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Citation of this paper

Estimates of protein fractions of various heat-treated feeds in ruminant production

Ho Thanh Tham, Ngo Van Man* and T R Preston**

Department of Animal Husbandry, College of Agriculture and Applied Biology, Cantho University, Cantho City, Vietnam
httham@ctu.edu.vn
*
Deparment of Animal Nutrition, Nong Lam University, Ho Chi Minh City, Vietnam
**
UTA - TOSOLY - Finca Ecológica, Morario - Guapota - AA # 48, Socorro, Santander, Santander del Sur, Colombia

Abstract

Leaves of Cassava (Manihot esculenta Crantz) (CLM), Sesbania (Sesbania grandiflora) (SG), Leucaena (Leucaena leucocephala) (LL), Gliricidia (Gliricidia sepium) (GS) and Water hyacinth (Eichorniacrassipes) (WH) were subjected to heating at 60 (control), 100 or 140oC for 2 hours. The experimental treatments were laid out in a 5x3 factorial arrangement with 3 replications. The protein fractions of the leaves were estimated using The Cornell Net Carbohydrate and Protein System (CNCPS) model.

Heating the leaves to temperatures of 140ºC for 2 hours reduced the proportion of the protein in the A (NPN) and B2 (some fraction B2 escapes to the lower gut) fractions and increased the B3 (slowly rumen degradable fraction). The highest value for the B3 fraction was in water hyacinth (44% of the total crude protein) while in the other leave it was less than 10%. By contrast, the B2 fraction was low in water hyacinth (less than 20%) and high in the other leaves and especially in cassava leaf meal where it accounted for 66% of the total crude protein.

The implication from these findings is that the B2 fraction may be a better indicator of the probable bypass protein value of leaves than the B3 fraction.

Key words: By-pass protein, cassava, CNCPS, Gliricidia, leaves, Leucaena, rumen degradation, Sesbania, Water hyacinth


Introduction

Protein requirements for high rates of growth in ruminants cannot be met solely from microbial protein synthesis in the rumen; therefore, supplementation with high quality rumen undegradable protein is necessary (Preston and Leng 1987; McNiven 2002). Due to the high cost of protein supplements, ways and means of protecting the protein from degradation in the rumen whilst retaining the high digestibility is an urgent priority (Leng 1991). Many experiments have demonstrated the beneficial effects of the technological processing of feeds, particularly heat treatment, introduced by Chalmers et al (1954) (cited by Manget 1997), in reducing the degradation of the crude protein in the rumen without decreasing digestibility in the small intestine (Aufrère et al 2001). For highly producing ruminants, heat treatment of protein supplements has been used for increasing the amount of dietary protein escaping rumen degradation, and to increase the amino acid pool entering the small intestine (Faldet et al 1991).

Various approaches are available to assess the ruminal degradability of protein in feedstuffs, which include in vivo, in sacco, and in vitro methods (Elwakeel et al 2007). The in vivo method is considered ideal for protein source evaluation because feeds experience normal digestion processes. With in vivo measures, microbial and endogenous N needs to be distinguished from dietary N, which can lead to difficulty in estimation of ruminal protein degradability (Vanzant et al 1996). Most importantly, in vivo measures of ruminal protein degradability are not practical for routine evaluation of feeds due to cost, timeliness, and the need for cannulated animals.

The in sacco method is the most widely used method for estimating ruminal protein degradation because it is less expensive and simpler than in vivo methods (Ørskov et al 1980). However, soluble protein and protein in small particles can leave the bags through the pores without complete degradation (Martin 2001).

The Cornell Net Carbohydrate and Protein System (CNCPS) (Chalupa et al 1991; Sniffen et al 1992) is one of the schemes developed for the fractionation of protein in feeds. There is some information on crude protein content of various feedstuffs collected in the Mekong Delta (Dung 1996), and on the fractions derived by the CNCPS system (Dung 2001). However, fractionation of the protein by the CNCPS system in cassava leaves, and the influence of heat treatment on these fractions, have not yet been reported.

The characterization of the CP fractions in the CNCPS system in this system is as follows:

Fraction A is non-protein nitrogen (NPN), B is true protein, and C is unavailable true protein or bound protein. Fraction B is further divided into three fractions (B1, B2, and B3) that are believed to have different rates of ruminal degradation. Fractions A and B1 are soluble in borate phosphate buffer and are rapidly degraded in the rumen. Fraction B2 is fermented in the rumen at lower rates than buffer-soluble fractions, and some fraction B2 escapes to the lower gut. Fraction B3 is believed to be more slowly degraded in the rumen than are Fractions B1 and B2 because of its association with the cell wall; a larger proportion of B3 is thus believed to escape the rumen. Fraction C is the ADIP, and is highly resistant to breakdown by microbial and mammalian enzymes, and it is assumed to be unavailable for the animal (see Table 1).


