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Rumen degradation of cowpea husks and rice straw: microscopic evaluation of degraded residues
Animal FeedScitwceand
Technology, 35 (1991)
309-320
309
Elsevier Science Publishers B.V., Amsterdam
Rumen degradation of cowpea husks and rice straw: microscopic evaluation of degraded residues Y. Nakashima,
E.A. Adebowale’ and Y. Misawa
Faculty ofAgriculfure, Iware L’niversrry. S18-8. &da. Morioka. .rapan
(Received 5 November 1990;accepted2 May 1991)
ABSTRACT Nakashima. Y., Adebowale,E.A. and Misawa, Y., 1991. Rumen degradation of cowpea husks and rice straw: microscopic evaluation of degraded residues. AnOn. Feed&i.
Technol., 35: 309-320.
A scanning electron microscope was used to observe the microanatomy and relative ease of tissue degradation ofair-drted cowpea husks and rice straw internodes treated (soaked for 24 h) with either 1% NaOH or 1% alkaline H202 solution and suspended, together with untreated samples, in nylon hags in ruminally iistulated sheep. Incubation periods were 8.24 and 48 h. Data revealed that cowpea husks were degraded faster hut less extensively than rice straw. Whereas parenchyma and cortex tissues in addition to the epidermis in the chemically treated cowpea husks were degraded within 8 h of incubation, only rice straw parenchyma (with phloem in the NaOH-treated material) was at the initial stage of degradation. After 24 h microbial activity, degradation was already slowing down in the cowpea husks whereas more tissues in the treated rice straw (eptdermis and lignified vascular bundle) were undergoing degradation. Portions of the lignitied structures in the treated rice straw appeared to be completely removed after 46 h incubation, whereas lignified vascular tissues in both treated and untreated cowpea husks were undegradcd. These results indicate that microanatomy, inherent characteristics ofroughages and chemical treatment affect digestibility by rumen microorganisms.
INTRCDUCTION
Leguminous and graminaceous forages differ in microanatomy (Mowat et al., 1969). Silica, a structural inhibitor which can form various types of phytoliths in grasses (Twiss et al., 1969), is almost absent in legumes. Sites, types and percentages of lignificstion, amount of vascular tissue and cortex also contribute to the differences. These inherent characteristics of cell walls affect digestibility by microorganisms (Akin and Burdick, 1975). For instance, Adebowale and Nakashima ( 1990) found that the initial degradation rate of cowpea husks was faster than that of grasses although potential dry matter ‘Present address: Institute of Agricultural Research and Training, P.M.B. 5029, lbadan, Nigeria.
Obafemi
Awolowo
sity.
0 1991 Elsevier Scimce Publishers B.V. All rights reserved 0377-8401/91/$03.50
Univer-
degradability of maize stover was greater at 96 h. Some chemical data relate compositional differ :nces of legumes and grasses to digestibility (Mowat et al., 1969; Wilkir,on et al., 1970, Adebowale and Nakashima, I99 1) but these data do not fully explain digestibility variances (Van Soest, 1968). Coen and Dehority ( 1970). in their investigations with gnotobiotic fermentations, found that the molecular orientation and complexity ofcertain cell wall constituents may affect forage degradation by rumen microbes. Dekker et al. ( 1972) supgested that factors such as cellulose crystallinity and chemical heterogeneity of hemicehulose molecules may influence cell wall digestibility. Research relating the anatomical traits of grasses to digestibility has been reported for some forages (Akin et al., 1973; Brazle and Harbers, 1977; Akin, 1988), but comparisons among chemically treated and untreated leguminous and graminaceous straws are few, if any. The objectives of this study were to observe the sequence of ruminal hydrolysis of chemically treated cowpea husks and rice straw with scanning electron photomicrographs of in vivo digestion, and thereby determine which structures resist digestion. M.ATERIAlS
AND METHODS
Crop residues and their treatment
Cowpea husks ( Vigna unguiculata, W.) and internodesof rice straw (Oryza safira, L.) were cut into about 5 mm lengths with a razor blade so that the cross-section could be observed. They were soaked in 50 ml of either 1% NaOH or 1%alkaline H,O, solution for 24 h. The pH of the alkaline H20r was adjusted to
11.5with NaOH. Untreated
samples
were soaked in distilled
water
for 24 h. Treated and untreated samples were then washed with distilled water under water suction, air-dried and used for microscopic observation.
