaced in contrast to those of greenpanic which were rough owing to attached undi-gested bundle sheath cells and jagged, brokensections of epidermis; this could influence ease ofseparation of particles from the digesta mass andflow from the rumen.

Anatomical differences between these grasseswere, therefore, important in the rate of widthreduction of leaf particles during digestion andfor the characteristics of the isolated fibre, but notfor length reduction of particles during digestion.

Introduction

Intake of forage is often limited by the resistanceof the plant tissues to breakdown and the amountof fibre present in these tissues (Minson 1987). Inthe first paper of this series (Wilson et al. 1988) itwas shown for leaf material that length of particlewas reduced by chewing but not by digestion.Whereas, width of particle was reduced by bothchewing and digestion. It was shown also thatthere was a more rapid reduction in particle widthby digestion for the temperate Italian ryegrass

66 J. R. Wilson et al.

{Lolium multiflorium) than for the tropical greenpanic {Panicum maximum var. trichoglume).

TTiis paper examines the anatomical character-istics of the leaves of these grasses and relatesthese characteristics to the observed differences inthe mode of breakdown of particles duringchewing and digestion. The species were chosenbecause of their basic difference in leaf anatomyassociated with the Cj (ryegrass) and Ca (greenpanic) photosynthetic pathway.

Materials and methods

The tropical grass, green panic {Panicum maxi-mum var. trichoglume cv, Petrie) and the temper-ate grass, Italian ryegrass {Lolium multiflorum cv.Tetila) were grown in adjacent areas at Samford,south-east Queensland and were harvested inMay 1986 when in active vegetative growth toprovide samples of pure leaf blade. The bladeswere cut accurately into 50 mm long pieces andwere fed fresh to two steers fistulated in theoesophagus and rumen. Four replicate samples ofeach species were given to the animals. Thechewed material was collected and sub-sampleswere retained for particle size and anatomicalanalysis; the remainder was weighed into nylon

bags and suspended in the rumen of the animalsfor 6, 12, 24, 48, 96 h and 3 weeks. Full details ofthe pastures, harvesting, feed preparation, parti-cle size analysis and digestion have been pre-viously published (Wilson et ai, 1988).

Anatomical characteristics of the original feedwere assessed on samples of blade excised fromthe midpoint of representative leaves. This mater-ial was fixed in formalin-acetic acid-alcohol(FAA), embedded in glycol methacrylate plasticand sectioned at 5 /jm. The proportion of thedifferent cell types in blade cross-section and thesize of the vascular and sclerenchyma bundles inmidrib and across the lamina were measured fromprojected, magnified images of the sections on anelectronic digitizer tablet or VDU screen.

Samples for electron and light microscopy weretaken after chewing and after the varying periodsof digestion. Sub-samples for transmission elec-tron microscopy (TEM) were fixed in a modifiedKarnovskys fixative of 3% glutaraldehyde 3%paraformaldehyde in 0-05 M phosphate buffer(pH 6-8-7 2), post-fixed in 2% osmium tetroxide,embedded in Spurr's resin, sectioned at 50-100nm, and stained with uranyl acetate followed bylead citrate. Transverse sections of particles wereexamined for all digestion times and longitudinal

TaUc I. Anatomical characteristics of green panic and ryegrass leaf blades (Means offour replicates exceptfoi fibre characterislics).

Attribute

Tissue proportions in leaf cross seclon (%):Mesophyll (+ air space)EpidermisVascular tissueBundle sheathSclerenchymaParenchyma (midrib)

Midrib characterislics (% of leaf value):cross sectional areaproportion of tough tissue (vasculartissue plus sclerenchyma)

Fibre characteristics (diameter in mm)t:midrib vascular bundles -central

—olhersclerenchyma strand —central

—otherlamina vascular bundles—major

—intermediate—minor

sclerenchyma strands—abaxial

Vascular bundledcnsity (no. mm ~' leaf width)

Green panic

30 8 ±0-522-5 ±0-1

7-0 ±0-224'6±0'7

l-7±0113-6±11

23-8±l-5

3O-3±3 7

0-1380-035 (0-027-0-043)0-1810-048 (0-027-O-061)0-093 (0-074-0-108)0-030 (0-024-0-040)0-019 (0-016-0-020)0-054 (0-042-0 062)

