NDF degradability of hays measured in situ and in vitro

Animal Feed Science and Technology 104 (2003) 201–208

Short communication

NDF degradability of hays measured in situ and in vitro M. Spanghero a,∗ , S. Boccalon a , L. Gracco a , L. Gruber b a

Department of Animal Production Science, Udine University, Via S. Mauro, 2 Pagnacco 33010, Italy b Federal Research Institute for Agriculture in Alpine Regions, Gumpenstein, Austria Received 10 June 2002; received in revised form 20 November 2002; accepted 20 November 2002

Abstract The NDF degradability (dNDF ) of hays measured by an in situ method (nylon bag technique) was compared with in vitro fermentation, using the DaisyII incubator (Ankom® , Tech. Co., Fairport, NY, USA). Eighteen hays were produced from mountain areas (about 700 m.s.l.) from plots subjected to different cutting frequencies (two to four cuts per season) and types of N fertilisation (slurry and slurry plus mineral). Hay samples from each cut were incubated (in situ) in the rumens of three cows (incubation times: 2, 4, 8, 16, 24, 48 and 72 h; sample weight: 15 mg/cm2 of free bag area) or inserted (in vitro) into three different digestion jars (jar volume: 2 l; bag size 5 cm × 3 cm; sample weight: 250 mg per bag) that were placed into the DaisyII incubator for 48 h. The forages showed large variation in effective dNDF (assumed rumen turn over rate: 3% per hour) which ranged from 38 to 43% for hays with two cuts to 58–65% for the hays with four cuts per season. The dNDF obtained in situ (Y, effective) and in vitro (X, after 48 h of incubation) were highly correlated (P < 0.01) and the regression equation was Y = 0.74(±0.05)X + 6.39(±2.90),

n = 18,

S.D. = ± 1.96,

r 2 = 0.94.

The variability (coefficient of variation, CV) of the in vitro measurements (among jar repeatability) was 2.8%, which is close to that generally found for some chemical analyses of feedstuffs and lower than that obtained for the in situ measures (among cow repeatability, CV: 3.7%). The DaisyII incubator produces repeatable in vitro dNDF data which are highly related to those obtainable with the reference in situ procedure. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Degradability; In situ; In vitro; NDF; Hays

Abbreviations: dNDF , NDF degradability Corresponding author. Tel.: +39-0432-650110; fax: +39-0432-660614. E-mail address: [email protected] (M. Spanghero). ∗

0377-8401/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0377-8401(02)00327-9

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1. Introduction The NDF degradability (dNDF ) of roughages is an essential parameter in predicting their energetic value. The cell wall components in these feedstuffs are the main nutritive constituents and the extent of their rumen degradation is the main factor that influences the energetic value (NRC, 2001). Moreover, dNDF has been used in models to estimate the physical fill of fibrous feeds in the rumen (Weisbjerg et al., 1990; Madsen and Hvelplund, 1994; Stensig et al., 1994b) and, therefore, the intake capacity of animals. Rumen degradability has been measured in several laboratories by the in situ nylon bag technique. This method requires the incubation of bags containing test feeds in several rumen cannulated animals fed standardised diets for appropriate time intervals. Despite efforts to harmonise the procedure, the technique still has a low reproducibility (Madsen and Hvelplund, 1994). Therefore, the method is not utilised for routine analyses in commercial laboratories. Recently, a simpler in vitro incubation technique to measure the dNDF has been introduced (DaisyII incubator, Ankom® , Tech. Co., Fairport, NY, USA). This methodology allows simultaneous incubation of a large number of samples (for a maximum of 96 bags per fermentation batch) and the degradability data obtained compare favourably with those acquired with the conventional Tilley and Terry’s (1963) method and the in situ technique (Robinson et al., 1999; Mabjeesh et al., 2000; Wilman and Adesogan, 2000). An important limitation is associated with the usage of rumen inoculum, but it has been demonstrated (Robinson et al., 1999) that rumen liquid can be safely stored for short periods (up to 48 h) to be sent to laboratories that lack donor animals. This trial had the objective to compare the dNDF of hays obtained after fermentation in the DaisyII incubator with an in situ method.

