Rumen Protozoal Degradation of Structurally Intact Forage Tissues

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1978, p /78/ $02.00/0 Copyright 1978 American Society for Microbiology Vol. 36, No. 3 Printed in U.S.A. Rumen Protozoal Degradation of Structurally Intact Forage Tissues HENRY E. AMOS* AND DANNY E. AKIN Field Crops Utilization and Marketing Research Laboratory, Science and Education Administration- Federal Research, United States Department ofagriculture, Athens, Georgia Received for publication 10 July 1978 The association with and digestion of intact leaf sections of cool- and warmseason grasses by cattle rumen protozoa were investigated by light and scanning electron microscopy and by in vitro dry matter disappearance studies. Within extensively degraded areas of mesophyll tissue in cool-season forages, almost all protozoa were Epidinium ecaudatum form caudatum, with maximum numbers at 4 to 10 h of incubation. However, few protozoa were found inside warm-season forage leaves. In in vitro dry matter disappearance studies of a series of incubations with and without 1.6 mg of streptomycin per ml, which inhibited the cellulolytic activity of the bacteria, and in comparison with uninoculated controls, rumen protozoa degraded 11.0 and 3.7 percentage units of orchardgrass and bermudagrass, respectively. Scanning electron microscopy showed that the tissues degraded in orchardgrass consisted of large amounts of mesophyll and portions of the parenchyma bundle sheath and epidermis; no tissue loss due to the protozoa was observed in bermudagrass. The relationship of these observations to forage digestion is discussed. Bacteria and protozoa of the rumen enable associated with alfalfa stems during digestion. ruminants to utilize fibrous feedstuffs which cannot be utilized by nonruminants. The rumen and degradation of intact tissues of various for- However, information on the association with bacteria have received the greatest study by ages by rumen protozoa is scarce. microbiologists and nutritionists, but little information is available on the digestion of fiber by to evaluate the contribution of rumen protozoa The objectives of the present work were: (i) protozoa. It has been shown that neither the to the degradation of intact leaf tissue of various holotrichs nor the entodiniomorphs are required grasses and (ii) to identify the specific grass for normal fermentation within the rumen (9, tissues removed by the protozoa during fermentation, using gravimetric analyses and SEM. 10, 18). In fact, in the absence of protozoa, rumen bacterial numbers increase and maintain cellulose and fiber digestion (9, 10). However, fermentation benefits have been shown in vitro and Microbial inoculum. Ingesta removed from the MATERIALS AND METHODS in vivo when both bacteria and protozoa exist in rumen of a cannulated steer maintained on a mixed the system. Yoder et al. (18) reported a digestion grass pasture were squeezed through four layers of coefficient of 60% for cellulose inoculated with cheesecloth into a preheated (39 C) vacuum bottle. The strained rumen fluid was used in the preparation rumen bacteria and protozoa but only 40 and 7% of two inocula: inoculum 1, (whole rumen fluid) prepared by diluting the strained rumen fluid 1:1 with with individual washed suspensions of bacteria and protozoa, respectively. Other research (8, 9) preheated (39 C) McDougall buffer (13) saturated has shown increased animal performance and with carbon dioxide, and inoculum 2 (washed rumen increased production of total volatile fatty acids bacteria), prepared as described (3). in faunated versus defaunated lambs. Klopfenstein et al. (12) reported that faunated lambs fed managed warm- and cool-season grasses that were Substrate. Leaf sections were prepared from well- high-energy diets digested more dry matter and harvested at approximately 28 days of age, frozen in nitrogen than did defaunated controls; no differences were found with low-energy diets. dry ice, and stored at -30 C until used. Cool-season species included Kentucky-31 (Ky-31) tall fescue (Festuca arundinacea Schreb.), Boone orchardgrass (Dactylis glomerata L.), and Clair timothy (Phleum pra- Recently, Bauchop and Clarke (5), using scanning electron microscopy (SEM), reported that tense L.), and the warm-season grasses were coastal large numbers of the rumen protozoan Epidinium ecaudatum form caudatum were closely cola bahiagrass (Paspalum notatum, Flugge), bermudagrass (Cynodon dactylon [L.] Pers.), Pensa- 513

