* Materials and methods

* Results

* Discussion

* Conclusion

* Awards

* Literature Cited

ABSTRACT: The DECREASE in the digestibility of forage is linked with an Increase in cell walls and lignification of Tissues As They mature. All forages Are Composed of a heterogeneous group of cell types with particular caracteristicas That determines the Availability of polysaccharides to the rumen S microorganisms. In the present paper the Degradation of Bromus auleticus Trin. ex Nees leaf WAS Tissues Studied at Different incubation times in the rumen. Were samples taken in two Phases phenological, vegetative and pre-flowering, Which Were Different submitted to in situ digestion times on two Argentine Holstein dry cows. Simultaneously, leaf blade Were sections stained with acid phloroglucinol stain to detect the Presence of lignin. Leaf anatomy Influenced the degree of tissue digestion, whether due to lignified cell walls or accessibility. After 24 h incubation in the rumen, only the xylem, sclerenchyma and undigested Were mestome sheath. The phloem and clorenchyma Were digested after 18 h in the pre-flowering phase and 24 h in the vegetative phase. The epidermis WAS Partially digested, and the abaxial epidermis WAS Better digested. The accessibility of carbohydrates in cell walls to the microflora of the rumen is limited by the chemistry of cell walls, and Malthus, highly Organized Tissues with lignified secondary walls s, such as sclerenchyma and xylem, Have a double barrier (physical and chemical) Which Prevent Their digestion.

Key words: leaf anatomy, lignin, xylem, sclerenchyma, digestibility.

SUMMARY: The decrease in digestibility of forage is associated with increased cell wall lignification of tissues as they mature. All forages are composed of a heterogeneous group of cell types with characteristics that determine the availability of polysaccharides to rumen microorganisms. In this study, the degradation of bromegrass leaf tissues Chaco (Bromus auleticus Trin. Ex Nees) to different ruminal incubation times. Samples were taken from leaf blades in two phenological stages: vegetative and pre-flowering, which were submitted at different times in situ digestion in two Argentine Holstein dry cows. Parallel leaf blade sections were stained with phloroglucinol to detect the presence of lignin. Leaf anatomy influenced the degree of tissue digestion, either by the lignified cell walls accessibility. After 24 h of ruminal incubation, but remained undigested xylem, sclerenchyma and mestomatica sheath. The phloem and chlorenchyma were digested after 18 h in the pre and 24 h in a vegetative state. The degradation of the epidermis was partial and abaxial was digested to a greater extent. The accessibility of cell wall carbohydrates by the rumen microflora is limited by the structural arrangement of each type of tissue, and the chemistry of cell walls, thus, highly ordered tissues and lignified secondary walls and sclerenchyma and xylem have a double physical and chemical barrier that prevents its digestion.

Keywords: leaf anatomy, lignin, xylem, sclerenchyma, digestibility.

INTRODUCTION: The ruminant feed obtained from the energy needed for metabolism by microbial fermentation of plant tissues (Akin, 1982). High milk yields or high daily liveweight gains require a large consumption of highly digestible forages and the cell walls. The structural organization of the tissues that make up the organs of the plants affect consumption through its effect on the rate of passage through the rumen, the size and nature of the particle produced, and also affects the dry matter digestibility ( MS) through the cell wall characteristics, which determine the availability of polysaccharides to rumen microorganisms (Wilson, 1993, Jung et al., 1996).

All forages are composed of a heterogeneous group of cell types, each of whom has a unique cell wall. In leaves, the cells are organized into specialized tissues that define three systems: the dermal, comprising the epidermis, an outer protective sheath that covers the body of the plant, the vascular, composed of the xylem as a driver of water and conductive phloem sap, and the fundamental, which includes the basic tissues of the plant with varying degrees of specialization as in the chlorenchyma and sclerenchyma. The latter, along the xylem is the main supporting tissue, thick walled and lignified (Esau, 1982). The decrease in the digestibility of forage is associated with increased cell wall and lignification of tissues as they mature (Chesson, 1993, Jung et al., 1996, Wilson and Hatfield, 1997). Very thin cell walls (0.1 to 0.2 mm), non-lignified primary and the chlorenchyma and phloem, not a problem for digestion (Wilson, 1993). Xylem and sclerenchyma, have secondary cell walls (1 to 3 mm thick), lignified, and constitute an obstacle to their degradation in the rumen (Wilson, 1997).