2. Material and methods 2.1. Origin of hay samples and chemical analyses Eighteen hay samples were provided by the Federal Research Institute for Agriculture in Alpine Regions (Gumpenstein, Austria) and were part of a research programme (Gruber et al., 1999) to evaluate effects of cutting frequency (two to four cuts per season) and type of N application (slurry and slurry plus mineral) on yield and quality of hay produced in mountain areas (about 700 m.s.l.). The forages grown in 1997 were used in this trial. Part of each sample was milled (Cyclotech, Tecator, 1 mm screen) and analysed for dry matter, ash, nitrogen, ether extract (methods 930.15, 942.05, 976.05 and 954.02, respectively of the Association of Official Analytical Chemists, 1998) and for the NDF and ADF content (Van Soest et al., 1991) using the Ankom apparatus (Ankom® , Tech. Co., Fairport, NY, USA). For the NDF analysis, samples were not treated with ␣-amylase, ND solution contained Na sulfite and residues were not corrected for residual ash.

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2.2. In situ trial Samples used for incubation were coarsely ground (4 mm screen) and 2.1–2.2 g was inserted into polyester bags (approximately 15 mg/cm2 of free bag area; porosity: 40 ␮m) which were incubated in the rumen of three non-lactating rumen-cannulated cows for 2, 4, 8, 16, 24, 48 and 72 h (two bags/incubation time for each cow). The cows were fed a diet composed of hay (Festuca Arundinacea, 6 kg per day) and a commercial compound feed (3 kg per day, 180 g CP/kg). After incubation, bags were immediately rinsed (cold rinse cycle in a washing machine for about 15 min), dried (48 h at 60 ◦ C) and weighed. Bag residues of the same forage were then pooled for each time of incubation and analysed for NDF with the procedure and apparatus used for forages. The trial lasted 6 weeks with three forages tested per week. 2.3. In vitro trial Samples used for in vitro incubation were finely ground (1 mm screen) and 250 mg was inserted in filter bags (size 5 cm × 3 cm) and sealed. Three digestion jars (2 l capacity) were filled with pre-warmed (39 ◦ C) buffer solutions (1330 ml of solution A: KH2 PO4 10 g/l, MgSO4 ·7H2 O 0.5 g/l, NaCl 0.5 g/l, CaCl2 ·2H2 O 0.1 g/l, Urea 0.5 g/l; 266 ml of solution B: Na2 CO3 15.0 g/l, Na2 S·9H2 O 1.0 g/l) and placed into DaisyII incubator following the procedure described in detail by Robinson et al. (1999). Rumen liquor was collected from two rumen cannulated cows fed the same diet as those used for the in situ incubations and 400 ml of filtered rumen liquor was introduced in each jar. For each hay, three bags were placed in three different jars. After 48 h of incubation, bags were removed and rinsed thoroughly with cold tap water and immediately analysed for NDF content with the procedure and apparatus used for forages. 2.4. Degradability data interpolation and statistical analysis The in situ degradability data were fitted (PROC NLIN SAS, 1999) to the following single exponential model with an initial lag time in degradation (McDonald, 1981; Dhanoa, 1988): D = A + B × [1 − exp−C(t−L) ] where D is the potential degradability, A the readily degradable fraction, B the potentially degradable fraction, C (h−1 ) the degradation rate constant, t (h) the incubation time and L (h) is the lag time. The iterative procedure adopted the following assumptions: L = t when t ≤ L, and L = L when t > L. The effective degradability (ED) has been calculated at a rumen passage rate (K) of 0.03 per hour, as follows:
 B ×C ED = A + × exp−KL C+K The effective in situ and the in vitro dNDF data obtained from each forage after incubation in different cows or fermentation jars, were statistically analysed (SAS, 1999; PROC GLM)

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with the monofactorial linear model: y = µ + αi + ε where µ is the overall mean, α fixed effect of different forage types (18 combinations obtained from different N fertilisations, cutting frequencies and number of cut, i = 1, 18), ε the √ residual error. The repeatability standard deviation (among cows or jars) was calculated as ε.

