In vitro digestibility

The estimation of animal digestibility of a feedstuff is usually achieved in one of three ways: in vivo, in sacco or in vitro. The first uses real animals in feeding trials and gives the most realistic results to which the other methods are correlated; the second method allows feed samples contained in small permeable plastic (e.g. nylon) bags to be inserted through a cannula into the rumen or another section of the digestive tract. The last method allows the digestion of feed samples to occur in the laboratory using digestive juices obtained from a fistulated animal, commercially obtainable enzymes, detergent solutions, or any combination of these, with the aim of imitating naturally occurring digestive processes.

Extractions using detergent plus enzyme

Neutral cellulase plus gamanase digestibility (NCGD) of feeding stuffs. This method originated at a time when compound feeds contained less starch and more digestible fibre and oil than when ME prediction equations were derived in 1985. In this method published by MAFF (1993b), using a fat-free sample, neutral detergent removes soluble cell contents, a-amylase dissolves starch, while cellulase/polysaccharase dissolves cellulose and hydrolyses any polysaccharides in the feed, and gamanase hydrolyses galactomannans which occur in palm kernel products.

Neutral detergent (plus amylase) fibre (NDF) of feeding stuffs. This other MAFF (1993c) method removes cell contents from the fat-free sample by boiling with neutral detergent solution. The a-amylase converts any starch (which would enhance the fibre content) to soluble sugars. The residue is designated neutral detergent (plus amylase) fibre; the abbreviation given is NDF, but this would confuse it with the Van Soest and Wine NDF. Perhaps ND(+A)F would be clearer.

Rumen liquor plus neutral detergent

To obtain the in vitro true digestibility, the residue from the first buffered rumen liquor stage of the Tilley and Terry (1963) procedure is digested with neutral detergent solution. The ordinary true digestibility is found by subjecting the faeces to neutral detergent digestion. The neutral detergent soluble non-cell-wall fraction of faeces equates to the endogenous and bacterial loss.

Tilley and Terry (1963) procedure

This eponymous method is widely used and as originally proposed or in modified form has served as a benchmark for other methods. In fact, it is often referred to simply as the in vitro digestibility. The first stage involves anaerobic incubation at 38°C in the dark with partially filtered rumen liquor which has been buffered with McDougall's artificial saliva solution, previously saturated with CO2.

After 48 h, 5 ml of M Na2CO3 is added to aid sedimentation immediately before centrifugation. Although mercuric chloride was added to inhibit bacterial activity, immediately centrifuging after 48 h rather than storing samples avoids this. It also avoids disposal of a toxic reagent. The supernatant is decanted into a fine nylon cloth filter and any particles returned to the tube. The particles on the rubber stopper and those adhering to the sides of the tube are washed down to the pellet which is then broken up before adding the acid pepsin solution. This is incubated for a further 48 h, then filtered through a porous alumina crucible (unpublished modification to the original method) before oven-drying, weighing, and possibly ashing.

In vitro calculations

The Tilley and Terry method (X correlates with in vivo results (Y) as follows: Y = 0.99X -1.01

One particular correction is advisable. Standards of known in vivo and in vitro values covering the lower and higher digestibility range (about 50% and 70% respectively) should be obtained, possibly from a research station, and included with the sample batch. Rumen liquor varies in potency from week to week, therefore a proportional adjustment must be made to enable comparison of results from analyses performed at different times. This variation does not equally affect the low and high standards; one may decrease and the other increase. A graph should be drawn relating the difference of the measured standard from the stated value to the concentration. This could be a positive or negative slope. The samples' measured digestibility should be corrected according to the corresponding adjustment read off the graph. A typical example is shown in Fig. 4.3.

Alternatively, a spreadsheet program such as Microsoft Excel may be used to achieve the correction automatically. A typical example is shown in Table 4.2.

Digestibility equations

There are several ways of expressing the in vitro rumen liquor digestibility of a sample: the DOMD or D-value, the DMD value and the OMD value. These are defined below:

1. The DOMD (D-value) is the digestible organic matter in dry matter: = OM sample - (OM residue - OM blank)

DM sample x 100%

where OM is the organic matter in the original dried and milled sample (sample minus sample ash), OM residue is the organic material in the residue

90.0

90.0

Correction (%)

Fig. 4.3. Typical graph for correcting measured sample dry matter digestibility (DMD) values in proportion to deviation of low and high standards from their declared values.

Table 4.2. Typical spreadsheet for correcting the measured sample dry matter digestibility values in proportion to deviation of low and high standards from their declared values.