The proportion of leaf tissue, the thickness of cell walls and lignification can be modified by environmental factors and agricultural management. It was noted that the advancement of sowing dates (Masciangelo et al., 2002), nitrogen fertilization (Van Arendonk et al., 1997, Garnier et al., 1999), and adequate water supply (Buxton, 1996 ) have a positive relationship with the amount of woody tissues and digestibility of forages.

The Chaco bromegrass (Bromus auleticus Trin. Ex Nees) is a C3 species, perennial, native to central Argentina with potential to be used as winter forage (Zuloaga et al., 1994) for the quality and quantity of forage produced (Olmos, 1993).

The aim of this work was to study the degradation of bromegrass leaf tissues Chaco in different ruminal incubation times in two phenological stages: pre-flowering vegetative.

MATERIALS AND METHODS

Plants were grown from a population of B. auleticus Lehmann (Department Castellanos, Santa Fe Province, Argentina, 31 ^0 32 lat. South 61 ^0 32 long.), the trial was conducted in 1994 in the Botanical Garden of the Faculty of Agricultural Sciences de Esperanza, Universidad Nacional del Litoral (UNL), Department Las Colonias, Santa Fe, Argentina.

Samples were taken from leaf blades of B. auleticus in two stages: one in May, when the plant was in a vegetative state (vegetative) and the second in October on a rebound of 20 days of growth, being induced for flowering plants (before flowering).

Digestion in situ testing

Fresh Harvested forage was cut with scissors in fractions of 1.5 to 2.5 cm (Frecentese and Stritzler, 1985). Subsequently 5 g samples were placed in each bag of polyester of 10 x 20 cm, with pores of 50 +/- 15 mm to be incubated within the rumen of two Holstein cows fitted with cannulas Argentino dry.

The cows were fed alfalfa hay (Medicago sativa L.) with 17% crude protein (CP) and 60% in vitro DM digestibility (IVDMD). Two incubations were performed per animal for 4, 8, 16, 24 h at the vegetative stage, and 6, 12, 18 and 24 h pre-flowering state.

Electron microscopic observation

After incubation time of each sample, approximately 2 mm sections cut by hand, mounted on plates and placed in an oven at 65 ^0 C for 12 h. The samples were coated with a thin gold film using a laboratory evaporator (Veeco Instruments Inc., model VE-300, Plainview, Long Island, NY, USA), under argon for observation and micrographs obtained with a microscope scanning electron (JSM-35C Jeol, Tokyo, Japan) at 25 kV. Above them was to determine the degree of degradation of the tissues to the state of the cell walls. Four categories were established: D: highly digested, the tissue is absent, AD: advanced digestion, there are undigested remains of walls, P: partially digested, the walls show little digestion; I: undigested, the walls show no signs of digestion and are intact.

Histochemical analysis

The films show that incubated in the rumen, were kept refrigerated at 4 ^0 C, then cut cross-sections were prepared with a freezing microtome (Leitz 1310, Wetzlar, Germany), which underwent the test of hydrochloric acid fluoroglucine detect lignin (Jensen, 1962). The positive reaction to tests is manifested by a red or pink as the lignification process more or less intense, respectively. A negative reaction with phloroglucinol, remain colorless, usually mark those parts of the parenchyma, showing a wide degradability (Ohlde et al., 1992).RESULTS

The intensity of microbial degradation on each leaf tissue at different times of incubation in the rumen are shown in Table 1.

Table 1. Degree of digestion of leaf tissue in vivo in pre-flowering vegetative states of Bromus auleticus with different periods of rumen digestion.

Table 1: Degree of digestion in vivo of the Bromus auleticus Tissues in leaf and pre-flowering vegetative states with Different ruminal digestion times.

AD: advanced digestion, D: digested, I: undigested, P: partially digested, if: no information.

Vegetative state

In leaf blades with 4 and 8 h of ruminal incubation there was no significant digestion of plant tissues, both easily digestible (chlorenchyma and phloem), and poor or slowly digestible (xylem, sclerenchyma, mestomatica sheath, parenchyma sheath and epidermis).