About 1 g of untreated or treated samples were weighed into nylon bags and incubated in the rumen of one sheep (Suffolk). The incubation periods were 8, 24 and 48 h. The samples were then withdrawn from the rumen, washed with distilled water under water suction, and air-dried. About 20 samples each of treated or untreated plant material were hydrolysed at each time interval. At least three random samples of each were observed under the microscope. One sheep weighing about 40 kg and fitted with a permanent rumen cannula were fed chopped orchard grass hay and a concentrate ration (7 : 3) at 2% of body weight. The concentrate ration was made up of 7 1%grain (maize, milo, barley and soyabean), 19% bran (wheatbran and corn gluten feed), 5% oil seed meal (soyabean meal and rape meal) and 5% molasses. The ration was
supplemented with (per 100 kg ration) 0.32 kg sodium chloride, 0.51 kg calcium dihydrogen phosphate and 0.20 kg mineral-vitamin, and was given once daily at 09:OOh. Scanning electron microscope studies
Samples were fixed in 2% parafonnaldehyde-2S%glutaraldehyde in 0.1 M cacodylate buffer and postfixed in buffered I?/0osmium tetroxide at 4°C. They were then dehydrated in an ethanol series (5 min, twice in 50, 70, 80, 90,95 and 100%) and dried in a critical point dryer (Type HCP-1, Hitachi, Tokyo, Japan). They were mounted on 15 mmx 5 mm aluminum stubs (type GM Nisshin Em, Tokyo, Japan), coated under vacuum with 150 nm gold-palladium in an ion coater (Giko Engineering, Tokyo, Japan) at 15 kV. RESULTS
Tables 1 and 2 summarize the microscopic data and present a comparison of the ease of degradation of cowpea husks and rice straw internodes as revealed by scanning electron microscopy (SEM). The stages of microbial degradation of the chemically treated and untreated cowpea husks and rice straw internodes after various incubation intervals are illustrated by scanning electron photomicrographs. No structural changes were found when the speciTABLE
I
Comparison Incubation
oftreated
and untreated
time
Tissue
cowpea
husks degraded
by rumen
microorganisms
types
(b)
‘D. Degraded. ‘I. Initial stageofdegradamm. “N. Non-degraded
Epidermis
Parenchyma
COIWX
Lignified vascular tissue
D’ D D
I’ D D
I D D
N’ N N
D
I D D
I
L? D
D D
N N N
3 D D
D D D
D D D
N N N
312
Y.NAKASHIMA ETAL.
TABLE 2 Comparion oftreated and unlrealed rice straw internode degraded by rumen microorganisms Incubation flme (h)
lbzfrcared 8 24 48
Tissue types Parenchyma
Phloem
Epidermis
I’ D’ D
NZ D D
N D D
!
N
D D
D D
N D D
N D D
NaOH-rreared 8 I 24 D 48 D ilk&w H,O, rrrared 8 1 24 D 48 D
Llgnitied vascular tissue
Sclerenchyma
‘I. Initial stage of degradation. ‘N. Non-degraded. ‘D, Degraded.
Fig. I. Scanning electron photomicrographs of cross-sections of (a) cowpea husk and (b) rice straw internode (control) soaked in distilled waler (without rumen microorganisms) for 24 h. (a) Epidermis (E) and vascular tissue (V) of cowpea husk arc intact, and cortex (Co) and Parenchyma (Par) lack rigidity. (Magnification, X 160). (b) Epidermis (E).xylem (X), phloem (P). parenchyma (Par) of rice straw are intact. (Magnification, X 260).
RUMEN
DEGR*DATI*N
OFCOWPE.4
HUSKS
AND
RlCE
STR.xW
313
mens were soaked in distilled water for 24 h (control), except that the cortex and parenchyma of the cowpea husks appeared to have lost some rigidity (Figs. laand lb). Incubation for 8 h
After 8 h of incubation, rumen microbial degradation of the untreated specimens was only slight when compared with the control specimens. The epidermis of the cowpea husks has been sloughed and degrading of the underlying cortex has started (Fig. 2a). The parenchyma was also under microbial attack. However, for rice straw internodes, only the parenchyma showed some microbial activity and the phloem was beginning to lose rigidity (Fig. 2b). When specimens were soaked in 1% NaOH for 24 h and incubated in the rumen of the sheep for 8 h, there was detectable degradation in the cortex and parenchyma of the cowpea husks and the phioem and parenchyma ofthe rice straw (Figs. 3a and 3b). A similar pattern was obtained for specimens soaked in 1% alkaline HzOz (Figs. 4a and 4b), although the parenchyma and cortex appeared to be more eroded in alkaline H,Oz-treated than NaOH-treated cowpea husks. The reverse was true for the rice straw.
Fig. ?. Scanning electron photomicmgraphs of untreated cowpea husk (a) and internode of rice straw (b) incubated in the sheep rumen for 8 h. (a) Rumen microcrganisms have denuded the epidermis (E) of cowpeahusk. and the cortex (Co) and the parenchyma (Par) are under attack. (Magnification, x 160). (b) Degradation is apparent only in the parenchyma (Par). of the ricestraw, and the phloem (P) is beginning to lo\e rigidity. (Magnification, X 190).
Fig. 3. Scanning electron photomicrographs of NaOH-treated cowpea husk (a) and internode of rice straw (b) incubated in the sheep rttmen for 8 h. (a) Cowpea husk cross-section shows partial breakdow. of cortex (Co) and parenchyma (Par). (Magnification, x95). (b) Crosssection of rice straw shows internode phloem (P) and parenchyma (Par) undergoing degradation. (Magnification, X300).
Fig. 4. Scanning electron photomicrographs of alkaline H,02-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 8 h. (a) Cross-section of cowpea husk shows residue of pareschyma: lignitied vascular tissue is under attack of mmen microorganisms. (Magnification, x 325 ). (b) Rice straw cross-section shows no tissue loss but some collapseofparenchyma (Par). (Magnification, x 170).