2 514 AMOS AND AKIN and Pangola digitgrass (Digitaria decumbens Stent). Leaf sections were prepared from the middle portion of the blade. In vitro digestion studies. Representative warmand cool-season grasses were evaluated for the association with and degradation of structurally intact tissues by the rumen protozoa. Experiment 1. Leaf blade sections of each warmand cool-season grass were incubated in 300 ml of inoculum at 39 C in a 500-ml Erlenmeyer flask. Carbon dioxide was bubbled through the incubation medium throughout the 22-h fermentation. Three leaf sections of each grass were removed at 4, 10, and 22 h and examined without washing by light microscopy (LM) for the presence of protozoal types within degraded areas of the leaf section. All protozoa within degraded areas of each section were counted. The presence of protozoa within degraded areas at each sampling time was confirmed by SEM from leaf sections prepared as previously described (1). Experiment 2. To specifically define the contribution of rumen protozoa to intact leaf tissue degradation, orchardgrass and coastal bermudagrass were used as substrates in a series of incubations at 39 C. Intact sections of leaf blades about 1 cm long were prepared for in vitro dry matter disappearance (IVDMD), and sections 2 to 3 mm long were prepared for evaluation by LM and SEM. The leaf sections were incubated with one of the following inocula: (i) inoculum 1; (ii) inoculum 1 plus 1.6 mg of streptomycin per ml; (iii) inoculum 2; (iv) inoculum 2 plus 1.6 mg of streptomycin per ml; (v) the buffer of Cheng et al. (7) as a control; and (vi) autoclaved rumen fluid as a control. Incubations were conducted in 50-ml centrifuge tubes containing 30 ml of each inoculum, closed with rubber stoppers which had been fitted with Bunsen valves. For IVDMD, incubations were conducted for 48 h in triplicate with 0.5 g of dry matter per tube and replicated once to give six observations per grass. Single tube incubations were used for evaluation by LM and SEM of bacterial and protozoal degradation of grass tissue. Six leaf sections were removed after 4, 10, and 24 h of incubation and examined by LM, and all protozoa within degraded areas were counted. These leaf sections were also prepared for SEM observation (1). RESULTS Although the rumen protozoal types were variable during sampling of digesta, often a relatively high proportion of a spined entodiniomorph was present in the inocula (Fig. 1). These APPL. ENVIRON. MICROBIOL. protozoa were identical to the E. ecaudatum form caudatum described previously (5, 11). In experiment 1, these protozoa, to the exclusion of most other types, preferentially associated with the cool-season grasses but did not associate with the warm-season grasses to any extent (Table 1, Fig. 2-7). After incubation of the coolseason grass, large numbers of E. ecaudatum form caudatum were found near to and inside the digested zones early in the fermentation period (i.e., 2 to 5 h); these protozoa had ingested chlorophylous material (Fig. 2 and 3, arrows). The number of entodiniomorphs, primarily comprised of E. ecaudatum form caudatum, that associated with and apparently degraded intact tissues of the cool-season grasses was maximum at 4 to 10 h and declined thereafter (Table 1). In some leaf sections of orchardgrass, the spined protozoa were so closely packed within the digested areas that an accurate count was impossible. The protozoa were not only associated in large numbers with leaf blades of the cool-season grasses but also preferentially attacked and degraded the mesophyll tissue (Fig. 4, 5) during early digestion. Portions of the epidermis and TABLE 1. Numbers ofprotozoa found within degraded zones of mesophyll tissue of cool- and warm-season grasses (experiment l)' Fermentation time (h) Grass type Cool season Orchardgrass TNCb Ky-31 fescue Clair timothy Warm season Coastal bermudagrass Pensacola bahiagrass Pangola digitgrass a Each value is the mean of three leaf sections for each grass at each sampling time examined using light microscopy. b TNC, E. ecaudatum form caudatum were so tightly packed within the mesophyll that an accurate count was impossible. FIG. 1. LM of holotrich and entodiniomorph protozoa in rumen fluid. The holotrichs resemble Isotricha and the entodiniomorphs are identical to E. ecaudatum form caudatum. x200. FIG. 2. LM of E. ecaudatum form caudatum present in large numbers in a degraded mesophyll area of a Ky-31 fescue leaf blade. Some protozoa contain ingested plant material (arrows). x 169. FIG. 3. LM of a Ky-31 fescue leaf blade with a large number of E. ecaudatum form caudatum inside and outside the leaf blade; many of the protozoa contain ingested plant material (arrows). x68. FIG. 4. SEM of a Ky-31 fescue leaf blade digested in vitro with rumen fluid for 2 h, showing large numbers of E. ecaudatum form caudatum within degraded mesophyll tissue. The parenchyma bundle sheaths (arrows) have not been degraded. X320.