After 16 h of ruminal incubation of samples at the vegetative stage, the disappearance of the mesophyll was advanced but not completed, and phloem began to be digested in some bundles. Parenchyma sheath of vascular bundles remained intact or slightly digested in the largest beams, while the children do their disappearance was more important. The adaxial epidermis appeared intact and in areas where that was absent was due to his detachment, while the abaxial had some degree of digestion in some cells being only external tangential walls without degrading others. In the marginal edge of the blade could be seen of the mesophyll (Figure 1 A and B).

Figure 1 .- Photomicrographs of scanning auleticus Bromus sheets: A and B, in a vegetative state under 16 h of ruminal digestion;

C, in a state of pre-flowering under 12 h of rumen digestion, D, state pre-flowering under 24 h of ruminal digestion.

Figure 1 .- Scanning microphotographs of Bromus auleticus blades: A and B, vegetative stage submitted to 16 h of ruminal digestion;

C, pre-flowering stage submitted to 12 h of ruminal digestion; D, pre-flowering phase submitted to 24 h of ruminal digestion.

AB, abaxial epidermis; AD, adaxial epidermis; E, sclerenchyma F, phloem; M, mesophyll; VM, pod mestomatica, VP, parenchyma sheath and X, xylem. The scale is 100 m m.

After 24 h incubation in the rumen, the only tissues that remained undigested were the xylem, sclerenchyma and mestomatica sheath.

State preflowering

After 6 h incubation in the rumen of the blades (Table 1), mesophyll cell walls had collapsed. The vascular bundles mestomatica sheath and sclerenchyma caps remained intact because of their walls lignified, while the sheath was found collapsed parenchyma. Further, in certain regions disappeared mesophyll, especially in areas close to the epidermis and the beam higher. The phloem was digested in some vascular bundles.

After 12 h of digestion, the mesophyll and phloem of the samples pre-flowering state completely disappeared. In the epidermis, the inner tangential walls of the larger cells had disappeared. Parenchyma sheath beam was partially digested, leaving as remnants of the inner tangential walls, possibly due to the high degree of lignification of the middle lamella that binds these cells with mestomatica sheath remained intact (Figure 1 C). Stomata did not suffer degradation.

The adaxial epidermis tissue was completely removed before flowering after 18 h of incubation, together with sclerenchyma caps associated with it. This was due to some type parenchyma cells, interposed between the cap and sheath adaxial mestomatica, to be digested allowed the separation of adaxial sclerenchyma caps and associated epidermis. Some parenchyma sheath cells remained attached to the mestomatica in the area adjacent to the sclerenchyma caps that are loose. In some regions were absent from the inner tangential walls of the epidermis.

Only xylem tissues remained surrounded by its sheath mestomatica and abaxial sclerenchyma cap after 24 h of digestion in situ. These units of fabric held together by the outer tangential walls of the epidermis (Figure 1 D). Intact epidermal spines were observed.

Regarding the test with phloroglucinol (Table 2), the xylem showed a strong positive reaction in both cases, the sheath mestomatica gave strong positive reactions in the pre and lowest in the vegetative state, which was deducted a progressive lignification of it. Sclerenchyma in both samples showed a reaction intermediate between the xylem and other tissues. In addition, there was a group of colorless parenchyma cells, corresponding to the parenchyma sheath were not stained with phloroglucinol, which are among the vascular bundle sheath mestomatica and the cap on the adaxial sclerenchyma.

Table 2. Phloroglucinol reaction to test for the detection of lignin in different tissues and pre-flowering vegetative leaf fresh state.

Table 2: Reaction to the phloroglucinol test to detect lignin in Different Leaf Tissues at the pre-flowering and vegetative stages.

*: Indicates a corresponding color or read.

V: vegetative; PF: pre-flowering.

DISCUSSION

The composition and molecular organization of cell walls is the major determinant in the rate and extent of degradation of cell walls isolated cells, however, that behavior is not observed in vivo (Chesson, 1993), because cells members of tissue and their structure may have more influence on its degradation in the rumen that the chemical composition of their own walls (Chesson, 1993, Wilson and Hatfield, 1997).

Sleper and Roughan (1984), after 3 h of incubation of leaf blade of Phleum pratense L., found that the mesophyll, the epidemis and phloem were highly degraded, leaving only the cuticle, sclerenchyma and xylem, whereas Chepica (Agrostis capillaris L.) almost no digestion evident after 6 h of incubation, despite being both C3. The highest rate of disappearance of P. La Plata was related to the higher proportion of intercellular spaces.