Fig. 5. Scanning electron pholomicrographs of untreated cowpea husk (a) and rice straw internode (b) incubated in the sheep rumen for 24 h. (a) Cross-section of cowpea husk shows the residue of parenchyma (Par) and dignified vascular tissue (V). (Magnification, x50). (b) Cross-section of rice straw consists of only sclerencbyma (S), xylem (X) with vascular bundle sheath (-) and the lumen (L). (Magnification, x40.5).
I-@. 6. Scanntng electron photomicrographs of cross-section of NaOH-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep rumen for 24 h. (a) Vascular tissue (V) of cowpea husk is under microbial attack. (Magnification, x345). (b) Only portions of the lignit&d vascular tissue (V), sclerenchyma (S) and inner portions of the epidermis (E) of the rice straw remain. (Magniiicalion, X80).
316
Fig. 7. Scanning electron photomicrographs of cross-section of alkaline H20,-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 24 h. (a) Vascular tissue (V) of cowpea husk is under microbial attack. (Magnification, x380). (b) Along with the inner portion of the epidermal cell wall (E), the remnants of the vascular bundle (V) and sclerenchyma (S) of the rice straw show the effects of attack by rumen microbes. (Magnification, X34.5).
Fig. 8. Scanning electron photomicrographs of cross-section of NaOH-treated cowpea husk (a) and rice swaw Internode (b) incubated in the sheep rumen for 48 h. (a) Cowpea husk shows lignitied vascular tissue (V) under microbial attack. (Magnification, X455). (b) Residue of large vascular bundles (LVB) and portions ofthe small vascular bundle (SVB) ofthe rice straw remain. (Magnification. x75).
Fig. 9. Scanning electron photomicrogmphs of cross-section of alkaline HzOrtreated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 48 h. (a) Cross-section of cowpea husk shows resistance of lignitied vascular tissue (V) to digestion. (Magnification, x410). (b) Cross-section of rice straw shows residues of sclerenchyma (S) and small vascular bundle (SVB). (Magnification, x95).
Incubation for 24 h
After 24 h of microbial activity, the cortex of untreated cowpea husks was completely degraded and only residues of the parenchyma were detectable (Fig. 5a). The vascular tissue did not show much visible microbial attack. For the untreated rice straw internodes, the parenchyma, phloem and outer portions of the epidermis were completely degraded (Fig. Sb). Essentially the only remaining tissues were the sclerenchyma, the lignified inner bundle sheath, the metaxylem vessels and the cutinized epidermis. Microbial degradation was more extensive for the treated specimens. For NaOH-treated cowpea husks (Fig. 6a), the lignified vascular tissue was under microbial attack and the parenchyma had been completely degraded. The lignified inner bundle sheath and the metaxylem of the rice straw had become less resistant to microbial attack, leaving only the sclerenchyma, including the small vascular bundle, intact (Fig. 6b). Similar observations were recorded for the alkaline H202-treated roughagas (Figs. 7a and 7b). These observations are supported by the microscopic data summary in Tables 1 and 2. Incubation for 48 h
After 48 h incubation, differences in the extent of degradation from the 24 h incubation were visible only in the treated samples, hence figures for the
318
Y.NAKASHIMA ETAL.
untreated specimens are not shown. There was further attack on the lignitied vascular tissues of NaOH-treated cowpea husks (Fig. 8a) and some erosion of the lignified inner bundle sheath and small vascular bundle of the rice straw internodes (Fig. 8b). Alkaline HzOz-treated roughages appeared to be degraded to a greater extent (Figs. 9a and 9b) than NaOH-treated ones, as more microbial damage was visible. DISCUSSION
Scanning electron photomicrographs of cowpea husks and rice straw internodes soaked in distilled water for 24 h (control) revealed no tissue removal although there was loss of structural rigidity in the cortex and parenchyma of the cowpea husks. Brazle and Harbers ( 1977) made a similar observation while working with alfalfa hay, which indicates that changes during digestion are a combination of microbial degradation and physical disruption by rumen motility. After 8 h of incubation in the rumen of the sheep, the epidermis of the legume had been sloughed and the parenchyma tissues of both materials were under microbial attack. Although chemical treatment gave rapid degradation of the cortex of the legume, this was not the case with the graminaceous roughage. This indicates that the epidermis and cortex of the cowpea husks were degraded faster than those of rice straw, whereas degradation of the parenchyma tissues did not differ between the roughages. This is a clear indication of differences in the susceptibility of the tissues of these materials to microbial attack, and confirms the conclusion of Mowat et al. ( 1969) and Akin and Burdick ( 1973) that forages differ in microanatomy. By 24 h of microbial activity, all cowpea husk tissues except the lignified vascular tissue were degraded and the effects of chemical treatment were