Using Chemical and Biological Approaches to Predict Energy Values of Selected Forages Affected by Variety and Maturity Stage : Comparison of Three Approaches

Two varieties of alfalfa (Medicago sativa L cv. Pioneer and Beaver) and timothy (Phleum pratense L cv. Climax and Joliette), grown at different locations in Saskatchewan (Canada), were cut at three stages [1=one week before commercial cut (early bud for alfalfa; joint for timothy); 2=at commercial cut (late bud for alfalfa; pre-bloom head for timothy); 3=one week after commercial cut (early bloom for alfalfa; full head for timothy)]. The energy values of forages were determined using three approaches, including chemical (NRC 2001 formula) and biological approaches (standard in vitro and in situ assay). The objectives of this study were to determine the effects of forage variety and stage of maturity on energy values under the climate conditions of western Canada, and to investigate relationship between chemical (NRC 2001 formula) approach and biological approaches (in vitro and in situ assay) on prediction of energy values. The results showed that, in general, forage species (alfalfa vs. timothy) and cutting stage had profound impacts, but the varieties within each species (Pioneer vs. Beaver in alfalfa; Climax vs. Joliette in timothy) had minimal effects on energy values. As forage maturity increased, the energy contents behaved in a quadratic fashion, increasing at stage 2 and then significantly decreasing at stage 3. However, the prediction methods-chemical approach (NRC 2001 formula) and biological approaches (in vitro and in situ assay) had great influences on energy values. The highest predicted energy values were found by using the in situ approach, the lowest prediction value by using the NRC 2001 formula, and the intermediate values by the in vitro approach. The in situ results may be most accurate because it is closest to simulate animal condition. The energy values measured by biological approaches are not predictable by the chemical approach in this study, indicating that a refinement is needed in accurately predicting energy values. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 228-236)


INTRODUCTION
Alfalfa (Medicago sativa L.) and timothy (Phleum pratense L.) are two important forages grown in western Canada for export.Among varieties, Pioneer and Beaver alfalfa and Climax and Joliette timothy are well adapted to climate and soil conditions of the region (Canadian Dehydrator's Association, 1990;Canadian Hay Association, 1999).However, the detailed nutritive values for these varieties affected by cutting stages are lacking.In NRC dairy (2001), there are no energy values for timothy and no effect of varieties and cutting stage on energy values for alfalfa.To produce top quality alfalfa and timothy, the need for this information is crucial.
In NRC dairy (2001), a summative approach is used to derive total digestible nutrients at 1× maintenance (TDN1x).In this approach, the concentrations (% DM) of truly digestible nutrients for each feed are estimated (Weiss et al., 1992) according to the equations in NRC dairy (2001).This is a standard method, also called chemical approach-which means using all chemical compositions to estimate energy values.However, the TDN1x value could also be obtained using biological approaches to the some extent.The truly digestible neutral detergent fibre (tdNDF) value could be determined by using a 48 h rumen in vitro (NRC, 2001) or in situ assay.
The objectives of this study were: (1) to provide detailed information on energy values for each variety of alfalfa and timothy cut at different maturity stages for Canadian forage export industry; and (2) to investigate relationship between the chemical (NRC 2001 formula) and the biological approaches (in vitro and in situ assay) on prediction of energy values.

Forages
The samples of the two varieties and three cutting times

Using Chemical and Biological Approaches to Predict Energy Values of Selected Forages Affected by Variety and Maturity Stage: Comparison of Three Approaches
(1=one week before the commercial cut: early bud for alfalfa varieties and joint for timothy varieties; 2=at the commercial cutting stage: late bud for alfalfa varieties and pre-bloom head for timothy varieties; 3=one week after the commercial cut: early bloom for alfalfa varieties and full head for timothy varieties) of alfalfa (Pioneer and Beaver) and timothy (Climax and Joliette), grown at different locations (each) in Saskatchewan in year 2001, were obtained from Elcan Forage Inc. (Saskatchewan, Canada).The detailed locations, growth and climate conditions, estimated maturity stages and sampling procedure were previously described by Yu et al. (2003a,b).