Table 1. Partition of protein fractions in feedstuffs

Fraction

Classification (*)

Abbreviation

Enzymatic degradation

Estimation or definition

Non-protein nitrogen

A

NPN

Not applicable

Not precipitated

True protein

-

TP

-

Precipitate with TCA

True soluble protein

B1

BSP

Fast

Buffer soluble but precipitable (TP-IP)

Insoluble protein

-

IP

-

Insoluble in buffer

Neutral detergent soluble protein

B2

IP-NDIP

Variable

Difference between IP and protein insoluble in neutral detergent (ND)

ND insoluble protein but soluble in acid detergent

B3

NDIP-ADIP

Variable to slow

Protein insoluble in neutral detergent but soluble in acid detergent

Insoluble in acid detergent

C

ADIP or ADIN

Indigestive

Includes heat-damaged protein and nitrogen associated with lignin

* According to Pichard and Van Soest 1977 and Van Soest 1994

The main purpose of the research described in this paper was to use the CNCPS model to estimate: (i) the protein fractions of cassava leaf meal, and other protein-rich leaves, some of which have been used for ruminants in the Mekong Delta of Vietnam; and (ii) the degree to which these fractions are affected by heat treatment.
 

Materials and methods

Location of the study area

The experiment was conducted in the animal nutritional laboratory of the Department of Animal Husbandry, College of Agriculture and Applied biology, Cantho University, Cantho City, Vietnam. The duration of this study was 3 months, from October 2006 to December 2006.

Treatments and design

The factors in a 5x3 factorial arrangement with three replications in a randomized complete block were:

Source of leaf materials:

Heat treatment during 2 hours at:

Samples

Cassava leaf meal was made from cassava leaves, which were bought in Tayninh province. The leaves were collected from the field after harvesting the roots and sun-dried for 2 to 4 days. Sun drying consisted of spreading the leaves on the ground and turning them over while exposed to the sun, resulting in cassava leaf meal for direct feeding or storage.

The other leaves were harvested in September 2006 from areas surrounding Cantho city. These plant protein sources were prepared in the same way as for cassava leaf meal, by drying under sunlight. After sun-drying, the leaves were heated in an oven maintained at temperatures of 60, 100 or 140oC for 2 hours. The heated samples were then ground to pass a 1 mm screen and stored in a freezer until analyzed.

Measurements and analysis

Samples of feed were determined for dry matter (DM) and crude protein (CP) using procedures described by AOAC (1990). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the procedure of Van Soest et al (1991). Crude protein fractionation was performed according to the Cornell Net Carbohydrate and Protein System (Figure 1 and Table 1).



A = Soluble in buffer;  B1 = soluble in buffer and precipitated by TCA; B3 = insoluble in buffer, insoluble in neutral detergent solution but soluble in acid detergent solution; B2 = insoluble in buffer but soluble in neutral and acid detergent solutions; C = insoluble in buffer and both neutral and acid detergent solutions


Figure 1.
 Analyses of crude protein fractions using borate phosphate buffer and acid detergent and neutral detergent solutions (Roe et al 1990; Sniffen et al 1992).

To separate the true protein (TP) and non-protein nitrogen (NPN) (Fraction A), trichloroacetic acid (TCA) was used according to the method described by Licitra et al (1996). The TP was separated from the NPN by precipitation with TCA (final concentration 10%). Filtering was done by gravity and NPN was calculated as the difference between total forage N and the N content of the residue after filtration.

Buffer soluble protein was defined as the true protein soluble in a borate-phosphate buffer at pH 6.7-6.8 (Krishnamoorthy et al 1982). Samples were filtered by gravity through Whatman #541 filter papers for subsequent Kjeldahl nitrogen analysis. The insoluble N fraction after filtration was defined as the buffer insoluble protein (IP) fraction. Soluble true protein (Fraction B1) was calculated as the difference between TP and IP.

Neutral detergent soluble protein (Fraction B2) was estimated as the difference between IP and protein insoluble in neutral detergent (NDIP). The amount of soluble fibre-bound CP (Fraction B3) was calculated as CP in NDF minus acid detergent insoluble CP.

Acid detergent fiber (ADF) was prepared according to Van Soest et al (1991) with filtering by gravity on 12.5 cm Whatman # 541 filter papers. Residual N x 6.25 on the filter paper (Acid detergent insoluble protein: ADIP) was classified as Fraction C.

The values of CP in all the fractions including NPN were calculated as g N x 6.25 kg-1 CP.

