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 140^oC 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 of are 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, CNCPS, heat treatment, protein fractions, rumen degradation.

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 2006). 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).

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:

• Cassava (Manihot esculenta, Crantz) (CLM),

• Sesbania (Sesbania grandiflora) (SG),

• Leucaena (Leucaena leucocephala) (LL),

• Gliricidia (Gliricidia sepium) (GS),

• Water hyacinth (Eichornia crassipes) (WH).

Heat treatment during 2 hours at:

• 60^oC,

• 100^oC,

• 140^oC.

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 140^oC 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:

Y_ijk = µ + A_i + B_j + AB_ij + e_ij

where Y_ijk is the protein fraction. µ the mean value, A_i the source of the leaf materials, B_j the effect of heating (60, 100 or 140^oC), AB_ij the interaction between leaf sources and heating level, and e_ij 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).

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

Non-protein nitrogen (NPN - Fraction A)

In the control treatment (60^oC) 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 (140^oC) the reductions were 12, 15, 19, 38 and 58% for SG, LL, WH, CLM and GS, respectively compared with the treatment at 60^oC.

Fraction B₁

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

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).

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).

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).

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.

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 et al (2000) for soybean meal and fish meal subjected to heat treatment of 135^oC for 30 minutes. Jirí et al (1990) reported that one of the first noticeable changes of proteins on heating (even at temperatures around 100^oC) 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 150^oC, 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).

Conclusions

• The implication from the findings of our study, and those of Dung (2001), are that the B₂ fraction may be a better indicator of the probable bypass protein value of leaves than the B­₃ fraction.

• Heating the leaves to temperatures of 140ºC for 2 hours reduced the proportion of the protein in the A (NPN) and B₂ fractions and increased the B₃ fraction. However, the relationship of these changes to the nutritive value of the crude protein in the leaves may well be negative.
   

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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