obliterated (Table I ). In the case of the rice straw, microbial activity resulted in degradation of the phloem and the outer portions of the epidermis. The treatment even subjected the lignitied vascular tissue to microbial attack, and it was completely degraded at 48 h, especially in the alkaline H,Oz-treated rice straw. Although Monson et al. ( 1972), Hanna et al. (1973) and Akin et al. ( 1973) indicated that cutinized epidermis and lignified tissue usually resist microbial degradation, this study shows that chemical treatment and differences in microanatomy could make these tissues available for microbial degradation. Rumen microorganisms denuded the epidermis of the cowpea husks, thereby exposing the underlying cortex to degradation, whereas that of the rice straw was undegraded despite treatment and 48 h of microbial activity. This rapid degradation of treated cowpea husk epidermis, parenchyma and cortex within only 8 h incubation and the corresponding high dry matter disappearance could explain the high degradation rate obtained for legumes in
RUMEN
DEGRADATION
OFCOWPEI\
HUSKS
Mm
RICE
319
STRW
rumen degradation studies (Adebowale and Nakashima, 1991). As the vascular tissue of treated rice straw was slowly degraded whereas that of cowpea husks was undegraded, it was possible for the dry matter disappearance of the former to be higher than that of the latter at 48 h incubation, thus agreeing with the observation of Donefer et al. ( 1960) that the initial in vitro digestion rate of legumes was faster than that of graminaceous forages although the extent of digestibility of grass was greater. It also confirms the suggestion of Akin et ai. ( i 975 ) that iignins differ. Akin ( 1988) found that chemical treatment made tissues which stained positive with chlorine sulphide (CS+ ) available for microbial attack whereas this was not the case with those that stained positive with acid phloroglucinol ( AP + ) . Staining of vascular bundle and sclerenchyma tissues for lignin with AP+ or CS+ may further elucidate the resistance of these tissues to microbial attack. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of Dr. K. Taniguchi (preparation of samples for SEM), and M. Niisato and A. Ueyama (electron microscope staff). E.A.A. was the recipient of a Japan Society for the Promotion of Science Fellowship.
REFERENCES Adebowal:, EA. and Nakashima, Y., i99i. Romea di-gradation of a legume by-product and some graminae roughages: effect of chemical pretreatment with or without celhdase preparation on dry matter and cell wall disappearance. Anim. Feed Sci. Technol., submitted. Akin, D.E., 1988. Biological structure of lignocellulose and its degradation in the rumen. Anim. Feed Sci. Technol., 21: 295-310. Akin,
D.E.
and Burdick,
D..
1973. Microanatomical
vealed by light and electron microscopy.
differences
of warm-season
grasses re-
Agron. J., 65: 533-537.
Akin, D.E. and Burdick, D., 1975. Percentageoftissue types in tropical and temperategrassleaf blades and degradation of tissues by rumen microorganisms. Crop Sci., 15: 66 l-668. Akin, D.E.. Amos, H.E., Barton, II, F.E. and Burdick, D., 1973. Rumen microbial degradation of grass tissue revealed by scanning electron microscopy. Agron. _I., 65: 825-828. Akin
D.E.,
Barton
II, F.E. and Burdick,
Bermuda and Kentucky-31 Food Chem., 23: 924-927. Brazle, F.K. and Harbers,
L.H.,
D..
1975. Scanning electron
tall fescue extracted 1977. Digestion
microscopy
of Coastal
with neutral and acid detergents. J. Agric.
of alfalfa hay observed by scanning electron
microscopy. .I. Anim. Sci., 46: 506-512. Coen, J.A. and Dehority, B.A., 1970. Degradation and utilization of hemicelhdose forages by pore cultures of rumcn bacteria. Appl. Microbic& 2U: 362-368.
from intact
Dekker. R.F.H., Richards, G.N. and Playne, M.I., 1972. Digestion of polysaccharide constituents of tropical pasture herbage in the bovine rumen. Part I. Townsville stylo (Sfykxanthes hurnilis). Carbohydr. Rcs., 22: 173-185. Donefer, E., Crampton, E.W. and Lloyd, L.E., 1960. Prediction of the r.utritive value index of a forage from in viva romcn fermentation datz. J. Anim. Sci., 19: 545-552.
320
Y. NAKAsHlMAETAL.
Hanna, W.W., Monson, W.G., and Burton, G.W., 1973. Histological examination of fresh forage leaves after in vitro digestion. Crop Sci., 13: 9X-102. Monson, W.G., Powell, J.B. and Burton, G.W., 1972. Digestion of fresh forage in rumen fluid. Agron. J., 64: 231-233. Mowat, D.N., Kwain, M.L. and Winch, J.E., 1969. Lignitication and in vitro cell wall digestibility of plant parts. Can. I. Plant Sci., 49: 499- 504. Twiss, P.C., Suess, E. and Smith, R.M., 1969. Morphological classification of grass phytoliths. Soil Sci. Sot. Am. Proc., 33: 109-l 15. Van Soest, P.J.; 1968. Structural and chemical characteristics which limit the nutritive value of forages. In: Forages: Economics/Quality. ASA Spec. Publ. 13. Am. Sot. Agron., Madison, WI, pp. 63-76. Wilkinson, S.R., Adams, W.E. and Jackson, W.A., 1970. Chemical composition and in viva digestibility of vertical layers of coastal bemmda grass ( (Cymdon dacfyh (L) Pers. ). Agron. J.. 62: 39-43.