3 VOL. 36, 1978 FORAGE TISSUE DIGESTION BY RUMEN PROTOZOA 515 Il 11 11I Downloaded from on March 9, 2019 by guest

4 516 AMOS AND AKIN APPL. ENVIRON. MICROBIOL. -*PIMA_ FIG. 5. SEM of orchardgrass leaf blades digested in vitro with rumen fluid for 5 h, showing preferential attack of the protozoa on the mesophyll cells (arrows). The phloem and mesophyll are intact where the protozoa have not attacked. The inset shows a mesophyll area totally filled with E. ecaudatum form caudatum. x320; inset, x512. FIG. 6. SEM of a bermudagrass leaf blade digested in vitro with rumen fluid for 5 h. Protozoa are rarely found within blades, but when present are found in the mesophyll tissue (arrow). x512.

5 VOL. 36, 1978 FORAGE TISSUE DIGESTION BY RUMEN PROTOZOA 517 Downloaded from a. on March 9, 2019 by guest FIG. 7. SEM of a bahiagrass leaf blade digested mn vitro with rumen fluid for 5 h. Few protozoa are present in the blade, but when present are found in degraded areas of the mesophyll. Two protozoa (arrows) are wedged within partially degraded tissues. x512. FIG. 8. LM of the association with and degradation of orchardgrass leaf blades digested in vitro with rumen fluid containing 1.6 mg ofstreptomycin per ml. (a) At 4 h, protozoa are degrading the edge ofthe blade. x200. (b) At 10 h, protozoa are within degraded zones of the blade. x200. (c) At 24 h, protozoa have degraded the mesophyll to a large extent in certain areas and are present deep within two blades (arrows). x80.