Rumen in vitro and in situ assay
Rumen in vitro digestibility NDF of the forages samples (ground through a 2 mm screen by Hammer Mill) after 48 h incubation was determined using the standard Tilley and Terry in vitro procedure (Marten and Barnes, 1980).
Rumen in situ digestibility of NDF of the forages samples (ground through a 2 mm screen by Hammer Mill) after a 48 h incubation were determined using the departmental standard in situ procedure in dairy cows (McKinnon et al., 1995) with polyester bags (10×20 cm; pore size of 53±10 µm, ANKOM Company, Fairport, NY).

Chemical analysis
The forage samples (ground through 1 mm screen by Retsch ZM-1) were analyzed for DM, ash, EE and N (AOAC, 1990).Acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) were analyzed according to the procedures of Van Soest et al. (1991).Acid detergent insoluble N (ADIN) and neutral detergent insoluble N (NDIN) were determined according to the procedures of Licitra et al. (1996).

Estimation of energy values
Gross energy (GE) values were measured, using a Parr adiabatic bomb calorimeter (Model 1200, Parr Instrument Co., Moline, IL).The TDN 1X , DE 1X (=digestible energy at 1×maintenance), DE p , ME p (=digestible energy at production level) and NE Lp (=net energy at production level) values were estimated from the NRC dairy (2001).The ME beef96 (=metabolizable energy), NE m beef96 (=net energy for maintenance) and NE g beef96 (=net energy for growth) values were estimated from the NRC beef (1996).The tdNDF content was calculated separately using three approaches as follows: 1) Using NRC ( 2001) formula (standard method)chemical approach:  where, IVNDFD stands for in vitro digestibility of NDF at a 48 incubation.
3) Using standard in situ approach-biological approach II: where, ISNDFD stands for in situ digestibility of NDF at a 48 incubation.

Statistical analysis
Statistical analyses were performed using the GLM procedure of SAS (1991).The model used for the analysis was: Y jk(i) =µ+F i +V j (a) +V j (t) +M k (a) +M k (t) +e ijk Where, Y kl(i) is an observation of the dependent variable for the variety j at maturity k in the forage i; µ is the population mean for the variable; F i is the forage type i, i=a, t; a is alfalfa, and t is timothy; V j (a) is the effect of forage variety nested within alfalfa; V j (t) is the effect of forage variety nested within timothy; M k (a) is the effect of forage maturity nested within alfalfa; M k (t) is the effect of forage variety nested within timothy; and e ijk is the random error associated with the observation jk(i).Contrasts were used to determine treatment differences (SAS, 1991).Relationships between chemical approach and biological approaches on predicted energy values were also evaluated by correlation and regression analysis with the GLM procedure of SAS (1991).Significance was declared at p<0.05.

Estimating energy values of alfalfa and timothy using chemical and biological approaches
Using NRC 2001 formula-chemical approach : The normal method to estimate energy value for a feed is to use NRC ( 2001) equations.This is also called a chemical approach, which is to use all chemical compositions to estimate energy values.The effects of variety and cutting stage on energy content of alfalfa and timothy using this approach are presented in Table 2.The results indicated that across all treatments, there were no significant differences (p>0.05) between the two forage species (alfalfa vs. timothy) for all estimated energy values (   There were no differences (p>0.05) between alfalfa (Pioneer vs. Beaver) and between timothy varieties (Climax vs. Joliette) for all estimated energy values.
However, the stage of cutting, in both forages, had significant effects on energy values.In alfalfa, energy values in stage 1, 2 and 3 were significantly different (p<0.05).The energy values at stage 2 were higher than that at stage 1, but as alfalfa advanced to stage 3, all energy values significantly declined (p<0.05).The highest energy values occurred at stage 2 and the lowest at stage 3. Similar results were found in timothy.Although, stage 1 and 2 were not different (p>0.05) for all estimated energy values.As maturity further increased to stage 3, the energy values of timothy were decreased (p<0.05).The above results indicated that as alfalfa and timothy maturity increased, the energy contents behaved in a quadratic fashion, increasing at stage 2 and then significantly decreasing at stage 3.
Using in vitro assay-biological approach I : Using a biological approach-in vitro assay, the effects of variety and cutting stage on energy content of alfalfa and timothy are presented in Table 3.The results indicated that across all treatments, there were significant differences (p<0.01) between the two forage species (alfalfa vs. timothy) for all estimated energy values (TDN 1X : 60 vs. 46% DM; DE 1X : 2.76 vs. 1.92;DE p : 2.54 vs. 1.76;ME p : 2.12 vs. 1.33;NE Lp : 1.30 vs. 0.74; ME beef96 : 2.26 vs. 1.57;NE m beef96 : 1.39 vs. 0.73; NE g beef96 : 0.81 vs. 0.20 Mcal/kg DM).These results are completely different from that estimated by NRC 2001 formula-chemical approach, in which no significant differences were found between alfalfa and timothy.The higher energy content in alfalfa species corresponds to higher IVNDFD48 in alfalfa (41 vs. 35%) (Table 5).The estimated energy content for alfalfa in this approach was slightly higher than the tabular values in the NRC (2001).

