Natioiial Institute of Animal Science, Copenhagen, Danmark

Apparent digestibility of crude fat determined in trials with calves and pigs in relation to analytical methods applied’

By CRETE THORBEK and S. HENCKEL

Receipt of M s . 13. J . 77

Introduction

The method of determining the content of crude fat in ieedstuffs by ether extraction (ethyl ether) have been applied for many years (Weende-method). Investigations made by BECKEK, KAKSTEN and OSLAGE (1953), BECKER (1956) and HENKEL and BECKEK (1960) clearly demonstrated that i n different kind of rouhages as grass, lucerne, silage and hay much higher values for crude fat, compared with the original Weende-method, could be obtained by refined extraction methods. The refined extrac- tion method was laborious and for practical purposes a method including HCI- hydrolysis before ether extraction, as proposed by STOLDT (1953), was recommended for crude fat determinations in feedstuffs. The validity of the EE-method applied on faeces was discussed by HARTFIEL (1960, 1965), who concluded from his experiments with calves, that the EE-method should be replaced by the (HCl+EE)-method i n digestibility trials. The same conclusion was drawn by THOMSEN (1971) and SUNDSTOL (1974a, 1974b) i n their comprehensive investigations concerning fat content i n faeccs from different species. I n the present investigations, including faeces from pigs and calves, the EE-method has been compared with the (HCl+EE)-method and thc (EE+ HC1tEE)-method with double ether extraction. The calorific values of faeccs- fat by the different methods have been compared. The crude fat content in different feedstuffs have been determined by the EE- and (HCI +EE)-method. The possibility of obtaining a constant conversion factor by the two methods have been investigated. The apparent digestibility of crude fat determined by the 3 different analytical methods have been compared.

Materials and methods

Mater ia ls : From an experiment with 8 growing pigs (20-90 kg, Series 0), concerning maintenance requirement, 48 samples of faeces from periods on low feeding level have been applied for this investigation. The samples, being mean samples from collection periods of 7 days, were analysed for their fat content according to different analytical

I Herrn Professor Dr. M. BECKER, ehemaliger Direktor des Instituts f u r TierernShrungslehre 311 der Universitiit Kiel, zu seinem 70. Gcburtstag gcwidmet.

Z. Tierphysial., Tierernihrg. 11. I~urrermirrelkde. 39 (1977). 48-55 @ 1977 VerIag Paul l’arey, Hzmhurg und Berlin lSSN 0044-3565 / ASTirl-Coden: ZTTFAA

Digestibility of crude f a t determined by d i f f e ren t methods 49

methods, as well as for nitrogen, crude fibre, N F E and energy. The feed compound in question consisted of barley with a protein supply of skim milk powder or protein mixture (2/3 soybean meal + 1/3 meat bone meal) with addition of vitamins + minerals and a daily supply of 45 g lard + 5 g soyaoil. The fat content of the rations decreased from 12 to 9 o /o crude fa t (Ether extract) during the periods.

From a similar investigation with 16 growing calves (100-250 kg, Series G and H ) 24 samples of faeces from periods on low feeding level (Ser. G) have been analysed together with 32 samples of faeces from periods on low or high feeding (Ser. H) . In both series the feed compound consisted of barley, oats, soyabean meal, linseed meal and barley straw, supplemented with dried sugar beet pulp (Ser. G) or grasspellets (Ser. H). The mean fat content was about 3.5 O/o (Ether extract) in all periods.

13 feedstuffs used in balance experiments with pigs and calves (1971-73) have been analysed for their fat content according to different analytical methods. The feedstuffs were analysed 7-8 times each depending of the numbers of balance periods. Analytical methods:, The crude fat content in faeces and feedstuffs was determined according to 3 different methods:

1. Ether extraction (EE), Weende method: The dried, milled sample was dried 18 hours in N,-atmosphere before ether extraction.