Spreadsheet for correction of digestibility values between batches

Measured value (%)

Correction required

High standard Low standard

Corrected value (%)

Sample 1 51.8

Sample 2 62.5

Sample 3 73.2

Let correction graph be y = mx + c y = correction to be applied m = slope x = measured sample value mx = Q.10

c = intercept on y-axis

Vc = corrected value; Vm = measured value

Spreadsheet formulas

Result

C8 52.200

((B4-C4)-(B5-C5))/(C4-C5)*C8+(D4-(C4/(C4-C5))*((B4-C4)-(B5-C5))) ((B4-C4)-(B5-C5))/(C4-C5)*C9+(D4-(C4/(C4-C5))*((B4-C4)-(B5-C5))) ((B4-C4)-(B5-C5))/(C4-C5)*C10+(D4-(C4/(C4-C5))*((B4-C4)-(B5-C5)))

For Sample 1 For Sample 2 For Sample 3

C8+E20 C9+E21 C10+E22

following digestion (residue weight minus ashed residue weight), OM blank is the organic matter in the rumen liquor itself, and DM sample is a dry matter determination done on a separate sample.

This equation must be translated into the actual weighings required so that a spreadsheet can be drawn up. Errors can easily occur in the calculations unless the individual steps are understood. The sample weight is 0.5000 g and the calculation formula and any spreadsheet must be designed for this and allow for the fact that the original ash is carried out on 1.0000 g. The residue of undigested sample contains four components of the calculation:

• undigested sample organic matter

• sample residue ash

• blank (rumen liquor) organic matter

We are interested in the first component, so need to subtract the other components. The residue from the rumen liquor blank contains both blank organic matter and blank ash. When this value is subtracted from the above we get the sum of sample organic matter plus sample ash.

After weighing the dried residue it is subsequently ashed and weighed. This gives an ash comprising:

• sample residue ash

A separate ashing of a rumen liquor blank sample gives a figure for blank ash. When subtracted from the above residue ash, the difference gives the sample residue ash. Subtracting this value from the sum of sample organic matter plus ash, leaves us with the undigested residue sample organic matter. Finally, this is subtracted from the original sample organic matter to give the amount of digestible organic matter, which is corrected for dry matter content of the sample and expressed as a percentage or as g kg-1 digestibility.

0.5 - original ash

sample residue -blank residue sample residue ash -blank residue ash

2. The OMD value is the organic matter digestibility: = OM sample (OM residue OM blank)

OM sample

0.5 - original ash

A sample residue (sample residue ash blank residue blank residue ash

0.5 - original ash

3. The DMD value is the dry matter digestibility:

= [sample (sample residue blank residue)]

sample

A suggested spreadsheet for the above calculations is shown in Table 4.3. The values for residues and ash are entered from results sheets printed with columns for crucible weights, etc., unless the laboratory is equipped with computerized balances, when a more sophisticated spreadsheet could be devised.

When planning for Tilley and Terry digestibilities, it is common practice to ensure that the sheep or cattle have been fed for a couple of weeks on a basal diet similar to the test samples to be analysed. This is to ensure a buildup of the appropriate rumen flora resulting in a corresponding optimal activity. Whether or not this is necessary is open to question, and this and other sources of error have been discussed by Ayres (1991). It is also customary not to feed the animal on the morning planned for extracting the rumen liquor.

4. True dry matter digestibility (True DMD) (Van Soest et al.,1966) This is expressed by the equation:

True DMD = {(% cell content in DM x 0.98) + (% digestible cell wall in DM)}

The % cell content in DM is (100 - % cell wall in DM), which is derived from (100 - NDF).

The % digestible cell wall in DM is the (% cell wall in DM - % indigestible cell wall in DM).

The % indigestible cell wall in DM is the residual DM after digestion in rumen liquor (48 h) followed by the neutral detergent procedure and expressed as % sample DM.

Various aspects of in vitro methods, from its first use in 1880 to the 1980s have been discussed by the author (Faithfull, 1984). In particular, the effect of pH on tannin complexes, phosphates and sulphides have been studied.

The concept of fistulated animals may seem abhorrent. It should be observed, however, that properly tended animals appear to be quite contented, and that their lifetime as an experimental animal is far longer than it would otherwise have been. Nevertheless, it is impossible to prevent the animal from knocking the cannula, and it is easy for leaks to occur causing irritation to the skin around it. It is also expensive to maintain such animals in an acceptable way, and to justify this if long periods exist between experiments. The procedure is favoured by experienced researchers as it facilitates comparison of results with earlier published work, and may give more consistent results over periods of time. However, improved within-batch precision, economy of time, money and convenience, and improved public

Table 4.3. Typical spreadsheet in Microsoft Excel for calculating the various digestibility values.

