Effect of Shredding Alfalfa Stems on Fiber Digestion Determined by In Vitro Procedures and Scanning Electron Microscopy 1

Alfalfa stems were shredded using a rotary macerator, were ammoniated, or were left untreated (control). Shredding increased extent of NDF digestion over ammoniat ion and control at all times from 12 to 72 h of in vitro incubation. Shredding also increased rate of disappearance of potential ly digestible NDF (.032 h 1 for control, .045 h 1 for ammoniated, and .089 h -1 for shredded). Ammoniat ion increased extent of NDF digestion after 36 h. Computed time required for 95% digestion of potential ly digestible NDF was 94 h for control, 66 h for ammoniated and 34 h for shredded stems. Scanning electron microscopy revealed that the most prominent effect of shredding was separation of lignified and unlignified cells. Shredding split stems into a number of fragments and damaged the external cuticular waxy layer and structural integrity. Ammoniat ion caused cracks in lignified vascular tissue. During in vitro incubations, shredded and ammoniated cell walls were heavily colonized by bacteria by 6 h. After 24 and 48 h, large Received June 30, 1987. Accepted December 16, 1987. ~Mention of commercial products in this paper does not constitute endorsement by the USDA o r the ARS. 2Present address: Department of Dairy Science, Kangweon National University, Chuncheon 200, K o r e a . 3present address: Department of Agronomy, Ohio State University, Columbus 43210. masses of bacteria were still at tached to the surface of pith and lignified vascular cell walls of shredded and ammoniated stems but not control stems. Lignified vascular tissue of control stems was not degraded and structural integrity was maintained. I N T R O D U C T I O N Forage cell walls are held together by matrix components containing hemicellulose and lignin. The ester and ether linkages between lignin and hemicellulose, and between hemicellulose are reinforced with hydrogen bonding, securing a tight structure of polymer chains (24). These chemical and structural properties differ in resistance to chemical and microbial attack. Alkali t reatment is the most common chemical method used to improve nutritive value of fibrous forages (17, 31, 32). Alkali is thought to dissociate the lignincarbohydrate complexes from the cell wall matrix, possibly by swelling (17, 28, 29) and hydrolyzing phenolic bonds with cell walls (14). Thus, alkali t reatment may overcome barriers to ruminal microbial at tachment and degradation of plant cell walls. Mechanical t reatment of forage also may hold promise for improving forage quality. A mechanical maceration ~reatment for use with freshly cut forage material was developed, which greatly enhances drying rate and reduces selective leaf loss of alfalfa (26). This t reatment resulted in stems being shredded into numerous fibrous pieces and in a severely damaged cuticle (25), yielding increased surface area and reduced resistance to moisture transfer from the stem (26). However, it was not determined how these changes affected feeding value of macerated forages. 1988 J Dairy Sci 71:1536-1545 1536 SHREDDING OF ALFALFA STEMS 1537 This study was conducted to evaluate effects of severe mechanical t reatment (shredding) of alfalfa stems on fiber digestion using in vitro techniques and scanning electron microscopy. Ammoniat ion of stems was included as a contrast control to compare the affect of shredding on fiber digestibility with that of ammoniation, since the positive effects of the latter are well-documented with grasses (3, 12, 34). M A T E R I A L S AND METHODS Third growth Vernal alfalfa (Medicago sativa) was hand-harvested at the late bud stage of maturity. Approximate ly 6 kg of stems were separated by hand from the leaves immediately after cutting. One-third of the harvested stems was set aside as a control; an equal amount was shredded mechanically through a stationary macerator designed by Shinners et al. (25). This process results in little or no loss of DM or soluble plant components. The remaining 2 kg of fresh stem was ammoniated at approximately 125 psi for 30 rain followed by sudden pressure release. All stems then were dried in a convection oven at 60°C for 72 h. Stems were ground with a Wiley mill using a 2-ram screen. In vitro digestibility of NDF was determined using the procedure of Goering and Van Soest (10) with the following modifications. Whole rumen contents were obtained from a ruminally cannulated, nonlacrating Jersey cow consuming an all alfalfa hay diet. Rumen fluid inoculum, enriched with particle-associated microorganisms was prepared by the method of Craig et al. (8). Approximate ly . S g of DM was added to each in vitro tube. Tubes were incubated at 39°C for 6, 12, 18, 24, 36, 48, and 72 h, treated with 1 ml saturated HgC12, then frozen for later analysis. Three incubations were run with triplicate blank and sample tubes for each time point within each incubation. Neutral detergent fiber, ADF, and acid detergent lignin (ADL) in alfalfa stems, as well as residual NDF in sample and blank in vitro tubes were determined according to the methods outlined by Goering and Van Soest (10). Nitrogen content of stems was determined by the Kjeldahl method (2) except that a copper catalyst (Kjeltabs, Tecator Inc., Herndon, VA) was used during digestion. Alfalfa stems for SEM study were harvested in a manner similar to those used in the in vitro incubations. Plants were of the same cultivar and cutting but from a different field. Sections were prepared for scanning electron microscopy (SEM) as follows. Intact stems from control, ammoniated, and shredded alfalfa were cut with a razor blade into lengths of 3 to 4 mm (for cross-sections) and 8 to 9 mm ( for longitudinal sections) from an internode at the midpoint of the stem. Both cross and longitudinal sections were prepared from each stem sampled. An equal number of both cross and longitudinal sections (40 sections per time point per treatment) of each sample were placed in large mesh, wire baskets (10 sections per basket) and incubated in tubes (four baskets per 100 ml in vitro tube) as described for 6, 24, and 48 h. Initial (0 h) samples, which were not exposed to rumen microorganisms, were fixed immediately for each treatment. After removal from incubations, wire baskets were rinsed in distilled water, and specimens were fixed for 24 h at room temperature in 2.5% (vol/vol) glutaraldehyde buffered at pH 7.2 with .1 M Sorenson's phosphate buffer (15). After fixation, samples were washed three times in the buffer without glutaraldehyde for 10 min each. Samples were then dehydrated in a series of ethanol washes (10 min each in 35, 50, 70, 85, and twice each in 95 and 100% vol/vol ethanol), then dried in a Samdri 780A critical point drier. Specimens were mounted on an aluminum holder with colloidal graphite adhesive, and coated with gold in a Hummer III sputter coater, before being observed and photographed using a JEOL 35-CF SEM (Jeol Ltd., Tokyo, Jpn) at 10 kV. Digestibility data were analyzed by least squares procedures as outlined by Steel and Torrie (30). In vitro NDF digestion data were analyzed using one-way ANOVA. Rates of NDF digestion were expressed on the basis of potentially digestible NDF as described by Smith et al. (27). Potentially digestible NDF was determined as the amount of NDF that had disappeared after 72 h of in vitro incubation. RESULTS A N D DISCUSSION