2. HCI-hydrolysis before ether extraction (HCI + EE), Stoldt method: 1-2 hours hydrolysis of the wet (original) sample with 125 ml 4n HCI under reflux. Filtration and washing with not water until cloride-free. Drying of filter and remanence in N,-atmosphere before ether extraction.

3. Ether extraction before the Stoldt (EE + HCI 4- EE): Method 1 + 2 with sum- mation of the two ether extracts.

Results

The fat content in faeces from pigs (Ser. 0) and from calves (Ser. G) was determined by the EE-method and the HC1 + EE)-method while in series H with calves the EE-

Table 1

Crude fat (O/o of DM) in faeces from pigs and calves determined by ether extraction (EE), HCI-hydrolysis before ether extraction (HCI + EE) or ether extraction before and after

HCI-hydrolysis (EE + HCI + EE). Mean of 8 animals in each period

Pigs 0 Pigs 0 Pigs 0 Pigs 0 Pigs 0 Pigs 0 Calves G Calves G Calves G Calves H Calves H Calves H Calves H

I Low 111 Low IV Low VI Low IX Low X Low I1 Low V Low

VII Low I High I1 Low I11 High IV Low

3.19 22.6 3.1 1 23.6 2.90 20.6 2.46 16.6 2.03 19.9 1.64 17.1 1.71 3.72 1.60 4.38 1.68 4.48 2.42 - 1.96 - 1.98 - 1.78 -

50 G. aorbek and S . Henckel

method was compared with the (EE + HCI + EE)-method. The results obtained are given in Table 1 as mean of 8 animals in each period.

The decreasing values of fat percentage in faeces from pigs during the periods independent of analytical methods applied, may be explained by the decreasing con- tent of crude fat in the feed compounds in question.

I t is clearly demonstrated that by using the EE-method very low values af crude fat i n faeces have been obtained. In the experiments with pigs the results by the EE- method were only 10-15 O/O of the values determined by the (HCI + EE)-method, while in calves the EE-method gave 35-45 O/O of the fat content measured by the (HCI + EE)-method as well as by the (EE + HCI + EE)-method.

In order to evaluate the validity of the different methods for crude fat deterniina- tion all the samples of faeces were analysed for nitrogen, crude fibre, NFE and energy. The individual figures obtained were used for calculation of the relationship between fat- and energy content according to the following regression model:

y = Energy in faeces, kcal = ax, + bx, i- cxQ + dx, x, = g protein ( N X 6.25) in faeces x, = g crude fibre i n faeces x , = g N F E in faeces x,, = g crude fat i n faeces, determined by the different methods.

The calculations were made partly with fixed factors for protein, crude fibre and N F E and partly as a multiple regression equation with determination of all factors simultaneously. As fixed factors the following values were applied: 5.7 kcal/g protein, 4 .2 kcal/g crude fibre and 4.2 kcalig NFE. The results obtained are shown i n Table 2.

where

Table 2

Energy factors for fat in faeces in relation to analytical methods applied for fat determi- nation. Regression coefficient calculated with fixed or variable factors for crude protein,

crude fibre and NFE

Pigs 0 EE Calves G EE Calves H EE

5.7 4.2 4.2 46.5 1.9 5.7 4.2 4.2 40.0 0.5

38.2 0.8 5.7 4.2 4.2 Pigs 0 (HCI 4- EE) 5.7 4.2 4.2 9.8 0.1 Calves G (HCl + EE) 5.7 4.2 4.2 17.9 0.4 Calves H (EE + HCI’+ EE) 5.7 4.2 4.2 15.7 0.5 Pigs 0 (HCI f EE) 6.7 0.4 3.7 0.3 3.9 0.2 9.8 0.3 Calves G (HCl + EE) 7.7 0.6 5.0 0.3 4.1 0.2 6.5 1.0 Calves H (EE + HCI f EE) 5.5 0.5 4.9 0.4 4.8 0.3 9.6 0.4

The relationship between fat determination by the EE-method and the energy content in 48 samples of faeces from pigs (Ser. 0) was found to be 46.5, being quite unacceptable. By the (HCI + EE)-method the regression coefficients for fat were 9.8 with fixed as well as with varible facors, beeing acceptable.