Technology, 35 (1991)
309-320
309
Elsevier Science Publishers B.V., Amsterdam
Rumen degradation of cowpea husks and rice straw: microscopic evaluation of degraded residues Y. Nakashima,
E.A. Adebowale’ and Y. Misawa
Faculty ofAgriculfure, Iware L’niversrry. S18-8. &da. Morioka. .rapan
(Received 5 November 1990;accepted2 May 1991)
ABSTRACT Nakashima. Y., Adebowale,E.A. and Misawa, Y., 1991. Rumen degradation of cowpea husks and rice straw: microscopic evaluation of degraded residues. AnOn. Feed&i.
Technol., 35: 309-320.
A scanning electron microscope was used to observe the microanatomy and relative ease of tissue degradation ofair-drted cowpea husks and rice straw internodes treated (soaked for 24 h) with either 1% NaOH or 1% alkaline H202 solution and suspended, together with untreated samples, in nylon hags in ruminally iistulated sheep. Incubation periods were 8.24 and 48 h. Data revealed that cowpea husks were degraded faster hut less extensively than rice straw. Whereas parenchyma and cortex tissues in addition to the epidermis in the chemically treated cowpea husks were degraded within 8 h of incubation, only rice straw parenchyma (with phloem in the NaOH-treated material) was at the initial stage of degradation. After 24 h microbial activity, degradation was already slowing down in the cowpea husks whereas more tissues in the treated rice straw (eptdermis and lignified vascular bundle) were undergoing degradation. Portions of the lignitied structures in the treated rice straw appeared to be completely removed after 46 h incubation, whereas lignified vascular tissues in both treated and untreated cowpea husks were undegradcd. These results indicate that microanatomy, inherent characteristics ofroughages and chemical treatment affect digestibility by rumen microorganisms.
INTRCDUCTION
Leguminous and graminaceous forages differ in microanatomy (Mowat et al., 1969). Silica, a structural inhibitor which can form various types of phytoliths in grasses (Twiss et al., 1969), is almost absent in legumes. Sites, types and percentages of lignificstion, amount of vascular tissue and cortex also contribute to the differences. These inherent characteristics of cell walls affect digestibility by microorganisms (Akin and Burdick, 1975). For instance, Adebowale and Nakashima ( 1990) found that the initial degradation rate of cowpea husks was faster than that of grasses although potential dry matter ‘Present address: Institute of Agricultural Research and Training, P.M.B. 5029, lbadan, Nigeria.
Obafemi
Awolowo
sity.
0 1991 Elsevier Scimce Publishers B.V. All rights reserved 0377-8401/91/$03.50
Univer-
degradability of maize stover was greater at 96 h. Some chemical data relate compositional differ :nces of legumes and grasses to digestibility (Mowat et al., 1969; Wilkir,on et al., 1970, Adebowale and Nakashima, I99 1) but these data do not fully explain digestibility variances (Van Soest, 1968). Coen and Dehority ( 1970). in their investigations with gnotobiotic fermentations, found that the molecular orientation and complexity ofcertain cell wall constituents may affect forage degradation by rumen microbes. Dekker et al. ( 1972) supgested that factors such as cellulose crystallinity and chemical heterogeneity of hemicehulose molecules may influence cell wall digestibility. Research relating the anatomical traits of grasses to digestibility has been reported for some forages (Akin et al., 1973; Brazle and Harbers, 1977; Akin, 1988), but comparisons among chemically treated and untreated leguminous and graminaceous straws are few, if any. The objectives of this study were to observe the sequence of ruminal hydrolysis of chemically treated cowpea husks and rice straw with scanning electron photomicrographs of in vivo digestion, and thereby determine which structures resist digestion. M.ATERIAlS
AND METHODS
Crop residues and their treatment
Cowpea husks ( Vigna unguiculata, W.) and internodesof rice straw (Oryza safira, L.) were cut into about 5 mm lengths with a razor blade so that the cross-section could be observed. They were soaked in 50 ml of either 1% NaOH or 1%alkaline H,O, solution for 24 h. The pH of the alkaline H20r was adjusted to
11.5with NaOH. Untreated
samples
were soaked in distilled
water
for 24 h. Treated and untreated samples were then washed with distilled water under water suction, air-dried and used for microscopic observation.