6 518 AMOS AND AKIN parenchyma bundle sheath were degraded during late digestion (24 to 48 h). Few protozoa were found within degraded areas of the warm-season grasses (Table 1), and these few protozoa were also found in degraded mesophyll areas (Fig. 6, 7). Often the protozoa in the incubation tubes containing warm-season grass blades were nonmotile and lacked ingested plant materials, indicating their inability to derive nutrients from these forages. In experiment 2, inoculum 1 (whole rumen fluid) had a high proportion of the spined protozoa as evaluated by LM. However, inoculum 2 (washed rumen bacteria) lacked the large entodiniomorphs. In inoculum 1, rumen protozoa again associated in large numbers with orchardgrass and reached a maximum at 10 h. With streptomycin, large numbers were found within extensively degraded regions at 24 h (Table 2, Fig. 8). These protozoa seldom associated with warm-season species (Table 2). The IVDMD of orchardgrass and bermudagrass incubated for 48 h is shown in Table 3 and APPL. ENVIRON. MICROBIOL. in Fig. 9 to 17. Orchardgrass was more digestible than the bermudagrass in inoculum 1 and in inoculum 2 when streptomycin was omitted (Table 3, Fig. 9, 11, 13, 15). Although in one study IVDMD was unusually high for bermudagrass TABLE 2. Numbers ofprotozoa within degraded zones of mesophyll tissue of orchardgrass and coastal bermudagrass (experiment 2) a Coastal bermudagrass at fermen- Orchardgrass at fer- Treatment mentation time (h): tation time (h): Inoculum 1b Inoculum 1 plus streptomycin' a Each value is the mean of six leaf sections for each grass at each sampling time examined using light microscopy. ' Strained rumen fluid diluted 1:1 with McDougall buffer. ' Streptomycin (1.6 mg/ml) was added to the inoculum at 0 time. TABLE 3. Percentage IVDMD from intact leaf sections of orchardgrass and coastal bermudagrass after 48 h of incubation (experiment 2)a Orchardgrass Coastal bermudagrass Treatment 1 2 Mean 1 2 Mean Inoculum lb 57.5 ± ± ± ± Inoculum 1 + strepto ± ± ± ± mycinm Autoclaved inoculum ± ± Inoculum 2d 42.7 ± ± ± ± Inoculum 2 + strepto ± ± ± mycinm Cheng buffer 16.8 ± ± ± a Each value is the mean of three observations for replicates 1 and 2. b Strained rumen fluid diluted 1:1 with McDougall buffer. cstreptomycin (1.6 mg/ml) was added to the inoculum at 0 time. d Washed rumen bacteria. FIG. 9. SEM of an orchardgrass leaf blade digested in vitro with rumen fluid for 48 h. Only lignified vascular bundles (L), sclerenchyma (S), and cuticle (C) remain. x320. FIG. 10. SEM of an orchardgrass leaf blade digested in vitro with rumen fluid containing 1.6 mg of streptomycin per ml for 48 h. Mesophyll cells (M) and portions of the epidermis (arrows) have been removed, indicating the action of the protozoa in degrading the fragile tissue. x320. FIG. 11. SEM of an orchardgrass leaf blade digested in vitro with washed rumen bacteria for 48 h. Lignified vascular bundles (L), sclerenchyma (S), and cuticle (C) remain. x320. FIG. 12. SEM of an orchardgrass leaf blade digested in vitro with washed rumen bacteria containing 1.6 mg of streptomycin per asl for 48 h. No tissues are lost, showing the inhibition of bacterial digestion by the antibiotic. X320. FIG. 13. SEM of a bermudagrass leaf blade digested in vitro with rumen fluid for 48 h. Phloem and mesophyu cells (arrows) are removed, parenchyma bundle sheath (P) and epidermis (E) are partially degraded, and lignified tissues (L) are intact. x320. FIG. 14. SEM of a bermudagrass leaf blade digested in vitro with rumen fluid containing 1.6 mg of streptomycin per ml for 48 h. No tissues are removed, indicating the lack of attack and tissue removal of bermudagrass by the protozoa. X320.