8-7 ±0-4

SignificanceRyegrass ofdifference

65-6 + 1-5 • • •22-8 ±0-9 NS

3-1+0-25-5±0-3 • "0 7±0-l • • •2-3 ±0-8 • • •

18-3±I-4 •

19-2±l-6 •

0-118

0-105

0'082 (0-055-O-I08)

0-044 (0-034-0-053)0-026 (0-015-0-036)

2-3±0-l

t Values and range found in a typical leaf.

Particle size reduction. 11 67

Set

Plate I . Lighi photomicrographs of fresh leaTblades for green panic (/". mfljt/mam var. trichoglturte) (a.) midnb, K 6 I , (b) minor andmajor vascular bundle, x 238; and ryegrass (Z,.mu/»:flo/-um)(c) midrib, >c55.and(d)majorvascular bundle, x 103.M,mesophyll ; VT,vascular tissue; BS, bundle sheath; E, epidermis; Sci, sclerenchyma.

sections of fibres for 96 h and 3 week-digestiontimes. For scanning electron microscopy (SEM),samples of the chewed boli, 6, 12 and 24 hdigested material fixed in FAA were washed in0-2 M cacodylate buffer, post-fixed with 1-5%osmium tetroxide in 0-1 M cacodylate buffer (pH7-2), critical point dried, glued onto an aluminiumstubb and coated with gold. The 48 h-, 96 h- and3-week digested particles from FAA were rinsedin arsenate buffer and placed directly onto astubb.

The size of particles in the 96 h-digested feedwas measured for two of the replicate samples.The particles, which were mostly isolated fibrestrands, were progressively separated and mea-sured as groups representing very coarse, coarse,medium and fine fibres. This classification offibres was based on differences in their width(Table 2), the different-sized groups of fibres werereadily distinguishable within the sample. Lengthwas measured for each individual fibre but width

(at X 60 magnification) was measured only for asample of 30-60 fibres from each size group.Separation of the fibres often necessitated break-age of the short transverse veins which link theparallel veins at intervals. Despite careful dissec-tion under a binocular microscope breakage ofthe longitudinal 'fine' fibre veins was unavoid-able, especially for ryegrass, so much so that thisgroup was measured for only one replicate. Forgreen panic there were also 'very fine' fibrespresent (0-006-0-014 mm diameter), but it wasnot possible to disentangle these without exten-sive breakage; a sample of 40 of these fibres wasmeasured from replicate I. Frequency distribu-tions of particle lengths were calculated andsignificance of the difference between meansdetermined by t-test. The relative contribution ofthe different fibre groups to the total particulatematter measured was calculated on a volumebasis, regarding the very coarse particles as'planks' with a thickness equivalent to that of the

68 / R. Wilson et al.

Plate 2. Scanning electron micrographs of chewed and digested leaf blades for green panic (P. maximum var, Irkhoghme) (a) chewedleaf, (b) 6 h, (c) 12 h, and (d) 24 h digested; and ryegrass {L. multiflorum) (e) chewed, (f) 6 h, (g) 12 h and (h) 24 h digested. Bar representsI mm (fl,c,e,f,g) or 0 i mm (b,d,h).

average thickness of the lamina (ex midrib), andthe other fibres as cylinders.

Results

Leaf anatomical characteristics

Compared with ryegrass, leaves of green panichad less than half the proportion of mesophylltissue and more than twice the proportion of thethicker-walled cells, viz. bundle sheath, vascular

tissue and sclerenchyma (Table 1, Plate 1). Themidrib ofgreen panic comprised a larger propor-tion of the leaf and contained a higher proportionof the leafs m«;hanical tissues than did thai ofryegrass. These differences can be equated todifferences in volume of the structures because ofthe parallel venation. The width of the fibrestrands within the leaf of each species, viz.vascular bundles (excluding bundle sheath) andsclerenchyma tissue, are shown in Table 1; the size

