Accepted Manuscript

Composition factors predicting forage digestibility by horses

T.L. Hansen, L.M. Lawrence

PII: S0737-0806(17)30497-5
DOI: 10.1016/j.jevs.2017.08.015
Reference: YJEVS 2373

To appear in: Journal of Equine Veterinary Science

Received Date: 16 June 2017

Revised Date: 14 August 2017
Accepted Date: 14 August 2017

Please cite this article as: Hansen TL, Lawrence LM, Composition factors predicting forage digestibility
by horses, Journal of Equine Veterinary Science (2017), doi: 10.1016/j.jevs.2017.08.015.

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4 Composition factors predicting forage digestibility by horses

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9 T.L. Hansen and L.M. Lawrence*

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12 Department of Animal and Food Sciences, University of Kentucky, Lexington, KY 40546 USA
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17 Corresponding author. Tel: 859-257-7509; Fax: 859-257-2534; EM: llawrenc@uky.edu

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19 Abstract

20 Forages play a key role in the equine diet; however, assessing the feeding value of

21 forages through in vivo digestibility studies is costly and time-consuming. The ability to evaluate

22 feeding value through a model relating chemical composition to forage digestibility could be a

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23 useful tool in the equine industry. The objective of this study was to relate forage chemical

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24 composition to in vivo dry matter digestibility (DMD) using previously published data, with the

25 expectation that forage DMD would be most significantly related to neutral detergent fiber

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26 (NDF) and acid detergent fiber (ADF) content of the forage. Simple linear regression results

27 indicated more variation in forage digestibility was explained by NDF and crude protein (CP)

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concentrations than ADF (r2 = 0.6017, 0.5402, and 0.4643 respectively). The best 2 variable
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29 model for equine forage DMD was related to NDF and CP concentrations (DMD = 65.81 +

30 0.7207×CP – 0.3514×NDF; DM basis; P<0.0001, R2 = 0.6690, adj. R2 = 0.6583). Model fit was
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31 marginally improved with the inclusion of 3 predictor variables. Comparison of these equations
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32 to independent data sets not used in model development showed promise in predicting forage
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33 digestibility within the range of forages used to generate the equations (CP: 2.9 – 22.6, ADF:

34 25.4 – 52.4, NDF: 32.8 – 83.3, DM basis). Further study is needed to expand the equations for a
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35 broader range of forages, but equine professionals and horse owners can use NDF and CP to

36 make relative comparisons of forage value with typical hays fed to horses.
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37 Keywords: diet, equine, regression model, hay

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38 Abbreviations: acid detergent fiber (ADF), adjusted multiple coefficient of determination (adj.

39 R2), coefficient of determination (r2), crude fiber (CF), crude protein (CP), digestible dry matter

40 (DDM), dry matter digestibility (DMD), hemicellulose (HEM), multiple coefficient of

41 determination (R2), neutral detergent fiber (NDF), organic matter (OM), percent (PER),
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42 predictive coefficient of variation (pred. R2), predictive residual sum of squares (PRESS),

43 relative feed value (RFV), relative forage quality (RFQ)

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44 1. Introduction

45 Under most conditions, forage constitutes an important part of the equine diet.

46 Digestibility is one of the factors that affects the feeding value of forage. The gold standard for

47 determining forage digestibility by horses is an in vivo digestibility trial; however, determining in

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48 vivo digestibility is costly, labor intensive, and returns information slowly. Thus, there has been

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49 considerable interest into alternative methods to evaluate forage quality such as in situ and in

50 vitro assays [1, 2], but these assays are also relatively slow to return information.

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51 Ruminant nutritionists and forage scientists have used forage chemical composition to

52 develop indices such as relative feed value (RFV) and relative forage quality (RFQ) [3, 4].

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These indices are largely based on NDF and ADF concentrations, with greater NDF and ADF
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54 concentrations indicating lower forage quality. These indices provide a virtually instantaneous

55 evaluation of forage quality for ruminants; however, it is unclear how these indices apply to
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56 horses. While there have been many studies on factors affecting digestibility of feeds by horses,
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57 studies that have attempted to assess the relationships between forage chemical composition and
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58 forage digestibility by horses have been limited by number or type of forages utilized [5].

59 Broader datasets are needed to clarify the relationships between chemical composition and
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60 forage digestibility by horses.

61 The objectives of this study were to determine if chemical composition could be used to
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62 predict forage digestibility by horses and if so, to develop a model for predicting DMD. Rather
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63 than conduct additional in vivo experiments, we utilized data from previously published studies

64 representing a wide range of forages. We hypothesized that forage NDF and ADF

65 concentrations would be the most useful predictors of forage digestibility, and with increasing

66 concentrations of NDF and ADF forage digestibility would decrease.