In the experiment with calves (Ser. G. and H.) the EE-method gave unacceptable regression coefficients of 38-40 as in the experiment with pigs. The (HC1 + EE)-

Digestibility of crude f a t determined b y different methods 51

method applied on 2 4 samples of faeces from Ser. G. gave a regressioncoefficient of 17.9, when using fixed factors. By calculations with variable factors a rather low value of 6.5 was found. In series H 3 2 samples of faeces were analysed by the (EE + HCI + EE)-method, using fixed factors in the calculation the coefficient for fat was 15.7, whereas variable factors gave on acceptable factor of 9.6.

Table 3

Crude fat in feedstuffs determined by ether extraction (EE) or ether extraction after HCI hydrolysis (HCI + EE)

EL (HCI + EE) (EE + HCI + EE) Feedstuffs ! I’ I O I o 1 SE 1 010 1 SE 1 @/a

Barley 8 1.93 0.045 2.57 0.041 75.1

Oats 7 4.44 0.024 5.72 0.051 77.6 Wheat bran 8 4.59 0.108 5.60 0.059 82.0 Meat bone meal 8 4.50 0.222 5.53 0.185 81.4 Herring meal 8 6.87 0.087 7.91 0.117 86.9 Soyabean meal 8 0.62 0.030 2.00 0.034 31 .O Sunflower meal 8 2.00 0.032 2.86 0.041 69.9 Linseed meal 8 5.62 0.024 6.81 0.036 82.5 Sugar beet pulp 7 0.44 0.022 1.;!1 0.040 36.4 Barley straw 8 1.04 0.058 1.50 0.077 69.3

Grass pellets 7 7.19 0.034 8.49 0.030 84.7

Wheat 8 1.96 0.049 2,46 0.042 79.7

Hay 8 1.11 0.053 1.87 0.102 59.4

13 different feedstuffs have been analysed 7-8 times according to the EE-method as well as the (HCI + EE)-method. The results are shown in Table 3.

The analytical accuracy, measured by the standard deviation of mean (SE), is iden- tical for the two methods in question. It is obvious that the content of crude fat

Table 4

Digestibility of crude fat in relation to analytical methods applied for fat determination

Crude fat digested I (EE + + EE (HCI + FE)

Species Ser. no, I 1 Crude fat digested I Crude fat digested I 1 I

Pigs 0 BA f MI 4 93.6 Pigs 0 12 95.2 Piis o 8 97.0 Pigs 0 BA f PR 4 95.4 Pigs Pigs Calves Calves Calves Calves Calves Calves Calves

0 0 G I1 G V G VII H I H I1 H I11 H IV

12 95.6 8 96.2 8 83.3 8 84.6 8 82.2 8 79.7 8 81.6 8 83.1 8 82.1

SD I @la SD I 010 1 SD

0.7 66.6 1.4 71.9 0.5 71.6 1.1 58.9 0.6 66.3 1.0 63.5 1.8 74.6 1.0 70.8 1.8 66.1 3.2 - 1.8 - 2.2 - 2.5 -

10.7 - 6.7 - 2.1 - 7.1 - 6.7 - 5.9 - 1.7 67.6 3.5 2.2 70.0 2.0 6.5 64.3 3.6

64.7 10.5 68.8 3.6 66.5 13.2 71.3 3.0

52 G. 7borbek and S. Henckel

measured by the (HCI + EE)-method ist greater than values obtained by the EE-method, but no constant ratio has been found. For grains and products of animal origin the ratio EE/(HCI + EE) is about 80 o/o, while the other feedstuffs varies in relation to their crude fat content from 31 to 85 o / o .

From the digestibility trials with pigs and calves, including 104 balances, the influ- ence on digestibility of crude fat in relation to analytical methods applied, have been investigated. The results are shown in Table 4.