The energy contents for timothy have no tabulated values recorded in NRC (2001).
There were no differences (p>0.05) between alfalfa varieties (Pioneer vs. Beaver), but significant differences (p<0.05) between timothy varieties (Climax vs. Joliette) for all estimated energy values (   The stage of cutting, in both forages, had significant effects on energy values.In alfalfa, energy values in stage 1 and 2 were not different (p>0.05),although the energy values at stage 2 were numerically higher than that at stage 1, but as alfalfa advanced to stage 3, all energy values significantly declined (p<0.05).Similar results were found in timothy.Stage 1 and 2 were not different (p>0.05) for all estimated energy values.As maturity further increased to stage 3, the energy values of timothy were decreased (p<0.05)(for example, TDN 1x : 42.3, 45.3, 40.7% for timothy cut at stage 1, 2 and 3, respectively).The above results again indicated that as alfalfa and timothy maturity increased, the energy contents behaved in a quadratic fashion, increasing at stage 2 and then significantly decreasing at stage 3.
Using in situ assay-biological approach II : Using the in situ assay -biological approach II, the effects of variety and cutting stage on energy content of alfalfa and timothy are in Table 4.It was found that across all treatments, there were significant differences (p<0.01) between the two forage species (alfalfa vs. timothy) for all estimated energy values (TDN 1X : 64 vs. 55% DM; DE 1X : 2.91 vs. 2.43; DE p : 2.67 vs. 2.23; ME p : 2.25 vs. 1.80;NE Lp : 1.39 vs. 1.08;ME beef96 : 2.39 vs. 1.99;NE m beef96 : 1.51 vs. 1.14;NE g beef96 : 0.91 vs. 0.58 Mcal/kg DM).These results are also different from that estimated by NRC 2001 formula-chemical approach, in which no significant difference was found between alfalfa and timothy.
There were no differences (p>0.05) between alfalfa varieties (Pioneer vs. Beaver) and between timothy (Climax vs. Joliette) for all estimated energy values.However, the stage of cutting, in both forages, had significant effects on energy values.In alfalfa, energy values in stage 1 and 2 were similar, but as alfalfa advanced to stage 3, all energy values significantly declined (p<0.05).Similar results were found in timothy.Stage 1 and 2 were not different (p>0.05) for all estimated energy values.As maturity further increased to stage 3, the energy values of timothy were decreased (p<0.05).
In general, using three approaches-no matter chemical approach or biological approaches, all detected that the cutting stage of alfalfa and timothy had significant effect on energy values, which were decreased with increasing maturity stage to 3 (Table 2, 3 and 4).However, using the  NRC 2001 formula, the energy values were estimated no significant differences between the two species (alfalfa and timothy) (Table 2).However, this was not the case when using biological approaches-in vitro and in situ assay, which detected significant differences between two species (Table 3 and 4).Using NRC 2001 formula, the energy values were not different between the varieties within each forage species (Table 2).These results were in agreement with the in situ results, but were in disagreement with the in vitro results (Table 3).