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67 2. Materials and Methods

68 2.1. Data Collection

69 In order to obtain a large range of values for in vivo forage digestibility by horses, a

70 literature search was conducted utilizing the search engine Google Scholar using the keywords

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71 “digestibility”, “horses”, and “forage”. Articles and abstracts were selected based on the use of

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72 total fecal collections in mature horses or ponies fed all forage diets. Studies using internal or

73 external markers, young horses (< 2 y of age), or those determined to have non-standard

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74 practices (use of cannulated horses, short dietary adaptation, or inadequate description of

75 calculations or methods) were excluded from analysis. Studies were included if a portion of the

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study fit all of the criteria (Example: For a study comparing forage digestibility between growing
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77 and mature horses, only the mature horse data were retained for analysis). Studies must have

78 reported relevant chemical components (NDF, ADF, and CP) as well as DMD. Additionally, the
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79 same criteria were applied to the proceedings from the Equine Nutrition and Physiology
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80 Symposium and Equine Science Symposium from 1968 through the year 2011. The literature
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81 search resulted in 26 studies that fit the specified criteria (Table 1).

82 The data collected from these studies are shown in Table 2. The amount of hemicellulose
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83 (HEM) in the forage was estimated by HEM = NDF-ADF. The percent of ADF in the NDF

84 portion of the forage (PER) was calculated as PER = ADF/NDF×100. In total, there were 70
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85 DMD mean values from 70 forage diets. There were 65 measurements of NDF concentrations,
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86 70 measurements of ADF concentrations, 69 measurements of CP concentrations, 65

87 measurements of HEM, and 65 measurements of PER. There were 65 DMD measurements with

88 a comprehensive report of forage composition (included each of NDF, ADF, and CP

89 concentrations).
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90 The mathematical relationships between diet digestibility and forage composition using

91 data from the selected studies were evaluated utilizing both simple and multiple linear

92 regression. Explanatory variables included NDF, ADF, and CP concentrations of the forages in

93 addition to calculated HEM and PER values. Multiple linear regression used combinations of

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94 these explanatory variables. Polynomial regression included the square of explanatory variables

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95 in multiple regression models. A classification variable for type of forage was not included due

96 to the small number of available forage diets for certain types of forages (only one measurement

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97 for warm-season legume forages, and less than 15 complete measurements each for cereal grain

98 hays, cool-season grasses, and cool-season legumes). In addition, a classification variable for

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methods used to analyze forage chemical composition was not included due to insufficient
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100 information in some papers.

101 2.2. Statistical Analysis and Model Validation

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102 Regression analysis was performed with the PROC REG procedure of SAS software
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103 (Version 9.3; SAS Institute, Inc., Cary, NC). Simple linear regression models were compared
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104 using the coefficient of determination (r2), whereas multiple linear regression models were

105 evaluated based on the adjusted R2 (adj. R2). The use of the adj. R2 in multiple regression
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106 models was necessary in order to account for a continual increase in the multiple coefficient of

107 determination when additional explanatory variables were added to the model.
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108 Regression models were tested with data from three equine digestibility studies (4
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109 different forages; Table 3) not included in the initial regression analysis data [31-33]. A paired t-

110 test was used to compare calculated DMD to in vivo values. Predictive ability of models was

111 determined by predicted residual sum of squares (PRESS) and predictive R2 (pred. R2) statistics.

112 3. Results
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113 Simple linear regression indicated all forage composition factors were significant to

114 predict in vivo DMD (Table 4); however, the best fit of the available data was achieved when

115 using NDF concentrations of forages (r2 = 0.6017; Figure 1). Crude protein concentrations

116 showed the second best fit to the available data (r2 = 0.5402). Less than 50% of the variation in

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117 DMD was explained by the ADF concentration of the forage (r2 = 0.4643; Figure 2).

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118 When using two predictor variables for multiple linear regression, the best fit for forage

119 composition to predict in vivo DMD was achieved (as determined by the adj. R2) with the

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120 inclusion of the predictor variables NDF and CP (Table 5). Dry matter digestibility was related

121 to NDF and CP concentrations by DMD = 65.81 + 0.7202×CP – 0.3514×NDF (DM basis;

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P<0.0001, R2 = 0.6690, adj. R2 = 0.6583). The addition of any third forage composition
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123 explanatory variable to that model did not increase the fit of the model as determined by the adj.

124 R2 nor were all three explanatory variables individually significant. However, formation of other
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125 three variable models increased the fit of the model to the data available. The model of best fit
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126 was achieved with CP, ADF, and PER predictor variables (DMD = 56.16 + 0.6080×CP –
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127 0.7163×ADF – 0.2560×PER; DM basis, P<0.0001, R2 = 0.6833, Adj.R2 = 0.6677).

