c07-08-0461 Grabber.indd

Conditioning and conservation methods may interact with polyphenols to alter forage crude protein (CP) solubility and degradability. In this study, forages with ~200 g CP kg−1 dry matter were roll conditioned or macerated, conserved as hay or silage, and then analyzed for CP fractions. Shifting from roll conditioning to maceration of polyphenol-free alfalfa (Medicago sativa L.) reduced buffer soluble protein (SP) with little effect on protease rumen-undegradable protein (RUP) and intestinal available protein (IAP, RUP minus acid-detergent insoluble CP). In birdsfoot trefoil (Lotus corniculatus L.), condensed tannin (CT) and maceration independently reduced SP and increased RUP to yield up to 62% more IAP in hay and 145% more IAP in silage than alfalfa. Based on results with trefoil, roll-conditioned forage with 70 to 120 g CT kg−1 CP or macerated forage with more modest CT levels should meet a 350 g RUP kg−1 CP target to support 35 kg d−1 milk yield by cattle. In roll-conditioned red clover (Trifolium pratense L.), o-quinones formed by polyphenol oxidase reduced SP and increased RUP to yield 50% more IAP in hay and 88% more IAP in silage than alfalfa. Surprisingly, maceration reduced SP, RUP, and IAP in clover. Following maceration, RUP and IAP in conserved clover and trefoil responded similarly to CT, suggesting that maceration disabled o-quinone protection of protein substrates. Although red clover has high RUP, low reported milk yields indicate o-quinones might depress IAP. U.S. Dairy Forage Research Center, USDA-ARS, Madison, WI 53706. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. Received 27 Aug. 2007. *Corresponding author ( John. [email protected]). Abbreviations: CP, crude protein; CT, condensed tannin; DM, dry matter; IAP, intestinal available protein; IRDP, insoluble rumendegradable protein; NPN, nonprotein N; RDP, rumen-degradable protein; RUP, rumen-undegradable protein; SP, soluble protein. Published in Crop Sci. 48:804–813 (2008). doi: 10.2135/cropsci2007.08.0461 © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . CROP SCIENCE, VOL. 48, MARCH–APRIL 2008 WWW.CROPS.ORG 805 fi ber intake, or increase reliance on high-input row crops. Therefore, increasing the proportion of alfalfa protein digested in the gastrointestinal tract should improve the performance and sustainability of farming systems based on ruminant livestock. In the future, the expression of protein-binding polyphenols such as condensed tannins (CT) and o-quinones in alfalfa should provide a sustainable approach for reducing wasteful proteolysis and increasing intestinal protein digestion by livestock (Sullivan and Hatfi eld, 2006; Xie et al., 2006). Direct ensiling studies have demonstrated that polyphenols in forage legumes can reduce proteolysis by up to 60% compared to alfalfa (Albrecht and Muck, 1991; Jones et al., 1995b). Studies with freeze-dried herbage also indicate that forage legumes with polyphenols undergo less ruminal proteolysis to provide up to threefold more plant protein for intestinal digestion than alfalfa (Broderick and Albrecht, 1997; Broderick et al., 2004). While excessive levels are detrimental (Min et al., 2003), moderate levels of CT (~25 g kg−1 dry matter [DM]) in fresh birdsfoot trefoil (Lotus corniculatus L.) can shift protein digestion to the intestine (Waghorn et al., 1987), increasing milk production and growth of ruminant livestock (Min et al., 1998; Woodward et al., 1999). Comparable levels of CT in ensiled birdsfoot trefoil can also enhance protein utilization and milk production of dairy cattle while mixed results were observed with ensiled red clover (Trifolium pratense L.) containing o-quinones (Broderick et al., 2001; Hymes-Fecht et al., 2005). In addition to other benefi ts (Hintz et al., 1999), increased severity of conditioning at cutting can shift protein fractions in alfalfa from rapidly to slowly degraded forms (Agbossamey et al., 1998). Although not reported, severe conditioning by mechanical maceration could enhance the release of CT from vacuoles in specialized cells (Lees et al., 1995) and their interaction with plant proteases and protein substrates. Similarly, mechanical maceration could facilitate the interaction of proteins and proteases with o-quinones formed by the action of chloroplastic polyphenol oxidase on o-diphenols stored in vacuoles (Sullivan et al., 2004). Conservation methods can also infl uence proteolysis in forages. Following conservation of alfalfa, NPN as a proportion of protein is typically 25% in hay compared to 50% or more in silage (Hristov and Sandev, 1998; Kohn and Allen, 1995). Switching from silage to hay conservation can also reduce rumen proteolysis and enhance microbial protein synthesis to improve animal production (Peltekova and Broderick, 1996; Vagnoni and Broderick, 1997). By contrast, the eff ect of hay vs. silage conservation on protein fractions in polyphenol-containing forages has received scant attention (Kraiem et al., 1990). In this study, forages containing CT or o-quinones or lacking these polyphenols were conventionally conditioned or macerated to vary the degree of cell breakage and the potential for polyphenol–protein interactions during conservation as hay or silage. Hays and silages were then treated with buff er, protease, and acid-detergent solutions to assess how forage polyphenols and methods of