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8 520 AMOS AND AKIN APPL. ENVIRON. MICROBIOL. Downloaded from FIG. 15. SEM of a bermudagrass leaf blade digested in vitro with washed rumen bacteria for 48 h. Phloem and mesophyll are removed (arrows), parenchyma bundle sheath (P) and epidermis (E) are partially degraded, and lignified tissues (L) are intact. x320. FIG. 16. SEM of bermudagrass leaf blade digested in vitro with washed rumen bacteria containing 1.6 mg of streptomycin per ml for 48 h. No tissues are removed, indicating the inhibition of bacterial digestion by the antibiotic. x320. FIG. 17. SEM of an orchardgrass leaf blade incubated in Cheng buffer for 48 h. No tissues are removed, but the fragile mesophyll (M) and some parenchyma bundle sheath cells (drrows) have lost structural rigidity. x320. in inoculum 1 with streptomycin, the micrographs indicated that extensive digestion of the mesophyll cells occurred in orchardgrass but not in bermudagrass (Fig. 10, 14), again indicating the preferential attack of orchardgrass by the rumen protozoa. Dry-matter loss in either forage incubated with inoculum 2 plus streptomycin was essentially the same as that of forage incubated in control buffer (cf. Fig. 11, 12, 16, and 17 and Table 3). That bacterial degradation did not occur in inocula with streptomycin is further shown by the fact that phloem, one of the most digestible tissues (2, 3), remained undegraded after 48 h (Fig. 10, 12, 14, 16). DISCUSSION Entodiniomorphs, primarily E. ecaudatum form caudatum, preferentially associated with and degraded mesophyll tissue of both cool- and warm-season grasses. The number of the epidi- on March 9, 2019 by guest

9 VOL. 36, 1978 nia found within degraded mesophyll areas was much higher with the cool-season grasses than with warm-season grasses. Several types of incubation were conducted in our laboratory in an attempt to determine the contribution of E. ecaudatum form caudatum to the degradation of intact grass leaf blades (Akin and Amos, unpublished data). The CuS04 method used by Slyter and Wolin (16) to defaunate continuous in vitro rumen fermenters was used initially; however, this level of CuS04 (0.1 mg/ml of inoculum) not only appeared to kill the protozoa but also markedly inhibited the attachment to and digestion of the grass tissue by the bacteria. The antibiotic chloramphenicol, at a concentration of 1,ug/ml of inoculum, was used in an attempt to inhibit bacterial growth. However, this level of antibiotic (one-tenth to one-twentieth the concentration normally used to inhibit bacterial growth and protein synthesis [14]), was much more effective against the protozoa than bacteria and resulted in a complete immobility of the protozoal population. In two unpublished fermentation studies, rumen protozoa were sedimented from the rumen fluid and washed as reported by Yoder et al. (18) and used to "enrich" the whole rumen fluid inoculum with protozoa; IVDMD of both orchardgrass and bermudagrass was slightly higher with the added protozoa. However, examination by electron microscopy of this protozoal preparation incubated in McDougall buffer with leaf sections showed numerous rumen bacteria attached to the protozoa and also bacteria degrading the plant cell walls. The increase in IVDMD may have been the result of increased bacterial numbers. Streptomycin (1.6 mg/ml) added to inoculum 2 was found to virtually eliminate the bacterial digestion of forage tissues (see Table 3 and Fig. 12 versus Fig. 17) and was used in experiment 2 to determine the contribution of the E. ecaudatum form caudatum to forage tissue degradation. These results indicated that the protozoa degrade intact leaf blades in the absence of bacteria. This concentration of streptomycin was approximately three times the level used by Sugden (17) but was totally effective in inhibiting the bacterial cellulolytic activity and did not inhibit the protozoa according to motility in these studies. The protozoa removed (i.e., digested) 11.0 and 3.7 percentage units more forage tissue than the average value of dry matter loss due to washout for autoclaved rumen fluid, washed rumen bacteria + streptomycin, and Cheng buffer for orchardgrass and bermudagrass, respectively (Table 3). SEM and LM indicated a clearer distinction between the attack FORAGE TISSUE DIGESTION BY RUMEN PROTOZOA 521 of orchardgrass and