3. Results and discussion The hays showed a large variability in chemical composition (Table 1) mainly due to different cutting frequencies and cutting numbers. Increasing the number of cuts per season allows grasses to be harvested at an earlier growth stage, and this affects the chemical composition and improves the nutritional value of the hays (Gruber et al., 1999). The CP content increased, from 76 to 166 g/kg DM, and the fibre content decreased from 667 to 470 g NDF/kg DM, comparing hays with two to those having four cuts per season. Also the ash content increased with higher cutting frequency because soil contamination is more pronounced in forages with high leaf: stem ratios and because leaves contain more minerals than stems (Minson, 1990). Within each cutting frequency, the first cut hay obtained after the spring growth generally had lower CP and higher NDF contents, with respect to the other cuts. These chemical modifications are similar to those recorded on a set of forages grown 2 years before on the same permanent grassland (Spanghero et al., 1999), with the only exception being the lower CP content and a slightly higher NDF content of hays used in present experiment. Table 1 also shows the kinetic rumen degradation parameters and effective degradability of NDF obtained in situ. In general, the hays had a low soluble fraction (<9%, excluding two hays) and a short lag time (<0.7 h). The potentially degradable NDF fraction was about 65% for the hays with two cuts per year and 70–80% in hays with more cuts per year. The rate of degradation increased with higher cutting frequency (3.6–4.8, 5.4–7.7 and 5.8–8.3% per hour, for hays with to two to four cuts per year). A rumen turn over rate of 3% per hour was used to calculate the effective degradability of NDF. Such a value has been adopted as an intermediate rate after examining experimental values obtained from cows after measurements of rumen evacuation (1.3–2.0% per hour, Stensig et al., 1994a; 2.1–3.4% per hour, Robinson et al., 1987; 1.8–2.3% per hour, Stensig and Robinson, 1997) or duodenal decline of a single dose of chromium mordanted forage (3.3–4.0% per hour, Robinson et al., 1987; 2.4–4.0% per hour, Stensig et al., 1994a). The effective degradability had low values, of 38–43%, for the two cuts per year hays, intermediate values (51–59%) for three cut hays and the highest values for those with four cuts (58–65%). The in vitro dNDF data, obtained after 48 h of fermentation in the DaisyII incubator, showed the same tendency with low (44–52%), intermediate (59–66%) and high (67–79%) values for hays cut two to four times per season, respectively.

Table 1 Chemical composition, in situ and in vitro NDF rumen degradability of hays Cutting frequency

Cut order

First

2

Second

3

First

3

Second

3

Third

4

First

4

Second

4

Third

4

Fourth

DM (g/kg)

Chemical composition (DM (g/kg)) CP

Ash

EE

NDF

ADF

A (%)

b

In vitro NDF degradability

B (%)

C (% per hour)

L (h)