Name

In vitro DOMD, OMD & DMD measurement

Corrected

Corrected

Sample

Sample

Sample

Fractional

S dry

Sample

Fractional

Sample

Residual

Residual

Residual

OMD

DOMD

DMD

Average

ID

wt.

DM

sample DM

wt.

ash %.

ash

OM

wt.

ash

OM

g/kg

g/kg DM

g/kg

1

0.5000

98.01

0.9801

0.4901

7.24

0.0355

0.4546

0.1891

0.0085

0.1775

610

565

614

=AVERA

2

0.5000

97.22

Formula =D5/100

=samplewt *E5

6.80

=G5/ 100*F5

=F5-G5

0.1773

0.0072

=J5-K5 -M19

=(I5-L5) /I5*1000

=(I5-L5) /F5*1000

=(F5-J5) /F5*1000

(M4:M5)

3

0.5000

98.25

0.9825

0.4913

8.43

0.0414

0.4498

0.1896

0.0088

0.1777

605

554

614

4

0.5000

97.56

0.9756

0.4878

7.48

0.0365

0.4513

0.1907

0.0079

0.1797

602

557

609

603

5

0.5000

98.18

0.9818

0.4909

6.61

0.0324

0.4585

0.1814

0.0076

0.1707

628

586

630

6

0.5000

97.73

0.9773

0.4887

7.53

0.0368

0.4519

0.1882

0.0081

0.1770

608

562

615

618

7

0.5000

97.66

0.9766

0.4883

7.96

0.0389

0.4494

0.1901

0.0077

0.1793

601

553

611

8

0.5000

97.94

0.9794

0.4897

8.01

0.0392

0.4505

0.1866

0.0083

0.1752

611

562

619

606

9

0.5000

98.12

0.9812

0.4906

8.22

0.0403

0.4503

0.1871

0.0080

0.1760

609

559

619

10

0.5000

96.97

0.9697

0.4849

7.87

0.0382

0.4467

0.1888

0.0077

0.1780

602

554

611

605

L1

0.5000

98.44

0.9844

0.4922

6.98

0.0344

0.4578

0.1903

0.0082

0.1790

609

567

613

L2

0.5000

97.89

0.9789

0.4895

7.34

0.0359

0.4535

0.1855

0.0090

0.1734

618

572

621

613

H1

0.5000

98.35

0.9835

0.4918

7.97

0.0392

0.4526

0.1899

0.0083

0.1785

606

557

614

H2

0.5000

97.17

0.9717

0.4859

6.84

0.0332

0.4526

0.1977

0.0076

0.1870

587

547

593

596

1

0.0040

0.0009

0.0031

2

0.0040

0.0009

=J19-K19

0.0031

The row for Sample 2 is used to display the formulae, which are normally hidden.

perception all point to alternative methods as being the way forward. One such method uses faecal liquor and has been discussed by Omed et al. (2000). Replacing the acid pepsin stage with biological washing liquid produced digestibilities very close to the known in vivo values for a variety of grasses, legumes and hays (Solangi, 1997). The two-stage pepsin-cellulase method (see below) is probably the best alternative to the Tilley and Terry procedure.

Cellulase digestibility

A convenient procedure for assessing the digestibility of forages is the cellulase digestibility technique. This was refined by Jones and Hayward (1973) at the Welsh Plant Breeding Station (WPBS) in Aberystwyth (since 1992, the Institute for Grassland and Environmental Research). It was later extended to a two-stage procedure with a pepsin pre-treatment (Jones and Hayward, 1975).

The pepsin removes protein from the cell walls and possibly modifies the cell wall polysaccharide in such a way as to render it more susceptible to attack by the cellulase enzyme. It also allows cellulases from different sources to be used with less effect from variation in enzyme activity. The single stage cellulase technique is suggested for screening in plant breeding programmes, but in this case, the higher activity enzyme from Trichoderma viride will yield a higher correlation with in vivo and in vitro digestibility. One might expect less precision when digesting with enzymes versus rumen liquor, because enzymes lack the ability of microorganisms in adapting to a substrate. Stakelum et al. (1988), however, found a similar accuracy in predicting in vivo digestibility when using the rumen liquor-pepsin, pepsin-cellulase or neutral detergent-cellulase methods.

0 0

Post a comment