bermudagrass in that few protozoa attacked the warm-season grass. The reasons for the association of epidinia with the cool-season grass are not clear and may be related to a number of factors inherent to the two types of grasses. One factor may be the amount of easily degradable tissue between vascular bundles which can be attacked by the protozoa. Akin and Burdick (4) reported that the mesophyll comprises more than 50% of the cross-sectional leaf area of the cool-season grasses and only about 25% in most of the warmseason grasses examined. Another factor may be the arrangement of mesophyll tissue, which differs considerably between the two types of grasses. The cool-season grasses are characterized by loose, irregularly arranged mesophyll cells containing a relatively large amount of air space between cells in comparison to the vascular bundles; the warm-season grasses are characterized by more densely packed mesophyll cells, which often are oriented radially around the vascular bundles (6, 15). The mesophyll of the cool-season grasses is one of the first tissues degraded by the rumen bacteria and disappears much earlier in the cool-season grasses than in warm-season grasses when incubated with the rumen bacteria (3), indicating a more inherent rigidity of mesophyll in warm-season species. The amount, arrangement, and rigidity of the mesophyll cells may influence the ability of the protozoa to invade the mesophyll tissue as well as the availability of nutrients contained therein. Starch grains are located in the mesophyll and parenchyma bundle sheath of cool-season grasses, whereas warm-season grasses have starch stored predominantly in the parenchyma sheath (6), which is a more rigid, slowly degraded tissue (4). The difference in availability of starch may influence the attack of particular grasses by protozoa, since the entodiniomorphs (including E. ecaudatum form caudatum) "avidly ingest starch," which probably serves as their chief source of carbohydrate (11). LITERATURE CITED 1. Akin, D. E Ultrastructure of rumen bacterial attachment to forage cell walls. Appl. Environ. Microbiol. 31: Akin, D. E., and H. E. Amos Rumen bacterial degradation of forage cell walls investigated by electron microscopy. Appl. Microbiol. 29: Akin, D. E., H. E. Amos, F. E. Barton II, and D. Burdick Rumen microbial degradation of grass tissue revealed by scanning electron microscopy. Agron. J. 66: Akin, D. E., and D. Burdick Percentage of tissue types in tropical and temperate grass leaf blades and degradation of tissues by rumen microorganisms. Crop Sci. 15: Bauchop, T., and R. T. J. Clarke Attachment of

10 522 AMOS AND AKIN the ciliate Epidinium Crawley to plant fragments in the sheep rumen. Appl. Environ. Microbiol. 32: Brown, V Leaf anatomy in grass systematics. Bot. Gaz. 119: Cheng, E. W., G. Hall, and W. Burroughs A method for the study of cellulose digestion by washed suspensions of rumen microorganisms. J. Dairy Sci. 7: Christiansen, W. M. C., R. Kawashima, and W. Burroughs Influence of protozoa upon rumen acid production and live weight gains in lambs. J. Anim. Sci. 24: Eadie, J. M., and J. C. Gill The effect of the absence of rumen ciliate protozoa on growing lambs fed on a roughage-concentrate diet. Br. J. Nutr. 26: Eadie, J. M., and P. N. Hobson Effect of the presence or absence of rumen ciliate protozoa on the total rumen bacterial count in lambs. Nature (London) 193: Hungate, R. E The rumen and its microbes. Academic Press Inc., New York. APPL. ENVIRON. MICROBIOL. 12. Klopfenstein, T. J., D. B. Purser, and W. J. Tyznik Effect of defaunation on feed digestibility, rumen metabolism and blood metabolites. J. Anim. Sci. 25: McDougall, E. I Studies on ruminant saliva. I. The composition and output of sheep's saliva. Biochem. J. 43: Mahler, H. R., and E. H. Cordes Biological chemistry. Harper and Row, New York. 15. Metcalfe, C. R Anatomy of the monocotyledons. I. Graminae. Oxford Press, London. 16. Slyter, L. L., and M. J. Wolin Copper sulfateinduced fermentation changes in continuous cultures of the rumen microbial ecosystem. Appl. Microbiol. 15: Sugden, B The cultivation and metabolism of oligiotrich protozoa from the sheep's rumen. J. Gen. Microbiol. 9: Yoder, R. D., A. Trenkle, and W. Burroughs Influence of rumen protozoa and bacteria upon cellulose digestion in vitro. J. Anim. Sci. 25: Downloaded from on March 9, 2019 by guest