The EE-method applied on pigs showed very high coefficients of digestibility of crude fat, about 96 O / o , while in the experiments with calves somewhat lower values, about 82 O/a, was found. By using the (HCI + EE)-method the digestibility coeffi- cients decreased to about 68 o / o for pigs and to 71 o / o for calves. If the (EE + HCI + EE)-method was applied on calves the digestibility of crude fat was found to be about 68 Q / o .

Discussion

The analytical accuracy of the EE- and (HCI + EE)-method was compared by means of the statistical method described by RASCH, LUDVIGSEN & THORBEK (1958), using the daily routine work with duplicate analysis in order to estimate the stand- ard deviation of the methods in question. From the experiment with pigs, including total sampling of faeces from 8 pigs in 6 balance periods, the standard deviations varied from 0.07-0.12 O / o (abs) for the EE-method and from 0.08-0.12 O/O for the (HCI + EE)-method, indicating the same analytical accuracy for the two methods, expressed in absolute terms. With the higher fat content found by the (HCI + EE)- method the relative standard deviation (CV O/O) is even lower for this method in spite of more analytical operations.

The very low content of EE-fat in faeces samples from pigs in the present investi- gation found to be only 10-15 O / o of the (HCI + EE)-fat, is in accordance with the results obtained by THOMSEN (1971), who found a mean value of 14O/o (9-280/0) EE- fat in 15 samples from piglets kept on feed compounds added 6 or 120/0 of tallow. In his comprehensive studies SUNDSTOL (1974a) has found, that the content of EE-fat could be less than 1/10 of the corresponding content of (HCI + EE)-fat i n faeces from pigs, and that the discrepancy between the two methods increases with increasing saturation of dietary fat.

In experiment with sheep SUNDSTO~L, (1974b) found that the (HCI + EE)-fat in faeces was roughly twice that of EE-fat. In 16 samples of faeces from cows THOMSEN (1971) found EE-values from 24-54 O / O of the HCI + EE)-fat, while in the present investigation including 56 samples of faeces from calves values of 35-45 O/o was found.

It is obvious that the very variable values for EE-fat in faeces in relation to (HCI + EE)-fat depends of species in question, amount of fa t in the feed compound and the composition of the dietary fat. Furthermore low figures for EE-fat may be caused by excess of C a + + in the faeces by which some of the fatty acids will be bound in a form inaccessible for dimrect ether extraction, PETTERSON (1964).

By using fixed factors for crude protein, crude fibre and N F E the regression coef- ficients was found to be 47 kcal/g EE-fat in faeces from pigs while by using the (HCI + EE)-method the regression coefficients came near to the theoretical value with 9.8 kcal/g fat with fixed as well as with variable factors, (Table 2). This is in

Digestibility of crude fat determined by different methods 53

accordance with SUNDSTOL (1974a) who, by using fixed energy factors as 5.72 for protein, 4.79 for' crude fibre and 4.17 for NFE, found 22.4 kcal/g EE-fat and 10.0 kcal/g (HCI + EE)-fat in experiments with pigs. In a slightly different way of cal- culation using fixed factors as in the present investigation and with the theoretical factor of 9.5 kcal/g fat, THOMSEN (1971) found a dif-ference of 22 O/O between deter- mined and calculated energy content in faeces from pigs by using the EE-method, while no significant difference was found by using the (HCI + EE)-method. It may be concluded that the EE-method, giving unreliable values for fat in faeces, should not be applied in digestibility trials with pigs, but the (EE + HC1)-method should be recommended.