128 Polynomial variables were not significant in the models when using one forage
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129 composition variable (for example, relating DMD to NDF and NDF2). Inclusion of multiple

130 composition variables in polynomial regression did not increase the fit of the model, nor were all
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131 predictor variables significant (data not shown).

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132 Using the independent data from Table 3, predictive ability was determined for the three

133 best fitting regression models (bolded in Table 5). Predictive statistics were marginally improved

134 for the models relating DMD to CP, ADF, and HEM (PRESS = 2012.55, pred. R2 = 0.6426) and

135 CP, ADF, and PER (PRESS = 2008.01, pred. R2 = 0.6434) compared to the two predictor model
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136 (PRESS = 2053.35, pred. R2 = 0.6353). There was no difference between observed in vivo

137 digestibility and model predicted digestibility (P> 0.1; Figure 3).

138 4. Discussion

139 Although all composition factors had a significant influence on DMD, this study found

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140 the best predictive factor for DMD was NDF concentration when using simple linear regression.

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141 Martin-Rosset et al. [5] reported digestible organic matter (OM) was related to CP, crude fiber

142 (CF), NDF, and ADF concentrations of legume and grass hays, again demonstrating the best

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143 linear fit was achieved when using NDF concentration to predict forage digestibility by horses

144 (r2 = 0.707 in that study). The coefficients of determination for all comparable equations were

145
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greater in that study than the current study. Martin-Rosset et al. [5] may have observed a better
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146 fit because those studies were all conducted at the same institution allowing for more consistency

147 of study methods. In the present study there were differences in analytical procedures as well as
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148 fecal collection procedures among studies. Additionally, in that study [5] most of the forages
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149 were cool-season grass hays, whereas this study uses a broader range of forage types.
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150 The relatively low coefficient of determination for ADF concentration to predict in vivo

151 DMD was unexpected because ADF has often been used to determine forage digestibility by
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152 ruminants. For example, the recommended equation to determine digestible DM (DDM) when

153 calculating RFV of a forage is DDM = 88.9 – 0.770×ADF (DM basis) [34]. However, it is
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154 important to consider that DDM equation has been shown to poorly predict DDM in some
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155 instances [4]. Rohweder et al. [3] also found forage ADF concentrations were useful to predict

156 alfalfa DMD, but the fit was poorer with grasses than legumes. Forage ADF concentrations may

157 not have fit the data well in the current study because the dataset used a wide variety of forages,

158 including warm- and cool-season grasses and legumes. Also, ADF concentrations had a
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159 relatively small range of values compared to NDF and CP and may have led to the poor fit

160 shown in this study.

161 Multiple linear regression with forage composition variables NDF and CP achieved a

162 better fit to the data than simple linear regression. The use of a third forage composition

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163 explanatory variable marginally increased the fit of the model, but in many instances all three

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164 explanatory variables in those models were not statistically significant. This observation could be

165 due to a correlation among composition variables within the forages. Models that performed well

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166 included CP and fiber composition variables. The models with the highest fit explained

167 approximately two-thirds of the variation associated with digestibility. It would be unlikely that

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forage chemical composition alone would explain all of the variation in digestibility as other
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169 factors such as level of intake and degree of processing could also influence digestibility.

170 Using the 3 bolded multiple regression equations in Table 5, the differences between the
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171 observed and predicted values for digestibility of forages in the independent data sets were
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172 minimal. Therefore, these equations show promise for estimating forage feeding value for
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173 horses. Only slight improvements were observed with the use of a multiple regression equation

174 with 3 dependent variables, indicating the two variable model relating NDF and CP
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175 concentrations to DMD is sufficient for assessing feeding value. More independent data sets are

176 needed in order to test the models for a range of forage composition values; however, this step is
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177 currently limited by the availability of additional applicable studies. Also, all of the forages used
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178 in the present study were dry, so additional studies are needed to examine the applicability of the

179 models to fresh and ensiled forages.

180 Agreement between predicted and observed values could potentially be improved if

181 equations could be made specific for the type of forage (cool-season grasses, warm-season
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182 grasses, legumes, etc.) as has been done in ruminant studies [3, 35]. However, categorical

183 analysis was not practical in this assessment due to the small sample size from the literature.

184 Once additional digestibility studies using pure legume or pure grass forages are published the

185 feasibility of equations for specific forage type (or genus) can be revisited. However, horses are

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186 often fed mixed hays containing both legumes and grasses; therefore, a model that predicts

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187 across multiple forage types could have practical value in the horse industry.