With respect to the rumen degradation of leaf tissue and pre-flowering vegetative state, B. auleticus followed the characteristic pattern of grasses: mesophyll and phloem (rapidly degraded)> epidermis and parenchyma bundle sheath (slowly degraded)> sclerenchyma (non-degraded slowly)> cuticle and lignified vascular tissue (not degraded) (Akin and Burdick, 1975; Magai et al., 1994).

Certain features of leaf anatomy influenced the degree of tissue digestion, either by their lignification or accessibility. As noted above, ribs remaining between the grooves of the sheet are closely related to the vascular bundles, and have similar size independent of the order containing the vascular bundle, while no development of ribs and grooves on the abaxial side . This caused the differential digestion of the epidermis, the abaxial being rapidly digested, probably due to a lower degree of cutinisation and lignification.

All the vascular bundles of prairie grass have double sheath, a continuous mestomatica and parenchyma, which is disrupted in the abaxial vascular bundles in the first and second order, which are extensions of the bundle sheath that communicate the abaxial sclerenchyma with the bundle sheath. These features parenchyma cells, and according to the test with phloroglucinol were not lignified, to be digested allowed the separation of adaxial sclerenchyma caps and associated epidermis. This allowed access to the parenchyma of the bundle sheath and parenchyma of the protoxylem, the metaxylem vessels being undigested.

Mesophyll, phloem, parenchyma sheath and the epidermis was digested faster in the samples in a vegetative state before flowering. Probably this behavior is due to the sample sheet at flowering up to 20 days of renewed growth. It was noted that the xylem, sclerenchyma and bundle sheath always remained mestomatica of indigestible, coinciding with the determinations of lignin with phloroglucinol. Grenet and Jamot (1989) found in ryegrass (Lolium multiflorum Lam.) That the sclerenchyma of the plates, which gave positive reaction with phloroglucinol, remained undigested after 8 h of incubation, together with the xylem and cuticle remained intact.

Lignified secondary wall in grasses is not completely indigestible, can be digested when it facilitates the access of rumen microorganisms to them. The poor digestibility observed is due to the limits imposed by the structure. The group formed by the middle lamella (LM) and the primary wall (PP) is the starting site of lignin deposition, which is indigestible (Wilson, 1993), and is one of the major constraints in the digestion of the thick lignified secondary walls. In turn, the LM cements adjacent cells, therefore the particles form large multicellular fibers, many of which have not broken ends, so they are completely indigestible.

Carbohibratos accessibility to cell walls by the rumen microflora is limited by the structural arrangement of each tissue and chemistry of cell walls (Wilson, 1993) thus highly ordered tissues and lignified secondary walls as sclerenchyma and the xylem, have a double physical and chemical barrier that prevents its digestion (Wilson and Hatfield, 1997). This seems to occur in woody tissues of Chaco bromegrass. According to histochemical tests, sclerenchyma in the pre-flowering vegetative state and is less lignified than the xylem and the sheath mestomatica, however after 24 h of digestion in situ remained undigested like them.

CONCLUSION

In B. auleticus, the secondary walls of xylem and lignified sheath mestomatica is more than the sclerenchyma, both in pre-flowering vegetative state. Despite this differential lignification, these tissues remained intact after 24 h of digestion.

ACKNOWLEDGMENTS

We thank M. Agronomist Sci Miriam Gallardo, the National Institute of Agricultural Technology, Agricultural Experimental Station Rafaela, which enabled the completion of the digestibility trials.

LITERATURE CITED

Akin, d.”A. 1982. Section to slide technique for study of anatomy and forage digestion. Crop Sci 22:444-446.

Akin, d.”A., and D. Burdick. 1975. Percentage of Tissues types in tropical and temperate grass leaf blades and degration of Tissues by rumen microorganims. Crop Sci 15:661-668.

Buxton, D.R. 1996. Quality-related Characteristics of forages as Influenced by plant environment and Agronomic factors. Anim. Feed Sci Technol. 59:37-49.

Chesson, A. 1993. Mechanistic models of forage cell wall Degradation. p. 347-375. In H.G. Jung et al. (Eds.). Forage cell wall structure and digestibility. ASA, CSSA, SSSA, Madison, Wisconsin, USA.