From the experiments with calves the regression coefficients varied from 38-40 kcal/g EE-fat, while the coefficients was 17.9 kcal/g (HCI + EE)-fat by using fixed factors. Calculated with variable factors a rather low regression coefficient of 6.5 kcal/g fat was found combined with an unacceptable energy factor, of 7.7 kcal for protein, indicating a strong correlation between the two variables. By using double ether extraction in series H the regression coefficient was 15.7 kcal/g (EE + HCI + EE)-fat with fixed factors. Calculated with variable factors the theoretical energy value for fa t was obtained with 9.6 f 0.4 kcal. The coefficients of 5.5 for protein, 4.9 for crude fibre and 4.8 for N F E are in accordance with KELLNER (1924) who found higher calorific values for crude fibre and N F E in faeces samples compared with feed- stuffs.

In his investigation with faeces from cows THOMSEN (1971) found a mean differ- ence of 15 O/O between determined and calculated energy content in faeces when using the EE-method, while the difference was reduced to 7.5 o/o with the (HCI + EE), method, indicating that still some of the fat was not determined by this method. SUNDSTOL (1974b) found by using fixed energy factors a regression coefficient of 24.1 kcal/g EE-fat and 13.3 kcal/g (HCI + EE)-fat in faeces from sheep and concludes that neither the EE-method nor the (HCI + EE)-method represent the total lipids of sheep faeces, but the latter is significantly better than the former. In faeces from rumi- nants, with some of the lipids being water soluble, the (HCI + EE)-method will not include the total amount of lipids, but the (EE + HCI + EE)-method should be preferred.

In the present investigation 13 different feedstuffs were analysed 7-8 times each for their fa t content according to the EE- or (HCI + EE)-method (Table 3). The EE- method always gave lower values than the (HCI + EE)-method as found by THOMSEN (1972) and by SUNDSTOL (1974a, 1974b) who in rations as in feedstuffs found that the EE-method gave values from 52-97 O/O of the values from the (HCL + EE)-method. From our investigation no constant conversion factor could be found, the ratio EE/ (HCI + EE) varied from 0.31-0.87 depending of the origin and fat content. I t is concluded that the (HCI + EE)-method should be preferred in order to express the total amount of crude fat in feedstuffs.

The question arises how much will the values for fat digestibility be influenced by the analytical methods applied? In our experiments including 48 balance periods with pigs fed barley + skim milk powder or protein mixture the EE-method gave a mean digestibility of fat about 96 O/o, while the (HCI + EE)-method gave 68 O/O. An appar- ent digestible of 96'0/0 is quite unacceptable, considering that an exretion of 11-15 g

54 G. 7horbek mid S. Henckrl

endogenous fat daily could be expected, FREEMAN et al. (1968) which would give a true digestibility of fa t above I O O o i o .

In the experiments with calves including 56 balance periods the EE-method gave a mean digestibility of fat about 82 O/o, being reduced to 71 O/o by using the (HCI + EE)-method, with further reduction to 68 O/o by the double ether extraction, (EE + HCI + EE). From an analytical point of view the last method should be applied in digestibility trials as it will include the water soluble lipids, but with a difference only about 3 o/o between this method and the (HCI + EE)-method it depends of the applicatica of the results wath method should be preferred.

Summary

1. Different analytical methods of determining crude fat in feedstuffs and faeces in digestibility trials with pigs and calves have been compared. The methods applied were ether extraction (EE), HCI-hydrolysis followed by ether extraction (HCI + EE), and ether extraction before and after HCI-hydrolysis (EE + HCI + EE).

2. The EE-method gave the lowest values of fat in faeces as in feedstuffs compared with the two other methods (Tble 1 and 3). No constant conversion factors could bc contained. The calorific value of fat in faeces, calculated by regressionanalysis varied from 38-46 kcal/g EE-fat, being quite unacceptable (Table 2). The EE- method should not be applied in digestibility trials giving too high figures for di- gestibility of fat (Table 4).

3. The (HCI + EE)-method applied on faeces from pigs gave an acceptable calorific value of 9.8 kcal/g fat and the apparent digestibility of fat was reduced from 96 "/o found by the EE-method to 68 o/o (Table 4), indicating that the (HCI + EE)-method should be preferred in digestibility trials with pigs.