188 5. Conclusions

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189 With a large range of in vivo DMD values from published equine forage digestibility

190 research, regression results indicated digestibility was related to forage chemical composition.

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Overall, forage digestibility was negatively correlated to fiber concentrations and positively
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192 correlated to CP concentrations. Crude protein and NDF concentrations can be used to quickly

193 assess forage composition and determine more digestible forages, and therefore, forages with
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194 greater feeding value. Further work is needed to determine if this model is applicable to forages
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195 with nutrient compositions beyond the range of forages used to create the models. Individual
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196 models for different forage types may better predict DMD, but the limited number of equine

197 digestibility studies does not allow for that analysis at this time. The current model has
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198 advantages of easily determined nutrient composition factors and a wide range of applicability.

199 Acknowledgements
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200 This is publication 15-07-035 of the Kentucky Agricultural Experiment Station and is
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201 published with the approval of the Director. This work is supported by the National Institute of

202 Food and Agriculture (Hatch).

203 Highlights

204 • Forage chemical composition can be used to estimate forage quality and digestibility.
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205 • More variation in forage digestibility was explained by NDF and CP values than ADF.

206 • Addition of a third regression variable marginally increased model predictive ability.

207 • Forage digestibility can be quickly assessed by NDF and CP concentrations.

208

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209 References

210 1. Koller BL, Hintz HF, Robertson JB, Van Soest PJ. Comparative cell wall and dry matter

211 digestion in the cecum of the pony and the rumen of the cows using in vitro and nylon

212 bag techniques. J Anim Sci 1978; 47:209-15

PT
213 2. Earing JE, Cassill BD, Hayes S.H, Vanzant E., Lawrence LM. Comparison of in vitro

RI
214 digestibility estimates using the DAISYII incubator with in vivo digestibility estimates in

215 horses. J Anim Sci 2010; 88: 3954-63.

SC
216 3. Rohweder DA, Barnes RF, Jorgensen N. Proposed hay grading standards based on laboratory

217 analyses for evaluating quality. J Anim Sci 1978; 47: 747-59.

218
U
4. Moore J, Undersander D. Relative Forage Quality: An alternative to relative feed value and
AN
219 quality index, in: Proc 13th Florida Rumininant Nutr Symp Gainesville FL 2002; pp 16-

220 31.
M

221 5. Martin-Rosset W, Andrieu J, Jestin M., Macheboef D, Andueza, D. Prediction of organic

D

222 matter digestibility of forages in horses using different chemical, biological and physical
TE

223 methods, in Saastamoinen, M., Fradinho, M.J., Santos, A.S., Miraglia, N. (Eds.), Forages

224 and Grazing in Horse Nutrition. Wageningen Academic Publishers, Wageningen,

EP

225 Netherlands, 2012. pp. 83-95.

226 6. Aiken GE, Potter GD, Conrad BE, Evans JW. 1989. Voluntary intake and digestion of Coastal
C

227 bermudagrass hay by yearling and mature horses. J Equine Vet Sci 1989; 9: 262-64.
AC

228 7. Bergero D, Miraglia N, Abba C, Polidori M. Apparent digestibility of Mediterranean forages

229 determined by total collection of faeces and acid-insoluble ash an internal marker. Livest.

230 Prod Sci 2004; 8: 235-38.

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231 8. Crozier JA, Allen VG, Jack NE, Fontenot JP, Cochran MA. Digestibility, apparent mineral

232 absorption, and voluntary intake by horses fed alfalfa, tall fescue, and Caucasian

233 bluestem. J Anim Sci 1997; 7: 1651-58.

234 9. Cuddeford D, Pearson RA, Archibald RF, Muirhead RH. Digestibility and gastrointestinal

PT
235 transit time of diets containing different proportions of alfalfa and oat straw given to

RI
236 Thoroughbreds, Shetland ponies, Highland ponies and donkeys. Brit Soc Anim Nutr

237 1995; 61: 407-17.

SC
238 10. Cymbaluk NF, Christensen DA. Nutrient utilization of pelleted and unpelleted forages by

239 ponies. Can J Anim Sci 1986; 66: 237-44.

240
U
11. Cymbaluk NF. Comparison of forage digestion by cattle and horses. Can J Anim Sci 1990;
AN
241 70: 601-10.

242 12. Drogoul C, Poncet C, Tisserand J. Feeding ground and pelleted hay rather than chopped hay
M

243 to ponies 1. Consequences for in vivo digestibility and rate of passage of digesta. Anim
D

244 Feed Sci Tech 2000; 87: 117-30.

TE

245 13. Dugan K, Pond K, Burns J, Fisher D. A comparison of intake and digestibility of Coastal

246 bermudagrass and flaccidgrass hays in horses, in: Proc 13th Equine Nutr Phys Sym
EP

247 Savoy, IL,1993. pp. 11

248 14. Eckert JV, Myer RO, Warren LK, Brendemuhl JH. Digestibility and nutrient retention of
C

249 perennial peanut and bermudagrass hays for mature horses. 2010; J Anim Sci 88:2055-
AC

250 61.