Statistical analysis

The effect of heat treatment on the protein fractions of samples was analyzed using the General Linear Model (GLM) option of the Minitab software (Minitab release 13.1 2000), as shown below:

Yijk = µ + Ai + Bj + ABij + eij

where:

Yijk is the protein fraction.
µ the mean value,
Ai the source of the leaf materials,
Bj the effect of heating (60, 100 or 140oC),
ABij the interaction between leaf sources and heating level, and
eij the error.
 

Results

Effect of heat treatment
Dry matter (DM) and crude protein (CP) content

After heating followed by grinding most of the samples heated at 60ºC tended to absorb more moisture than those heated at 100 and 140ºC (Table 2).


Table 2.  Effect of heating on residual DM (g/kg feed) in leaf samples

 

60oC

100oC

140oC

SEM

P

CLM

869a

907b

907b

3.6

0.001

SG

904a

941b

962c

2.7

0.001

LL

901

905

914

5.2

0.261

GS

893a

907ab

909b

2.5

0.007

WH

894a

910b

961c

3.7

0.001

abc Mean within rows with differing superscript letters are significantly different (P<0.05).


Heating at high temperature (100 and 140oC) appeared to have very little effect on total crude protein content with slight reductions at 140oC for some of the leaves (Table 3).


Table 3.  Effect of heating on crude protein in leaf samples (g/kg DM)  

 

60oC

100oC

140oC

SEM

P

CLM

255a

242b

242b

1.81

0.004

SG

318a

316a

301b

2.33

0.004

LL

302a

296ab

289b

2.01

0.012

GS

217

212

210

5.23

0.695

WH

246

244

240

2.41

0.355

abc Mean within rows with differing superscript letters are significantly different (P<0.05).

Non-protein nitrogen (NPN - Fraction A)

In the control treatment (60oC) the highest value for NPN was in SG (465 g/kg CP) and the lowest one was in CLM (114 g/kg CP) (Table 4) This component decreased markedly with the gradual increase of heating temperature. In the high temperature treatment (140oC) the reductions were 12, 15, 19, 38 and 58% for SG, LL, WH, CLM and GS, respectively compared with the treatment at 60oC.


Table 4.  Effect of heating on Fraction A (NPN) in leaf samples (g/kg CP)

 

60oC

100oC

140oC

SEM

P

CLM

114a

111a

71b

4.81

0.001

SG

465a

452a

411b

8.47

0.010

LL

390a

375a

333b

5.8

0.001

GS

186a

170a

78b

8.89

0.001

WH

142a

133ab

115b

6.01

0.050

abc Mean within rows with differing superscript letters are significantly different (P<0.05)

Fraction B1

The fraction B1 was decreased markedly by heating to 140oC (Table 5) with major differences among the leaves, the ranking from high to low being WH>GS>LL>CLM>SG.


Table 5.  Effect of heating on Fraction B1 (g/kg CP) in leaf samples

 

60oC

100oC

140oC

SEM

P

CLM

42

29

3

20.54

0.453

SG

19

10

7

13.42

0.878

LL

126a

42b

25b

5.85

0.001

GS

138a

134a

57b

17.17

0.027

WH

155a

67b

62b

10.45

0.001

abc Mean within rows with differing superscript letters are significantly different (P<0.05)

Fraction B2

Values for fraction B2 were higher than for B1, and also declined with increase in temperature except in the case of leucaena (Table 6).


Table 6.  Effect of heating on Fraction B2 (g/kg CP) in leaf samples

 

60oC

100oC

140oC

SEM

P

CLM

663a

651a

359b

16

0.001

SG

434a

450a

309b

15.76

0.001

LL

421a

497b

545b

10.96

0.001

GS

553a

503a

244b

13.98

0.001

WH

213a

263b

15c

11.28

0.001

abc Mean within rows with differing superscript letters are significantly different (P<0.05)

Fraction B3

The B3 fraction, which is considered to be the most important as a potential source of bypass protein, was increased markedly by heat treatment in all the leaves with the exception of leucaena which showed only minor changes due to heating (Table 7).


Table 7.  Effect of heating on Fraction B3 (g/kg CP) in leaf samples

 

 

60oC

100oC

140oC

SEM

P

 

CLM

93a

109a

429b

24.13

0.001

 

SG

45a

54a

241b

5.06

0.001

 

LL

93

96

100

5.19

0.669

 

GS

22a

84b

494c

9.53

0.001

 

WH

437a

485b

760c

5.06

0.001

 

abc Mean within rows with differing superscript letters are significantly different (P<0.05)

Fraction C

The results of heat treatment on fraction C were variable with increases for CLM and GS but little change in the other leaves (Table 8).