Particle size reduction. II 69

Plate 3. l.i^hi phohjiiiiL-rogiaph.^orchewed and digested leaf blade for green panic (/".maxiffium var. incfto^/ume) (a) 24 h digestion,minor vascular bundles ( = very (ine fibres) with rough surface due to attached bundle sheath cells and their undigested radial walls,X 70, (b) 96 h digestion, solid leaf piece, >; 50, NB. detached "very fine" fibres (1) unbroken for full length of leaf segment (c) 96 hdigestion, isolated major bundle-f-sclerenchyma wiih jagged, torn epidermis showing sinuous walls of the Intercostal cells, v 75; andryegrass (L, multiflorum) (d) 12 h digested, epidermal sheet showing straight-side walls of intercostal cells, x 200, and (e) % h digestion,isolated major bundles with smooth surface, x 22.

of fibre strands covered a wide, but similar, rangein both species.

The mesophyl! of green panic is more denselypacked than that of ryegrass (Plate la, b). Inaddition, in green panic the adaxial and abaxialepidermis is connected to the vascular bundles bythick-walled sclerenchyma and bundle sheathcells (Plate Ib) whereas in ryegrass thin-walledmesophyll cells separate the vascular bundlesfrom the epidermis (Plate Id).

Tissue breakdown and fibre particle characteristics

During chewing, ryegrass leaf split longitudinallymore easily than green panic leaf, as illustrated bythe SEM photographs of the broken end of thechewed particles (Plate 2a, e). The epidermal cellsof the two species differed in structural arrange-ment with their long cells in the region between

the vascular strands (intercostal cells) havingsinuous (dove-tailed) side walls in green panic(Plate 3c) compared to straight side walls inreygrass (Plate 3d).

During digestion, further reduction in particlewidth occurred in both species but was morerapid in the ryegrass (Plate 2). This faster rate ofwidth reduction in ryegrass was associated withthe easy detachment of the epidermis from thevascular bundles when the mesophyll cells weredigested, so that after 12 h of digestion theremaining particles of ryegrass consisted almostentirely of isolated vascular and sclerenchymastrands (Plate 2g). By 24 h some of the vascularstrands had been reduced to the main xylemelements (Plate 2h).

In contrast to ryegrass, the particles of greenpanic remained relatively intact up to 12 h ofdigestion (Plate 2a-c), even though TEM sectionsshowed a similar digestion of mesophyll in this

70 / R. Wilson et al.

0 8

0-6

0-4

0-2

0

Very coarse

(a) Ryegrass

Coorse Medium Fine

IL I . .

0-8

0-6

04

0-2

(b) Green panic

L

Size classes (mrTi)Figure 1. Frequency distribution of lengths of fibre particles, divided into groups based on width,from leaf blades of ryegrass (L. multiflorum) and green panic {P. maximum var. irichoglume) forchewed fresh feed after 96 h of digestion in nylon bags in the rumen of cattle. Error bars are2 X standard error of the means of two rqilicate feeds.

Table 2. Panicle dimensions and relative contribution on a volume basis of different groups of fibres in samples of chewed green panicand ryegrass leaf afler digestion for 96 h (values from two replicate feeds)

Type offibre particle

Very coarseCoarseMedium^Fine*Mean particle sizeafter chewing

No, particlesmeasured'

100261594

1702

Green panic

Length Width(mm) (mm)

8-09±0'63 O-874±0'O405-01 ±0-22 0-I30±0-0035-70 + 0-17 0-082±0-0024'I2±O'O9 0-048±0-002

5-42+0-08 1-57 + 0-03

Relativecontribution

0'620-130-130 12

No, particlesmeasured

777851813

Ryegrass

Length(mm)

12-44+0-3010-20±0-275-24 + 0-17'

11 26 ±0 35

Width(mm)

0-108 ±0-0020-066 ±0-0020-044 ±0-002

2-37 ±0-08

Relativecontribution^

00-580-180-24

' For length only; width was measured on 30-60 fibre strands in each size group.Relative contribution based on replicate I data only,

^ Solid piecesof leaf comprising an average of four vascular strands,* Single, isolated vascular and sclerenchyma strands,^ Replicate I only.