251 15. Holland JL, Kronfeld DS, Sklan D, Harris PA. Calculation of fecal kinetics in horses fed hay

252 or hay and concentrate. J Anim Sci. 1998; 76: 1937-44.

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253 16. Hussein HS, Vogedes LA, Fernandez GC, Frankeny RL. Effects of cereal grain

254 supplementation on apparent digestibility of nutrients and concentrations of fermentation

255 end-products in the feces and serum of horses consuming alfalfa cubes. J Anim Sci 2004;

256 82:1986-96.

PT
257 17. Lieb S, Mislevy P. Comparative intake and nutrient digestibility of three grass forages:

RI
258 Florakirk and Tifton 85 bermudagrass and florona stargrass to coastal bermudagrass, in:

259 Proc 17th Equine Nutr Phys Symp Lexington, KY, 2001; p. 390

SC
260 18. Miraglia N, Bergero D, Polidori M, Peiretti PG, Ladetto G. The effects of a new fibre-rich

261 concentrate on the digestibility of horse rations. Livest Sci 2006; 100:10-13.

262
U
19. Miyaji M, Ueda K, Kobayashi Y, Hata H, Kondo S. Fiber digestion in various segments of
AN
263 the hindgut of horses fed grass hay or silage. Anim Sci J 2008;79: 339-46.

264 20. Moore-Colyer MJS, Morrow HJ, Longland AC. Mathematical modeling of digesta passage
M

265 rate, mean retention time and in vivo apparent digestibility of two different lengths of hay
D

266 and big-bale grass silage in ponies. Brit J Nutr 2003; 90:109-18.
TE

267 21. Ordakowski-Burk AL, Quinn RW, Shellem TA, Vough LR. Voluntary intake and

268 digestibility of reed canarygrass and timothy hay fed to horses. J Anim Sci.2006;
EP

269 84:3104-09.

270 22. Ott E. Influence of level of feeding on digestion efficiency of the horse, in: Proc 7th Equine
C

271 Nutr Phys Symp Warrenton, VA, 1981; pp. 37-47.

AC

272 23. Pagan JD, Jackson SD. Digestibility of long stem alfalfa, pelleted alfalfa or an

273 alfalfa/bermuda straw blend pellet in horses, in: Proc. 12th Equine Nutr Phys Symp

274 Calgary, Alberta, Canada, 1991; pp. 29-32

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15

275 24. Pearson RA, Archibald R, Muirhead, RH. The effect of forage quality and level of feeding on

276 digestibility and gastrointestinal transit time of oat straw and alfalfa given to ponies and

277 donkeys. Brit J Nutr 2001; 85:599-06.

278 25. Pearson RA, Archibald RF, Muirhead RH. A comparison of the effect of forage type and

PT
279 level of feeding on the digestibility and gastrointestinal mean retention time of dry

RI
280 forages given to cattle, sheep, ponies and donkeys. British journal of nutrition.

281 2006;95(01):88-98.

SC
282 26. Potts LB, Hinkson JJ, Graham BC, Löest CA, Turner JL. Nitrogen retention and nutrient

283 digestibility in geldings fed grass or alfalfa hay or alfalfa cubes. Journal of Equine

284
U
Veterinary Science. 2009 May 31;29(5):487-8.
AN
285 27. Sponheimer M, Robinson T, Roeder B, Hammer J, Ayliffe L, Passey B, Cerling T, Dearing

286 D, Ehleringer J. Digestion and passage rates of grass hays by llamas, alpacas, goats,
M

287 rabbits, and horses. Small Ruminant Research. 2003 May 31;48(2):149-54.
D

288 28. Staniar WB, Bussard JR, Repard NM, Hall MH, Burk AO. Voluntary intake and digestibility
TE

289 of teff hay fed to horses. J Anim Sci 2010; 88:3296-03.

290 29. Sturgeon LS, Baker LA, Pipkin JL, Haliburton JC, Chirase NK. The digestibility and mineral
EP

291 availability of matua, bermudagrass, and alfalfa hay in mature horses. J Equine Vet Sci

292 2000;20: 45-48.

C

293 30. Thompson K, Jackson S, Baker J. Apparent digestion coefficients and associative effects of
AC

294 varying hay:grain rations fed to horses. Nutr Rep Int 1984; 30: 189-97.