Table 8.  Effect of heating on fraction C (g/kg CP) in leaf samples

 

60oC

100oC

140oC

SEM

P

CLM

88a

100a

138b

3.18

0.001

SG

36

34

32

1.25

0.134

LL

37a

55b

60b

2.04

0.001

GS

101a

109ab

128b

4.51

0.016

WH

54

53

48

1.8

0.128

abc Mean within rows with differing superscript letters are significantly different (P<0.05)

Comparisons among the leaves

There were major differences among the leaves for the different crude protein fractions (Tables 2 through 8). For the control treatment at 60ºC (Figure 2) the A fraction (NPN) showed high values for sesbania and leucaena and low values for the other leaves. In contrast, the B3 fraction accounted for only a small proportion of the crude protein in all the leaves except for water hyacinth for which it accounted for almost half of the crude protein. For the B2 fraction the highest values were for cassava leaf meal, the lowest were for water hyacinth with intermediate values for gliricidia, sesbania and leucaena.



Fraction C is the insoluble fraction, B1 is the soluble fraction, B2 is the slowly fermented fraction and B3 the fraction not fermented in the rumen but supposedly digestible in the intestine.
 

Figure 2.  Fractionation of the protein in leaves (heated for 2 hours at 60ºC) according to the CNCPS model.

Discussion

Effect of heating

The reduction in the NPN component due to heating presumably was due to the loss of amino acids as observed by Kari Lj°kjel et al (2000) for soybean meal and fish meal subjected to heat treatment of 135oC for 30 minutes. Jirí Davidek et al (1990) reported that one of the first noticeable changes of proteins on heating (even at temperatures around 100oC) is the loss of labile amino acids such as cystine and lysine. Lysine is one of the most temperature sensitive amino acids; it is often destroyed at levels 5 to15 times higher than the other amino acids (Dakowski et al 1996).

The low values for the B1 fraction agree with the report of Pichard and Van Soest (1977), that in harvested forages the B1 fraction of the total protein is low Caballero et al (2001) indicated that B1 increased with maturity of forages and decreased from fresh to dry which is in agreement with the results of the current study.

The increase in the B3 fraction at the expense of B2 as a result of heating, was probably due to the denaturation of the proteins as this has been shown to reduce their solubility (Van Soest 1994). Heat treatment has been shown to differ in its efficacy with different protein meals. When groundnut cake was heated at 150oC, protein solubility was reduced from 23.6 to 10.4 percent. However, in soybean meal, under similar conditions, the reduction in solubility was only from 14.3 to 10.3 percent (Walli 1995 cited by Manget 1997). The effect of heating on leaf proteins is likely to be different according to the degree of cross linkages with the fibre (Van Soest 1994) and the formation of insoluble complexes with compounds such as tannins (Barry et al 2001).

Effect of source of leaves

In the CNCPS model, fraction B3 is believed to be more slowly degraded in the rumen than are fractions B1 and B2 and is thus believed to escape the rumen fermentation (Sniffen et al 1992). However, in our study the highest value for the B3 fraction was in water hyacinth (44% of the total crude protein) while in the other it was less than 10%. By contrast, the B2 fraction was low in water hyacinth (less than 22%) and high in the other leaves and especially in cassava leaf meal where it accounted for 66% of the total crude protein. Similar values for B3 and B2 in water hyacinth (37% and 24%, respectively) and in Sesbania grandiflora (10% and 50% for B3 and B2, respectively) were reported by Dung (2001) (Figure 3). Sesbania grandiflora has been shown to support growth rates in goats of over 100 g/day (Nhan, 1998) while in Paper I of this thesis, growth rates in cattle were linearly increased when cassava leaf meal was used as the supplement to urea-sprayed rice straw. Using the in vitro pepsin-pancreatin technique to evaluate rumen undegraded protein of cassava hay, Promkot and Wanapat (2003) reported that in cows fed with urea-treated rice straw, the value of rumen undegradable protein (expressed as % of total CP) was 45.4 and very similar to that in cottonseed meal which is known to be one of the best sources of bypass protein for growing cattle (Zhang Weixian et al 1994).

It is relevant to note that the CNCPS model did not provide realistic predictions of milk production when a forage (alfalfa silage) was the sole dietary ingredient (Aquino et al 2003).



SGM (Sesbania grandiflora, mature), SGY (Sesbania grandiflora, young); WHY (Water hyacinth, young), WHM (Water hyacinth, mature); BGA (Brewer's grain, artisanal), BGI (Brewer's grain, industrial); FMI (Fish meal, industrial), FME (Fish meal, export).
 

Figure 3.  Fractionation of the protein in leaves and protein meals according to the CNCPS model (Dung 2001).

Conclusions

Acknowledgements

Financial support from SIDA-SAREC is grateful acknowledged and the authors would like to thank Ms. Nguyen Thi Ngan and Mr. Nguyen Thiet for their help in laboratory analyses.


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