Species to that in ryegrass. After 24 h, the particlesof green panie still mainly comprised multiplevascular strands held together by the epidermiswhich remained attached to the vascular bundles(Plate 2d), These multivascular strands weregradually reduced to isolated fibre bundles withfurther digestion, but even after 96 h, 0 04 of theparticles present (Table 2) in green panic were stillof this type (Plate 3b).

The isolated vascular bundles (fibre particles)differed in surface characteristics between thespecies. The ryegrass bundles were left with asmooth surface (Plate 3e) because the sheath ofcells surrounding them within the leaf comprisedthin-walled mesophyll cells (Plate lc, d) that werecompletely digested (Plate 2g). In contrast, thevascular bundles of green panic were rough-surfaced because of undigested bundle sheath

Particle size reduction. II 71

Plate 4. Scanning electron micrographs of chewed leaf blades digested in the rumen for 3 weeks for (a-d) green panic {P. maximum var.trichoglume) and (c-g) ryegrass {L. muliiflorum). (a) and (e) main midrib vascular bundle and/or scierenchyma, (b) major vascularbundle with mcstomc sheath ceils stripping away, (c) and (0 isolated metaxylem vessels from major vascular bundles, (d) and (g)metjuylem vessel from minor vascular bundles. Bar represents 0-1 mm (a,b,c,e,0 or 10 ;«n (d.g).

cells, and/or the projecting, partly-digested radialwalls of these cells (Plate 3a) which were stillattached. SEM and TEM indicated that digestionof the bundle sheath cells in green panic beganonly after 12 h in the rumen and was still notcomplete after 48 h. Many of the isolated bundlesof green panic had torn, jagged pieces of epider-mis firmly attached to them (Plate 3c).

Particle size after 96 h digestion

To determine whether the diameter of the vascu-lar bundle or sclerenchyma fibres infiuenced theirresistance to breakdown In length during diges-tion, groups ot ditterent-sized fibres were mea-sured after 96 h of digestion. The mean lengths ofall fibre groups after 96 h digestion, except the

72 J. R. Wilson et al.

Plate 5. Transmission electron micrographs of longitudinal sections of fibres digested for 3 weeks for (a-b) green panic {P. maximumvar, trichoglume) and (c-d) ryegrass {L. multiflorum) (a) and (c) main xylem elements, (b) and (d) sclerenchyma fibre bundle, fungalhyphae (F). Magnification y2813(a-c), y2360(d).

fine fibres of ryegrass, were not consistently lowerthan the mean particle-lengths of the chewedmaterial before digestion (Table 2). The coarseand medium fibre groups had relatively similarmean lengths (Table 2) and distribution oflengths(Figure 1) but the fibres in the fine group had ahigher proportion of shorter particles {P < 0-001)than the former two groups. Breakage duringseparation could have accounted for much of thisdifference, especially in ryegrass because its finefibres broke more easily than those ofgreen panic.The forty 'very fine' green panic fibres measuredranged up to 18 mm long with 0-3 of them in the15-18 mm range; visually, these fibres wereequally as long as any of the coarser fibres but thetransverse vein connections to the stronger fibresmade their separation very difficult.

Although there were many fewer coarse fibresthan medium plus fine fibres, the former com-prised the bulk of the particulate matter byvolume (Table 2), and probably also by weight,because all fibres comprise similar cell types andshould not differ greatly in specific gravity.

Particle widths of these isolated vascular orsclerenchyma strands were generally < 0-1 mm indiameter (Table 2). Comparison of particlewidths with data in Table 1 indicates that thecoarse fibres largely originated from the vascularand sclerenchyma strands in the midrib and themedium fibres from the major vascular bundles inthe lamina. The fine fibres were of similar size tothe abaxial sclerenchyma strands, the largerintermediate vascular bundles of the lamina ingreen panic, and the minor vascular bundles inryegrass. The very fine fibres in green panic werethe minor vascular bundles with a diameter equalto 1-2 xylem vessels (Plate 4d}.