4. In faeces from calves a smaller or greater part of the lipids will be soluble in water and thereby not determined by the (HCI + EE)-method. Using a double ether extraction, (EE + HCI + EE) an acceptable calorific value of 9.6 kcal/g fat was obtained (Table 2). The difference in digcstibility of fat between the two methods was 3 o/o, not being significant.

Zusammenfassung

Scheinbare Verdaulichkeit des Rohfettes bestimmt in Verdauungsversuchen an Schweinen und Kalbern mit unterschiedlichen Analysenverfahren

1. Drei verschiedene Analysenverfahren zur Bestimmung der scheinbareii Rohfettver- daulichkeit in Verdauungsversuchen mit Schweinen und Kalbern wurden verglichen. Die folgenden Methoden wurden angewandt: Ather-Extraktion (EE), HCL-Auf- schlufl und anschlieflende Ather-Extraktion (HCL + EE), zweifache Ather-Extrak- tion vor und nach HCL-Aufschlufl (EE + HCI + EE).

2. Die EE-Methode ergab die niedrigsten Fettwerte sowohl im Kot als auch in Futter- mitteln im Vergleich zu den beiden anderen Methoden. Es konnte kein konstanter Umrechnungsfaktor ermittelt werden. Der Kalorienwert des Fettes im Kot, berech- net mit Hilfe einer Regressionsanalyse, variierte von 38-46 Kcal/g EE-Fett, was

Digestibility of crude fat determined by different methods 55

nicht befriedigend ist. Die EE-Methode sollte also nicht fur Verdauungsversuche benutzt werden, da sie zu hohe Werte fur die scheitibare Verdaulichkeit des Futter- fettes liefert.

3. Die (HC1 + EE-)Methode, bei Kotproben von Schweinen angewandt, ergab be- friedigende Kalorienwerte von 9,s kcal/g Fett und eine scheinbare Verdaulichkeit von 68 O / o . Nach der EE-Methode betrug die scheinbare Verdaulichkeit 96 O/O und lag dami,t zu hoch. Die Anwendung der (HCI + EE-)Methode scheint fur Verdau- ungsversuche mit Schweinen geeignet zu sein.

4. Im Kot von Kalbern ist ein schwankender Anteil der Lipide wasserloslich und da- her nicht durch die (HCl + EE-)Methode erfaflbar. Bei Anwendung der zweifa- chen Ather-Extraktion (EE + HCI + EE) wurde ein befriedigender Wert von 9,6 kcal /g Fett erreicht. Der Unterschied in der Verdaulichkeit des Fettes lag bei beiden Methoden bei 3 o / o und war nicht signifikant.

References

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Berlin: Paul Parey, 674 p. p. S. PETTERSON, A., 1964: Lantbrukshoglskolans medd., Ser. A., Nr. 16, Uppsala. 9. RASCH, G.; LUDVIGSEN, J.; THORBEK, G., 195s: Proc. 1st. Symp. on Energy Metabolism,

Copenhagen (1958). E A A P Publ. No. 8, 163. Statens Husdrybrugsudvalg, Copenhagen. 10. STOLDT, W., 1952: Fette, Seifen, Anstrichmittel. 54, 206 11. SUNDSTOL, F., 1974a: Meld. 159. Inst. husdyrernzring og fbringslcere, Norges landbr.

hogsk. 32 pp. 12. SUNDSTOL, F., 1974b: Meld. 161. Inst. husdyrernzring 08 fbringslaxe, Norges landbr.

hogsk. 31 pp. 13. THOMSEN, K. V., 1971: Ugeskr. Agron., 116 nr. 26/27, 568 og nr. 29130, 613. 14. THOMSEN, K. V., 1972: Foderstofanalysen. Ugeskr. Agron. Hort., 1, 316.

Authors’ address: GRETE THORBEK, National Institute of Animal Science, Rolighedsvej 25, DK-1958, Copenhagen S. HENCKEL, Regnecentralen A/S, Falkoneralle 1, DK-2000, Copenhagen