Effective degradabilityb

1 2 1 2

921 924 914 923

78 76 104 92

57 54 66 67

14 16 23 23

657 667 582 564

412 406 330 319

2.0 4.7 1.3 3.3

61.7 65.6 66.5 66.2

4.3 3.6 4.8 4.5

0.58 0.48 0.08 0.28

37.6 40.1 41.7 42.6

44.0 44.3 47.8 51.9

1 2 1 2 1 2

918 924 919 921 903 910

123 120 120 112 146 133

69 73 87 84 91 96

19 21 22 28 25 28

608 602 589 570 470 491

354 364 329 326 272 279

0.0 0.1 3.9 4.0 3.8 8.8

77.7 78.5 73.7 72.8 76.3 72.5

6.0 5.7 5.4 6.9 7.7 6.4

0.55 0.27 0.01 0.08 0.01 0.27

51.0 51.1 51.4 54.7 58.6 57.7

64.8 58.5 60.4 62.6 66.5 65.9

1 2 1 2 1 2 1 2

909 905 906 910 911 913 904 910

137 134 164 152 139 125 164 166

85 90 101 102 93 94 95 123

24 18 24 13 20 20 25 21

579 570 527 547 542 517 472 526

334 338 259 271 267 263 233 280

4.1 2.1 5.8 15.7 8.7 4.9 0.7 11.4

79.8 80.0 77.6 69.0 75.8 79.2 80.6 67.2

7.0 7.0 8.3 7.8 5.8 6.9 7.9 7.2

0.45 0.32 0.21 0.68 0.39 0.12 0.22 0.24

58.8 57.6 62.2 64.7 57.8 59.3 58.3 58.4

69.3 68.0 75.2 78.5 72.9 71.4 74.1 67.3

53.5 8.05

63.5 10.51

1.96

1.79

Average Standard deviation (S.D.) Residual S.D. a

In situ NDF degradability parameters

M. Spanghero et al. / Animal Feed Science and Technology 104 (2003) 201–208

2

Fertilisationa

1: slurry; 2: slurry plus mineral N. Calculated assuming a ruminal outflow rate of 3.0 % per hour. 205

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Fig. 1. Relationships between effective in situ degradability (y) and content (䊐, - - -) or in vitro degradability ( , —) of hay NDF.

The dNDF obtained in situ (Y, effective) and in vitro (X, after 48 h of incubation) were highly correlated (P < 0.01, Fig. 1) and the regression equation was as follows: Y = 0.74(±0.05)X + 6.39(±2.90),

n = 18,

S.D. = ±1.96,

r 2 = 0.94

On the contrary, the NDF content of hays accounted for only about half of the variability of the in situ degradability (r 2 = 0.53, Fig. 1). The DaisyII incubator has been examined recently. For example, Wilman and Adesogan (2000) obtained a good correlation (r 2 = 0.92–0.95) with the true DM digestibility measured with the conventional Tilley and Terry’s (1963) method (TT) for 72 combinations of forage species, plant parts, milling sizes and field replications. Mabjeesh et al. (2000) performed the same comparison (DaisyII incubator versus TT method) on 17 concentrate ingredients and obtained a satisfactory relationship (r 2 = 0.81), even though the DaisyII incubator gave higher values for some energy concentrates and protein supplements. The comparison between dNDF measured in situ and with the DaisyII incubator is limited to the work of Robinson et al. (1999), who reported an overestimation of the in situ data over the in vitro degradation after 48 h of incubation (77.4 versus 72.2% NDF, as averages of eight feeds). The present research confirms this finding since the in vitro data were, on average (Table 1), about 19% higher than the calculated effective dNDF . Mabjeesh et al. (2000) reported satisfactory precision of the technique (higher than that of TT method), while the contrary was found by Wilman and Adesogan (2000). In the present experiment, the repeatability of the measurements (among jar coefficient of variation: 2.8%) was satisfactory and similar to that generally found for other chemical analysis (e.g. NDF;

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Bovera et al., submitted for publication). For in situ effective degradability, we only found a slightly higher value of repeatability (among cow coefficient of variation: 3.7%), but the common NDF analysis on the pool residue from bags incubated in different cows could have reduced the variability between animals.

4. Conclusions The DaisyII apparatus appears to be an appropriate technique as it is able to produce repeatable in vitro dNDF data highly correlated to those obtainable with a reference in situ procedure. The apparatus allows simultaneous incubation of a large number of samples (for a maximum of 96 bags per fermentation batch), giving advantages in terms of labour consumed and costs per determination. However, present results have been obtained within an homogeneous category of ingredients (i.e. hays from the same mountain area) and need to be verified with a more variable set of fibrous feeds.

