c07-05-0310 Coblentz.indd

Alfalfa (Medicago sativa L.) proteins ingested by dairy cows typically degrade at rapid rates and exhibit extensive ruminal degradability. Although the effects of conservation method (hay or silage) on these characteristics have been evaluated extensively, agronomic factors, such as harvest timing, have not. Our objective was to quantify rumen degradable protein (RDP) for ‘Affi nity’ alfalfa harvested over a range of ages (0, 5, 10, 15, and 20 d following Stage 2) within each of four harvest periods (spring, early and late summer, and fall). For 2004, there were no interactions (P ≥ 0.372) between harvest period and days within harvest period for any protein component. Crude protein (CP), neutral-detergent soluble CP (NDSCP; g kg–1 dry matter [DM]), and RDP (g kg–1 DM) declined in a quadratic (P ≤ 0.026) relationship with days following Stage 2. A quadratic (P = 0.002) pattern also was observed for rumen undegradable protein (RUP), but the overall range was small (60.4–66.5 g kg–1 DM). On a CP basis, RDP declined linearly (P < 0.001) from 720 to 659 g kg–1 CP during 2004. For 2005, there were interactions (P ≤ 0.020) of harvest period and days within period for all protein-related response variables, but trends over time within each harvest period generally were similar to those observed in 2004. Overall, RDP declined as alfalfa plants aged within harvest period, but these responses were due pri marily to reduced concentrations of CP within the cell-soluble fraction. W.K. Coblentz, USDA-ARS, U.S. Dairy Forage Research Center, 8396 Yellowstone Dr., Marshfi eld, WI 54449; G.E. Brink and N.P. Martin, USDA-ARS, U.S. Dairy Forage Research Center, Madison, WI 53706; D.J. Undersander, Dep. of Agronomy, University of Wisconsin, Madison, WI 53706. Mention of trade names or commercial products is solely for the purpose of providing specifi c information about scientifi c procedures and/or subsequent evaluation of results, and does not imply any recommendation or endorsement by the USDA. Received 1 June 2007. *Corresponding author ([email protected]). Abbreviations: CP, crude protein; DM, dry matter; ES, early-summer harvest period; FA, fall harvest period; GDD, growing degree days; LS, late-summer harvest period; NDF, neutral-detergent fi ber; NDICP, neutral-detergent insoluble CP; NDSCP, neutral-detergent soluble CP; RDP, rumen degradable protein; RUP, rumen undegradable protein; SP, spring harvest period. Published in Crop Sci. 48:778–788 (2008). doi: 10.2135/cropsci2007.05.0310 © 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 779 storage as hay reduced protein degradation rate from 0.171 to 0.075 h–1, and increased RUP from 240 to 397 g kg–1 crude protein (CP), relative to freeze-dried standing forage. Several studies have shown that ruminal degradation of forage proteins from alfalfa hay can be limited by externally applied heat treatment (Broderick et al., 1993; Yang et al., 1993) or by spontaneous heating during storage (Coblentz et al., 1997). However, these increases in RUP can be complicated by concurrent increases in acid detergent insoluble CP (Broderick et al., 1993; Yang et al., 1993; Coblentz et al., 1996), which is assumed to have low bioavailability (Licitra et al., 1996). Accumulation of aciddetergent insoluble CP also occurs in response to spontaneous heating during ensiling, a process that is common in drier silages (Rotz and Muck, 1994). While these research initiatives have focused heavily on conservation as hay or silage, eff ects of agronomic management factors, such as harvest timing, are less clear. Using in situ methodology, Hoff man et al. (1993) found that RDP (g kg–1 CP) decreased with plant maturity for alfalfa, red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.) harvested at the late vegetative, late bud, and midbloom stages of growth; however, this response was largely the result of changing proportions of soluble, slowly degraded, and unavailable fractions within the total pool of CP rather than a less rapid degradation rate for more mature forages. Cassida et al. (2000) reported some increases in RUP (g kg–1 dry matter [DM]) with plant maturity for alfalfa, red clover, and birdsfoot trefoil forages; however, these responses were sharper when RUP was reported on a gram per kilogram CP basis. In contrast, Broderick et al. (1992) used an in vitro technique that prevents the uptake of protein degradation products by microbes for subsequent protein synthesis (Broderick, 1994) and found no relationship between plant maturity and either degradation rate or RUP (g kg–1 CP) for 89 alfalfa forages harvested over a range of maturities, cuttings, and