Physiochemical Characteristics and Molecular Structures for

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Physiochemical Characteristics and Molecular Structures for Digestible Carbohydrates of Silages Basim Refat, Luciana Louzada Prates, Yaogeng Lei, David Christensen, John J. McKinnon, and Peiqiang Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01032 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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Journal of Agricultural and Food Chemistry

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Physiochemical Characteristics and Molecular Structures for

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Digestible Carbohydrates of Silages

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Basim Refat, Luciana L. Prates, Yaogeng Lei, David A. Christensen,

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John J. McKinnon, Peiqiang Yu*

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Department of Animal and Poultry Science, College of Agriculture and Bioresources

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University of Saskatchewan, Saskatoon, S7N 5A8, SK, Canada

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*Corresponding author:

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Peiqiang Yu, PhD. Professor & Ministry of Agriculture Strategic Research Chair Department of Animal and Poultry Science College of Agriculture and Bioresources, University of Saskatchewan, Room 6D10, 51 Campus Drive, Saskatoon, SK, Canada, S7N 5A8 Tel: (306) 966 4150 Email: [email protected]

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ABSTRACT

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The main objectives of this study were (1) to assess the magnitude of differences among

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new barley silage varieties (BS) selected for varying rates of in vitro NDFD digestibility

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(ivNDFD; Cowboy BS with higher ivNDFD , Copeland BS with intermediate ivNDFD

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and Xena BS with lower ivNDFD) as regards to their physiochemical features (energy),

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carbohydrate nutrient fractions and rumen digestive contents in dairy cows in comparison

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with a new corn silage hybrid (Pioneer 7213R), and (2) to identify the pattern of

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interactive association between the molecular structure of carbohydrates and digestible

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carbohydrates content. The carbohydrates-related molecular structure spectral data was

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measured using advanced vibrational molecular spectroscopy (FT/IR). In comparison to

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BS, corn silage showed a significant (P < 0.05) high level of starch, energy content and

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higher degradation of DM. Cowboy BS had lower feeding value (higher indigestible fiber

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content and lower starch content) and lower DM degradation in the rumen compared to

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other BS varieties (P < 0.05). The spectral intensities of molecular carbohydrates (P
95% of remaining observed variance. Functional groups of

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ligneous (ca. 1525- 1487 cm-1), cellulosic compounds (ca. 1292-1189 cm-1), structural

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CHO (ca. 1487- 1189 cm-1), total CHO (ca. 1189-909 cm-1) and non-structural CHO (ca.

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909- 880 cm-1) were analyzed using multivariate molecular spectral analysis

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Chemical Analysis

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Dry matter (method 930.15), ash (method 942.05), and crude fat (method 2003.05) were

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analyzed according to AOAC.

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using a Leco FP 528 Nitrogen Combustion Analyzer (Leco, St Joseph, MI). The methods

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described in Van Soest et al. and AOAC (method 2002.04) combined with an ANKOM

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A200 filter bag technique (ANKOM Technology Corp., Fairport, NY, USA) were used to

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determine neutral detergent fiber.

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stable α- amylase before neutral detergent extraction. Acid detergent fiber (ADF; method

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973.18) and acid detergent lignin (ADL; method 973.18) were analyzed according to

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AOAC

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(ANKOM Technology Corp., Fairport, NY, USA). Ethanol soluble carbohydrate and

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starch were determined using the methods described by Hall et al. 31 The iNDF content of

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each feed sample was determined following in situ incubations for 288h in the rumen

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using two lactating dairy cows.32 Two experimental runs were carried out. Around 3 g of

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ground sample (1-mm) were weighed in triplicate into 5 × 10 cm size custom made in

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For estimation of CP (N × 6.25), N was determined

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This method was combined with adding with heat-

. This method was combined with an ANKOM A200 filter bag technique



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situ bags (6 µm pore size, part no. 07 – 6/5, Sefar America Inc., Depew, NY) and were

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randomly assigned to cows. After removal from the rumen, the bags were rinsed with

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cold water, oven dried at 55ºC for 48 h. Subsequently, the NDF of residues was analyzed.

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Statistical Analysis

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Statistical analyses were performed using the PROC MIXED procedure of SAS 9.4 (SAS

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Institute, Cary, NC). Chemical profile was analyzed using completely randomized design.

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The model used for analyzing this design was as follow: Yij = µ+ Ti + eij, where Yij is an

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observation of the dependent variable; µ is the population mean for the variable; Ti is the

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treatment effect, as a fixed effect, eij are the random error associated with the observation

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ij.

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In situ degradability of DM and NDF were analyzed using a randomized complete

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block design. The model used for this design was as follow: Yijk = µ+ Ti + Bj +eijk, where

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Yijk is an observation of the dependent variable ij; µ is the population mean for the

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variable; Ti is the treatment effect, as a fixed effect, Bj is a block effect with in situ

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animals, as a random effect, and eijk is the random error associated with the observation

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ij.