295 31. Clauss M, Schiele K, Ortmann S, Fritz J, Codron D, Hummel J, Kienzle E, The effect of

296 very low food intake on digestive physiology and forage digestibility in horses. J Anim

297 Phys Anim Nutr 2014; 98:107-18.

 ACCEPTED MANUSCRIPT
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298 32. Jensen RB, Austbø D, Bach Knudsen KE, Tauson AH. The effect of dietary carbohydrate

299 composition on apparent total tract digestibility, feed mean retention time, nitrogen and

300 water balance in horses. Animal. 2014; 8: 1788-96.

301 33. Miyaji M, Ueda K, Hata H, Kondo S. Effect of grass hay intake on fiber digestion and

PT
302 digesta retention time in the hindgut of horses. J Anim Sci 2014; 92:1574-81.

RI
303 34. Undersander DJ, Mertens DR, Thiex NJ. Forage analyses procedures. National Forage

304 Testing Association, Omaha, NE. 1993.

SC
305 35. Carro MD, López S, Gonález JS, Ovejero FJ. Comparison of laboratory methods for

306 predicting digestibility of hay in sheep. Small Ruminant Research 1994; 14: 9-17.

307
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309 Table 1

310 Summary of studies used in regression analysis for determining the relationships between forage

311 chemical composition and in vivo forage digestibility.

Animal Fecal

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Study Description of Forage(s) n Gender
Type Collection (d)
Aiken et al.
Bermudagrass hay1 4 Horse Geldings 4
[6]
1
Bergero et al. Natural meadow hay

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4(5)a Horse Geldings 6
[7] Sainfoin hay1
Alfalfa hay1
Crozier et al.
Caucasian bluestem1 6 Horse Geldings 5
[8]
Tall fescue hay1

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Horses
Cuddeford et Molassed dehydrated alfalfa
8 and Geldings 7
al. [9] hay2
Ponies
Alfalfa hay1,3

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Cymbaluk
Barley hay3
and
Brome grass hay1 4 Pony Geldings 6
Christensen
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Oat hay1,3
[10]
Slough hay1
Alfalfa hay1,3
Altai wildrye hay1
Bromegrass hay1
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Cymbaluk
6 Pony Geldings 4
[11] Crested wheatgrass hay1
Kentucky bluegrass hay1
Reed canary grass hay1
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Drogoul et 50/50 Mix Lucerne and

10 Pony Geldings 6
al. [12] Cocksfoot2,3
Dugan et al. Coastal bermudagrass hay1
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4 Horse Mares 5
[13] Flaccidgrass hay1
Earing et al. Alfalfa hay4
4 Horse Geldings 5
[2] Timothy hay4
Coastal bermudagrass hay1
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Eckert et al.
Perennial peanut hay1 6(5)b Horse Geldings 4
[14]
Tifton- 85 bermudagrass hay1
Holland et al.
Alfalfa/orchardgrass mix hay1 4 Horse Geldings 10
[15]
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Hussein et al.
Alfalfa hay4 4 Horse Geldings 5
[16]
1
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Coastal bermudagrass
Lieb and Florakirk bermudagrass hay1
4 Horse Geldings 5
Mislevy[17] Florona stargrass hay1
Tifton-85 bermudagrass hay1
Miraglia et
Meadow hay1 4 Horse Geldings 6
al. [18]
Geldings
Miyaji et al.
Timothy hay1 3 Horse and 4
[19]
Mares
Moore-
Timothy/perennial ryegrass
Colyer et al. 4 Pony Geldings 7
hay1
[20]
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312 Table 1 (Continued)

Ordakowski-
Reed canary grass hay1 Timothy
Burk et al. 6(5)b Horse Geldings 4
hay1
[21]
Ott [22] Bermudagrass hay1 4 Horse Geldings 5
Alfalfa hay1,3
Pagan and

PT
Alfalfa/bermudagrass straw 4 Horse Geldings 5
Jackson [23]
blend3
Pearson et al. Molassed dehydrated alfalfa hay2
4 Pony Geldings 7
[24] Molassed dehydrated oat straw2

RI
Molassed dehydrated alfalfa hay2
Pearson et al. Barley straw2
4 Pony Geldings 7
[25] Mixed grass hay1
Mixed grass hay1

SC
Alfalfa hay1
Potts et al.
Alfalfa cubes4 6 Horse Geldings 4
[26]
Bermudagrass hay1
Sponheimer Smooth bromegrass hay1 Not
4 Horse 5

U
et al. [27] Bermudagrass hay1 reported
Staniar et al.
Teff hay1 6 Horse Mares 3
[28]
AN
Alfalfa hay1
Sturgeon et
Bermudagrass hay1 6 Horse Geldings 4
al. [29]
Matua hay1
Thompson et
Alfalfa hay1
M

4 Horse Geldings 5
al. [30]
1
313 Long stem forage
2
314 Chopped forage
D

3
315 Pelleted forage
4
316 Cubed forage
TE

a
317 One horse added to the study for a period
b
318 One horse removed from the study for a period
319
320
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321 Table 2

PT
322 Nutrient composition means, ranges, and dry matter digestibility from studies used for model development.