years. Therefore, the relationship between harvest timing and/or plant maturity and subsequent estimates of RUP or RDP remains unclear, and may be confounded strongly by climatic conditions during growth (Griffi n et al., 1994; Cassida et al., 2000). Currently, the in situ procedure (Vanzant et al., 1998), corrected for microbial contaminant N, is the most common method used for evaluating relative proportions of RDP and RUP in ruminant feedstuff s; however, in vitro procedures that utilize semipurifi ed proteolytic enzymes have been developed as routine laboratory techniques for the estimation of these protein fractions in forages (Krishnamoorthy et al., 1983; Licitra et al., 1998; Coblentz et al., 1999). Generally, single-endpoint enzymatic techniques can more easily accommodate the sample numbers generated from plot-type studies than full time-course kinetic evaluations by in situ methodologies. Our primary objective for this study was to utilize a preparation of Streptomyces griseus protease to assess the eff ects of harvest period and days within harvest period on concentrations of RDP and RUP for alfalfa forages harvested at Prairie du Sac, WI, during 2004 and 2005. MATERIALS AND METHODS Plot Management During August 2003, a Richwood silt loam (fi ne-silty, mixed, superactive, mesic Typic Argiudoll) soil, located near Prairie du Sac, WI, was amended to meet soil-test recommendations for P, K, and pH, and ‘Affi nity’ alfalfa was then drilled into a prepared seedbed at a rate of 22 kg ha–1. Four 7.6 by 6.1 m plots were established in each of four fi eld blocks. After establishment, soil tests were taken annually, and amendments were applied as needed to meet soil test recommendations of the University of Wisconsin Cooperative Extension Service. During the course of the study, potato leafhoppers (Empoasca fabae Harris) were controlled as needed with applications of lambda-cyhalothrin {[1α(S*),3α(Z)]-( ± )-cyano-(3-phenoxyphenyl)methyl-3-(2chloro-3,3,3-trifl uoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate} at a rate of 0.024 kg a.i. ha–1. Beginning in the spring of 2004, the four plots within each block were assigned randomly to one of four harvest periods (spring [SP], early summer [ES], late summer [LS], and fall [FA]). For the SP period, one 1.5 by 6.1 m strip from each plot was harvested when plant maturity reached Stage 2 (Kalu and Fick, 1981). At this time, designated as Day 0, stem length was >0.30 m, but no buds, fl owers, or seedpods were visible. Subsequently, additional 1.5 by 6.1 m strips were assigned randomly throughout the plot, and harvested at 5-d intervals for a total of fi ve strips per plot (0, 5, 10, 15, and 20 d). Although there was some variability between harvest periods, sampling dates were structured such that Day 10 generally coincided with a one-tenth bloom stage of development. In addition, this 20-d harvest window within each harvest period represented a realistic range of time over which most producers would typically harvest alfalfa in Wisconsin. While harvesting during the SP period, plots designated for the ES, LS, and FA harvest periods were clipped at 1/10 bloom, but no samples or data were collected. These harvest procedures were then repeated later during the growing season as plots assigned to the ES, LS, and FA harvest periods reached Stage 2, as defi ned previously. All sampling dates and growing degree days (GDD) accumulated within each harvest period during 2004 and 2005 are reported in Table 1. Growing degree days were calculated daily by subtracting 5°C from the average of the maximum and minimum temperatures for that day, and then summing over days within each harvest period. Some discussion of postsampling management also is warranted. Following data collection from the 0, 5, 10, 15, and 20-d strips of the SP plot, regrowth from all SP strips was allowed to reach a minimum of one-tenth bloom before the next (ES) harvest. At that time, no data were taken, and all harvested forage from the SP plot was discarded. For each subsequent harvest period (LS and FA), the entire SP plot was harvested at one-tenth bloom, but no data were recorded. Identical postsampling procedures were used for plots designated for data 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 . 