Indigestible structures after 3 weeks digestion

The intractable anatomical elements after threeweeks digestion were identical in appearance forboth species (Plate 4). The only major changefrom the shorter digestion times was the splittingaway of strands of mestome sheath cells andprotoxylem vessels (Plate 4b) to leave many fibresas only metaxylem vessels with or without some

Particle size reduction. II 73

tracheary elements in between (Plates 4c, d, f, g).The surface view of the fibres by SEM shows noevidence of digestive erosion that could lead tobreakage laterally, nor is there evidence from thelongitudinal sections of fibres under TEM thatmicrobial digestion of the walls would be suffi-cient to cause breakage of either xylem elements(Plate 5a, c) or sclerenchyma strands (Plate 5b, d)in either species.

Discussion

This paper has identified important links betweenleaf anatotny and the resistance of leaf to break-down and the amount and characteristics of thefibres produced after chewing and digestion.

The leaves ofgreen panic were more rigid thanthose of ryegrass, but nevertheless the formerspecies showed much greater particle breakdownduring chewing (Wilson et al., 1988). Possibly,leaf rigidity is associated with the 'brittleness'factor described by Ulyatt (1983). Alternatively,the higher proportion of thick-walled cells couldhave resulted in the need for more chewing pergram of leaf. If leaf rigidity, as seems likely, iscaused by the dense packing of mesophyll, thehigh number and close spacing of veins, and thehigh proportion of thick-walled cells in a leaf,then the leaves of most tropical grasses will havethis characteristic because this structure is linkedto the C4 anatomy (Laetsch, 1974; Wilson et al.,1983). C« grasses also have a higher proportion ofsclerenchyma than Cj grasses (Akin and Burdick,1975) and this would result in a much greater leafstiffness (Vincent 1982).

Reduction in particle width required the split-ting and shedding of the epidermis. This occurredmore rapidly in ryegrass than green panic; in theformer species it started at the ends of leaf piecesexposed by chewing and was essentially completeafter only 6-12 h of digestion. Two anatomicalfeatures are suggested as contributing to thisdifference between the two grasses.

The first feature is that the walls of theintercostal cells of the epidermis of ryegrass arestraight-sided and those of green panic sinuous.We postulate that the sinuous 'dovetail' construc-tion gives great strength to the epidermis andaccounts for the observation that the epidermis ofgreen panic split with difficulty and only bytearing across the cell walls (Plate 3c). Theryegrass leaf split more readily because adjacentepidermal cells parted cleanly at the middle

lamella. This distinction in epidermal construc-tion is a feature of taxonomic difference betweengenera of the temperate grass sub-family Pooi-deae and those of the tropical grass sub-familiesPanicoideae and Chloridoideae. Most of thecommonly used temperate grass genera {Agrostis,Avena, Bromus, Holcus, Lolium, and Phalaris)have straight-sided epidermal walls, whereas tro-pical grass genera almost invariably have sinuouswalls (Watson and Dallwitz 1980). The tropicalgrasses thus have an inherent disadvantage in thisaspect of particle breakdown which may bedifficult to overcome. Support for the importanceof this wall characteristic is provided by theobservations of Sakurai (1963) that epidermalremoval with chewing occurred easily in ryegrass,oats and Secale species and with difficulty inZoysia, Miscanthus, Zea and Dactylis. Theformer group, except Secale, have straight-sideepidermal cell walls and the latter group havesinuous walls.

The second feature is the presence or absence ofcellular 'girder' construction (Clifford and Wat-son 1977) in which the epidermis is firmly linkedto the vascular bundles via thick-walled scleren-chyma fibres. In Lolium, and some other temper-ate grass genera, this structure is absent and theepidermis/sclerenchyma is separated from thevascular tissue only by thin-walled, easilydigested mesophyll cells. In green panic and manyother tropical grasses, the epidermis/scleren-chyma is linked either directly to the vasculartissue or to the thick-walled bundle sheath cells.These cells are digested only slowly (Hastert et al.1983) and the epidermis cannot easily be shed.This difference in epidermal attachment wouldaccount for the observation of Pond et ai (1984)that the epidermis of ryegrass was less firmlyattached to the leaf blade than that of coastalbermudagrass. The absence of a girder structureis more prevalent in the temperate Pooideae thanin the tropical Chloridoideae and Panicoideae(Watson et ai, 1985). Nevertheless, girder ar-rangement is a highly variable feature (Watsonand Dallwitz, 1980); and ihis offers good poten-tial for finding anatomical variants with a struc-ture conducive to rapid epidermal shedding.