RI
Warm- Warm-
Cool-Season Cool-Season
Cereal Season Season Mixed
Variable Grass Legume All Forages
Forages1

SC
Grain hays Grass Legume
Forages Forages
Forages Forages
n 5 16 17 18 1 8 65

U
NDF % DM 73.8 ± 2.3 66.5 ± 1.4 43.4 ± 1.8 72.0 ± 1.9 46.0 59.3 ± 2.9 61.4 ± 1.7
Range 68.9 – 82.3 59.5 – 76.5 32.8 – 55.0 56.4 – 83.3 - 50.3 – 70.6 32.8 – 83.3

AN
n 6 18 19 18 1 8 70
ADF % DM 45.9 ± 2.6 39.4 ± 0.9 32.8 ± 1.0 37.5 ± 1.0 34.0 34.9 ± 1.6 37.1 ± 0.7

M
Range 34.4 – 52.4 33.2 – 45.9 25.4 – 43.0 28.9 – 43.0 - 30.3 – 40.9 25.4 – 52.4
n 6 18 18 18 1 8 69
CP % DM 5.8 ± 1.3 9.7 ± 0.7 17.4 ± 0.8 9.7 ± 0.7 11.0 13.9 ± 0.9 11.9 ± 0.6

D
Range 2.9 – 11.7 5.9 – 17.1 10.1 – 22.6 6.3 – 16.4 - 10.2 – 16.6 2.9 – 22.6

TE
n 5 16 17 18 1 8 65
HEM3 % DM 25.6 ± 1.3 27.6 ± 1.2 11.2 ± 1.3 34.5 ± 1.4 12.0 24.4 ± 1.6 24.4 ± 1.3
Range 22.8 – 29.9 17.8 – 36.5 3.3 – 22.0 19.6 – 43.3 - 198 – 311 3.3 – 43.3
EP
n 5 16 14 18 1 8 65
4
PER 65.3 ± 1.2 58.6 ± 1.3 75.2 ± 2 52.3 ± 1.3 73.9 58.9 ± 1.3 62.0 ± 1.3
C

Range 62.0 – 68.1 51.2 – 70.2 54.6 – 89.9 45.3 – 65.2 - 53.2 – 65.0 45.3 – 89.9
n 6 18 19 18 1 8 70
AC

DMD 43.0 ± 3.2 48.3 ± 1.3 62.5 ± 1.3 48.2 ± 1.5 65.0 55.7 ± 2.6 52.8 ± 1.1
Range 31.0 – 54.8 38.5 – 58.7 54.4 – 72.8 37.7 – 60.6 - 48.0 – 67.0 31.0 – 72.8
1
323 Mixed forages are diets consisting of two or more forage types.
2
324 Mean ± SE
3
325 HEM = NDF – ADF
4
326 PER = ADF/NDFx100
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327 Table 3

328 Study summary and forage nutrient composition for data sets not included in model development

329 and used to evaluate predictive ability of regression models.

Hay Nutrient Composition Animal Fecal

PT
Study n Gender
(DM basis) Type Collection (d)
Grass hay 1: CP 10.5, ADF 36.0,
Clauss et al. NDF 67.6 Geldings
4 Pony 5
[31] Grass hay 2: CP 5.8, ADF 38.6, and Mares

RI
NDF 69.5
Jensen et al. Timothy hay: CP 10.4, ADF 37.6,
4 Horse Geldings 5
[32] NDF 68.5

SC
Miyaji et al. Timothy hay: CP 11.6, ADF 39.0,
4 Horse Geldings 5
[33] NDF 70.3
330

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331 Table 4

332 Simple linear regression models using forage composition to predict forage digestibility.

Root
Modela P-Value r2 Adj. R2
MSE

PT
DMD = 85.42 – 0.5324×NDF < 0.0001 5.966 0.6017 0.5954

DMD = 35.85+ 1.432×CP < 0.0001 6.411 0.5402 0.5329

RI
DMD = 93.38 – 1.100×ADF < 0.0001 6.919 0.4643 0.4558

SC
DMD = 67.27 – 0.5938×HEM < 0.0001 7.196 0.4206 0.4114

DMD = 24.72 + 0.4523×PER < 0.0001 8.088 0.2680 0.2564

a
333 Forage composition factors included NDF, ADF, and CP concentrations (DM basis) and