780 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, MARCH–APRIL 2008 was calculated as CP − NDICP, where NDICP was expressed as a proportion of whole-plant DM. In Vitro Incubation in Prepared Protease Solution The in vitro protease procedures used in this study were similar to those described by Krishnamoorthy et al. (1983), Licitra et al. (1998), and Coblentz et al. (1999). Streptomyces griseus protease (P-5147; Sigma Chemical Co., St. Louis, MO) contained 4.5 enzyme activity units per milligram of solid, where one activity unit of enzyme was able to hydrolyze casein to produce color equivalent to 1.0 μmol (181μg) of tyrosine per minute at pH 7.5 and 37°C. Sample size for all incubations was set on the basis of a common N content (15 mg) within each incubation fl ask; therefore, the actual sample weight was adjusted for CP concentration, and varied somewhat across forages. Each forage sample was incubated in a water bath for 1 h at 39°C in 40 mL (pH 8.0) of borate–phosphate buff er (Krishnamoorthy et al., 1983). Following the 1-h buff er incubation, 10 mL of prepared protease solution containing 0.33 activity units mL–1 of S. griseus protease was added to each fl ask, yielding a fi nal enzyme activity concentration of 0.066 activity units mL–1 in the incubation medium. Flasks were covered with aluminum foil, swirled daily, and incubated for 48 h at 39°C. One milliliter of sodium azide (1%, w/v) was added to each incubation fl ask as an antimicrobial agent. Following incubation, samples were immediately fi ltered through preweighed (dry basis) Whatman no. 541 fi lter paper (Whatman International Ltd., Maidstone, UK). Residues were washed with approximately 400 mL of deionized water (20°C), and dried in a gravity convection oven at 100°C; these residues were then analyzed for CP by the macro-Kjeldahl technique described previously. Single timepoint estimates of RDP were calculated as RUP (g kg–1 DM) = (g residual CP/g initial DM) × 1000, and RDP (g kg–1 DM) = CP − RUP. Estimates of RDP also were expressed on the basis of total plant CP; calculations were made by RDP (g kg–1 CP) = [RDP (g kg–1 DM)/CP] × 1000. Incubation fl asks containing each forage sample were evaluated by the S. griseus protease procedure in each of two separate runs. Values from each run were averaged to yield the fi nal RUP and RDP values for each forage replicate. Statistical Analysis Originally, year was included within the statistical analysis as a sub-subplot term. However, there were numerous interactions (P < 0.05) of year with other treatment eff ects; therefore, year was dropped from the model, and each year was evaluated independently. This analytical approach precludes evaluation of certain carryover eff ects of treatment; among these, the most important are comparisons across years of plots with the same combination of harvest period and days within period. However, given the emphasis on independence across both harvests and years that was build intentionally into the experimental design, it is far more likely that diff erences between years can be attributed to climatic variability, and that climatic eff ects would dwarf any potential carryover eff ects of treatment. Within year, data were analyzed by PROC GLM of SAS (SAS Institute, 1990) as a split-plot experiment with harvest collection during the ES or LS harvest periods. These procedures were designed to minimize any carryover eff ects from previous harvests or years, and maximize the statistical independence of each harvest period. The study was conducted over 2 yr (2004 and 2005); therefore, a total of eight harvest periods were included in the experiment. Within block, plot assignments for individual harvest periods (SP, ES, LS, and FA) and sampling dates within harvest period were maintained without additional randomization between years. Sample Preparation and Analysis All harvested alfalfa forages were dried for 48 h under forced air at 50°C, and ground subsequently through a Wiley mill (Arthur H. Thomas, Philadelphia, PA) equipped with a 1-mm screen. Concentrations of CP in each sample were quantifi ed by a macro-Kjeldahl technique (Association of Offi cial Analytical Chemists, 1998), where CP was calculated by multiplying the percentage of N in each forage sample by 6.25. Samples were then analyzed for neutral-detergent fi ber (NDF) using the batch procedures outlined by ANKOM Technology Corporation (Fairport, NY). Sodium sulfi te and heat-stable α-amylase were not included in the NDF solution. Following incubation in neutral detergent, neutral-detergent insoluble CP (NDICP) was determined by analyzing insoluble fi brous residues for concentrations of CP using the same macro-Kjeldahl technique described for quantifi cation of whole-plant concentrations of CP. Concentrations of NDICP were reported as both proportions of whole-plant DM and CP. Neutral detergent soluble CP Table 1. Harvest dates and growing degree days (GDD) within spring (SP), early-summer (ES), late-summer (LS), and fall (FA) harvest periods for 2004 and 2005. Harvest period Days 2004 2005