Once the leaf has disintegrated through split-ting or shedding of the epidermis, the character-istics of particles are those of the individualvascular or sclerenchyma fibre strands. Thisstudy reveals some' important features of thesefibres.

74 J. R. Wilson et al.

First, all the fibre particles of both species are< 0 2 mm in diameter but despite this, and thefact that some fibres may be reduced to only oneor two xylem elements, it is clear that digestionalone caused little reduction in their length.Because of the multi-celled nature of most of thevascular fibres and the laminated beam structureof the sclerenchyma fibres (Plates 5), points ofextensive digestive erosion of the walls wouldhave to be coincident at the same location acrossa number of walls for a fibre strand to breakacross its length solely due to digestion. TTie highresistance to length reduction was shown by allthe fibre classes from very fine (0 06 mm) to large(0-18 mm), and was similar for both the tropicaland temperate grass, and the material was of highdigestibility (Wilson et al., 1988), We hypothe-size, therefore, that the ineffectiveness of diges-tion alone to reduce particle length is likely toapply universally to all fibres from leaves of anygrass and that there is unlikely to be anatomicalvariation that will have any major effect on thissituation. Thus, chewing during rumination willbe of paramount importance for length reductionof even the finest fibres within a leaf.

Second, the isolated fibres of ryegrass aresmooth-surfaced whereas those ofgreen panic arerough because of the attached patches of bundlesheath cells or their undigested radial walls andthe presence of torn and jagged sheets of epider-mis. These features ofgreen panic fibres are likelyto be common to most C4 grasses, and may affectpacking density of the particles in the rumen. Theexternal roughness of the typical C* fibre particlecould increase also the resistance to independentflow from the fibre raft in the rumen. Particlesneed to be separated from the digesta mass in therumen and progress to the reticulum before theycan pass from the reticulo-rumen (McBride et ai,1984). Both surface roughness and the long lengthof the fibres prior to rumination may be signifi-cant entanglement factors which reduce ease ofparticle movement.

Third, the isolated vascular fibres of greenpanic and other C< grasses may have a volumeabout three times greater than that of fibres fromCj grasses like ryegrass because of the presence ofthe thick-walled, slowly digested bundle sheathcells. Additionally, these bundle sheath cellscontinue to digest for up to 48 h or longer in therumen, possibly resulting in entrapped gas bub-bles which could lower the functional specificgravity of the fibres. This could affect the ability

of these particles to pass from tbe rumen (Ken-nedy and Murphy, 1988).

In conclusion, tropical grass leaves have anato-mical features either associated with the C4 versusC3 photosynthetic pathway or as taxonomiccharacteristics of the Panicoid and Chloridoidversus Pooid sub-families which affect the quan-tity of fibre and the resistance of the leaf tophysical and microbial breakdown by the animal.These are: the high number of fibre elements perleaf; the large fibre diameter due to the bundlesheath cells; differences in epidermal construc-tion; and the surface roughness of the isolatedfibre strands. Further work will be required todetermine whether variation in these character-istics exists between tropical grasses, and it can beexploited to increase their intake by ruminants.

Acknowledgments

The senior author would like to acknowledgefinancial assistance received from the UnitedStates Department of Agriculture through ascientific agreement with the CommonwealthScientific and Industrial Research Organizationwhich allowed him to visit ihe USDA RussellResearch Centre, Athens, Georgia, USA toundertake the light and electron microscopicstudies reported here.

We would like to thank Mrs G. Meiburg, Mr P.Tuckett and Mr B. Smith, CSIRO Division ofTropical Crops and Pastures, Brisbane, Australiafor assistance with the experiment and preprationof samples for anatomy, and Mr W. Rigsby andMiss D. Smith, USDA Russell Research Centre,for help, respectively, with the electron micros-copy and some of the photography.

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{Received 23 March 1988; revised 25 July 1988)