Comparisons of digestibility and total digestible NDF obtained from chemical and biological approaches
Table 5 presents the results of digestibility of NDF and total digestible NDF (tdNDF) obtained from three methods.Using NRC 2001 formula, the estimated digestibility of NDF and tdNDF were higher (p<0.01) in timothy than in alfalfa.However, using biological approach-in situ assay, the digestibility of NDF of alfalfa and timothy were not significantly different (p>0.05).Using in vitro assay, the digestibility was higher in alfalfa (p<0.05).The results from three methods were not in agreement with each other.Using biological approaches-in vitro and in situ assay, the SEM for the digestibility was higher, compared with that using NRC 2001 formula (2.9 and 2.8 vs. 1.6).
The tdNDF obtained using three approaches were also different between the methods (Table 5).For alfalfa, the highest tdNDF was measured by the in situ assay (24% DM), followed by in vitro assay (20% DM).The lowest tdNDF was estimated by NRC 2001 formula (15% DM).For timothy, the highest tdNDF was estimated by in situ method (38% DM), followed by NRC formula method (36% DM).The lowest tdNDF was by the in vitro assay (24% DM).
The Pearson correlation coefficients (R) between chemical approach (NRC 2001 formula) and biological approaches (in vitro and in situ assay) on digestibility of NDF, total digestible NDF and TDN1x are presented in Table 6.The R values between NRC 2001 formula and in vitro and in situ on digestibility of NDF and tdNDF were >0.65.However, the R values between NRC 2001 formula and in vitro or in situ method on TDN1x were lower (0.03 and 0.34, respectively).

Relationship of energy values predicted from chemical and biological approaches
Regression was used to evaluate the relationship between TDN1x values estimated from NRC 2001 formula and in vitro or in situ methods.Statistical analysis results show that: When not considering feed species (alfalfa and timothy) as a covariate in estimated TDN1x, the relationship was very poor (with low R 2 <0.15).The results are following: 1) TND1x In vitro =43.25 (±55.35,p>0.05)+TDN1xNRC formula ×0.15 (±0.99, p>0.05) Where, P=0.8834, RSD=9.98,R 2 =0.001.

2) TND1x
In situ =-0.23 (±35.22,p>0.05)+TDN1xNRC formula ×1.07 (±0.63, p>0.05) Where, P = 0.1044, RSD=6.35,R 2 =0.12However, when considering feed species (alfalfa and timothy) as a covariate, the relationship was improved with very high R 2 >0.70.The predictive equations were: 1) TND1x  The above results indicate that forage type has profound impact on predictability of energy values of between NRC 2001 formula and the in situ or in vitro methods.This means that the energy values from the biological approaches were not predictable by chemical approach.
The NRC formula method is to use chemical compositions of a feed to estimate total digestible nutrients and energy values in animals.In vitro and in situ methods, to some extent, are biological methods.They actually measure total digestible nutrients of a feed, not by estimation.Therefore, biological approaches are more accurate to determine energy values for a feed.Compared in vitro and in situ results, it is not surprised to find in situ results were higher than in vitro results.This is because that in situ method is closest to simulate animal condition.
However, it needs to be mentioned that in this study only two forage species with two different varieties cut at three different times were evaluated.To make a final conclusion, large feed samples with a wide range of chemical composition are needed to be evaluated by these approaches: chemical and biological.

CONCLUSION
In general, forage species and cutting stage had profound impacts, but the varieties (Pioneer vs. Beaver; Climax vs. Joliette) had minimal effects on energy values.However, the prediction methods-chemical approach (NRC 2001 formula) and biological approaches (in vitro and in situ assay) had great influences on energy values.The highest prediction values were found by using in situ approach, the lowest prediction value by using NRC formula, and the intermediate values by in vitro approach.The in situ results may be most accurate because it is closest to simulating animal condition.Regression analysis show that the energy values measured by biological approaches are not predictable by chemical approach, indicating that a refinement is needed in accurately predicting energy values.

Table 1 .
Effect of variety and cutting stage on chemical composition of alfalfa and timothy

Table 3 .
Energy values of the forages (alfalfa and timothy varieties cut at three stages) estimated using in vitro assay

Table 4 .
Energy values of the forages (alfalfa and timothy varieties cut at three stages) estimated using in situ assay

Table 5 .
Comparisons of digestibility of NDF and total digestible NDF (tdNDF) obtained from three methods: NRC 2001 formula; in vitro and in situ assay The results indicate that the chemical or biological methods had great influences on estimation of energy values.Comparison of three approaches, the highest prediction values were found by using in situ approach and the lowest prediction values by using NRC 2001 formula, the intermediate values by in vitro approach (with total