conditioning and conservation aff ect protein fractions and the potential performance of livestock. MATERIALS AND METHODS Forage Production Base, low, and high CT populations of ‘NC-83’ birdsfoot trefoil (Miller and Ehlke, 1996), ‘Cinnamon’ or ‘Marathon’ red clover, and a highly dormant alfalfa (‘ZG9910’) derived from Spredor 3 and Pioneer 5151 ( J. Moutray, personal communication, 2002) were conventionally seeded on 10 Aug. 2001 and 18 Apr. 2002 near Prairie du Sac, WI. The Richwood silt loam soil (fi nesilty, mixed, superactive, mesic Typic Argiudoll) at the site had very high levels of available P (74 kg ha−1) and exchangeable K (292 kg ha−1), adequate levels of B and S, and a pH of 7.1. The year following seeding, spring growth was harvested between 13 June and 20 June in 2002 and between 29 May and 2 June in 2003. Summer regrowth was harvested about 40 d after the spring cuttings. In 2002, harvest of spring growth was delayed until slowly maturing birdsfoot trefoil reached late bud stage while red clover and alfalfa were at 10 to 50% fl owering. Summer regrowth in 2002 was harvested at 10 to 50% fl owering. In 2003, forages were harvested at late bud for spring growth and at 10% fl owering for summer regrowth. Between late April and harvest of spring growth, average temperatures were similar both years (12°C), but precipitation was greater in 2002 (26 cm) than in 2003 (11 cm). Summer regrowth in 2002 occurred under warmer and drier conditions (22°C average temperature, 6.5 cm precipitation) than in 2003 (18°C average temperature, 13 cm precipitation). At each harvest, plots were cut near midday at a 5-cm height and weeds were removed by hand. A subsample of herbage was frozen in liquid N and subsequently freeze-dried for analysis of CT. Fresh herbage was then conditioned (Fig. 1) by passage through intermeshing rubber rolls set at manufacturer specifi cations (New Holland, New Holland, PA) or by rotaryimpact maceration (Kraus et al., 1999). Conditioned herbage was then wilted on plastic mesh screens (1-mm openings) in forced-draft ovens run at 35°C from 0900 to 1800 h; ovens were turned off at night. After drying for about 24 h to a DM content of 350 g kg−1, macerated herbage (700 g) and coarsely chopped roll-conditioned herbage (500 g) were ensiled in 1-L glass canning jars. Herbage remaining on screens was ovendried at 35°C as hay. After incubating at room temperature for 90 d, silages were removed from jars, frozen in liquid N, and freeze-dried. Dried hay and silage samples were ground through a cyclone mill (2-mm screen) for analysis. Forage Analyses Laboratory assays were run in duplicate. Cell breakage in rollconditioned, chopped, and macerated herbage wilted to 35% DM was estimated by a conductivity index method (Kraus et al., 1999). Ground samples were analyzed for DM by drying R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 806 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, MARCH–APRIL 2008 were suspended and pelleted (4500 × g, 5 min) thrice with 35 mL of ice-cold water and then transferred with water to glassfi ber fi lters. Residues and fi lters were analyzed by combustion to estimate RDP, with correction for fi lter N. Insoluble RDP (IRDP) was calculated as RDP minus SP. Subtracting RDP from CP provided an estimate of rumen-undegradable protein (RUP) fl owing to the gastrointestinal tract. Finally, the quantity of intestinal available protein (IAP) was estimated as RUP minus “indigestible” aciddetergent insoluble protein (Aufrere and Guerin, 1996), which was determined on samples (0.5 g) sequentially extracted in fi lterbags with neutral detergent (run without amylase, sodium sulfi te, acetone washing, or oven drying) followed by acid detergent using a fi ber analyzer (ANKOM Technology, Macedon, NY). Acid detergent residues and fi lter bags were analyzed by combustion to estimate acid-detergent insoluble protein, with correction for fi lter bag N. Statistical Design and Analyses The experimental design was a randomized complete block with four fi eld replications and a split-split plot arrangement of treatments. Forage entry was the main plot, conditioning level was the subplot, and conservation method was the subsubplot. Data were analyzed using PROC MIXED (SAS Institute, 2003). Forage genotype, conditioning level, conservation methods, harvests, and years were fi xed eff ects. Blocks within years and associated interactions were random eff ects. Pairwise comparisons of least square means were performed when a signifi cant F-test was detected at P ≤ 0.05. Diff erences mentioned in the text were signifi cant at P ≤ 0.05. The responses of protein fractions in birdsfoot trefoil to CT were tested by regression analyses (Neter et al., 1990; SAS Institute, 2003). A reduced model (i.e., roll conditioned and macerated birdsfoot trefoil had the same slope and intercept) was rejected if it signifi cantly increased error sums of squares (P ≤ 0.05) compared to the full model (i.e., roll conditioned and macerated birdsfoot trefoil had unique slopes and/or intercepts). If the reduced model was rejected, then an indicator variable was added to the equation to test whether the intercept and/or slope of roll-conditioned trefoil diff ered from macerated trefoil at P ≤ 0.05. Similar approaches were used to test whether protein fraction responses to CT diff ered between red clover and birdsfoot trefoil.