About 1 g of untreated or treated samples were weighed into nylon bags and incubated in the rumen of one sheep (Suffolk). The incubation periods were 8, 24 and 48 h. The samples were then withdrawn from the rumen, washed with distilled water under water suction, and air-dried. About 20 samples each of treated or untreated plant material were hydrolysed at each time interval. At least three random samples of each were observed under the microscope. One sheep weighing about 40 kg and fitted with a permanent rumen cannula were fed chopped orchard grass hay and a concentrate ration (7 : 3) at 2% of body weight. The concentrate ration was made up of 7 1%grain (maize, milo, barley and soyabean), 19% bran (wheatbran and corn gluten feed), 5% oil seed meal (soyabean meal and rape meal) and 5% molasses. The ration was
supplemented with (per 100 kg ration) 0.32 kg sodium chloride, 0.51 kg calcium dihydrogen phosphate and 0.20 kg mineral-vitamin, and was given once daily at 09:OOh. Scanning electron microscope studies
Samples were fixed in 2% parafonnaldehyde-2S%glutaraldehyde in 0.1 M cacodylate buffer and postfixed in buffered I?/0osmium tetroxide at 4°C. They were then dehydrated in an ethanol series (5 min, twice in 50, 70, 80, 90,95 and 100%) and dried in a critical point dryer (Type HCP-1, Hitachi, Tokyo, Japan). They were mounted on 15 mmx 5 mm aluminum stubs (type GM Nisshin Em, Tokyo, Japan), coated under vacuum with 150 nm gold-palladium in an ion coater (Giko Engineering, Tokyo, Japan) at 15 kV. RESULTS
Tables 1 and 2 summarize the microscopic data and present a comparison of the ease of degradation of cowpea husks and rice straw internodes as revealed by scanning electron microscopy (SEM). The stages of microbial degradation of the chemically treated and untreated cowpea husks and rice straw internodes after various incubation intervals are illustrated by scanning electron photomicrographs. No structural changes were found when the speciTABLE
I
Comparison Incubation
oftreated
and untreated
time
Tissue
cowpea
husks degraded
by rumen
microorganisms
types
(b)
‘D. Degraded. ‘I. Initial stageofdegradamm. “N. Non-degraded
Epidermis
Parenchyma
COIWX
Lignified vascular tissue
D’ D D
I’ D D
I D D
N’ N N
D
I D D
I
L? D
D D
N N N
3 D D
D D D
D D D
N N N
312
Y.NAKASHIMA ETAL.
TABLE 2 Comparion oftreated and unlrealed rice straw internode degraded by rumen microorganisms Incubation flme (h)
lbzfrcared 8 24 48
Tissue types Parenchyma
Phloem
Epidermis
I’ D’ D
NZ D D
N D D
!
N
D D
D D
N D D
N D D
NaOH-rreared 8 I 24 D 48 D ilk&w H,O, rrrared 8 1 24 D 48 D
Llgnitied vascular tissue
Sclerenchyma
‘I. Initial stage of degradation. ‘N. Non-degraded. ‘D, Degraded.
Fig. I. Scanning electron photomicrographs of cross-sections of (a) cowpea husk and (b) rice straw internode (control) soaked in distilled waler (without rumen microorganisms) for 24 h. (a) Epidermis (E) and vascular tissue (V) of cowpea husk arc intact, and cortex (Co) and Parenchyma (Par) lack rigidity. (Magnification, X 160). (b) Epidermis (E).xylem (X), phloem (P). parenchyma (Par) of rice straw are intact. (Magnification, X 260).
RUMEN
DEGR*DATI*N
OFCOWPE.4
HUSKS
AND
RlCE
STR.xW
313
mens were soaked in distilled water for 24 h (control), except that the cortex and parenchyma of the cowpea husks appeared to have lost some rigidity (Figs. laand lb). Incubation for 8 h
After 8 h of incubation, rumen microbial degradation of the untreated specimens was only slight when compared with the control specimens. The epidermis of the cowpea husks has been sloughed and degrading of the underlying cortex has started (Fig. 2a). The parenchyma was also under microbial attack. However, for rice straw internodes, only the parenchyma showed some microbial activity and the phloem was beginning to lose rigidity (Fig. 2b). When specimens were soaked in 1% NaOH for 24 h and incubated in the rumen of the sheep for 8 h, there was detectable degradation in the cortex and parenchyma of the cowpea husks and the phioem and parenchyma ofthe rice straw (Figs. 3a and 3b). A similar pattern was obtained for specimens soaked in 1% alkaline HzOz (Figs. 4a and 4b), although the parenchyma and cortex appeared to be more eroded in alkaline H,Oz-treated than NaOH-treated cowpea husks. The reverse was true for the rice straw.
Fig. ?. Scanning electron photomicmgraphs of untreated cowpea husk (a) and internode of rice straw (b) incubated in the sheep rumen for 8 h. (a) Rumen microcrganisms have denuded the epidermis (E) of cowpeahusk. and the cortex (Co) and the parenchyma (Par) are under attack. (Magnification, x 160). (b) Degradation is apparent only in the parenchyma (Par). of the ricestraw, and the phloem (P) is beginning to lo\e rigidity. (Magnification, X 190).
Fig. 3. Scanning electron photomicrographs of NaOH-treated cowpea husk (a) and internode of rice straw (b) incubated in the sheep rttmen for 8 h. (a) Cowpea husk cross-section shows partial breakdow. of cortex (Co) and parenchyma (Par). (Magnification, x95). (b) Crosssection of rice straw shows internode phloem (P) and parenchyma (Par) undergoing degradation. (Magnification, X300).
Fig. 4. Scanning electron photomicrographs of alkaline H,02-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 8 h. (a) Cross-section of cowpea husk shows residue of pareschyma: lignitied vascular tissue is under attack of mmen microorganisms. (Magnification, x 325 ). (b) Rice straw cross-section shows no tissue loss but some collapseofparenchyma (Par). (Magnification, x 170).