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The ATR-FT/IR spectroscopic data were analyzed using a completely randomized

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design model with subsampling (spectra reading; n= 5 scans): Yij = µ+ Ti + eij, where Yij

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is an observation of the dependent variable ij (chemical functional groups such as; µ is

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the population mean for the variable; Ti is the treatment effect, as a fixed effect, eij are the

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random error associated with the observation ij. The significance was declared at P
0. 1; Table 1). Starch content of corn silage was higher

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compared with all barley forages (26 vs 12 % DM, P < 0.01), while NDF was lower in

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corn silage when compared with all barley forages (39 vs 46 % DM, P < 0.01). Lignin

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content was higher (5 %DM) in Copeland BS and Cowboy BS, intermediate in Xena BS

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(4.5% DM), and lower in corn silage (3% DM). Indigestible fiber fraction as expressed

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%NDF was higher in Cowboy BS (46 %NDF), intermediate in Copeland BS (42 %NDF)

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and lower in Xena BS and corn silage (averaged 35%NDF).

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Carbohydrate Fractions and Predicted Carbohydrate Degradation Fractions

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Table 2 shows the carbohydrate subfractions of barley silages compared with corn silage.

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There was no significant difference (P > 0.10) in CB3 (available NDF% CHO) among

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barley silage varieties (averaged 35 % CHO; Table 2). Significant differences were

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detected among barley silages for CC with Xena barley silage lower at 20% CHO, CDC

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Copland intermediate at 22% CHO, and CDC Cowboy higher at 30%. Corn silage had a

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relatively similar CB3 (P > 0.10) and higher CB1 (starch, % CHO) compared with all

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barley silages (P < 0.01). However, the average CC fraction was lower in corn silage

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(16% CHO) compared to the average across barley silages (P < 0.05).

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The results of the current study showed corn silage have a higher RDCB1 (20%

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DM; Table 2) than all barley silage varieties (10% DM). There were significant

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differences among barley varieties in RDCB3, being higher in Cowboy and Copeland BS

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(averaged 11% DM), and lower in Xena BS and corn silage (averaged 6% DM). The


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rumen degradation of total CHO was higher in corn silage (38% DM), intermediate in

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Copeland BS (34 % DM) and lower in Cowboy and Xena BS (averaged 29 % DM).

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Digestible Nutrients and Energy Values

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Cowboy BS had a significantly higher tdNDF (P < 0.05; Table 2) when compared with

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Copeland BS (26 vs 22 %DM). The tdNFC was higher in corn silage compared to

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Cowboy BS (44 vs. 29 % DM, respectively; P < 0.05). Corn silage had the greatest

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TDN1x (71.0% DM) and Cowboy BS had the lowest level (62 % DM), whereas Copeland

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and Xena BS had intermediate TDN1x values (averaged 64 % DM).

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The energy estimates were higher in corn silage (2.8, 2.4, and 1.5 Mcal/kg of DM

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for DE3×, ME3×, and NEL3×, respectively), intermediate in Copeland BS (2.6, 2.2, and 1.4

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Mcal/kg of DM for DE3×, ME3×, and NEL3×, respectively) and lower in Xena and

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Cowboy BS (2.5, 2.1, and 1.3 Mcal/kg of DM for DE3×, ME3×, and NEL3×, respectively).

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Rumen Degradation Kinetics of Nutrients in Barley and Corn Silages

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The results showed there were no significant differences in degradation rates of DM

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between all silages (Kd, 3.7%/h, P > 0.10; Table 3). The soluble fraction of DM was

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significantly different amongst all silages (P < 0.05), being higher in Xena (34%),

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intermediate in Copeland BS (29%) and lower in Cowboy BS and corn silage (23%). On

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the other hand, corn silage had the highest value for rumen slowly degradable DM

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fraction (52%), compared with Xena BS (39%). There were no significant differences

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among barley silage varieties in D fraction of DM (averaged 40%), however the U

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fraction was significantly higher in Cowboy BS (37%) compared to corn silage (25%).



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Among three barley forages, Xena BS had greater (P < 0.05) EDDM (50 %) compared to

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Cowboy BS (43 %), while corn silage had similar EDDM compared to barley silages.

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There were no significant differences in degradation rates of NDF between all

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silages (Kd, 3.2%/h; P > 0.10; Table 3). The lag time was significantly (P < 0.05; Table

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3) longer in corn silage (9h), intermediate in Xena barley silage (6h) and shorter in

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Copeland and Cowboy barley silages (averaged 3h). The undegradable fraction of NDF

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was significantly lower in Xena BS (26%), intermediate in Copeland BS (36%) and

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higher in Cowboy BS (44%). The effective degradation of NDF was significantly lower

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(P < 0.05) in Copeland BS (23%) than Xena BS (35%).