Esau, K. 1982. Anatomy of Seed Plants. 512 p. Editorial Southern Hemisphere, Buenos Aires, Argentina.

Frecentese, M.A., and N.P. Stritzler. 1985. Differential attack the bovine rumen flora on the leaf tissues of summer grasses. Rev Arg Prod Anim. (Argentina) 5:531-540

Garnier, E., J.L. Salager, G. Laurent, and L. Sonia. 1999. Relationships Between photosynthesis, nitrogen and leaf structure in 14 grass species and Their dependence on the basis of expression. New Phytol. 143:119-129.

Grenet, E., and J. Jamot. 1989. Kinetic study of the rumen microbial Degradation of lucerne and Italian ryegrass Observed by scanning electron microscopy. In R. Jarrige (ed) XVI International Grassland Congress. October 4-11. French Grassland Society, Nice, France.

Jensen, W.A. 1962. Botanical histochemistry. Principles and practice. 408 p. W.H. Freeman & Company, San Francisco, California, USA.

Jung, H., D.R. Buxton, R.D. Hatfield, D.R. Mertens, J. Ralph, and P.J. Weimer. 1996. Genetic manipulation of cell wall. Informational conference with dairy and forage industries. U.S. Dairy Research Center. Available in: http://www.dfrc.wisc.edu/Research_Summaries/ind_meet/dfrc2.pdf. Accessed 17 December 2002.

Magai, M.M., d.”A. Sleper, and P.R. Beuselinck. 1994. Degradation of three warm season grasses Prepared in a cellulase solution. Agron. J. 86:1049-1053.

Masciangelo, P., J.C. Tivano and J.C. Ramos. 2002. Influence of planting date on the quantitative anatomy of a leaf silage corn hybrid. VII Conference of Young Researchers. October 2002. Universidad Nacional del Litoral, Santa Fe, Argentina.

Ohlde, G.W., S.D. Akin, K. Becker, L.L. Kigsby, and C.E. Lyon. 1992. Diference in rumen bacterial Degradation of morfological Fractions in eight cereal straws and the effect of digestion on Different types of Tissues and mechanical properties of straw stalks. Anim. Feed Sci Technol. 36:173-186.

Olmos, F. 1993. Bromus auleticus. 30 p. Technical Series No. 35. National Agricultural Research Institute, Montevideo, Uruguay.

Sleper, d.”A., and P.G. Roughan. 1984. Histology of Several cool-season forage grasses digested by cellulase. N.Z. J. Agric. Res 27:161-79.

Van Arendonk, J.J., G.J. Nieman, J.J. Boon, and H. Lambers. 1997. Effects of nitrogen supply on the anatomy and chemical composition of leaves of four grass species Belonging to the genus Poa, as Determined by image-processing analysis and pyrolysis-mass spectrometry. Plant Cell Environ. 20:881-897.

Wilson, J.R. 1993. Organisation of forage plant tissues. p. 1-32. In H.G. Jung et al. (Eds.). Forage cell wall structure and digestibility. ASA, CSSA, SSSA, Madison, Wisconsin, USA.

Wilson, J.R. 1997. Structural and anatomical traits of forages Influencing Their nutritive value for ruminants. p. 173-208. In J.A. Gomide (ed.) Anais do International Symposium on Pasteje em produc~ao animal. Animal Husbandry Department. Federal University of Vicosa. 4-6 november 1997. Brazil.

Wilson, J.R., and R.D. Hatfield. 1997. Structural and chemical changes file DURING types of stem cell wall development: Consequence for Degradation fiber rumen microflora. Aust. J. Agric. Res 48:165-180.

Zuloaga, F.O., G.S. Nicora, Z.E. Rugolo, O. Morrone, J. Pensiero, and A.M. Cialdella. 1994. Catalogue of the family Poaceae in Argentina. 178 p. Missouri Botanical Garden, St. Louis, Missouri, USA.

Milagros Gasser1, July Ramos1, Abelardo Vegetti1 and Juan Carlos Tivano1

1 Universidad Nacional del Litoral, Faculty of Agricultural Sciences. Kreder 2805, CP 3080 Esperanza, Santa Fe, Argentina.