U
334 calculated values HEM (HEM = NDF – ADF) and PER (PER = ADF/NDF×100).
335
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336 Table 5

337 Multiple linear regression models using forage composition to predict forage digestibility.

Root
Modela P-Value R2 Adj. R2
MSE
DMD = 56.16 + 0.6080×CP –

PT
< 0.0001 5.407 0.6833 0.6677
0.7163×ADF – 0.2560×PER
DMD = 72.85 + 0.6501×CP –
< 0.0001 5.409 0.6830 0.6674
0.5661×ADF – 0.2808×HEM

RI
DMD = 72.85 + 0.6501×CP –
< 0.0001 5.409 0.6830 0.6674
0.2808×NDF – 0.2853×ADFb
DMD = 72.85 + 0.6501×CP –

SC
< 0.0001 5.409 0.6830 0.6674
0.5661×NDF + 0.2853×HEMb
DMD = 80.14 + 0.6931×CP –
< 0.0001 5.444 0.6789 0.6631
0.4412×NDF – 0.1370×PERb

U
DMD = 63.95 + 0.6214×CP –
0.1331×NDFb – 0.5141×ADFb + < 0.0001 5.444 0.6842 0.6631
0.1391×PERb
AN
DMD = 65.81 + 0.7207×CP –
< 0.0001 5.483 0.6690 0.6583
0.3514×NDF
DMD = 66.58 – 0.9966×ADF +
M

< 0.0001 5.716 0.6402 0.6286

0.3707×PER
DMD = 66.14 – 1.000×ADF +
< 0.0001 5.763 0.6402 0.6225
0.00725×HEMb + 0.3769×PERb
D

DMD = 66.14 + 0.00725×NDF –

< 0.0001 5.763 0.6402 0.6225
1.007×ADF + 0.3769×PERb
TE

DMD = 66.14 – 1.000×NDFb –

< 0.0001 5.763 0.6402 0.6225
1.007×HEM + 0.3769×PERb
DMD = 92.62 – 0.8092×ADF –
< 0.0001 5.791 0.6307 0.6188
EP

0.4084×HEM
DMD = 92.62 – 0.4009×ADF –
< 0.0001 5.791 0.6307 0.6188
0.4084×NDF
DMD = 92.62 – 0.8092×NDF +
C

< 0.0001 5.791 0.6307 0.6188

0.4009×HEM
DMD = 62.94 – 0.5921×ADF +
AC

< 0.0001 5.8515 0.6230 0.6108

0.9890×CP
DMD = 102.4 – 0.6357×NDF –
< 0.0001 5.895 0.6173 0.6050
0.1709×PERb
DMD = 47.58 + 1.053×CP –
< 0.0001 5.970 0.6075 0.5949
0.2970×HEM
DMD = 27.47 + 1.238×CP + 0.1720×PER < 0.0001 6.256 0.5690 0.5551
DMD = 128.7 – 1.298×HEM –
< 0.0001 6.762 0.4964 0.4802
0.7138×PER
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a
338 Forage composition factors included NDF, ADF, and CP concentrations (DM basis) and
339 calculated values HEM (HEM = NDF – ADF) and PER (PER = ADF/NDF×100).
b
340 Variable not significant (P>0.1)
341

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342 Fig. 1. Influence of neutral detergent fiber (NDF) concentration on dry matter digestibility

343 (DMD; DMD = 85.42 – 0.5324*NDF; r2 = 0.6017).

80
70

PT
60
50

RI
DMD (%)

40

SC
30
20

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10
0
AN
0 20 40 60 80 100
NDF (DM basis)
344
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345 Fig. 2. Influence of acid detergent fiber (ADF) concentration on dry matter digestibility (DMD;
346 DMD = 93.38 – 1.100*ADF; r2 = 0.4643).
80
70
60

PT
50
DMD (%)

40 DMD = 93.38 - 1.100×ADF

RI
r² = 0.4643
30
20

SC
10
0

U
0 10 20 30 40 50 60
AN
NDF (DM basis)
347
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348 Fig. 3. Comparison of regression model predicted and observed digestibility for independent data

349 sets. Observed error bars show SEM and predicted error bars show 95% confidence interval for

350 each hay.

PT
Observed CP,NDF CP,ADF,PER CP,ADF,HEM
60

RI
50
Digestibility (%)

40

SC
30

20

U
10
AN
0
Grass Hay 2 [31] Grass Hay 2 [31] Timothy Hay [32] Timothy Hay [33]
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