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Carbohydrate Molecular Spectroscopic Features of Barley and Corn Silage with

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Different Digestible Carbohydrates Content

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Peak height and area values of ligneous compounds in all forages were not significantly

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different (P > 0.1; Table 4). Also, all forages had similar structural carbohydrates peak

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area (STC_A; averaged 20 cm-1, P > 0.10). There were three spectral peaks within the

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STC_A centered at approximately ca. 1416, 1372, and 1320 cm−1. Xena BS had higher

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peak height at 1416 than corn silage and Copeland BS, while corn silage had higher peak

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height at 1372 cm−1 than Copeland BS (P < 0.05). The CEC peak area or height absorbed

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intensities for barley and corn silages were similar (P > 0.10). The total CHO peak area

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values were similar for all forages (TC_A; region and baseline ca. 1189-909 cm-1). Also,

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all forages had the same absorbed intensities at 1152, 1100, and 1030 (P > 0.1). The non-

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structural CHO peak area (NSTC_A), peak height H_898, and NSTC spectral ratios were

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significantly lower in corn silage compared with all barley silage varieties (P < 0.05).


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Multivariate Analysis of Carbohydrate Molecular Spectral Profiles related to

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Barley and Corn Silages

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Multivariate molecular spectral analysis related to fingerprint regions: (1) ligneous

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compounds fingerprint region: ca. 1525–1487 cm-1, (2) structural carbohydrate

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fingerprint region: ca. 1487–1189 cm-1, (3) total carbohydrates fingerprint region: ca.

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1189–909 cm-1, and (4) non-structural carbohydrates fingerprint region: ca. 909–880 cm-1

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are displayed in Fig 1:4. Results from CLA indicated no cluster classes of these regions

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could be discriminated from each other. For PCA analysis, the first principal component

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explained 96.5%, 97.5%, 98.3, and 99.8% of the variation, respectively. The principal

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components (PCs) for all forages (corn and barley silages) were overlapped in STC, TC,

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and NSTC regions. For the ligneous region, three separated classes related to each barley

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silage were distinguished.

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Carbohydrate Molecular Structural Profiles in Relation to Digestible Carbohydrate

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Contents

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Spectral intensity of NSTC_A had a negative correlation with starch (r = −0.76, P = 0.03;

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Table 5), and NSC content (r = −0.76, P = 0.03). Fiber fractions were significantly

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correlated with the molecular structure intensities of STC, where spectra area intensity of

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STC had a negative correlation with ADF content (r = −0.72, P = 0.05). There were not

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significant correlations (P > 0.05) between carbohydrate molecular structural profiles in

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relation to iNDF. For CNCPS carbohydrates fractions, only NSTC_A was positively

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correlated with CC fraction (r = 0.86, P = 0.01) and positively correlated with CC 17 ACS Paragon Plus Environment


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fraction (r = 0.86, P = 0.01). For energy values, TDN1x and NELp3x were negatively

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correlated with amide NSTC_A (r = −0.73, P < 0.05). Ligneous compounds spectral

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intensity was positively related to rate and extent of NDF degradation in the rumen (P
= 0.00 6 5 4

W

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Factor 2: .97%

2

W

W W X

P

1

W

X

X W X XW P W W P

0 P

-1 -2

P X

CC

X P P

X P C CC

P

C

C C C

P

-3 C -4 -5 -6 -7 -50

753 754 755 756 757

-40

-30

-20

-10

0

10

20

30

40

50

Factor 1: 98.28%

Figure 3. Multivariate spectral analyses of different forages: comparison between corn and barley silages: (a) Cluster analysis. Select spectral region: TC region (ca. 1189-909 cm-1). Cluster method: Ward's algorithm; (b) Principal component analysis. Scatter plots of the first principal components (PC1) vs the second principal component (PC2).

758


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(a)

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Tree Diagram for 39 Cases Ward`s method Squared Euclidean distances C W W W C X C P X X C W W W W P P P X C C W W X P P C W P P X X P X X P C C C 0.00

760 761

0.02

0.04

0.06

0.08

0.10

Linkage Distance


0.12

0.14

0.16


762 763

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(b) Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 0.8 0.6 0.4

W W

X X X W PW

X Factor 2: .11%

0.2

W C

-0.2

W W

C

PP 0.0

W C C

-0.4

P

X PC

P

P CC

P W X P

X

XC X

C C

P -0.6 -0.8 -1.0 -20

764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783

-15

-10

-5

0

5

10

15

20

Factor 1: 99.79%

Figure 4. Multivariate spectral analyses of different forages: comparison between corn and barley silages: (a) Cluster analysis. Select spectral region: NSTC region (ca. 1909880 cm-1). Cluster method: Ward's algorithm; (b) Principal component analysis. Scatter plots of the first principal components (PC1) vs the second principal component (PC2).


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