Fig. 5. Scanning electron pholomicrographs of untreated cowpea husk (a) and rice straw internode (b) incubated in the sheep rumen for 24 h. (a) Cross-section of cowpea husk shows the residue of parenchyma (Par) and dignified vascular tissue (V). (Magnification, x50). (b) Cross-section of rice straw consists of only sclerencbyma (S), xylem (X) with vascular bundle sheath (-) and the lumen (L). (Magnification, x40.5).
I-@. 6. Scanntng electron photomicrographs of cross-section of NaOH-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep rumen for 24 h. (a) Vascular tissue (V) of cowpea husk is under microbial attack. (Magnification, x345). (b) Only portions of the lignit&d vascular tissue (V), sclerenchyma (S) and inner portions of the epidermis (E) of the rice straw remain. (Magniiicalion, X80).
316
Fig. 7. Scanning electron photomicrographs of cross-section of alkaline H20,-treated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 24 h. (a) Vascular tissue (V) of cowpea husk is under microbial attack. (Magnification, x380). (b) Along with the inner portion of the epidermal cell wall (E), the remnants of the vascular bundle (V) and sclerenchyma (S) of the rice straw show the effects of attack by rumen microbes. (Magnification, X34.5).
Fig. 8. Scanning electron photomicrographs of cross-section of NaOH-treated cowpea husk (a) and rice swaw Internode (b) incubated in the sheep rumen for 48 h. (a) Cowpea husk shows lignitied vascular tissue (V) under microbial attack. (Magnification, X455). (b) Residue of large vascular bundles (LVB) and portions ofthe small vascular bundle (SVB) ofthe rice straw remain. (Magnification. x75).
Fig. 9. Scanning electron photomicrogmphs of cross-section of alkaline HzOrtreated cowpea husk (a) and rice straw internode (b) incubated in the sheep mmen for 48 h. (a) Cross-section of cowpea husk shows resistance of lignitied vascular tissue (V) to digestion. (Magnification, x410). (b) Cross-section of rice straw shows residues of sclerenchyma (S) and small vascular bundle (SVB). (Magnification, x95).
Incubation for 24 h
After 24 h of microbial activity, the cortex of untreated cowpea husks was completely degraded and only residues of the parenchyma were detectable (Fig. 5a). The vascular tissue did not show much visible microbial attack. For the untreated rice straw internodes, the parenchyma, phloem and outer portions of the epidermis were completely degraded (Fig. Sb). Essentially the only remaining tissues were the sclerenchyma, the lignified inner bundle sheath, the metaxylem vessels and the cutinized epidermis. Microbial degradation was more extensive for the treated specimens. For NaOH-treated cowpea husks (Fig. 6a), the lignified vascular tissue was under microbial attack and the parenchyma had been completely degraded. The lignified inner bundle sheath and the metaxylem of the rice straw had become less resistant to microbial attack, leaving only the sclerenchyma, including the small vascular bundle, intact (Fig. 6b). Similar observations were recorded for the alkaline H202-treated roughagas (Figs. 7a and 7b). These observations are supported by the microscopic data summary in Tables 1 and 2. Incubation for 48 h
After 48 h incubation, differences in the extent of degradation from the 24 h incubation were visible only in the treated samples, hence figures for the
318
Y.NAKASHIMA ETAL.
untreated specimens are not shown. There was further attack on the lignitied vascular tissues of NaOH-treated cowpea husks (Fig. 8a) and some erosion of the lignified inner bundle sheath and small vascular bundle of the rice straw internodes (Fig. 8b). Alkaline HzOz-treated roughages appeared to be degraded to a greater extent (Figs. 9a and 9b) than NaOH-treated ones, as more microbial damage was visible. DISCUSSION
Scanning electron photomicrographs of cowpea husks and rice straw internodes soaked in distilled water for 24 h (control) revealed no tissue removal although there was loss of structural rigidity in the cortex and parenchyma of the cowpea husks. Brazle and Harbers ( 1977) made a similar observation while working with alfalfa hay, which indicates that changes during digestion are a combination of microbial degradation and physical disruption by rumen motility. After 8 h of incubation in the rumen of the sheep, the epidermis of the legume had been sloughed and the parenchyma tissues of both materials were under microbial attack. Although chemical treatment gave rapid degradation of the cortex of the legume, this was not the case with the graminaceous roughage. This indicates that the epidermis and cortex of the cowpea husks were degraded faster than those of rice straw, whereas degradation of the parenchyma tissues did not differ between the roughages. This is a clear indication of differences in the susceptibility of the tissues of these materials to microbial attack, and confirms the conclusion of Mowat et al. ( 1969) and Akin and Burdick ( 1973) that forages differ in microanatomy. By 24 h of microbial activity, all cowpea husk tissues except the lignified vascular tissue were degraded and the effects of chemical treatment were obliterated (Table I ). In the case of the rice straw, microbial activity resulted in degradation of the phloem and the outer portions of the epidermis. The treatment even subjected the lignitied vascular tissue to microbial attack, and it was completely degraded at 48 h, especially in the alkaline H,Oz-treated rice straw. Although Monson et al. ( 1972), Hanna et al. (1973) and Akin et al. ( 1973) indicated that cutinized epidermis and lignified tissue usually resist microbial degradation, this study shows that chemical treatment and differences in microanatomy could make these tissues available for microbial degradation. Rumen microorganisms denuded the epidermis of the cowpea husks, thereby exposing the underlying cortex to degradation, whereas that of the rice straw was undegraded despite treatment and 48 h of microbial activity. This rapid degradation of treated cowpea husk epidermis, parenchyma and cortex within only 8 h incubation and the corresponding high dry matter disappearance could explain the high degradation rate obtained for legumes in
RUMEN
DEGRADATION
OFCOWPEI\
HUSKS
Mm
RICE
319
STRW
rumen degradation studies (Adebowale and Nakashima, 1991). As the vascular tissue of treated rice straw was slowly degraded whereas that of cowpea husks was undegraded, it was possible for the dry matter disappearance of the former to be higher than that of the latter at 48 h incubation, thus agreeing with the observation of Donefer et al. ( 1960) that the initial in vitro digestion rate of legumes was faster than that of graminaceous forages although the extent of digestibility of grass was greater. It also confirms the suggestion of Akin et ai. ( i 975 ) that iignins differ. Akin ( 1988) found that chemical treatment made tissues which stained positive with chlorine sulphide (CS+ ) available for microbial attack whereas this was not the case with those that stained positive with acid phloroglucinol ( AP + ) . Staining of vascular bundle and sclerenchyma tissues for lignin with AP+ or CS+ may further elucidate the resistance of these tissues to microbial attack. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of Dr. K. Taniguchi (preparation of samples for SEM), and M. Niisato and A. Ueyama (electron microscope staff). E.A.A. was the recipient of a Japan Society for the Promotion of Science Fellowship.
REFERENCES Adebowal:, EA. and Nakashima, Y., i99i. Romea di-gradation of a legume by-product and some graminae roughages: effect of chemical pretreatment with or without celhdase preparation on dry matter and cell wall disappearance. Anim. Feed Sci. Technol., submitted. Akin, D.E., 1988. Biological structure of lignocellulose and its degradation in the rumen. Anim. Feed Sci. Technol., 21: 295-310. Akin,
D.E.
and Burdick,
D..
1973. Microanatomical
vealed by light and electron microscopy.
differences
of warm-season
grasses re-
Agron. J., 65: 533-537.
Akin, D.E. and Burdick, D., 1975. Percentageoftissue types in tropical and temperategrassleaf blades and degradation of tissues by rumen microorganisms. Crop Sci., 15: 66 l-668. Akin, D.E.. Amos, H.E., Barton, II, F.E. and Burdick, D., 1973. Rumen microbial degradation of grass tissue revealed by scanning electron microscopy. Agron. _I., 65: 825-828. Akin
D.E.,
Barton
II, F.E. and Burdick,
Bermuda and Kentucky-31 Food Chem., 23: 924-927. Brazle, F.K. and Harbers,
L.H.,
D..
1975. Scanning electron
tall fescue extracted 1977. Digestion
microscopy
of Coastal
with neutral and acid detergents. J. Agric.
of alfalfa hay observed by scanning electron
microscopy. .I. Anim. Sci., 46: 506-512. Coen, J.A. and Dehority, B.A., 1970. Degradation and utilization of hemicelhdose forages by pore cultures of rumcn bacteria. Appl. Microbic& 2U: 362-368.
from intact
Dekker. R.F.H., Richards, G.N. and Playne, M.I., 1972. Digestion of polysaccharide constituents of tropical pasture herbage in the bovine rumen. Part I. Townsville stylo (Sfykxanthes hurnilis). Carbohydr. Rcs., 22: 173-185. Donefer, E., Crampton, E.W. and Lloyd, L.E., 1960. Prediction of the r.utritive value index of a forage from in viva romcn fermentation datz. J. Anim. Sci., 19: 545-552.
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Y. NAKAsHlMAETAL.
Hanna, W.W., Monson, W.G., and Burton, G.W., 1973. Histological examination of fresh forage leaves after in vitro digestion. Crop Sci., 13: 9X-102. Monson, W.G., Powell, J.B. and Burton, G.W., 1972. Digestion of fresh forage in rumen fluid. Agron. J., 64: 231-233. Mowat, D.N., Kwain, M.L. and Winch, J.E., 1969. Lignitication and in vitro cell wall digestibility of plant parts. Can. I. Plant Sci., 49: 499- 504. Twiss, P.C., Suess, E. and Smith, R.M., 1969. Morphological classification of grass phytoliths. Soil Sci. Sot. Am. Proc., 33: 109-l 15. Van Soest, P.J.; 1968. Structural and chemical characteristics which limit the nutritive value of forages. In: Forages: Economics/Quality. ASA Spec. Publ. 13. Am. Sot. Agron., Madison, WI, pp. 63-76. Wilkinson, S.R., Adams, W.E. and Jackson, W.A., 1970. Chemical composition and in viva digestibility of vertical layers of coastal bemmda grass ( (Cymdon dacfyh (L) Pers. ). Agron. J.. 62: 39-43.