Acknowledgements Financial support of this research was provided by the Italian Ministry of Education, University and Research (PRIN 2000 Project: Characterisation of Feeds by Means of In Vitro Rumen Degradation Kinetics of Carbohydrates and Proteins. Project coordinator: Prof. M. Antongiovanni). References Association of Official Analytical Chemists, 1998. Official Methods of Analysis, 16th ed. AOAC, Arlington, VA. Bovera, F., Spanghero, M., Crovetto, M., Masoero, F., Buccioni, A. Repeatability and reproducibility of the analytical determinations of the CNCPS system. Ital. J. Anim. Sci., submitted for publication. Dhanoa, M.S., 1988. On the analysis of dacron bag data for low degradability feeds. Grass Forage Sci. 43, 441–443. Gruber, L., Steinwidder, A., Stefanon, B., Steiner, B., Steinwender, R., 1999. Influence of grassland management in Alpine regions and concentrate level on N excretion and milk yield of dairy cows. Livestock Prod. Sci. 61, 155–170. Mabjeesh, S.J., Cohen, M., Arieli, A., 2000. In vitro methods for measuring the dry matter digestibility of ruminant feedstuffs: comparison of methods and inoculum source. J. Dairy Sci. 83, 2289–2294. Madsen, J., Hvelplund, T., 1994. Prediction of in situ protein degradability in the rumen. Results of a European ringtest. Livestock Prod. Sci. 39, 201–212. McDonald, I., 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci. Camb. 96, 251–252. Minson, D.J., 1990. Forage in Ruminant Nutrition. Academic Press, San Diego, USA, pp. 181–185. National Research Council, 2001. Nutrient Requirements of Dairy Cattle, seventh revised ed. National Academy of Sciences, Washington, DC. Robinson, P.H., Tamminga, S., Van Vuuren, A.M., 1987. Influence of declining level of intake and varying the proportion of starch in the concentrate on rumen ingesta quantity, composition and kinetics of ingesta turnover in dairy cows. Livestock Prod. Sci. 17, 37–62. Robinson, P.H., Campbell, M., Fadel, J.G., 1999. Influence of storage time and temperature on in vitro digestion of neutral detergent fibre at 48 h, and comparison to 48 h in sacco neutral detergent fibre digestion. Anim. Feed Sci. Technol. 80, 257–266.

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SAS, 1999. What’s New in SAS Software in Version 7 and the Version 8 Developer’s Release. SAS Institute Inc., Cary, NC. Spanghero, M., Gruber, L., Stefanon, B., Susmel, P., 1999. The estimation of the rumen rate of passage of dietary NDF from degradability and digestibility data in cows. Livestock Prod. Sci. 60, 71–79. Stensig, T., Robinson, P.H., 1997. Digestion and passage kinetics of forage fiber in dairy cows as affected by fiber-free concentrate in the diet. J. Dairy Sci. 80, 1339–1352. Stensig, T., Weisbjerg, M.R., Hvelplund, T., 1994a. Estimation of ruminal digestibility of NDF from in situ degradation and rumen fractional outflow rate. Acta Agric. Scand., Sect. A, Anim. Sci. 44, 96–109. Stensig, T., Weisbjerg, M.R., Madsen, J., Hvelplund, T., 1994b. Estimation of voluntary feed intake from in situ degradation and rate of passage of DM or NDF. Livestock Prod. Sci. 39, 49–52. Tilley, J.M.A., Terry, R.A., 1963. A two-stage method for the in vitro digestion of forage crops. J. Br. Grassland Soc. 18, 104–111. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods of dietary fiber, neutral detergent fiber and non-polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Weisbjerg, M.R., Bhargava, P.K., Hvelplund, T., Madsen, J., 1990. Use of degradation curves in feed evaluation, Report no. 679. National Institute of Animal Science, Frederiksberg, DK. Wilman, D., Adesogan, A., 2000. A comparison of filter bag methods with conventional tube methods of determining the in vitro digestibility of forages. Anim. Feed Sci. Technol. 84, 33–47.

Further Reading Madsen, J., Stensig, T., Weisbjerg, M.R., Hvelplund, T., 1994. Estimation of the physical fill of feedstuffs in the rumen by the in situ degradation characteristics. Livestock Prod. Sci. 39, 43–47.